EGF-induced Vacuolar (H+)-ATPase Assembly: A Role in
Signaling via mTORC1 Activation
by
Yanqing Xu
Faculty of Medicine
Division of Experimental Medicine
McGill University
Montreal, Quebec, Canada
January 2012
A thesis submitted to McGill University in partial fulfillment of the
requirements of the degree of Doctor of Philosophy
© Yanqing Xu, 2012
Abstract
Using proteomics and immunofluorescence we demonstrated epidermal
growth factor (EGF) induced recruitment of extrinsic V1 subunits of the vacuolar
(H+)-ATPase (vATPase) to rat liver endosomes (ENs). This was accompanied by
reduced vacuolar pH. Bafilomycin, an inhibitor of vATPase, inhibited EGFstimulated DNA-synthesis and mammalian target of rapamycin complex 1
(mTORC1) activation as indicated by a decrease in 4E-BP1 phosphorylation, and
p70S6K phosphorylation and kinase activity. There was no corresponding
inhibition of EGF-induced Akt and Erk activation. Chloroquine, a neutralizer of
vacuolar pH, mimicked bafilomycin’s effects. Bafilomycin did not inhibit the
association of mTORC1 with Raptor nor did it affect AMPK activity. Rather, the
intracellular concentrations of essential but not non-essential amino acids, were
decreased
by
bafilomycin
in
EGF-treated
primary
rat
hepatocytes.
Cycloheximide, a translation elongation inhibitor, prevented the effect of
bafilomycin on amino acids levels and completely reversed its inhibition of EGF
induced mTORC1 activation. In vivo administration of EGF stimulated the
recruitment of Rheb but not mTOR to endosomes and lysosomes. This was
inhibited by chloroquine treatment. Our results suggest a role for vacuolar
acidification in EGF signaling to mTORC1.
ii
Résumé
A l’aide des techniques de Protéomique et d’immunofluorescence, nous avons
démontré le recrutement des sous-unités extrinsèques V1 de l’(H+)-ATPase
(vATPase) par les endosomes du foie de rat suite à une stimulation à l’EGF
(Epidermal Growth Factor). Ce recrutement s’accompagne d’une réduction du
pH vacuolaire. L’utilisation de la Bafilomycine, un inhibiteur de la vATPase,
inhibe la synthèse d’ADN et de l’activation de mTORC1 (mammalian target of
rapamycin complex 1), indiqués par la diminution de la phosphorylation de 4EBP1, et de la phosphorylation et de l’activité enzymatique de p70S6k. Toutefois,
nous n’avons pas observé d’inhibition de l’activation de Akt et de Erk également
induites par l’EGF. La Chloroquine, un neutralisateur du pH vacuolaire, imite les
effets vus avec la Bafilomycine. Dans des hepatocytes primaires de rat traités
avec l’EGF, la Bafilomycine n’inhibe pas l’association de mTORC1 avec Raptor,
pas plus qu’elle n’affecte l’activité de AMPK, par contre elle diminue les
concentrations intracellulaires des acides aminés essentiels, mais pas des acides
aminés non-essentiels. La Cycloheximide, un inhibiteur de l’élongation de la
traduction, prévient les effets de la Bafilomycine sur les niveaux d’acides aminés,
et renverse complètement son effet inhibiteur sur l’activation de mTOR induite
par l’EGF. De plus, l’administration d’EGF in vivo induit le recrutement de Rheb
dans les endosomes et les lysosomes, mais pas celui de mTOR, et cette induction
est inhibée par l’administration de Chloroquine. Dans l’ensemble, nos résultats
suggèrent donc un rôle pour l’acidification vacuolaire dans la signalisation de
l’EGF.
iii
Acknowledgements
First and foremost, I express my gratitude to my supervisor Dr. Barry I.
Posner for his mentorship, patience and unwavering support which have allowed
me to develop my skills and my research. Over the course of my Ph.D. studies,
his unceasing interest and enthusiasm for science have been a source of both
inspiration and motivation.
A well-deserved and special thanks go to my thesis committee members and
advisors, Dr. Geoffrey N. Hendy, Dr. Mark Trifiro, Dr. Louise Larose and Dr.
Vincent Giguere, for all the insightful discussions and guidance they have given
me over the years.
A warm word of thanks goes to Dr. Stephanie Chevalier. Not only has her
advice and insight proven invaluable regarding my amino acid study project, but it
has also been a delight to interact with her.
Heartfelt thanks go to Dr. Posner’s technicians, Gerry Baquiran and Victor
Dumas, for their friendship and technical support. I especially appreciate Victor‘s
help with the figures in this thesis. I would also like to extend my gratitude to
Ginette Sabourin of the McGill Nutrition Center, for her help with the HPLC
analysis of amino acids in hepatocytes.
My most sincere thanks go to our laboratory manager, Mary Lapenna, for the
assistance, kindness and professionalism she has shown me during all these years.
iv
During my time in Dr. Posner’s laboratory, I have been blessed with a
friendly and motivating group of people on a daily basis. I would like to thank the
previous as well as current fellows and students: Dr. Amanda Parmar, Dr.
Alejandro Balbis, Dr. Emmanuelle Roux, Ye Wang and Jingwen Song. Their
friendship and support have made my work at the laboratory a rewarding
experience. Thank you in particular to Dr. Amanda Parmar, who initiated this
project, for her inspiration.
Thank you to my good friend Leonard Leung for his friendship and moral
support, and I wish him all the best for his Ph.D. studies.
Last but not least, my wholehearted thanks go to my family, my parents and
sisters, for their endless love and support throughout my life. Without the
understanding, support and encouragement my parents have shown me, I could
never have completed my Ph.D. studies, and therefore I would like to dedicate
this thesis to them.
v
Preface
This thesis is presented in the traditional format in accordance with the
guidelines of the National Library of Canada. As chapters for this thesis, I have
included Chapter 1 as an Introduction and literature review. Chapter 2 present the
Methods and Materials. Chapters 3 presents Results obtained. Chapter 4 presents my
Discussion and summary. Finally, Chapter 5 presents Contribution to original knowledge.
The references appear at the end of the thesis.
The data presented in this thesis is the original work of the candidate which
has been submitted:
Yanqing Xu, Amanda Parmar, Emmanuelle Roux, Alejandro Balbis,
Victor Dumas, Stephanie Chevalier and Barry I. Posner. EGF-induced
Vacuolar (H+)-ATPase Assembly: A Role in Signalling via mTORC1
Activation. (Submitted for publication)
The candidate was also involved in another project whose research is not
presented in this thesis, but has contributed to the following journal article:
Evgeny Kanshin, Michael Fedjaev, Yanqing Xu, Katerina Vetrogon,
Marcos R. DiFalco, Mila Ashmarina, Ilya Nifant’ev, Barry I. Posner and
Alexey V. Pshezhetsky. Global analysis of protein phosphorylation
networks in insulin signaling by sequential enrichment of phosphoproteins
and phosphopeptides. Mol. BioSyst., 2012, 8 (5), 1461 – 1471.
- Did the phosphoprotein analysis and data validation
vi
Table of Contents
Abstract………………………………………………………………………....ii
Résumé……………………………………………………………………….....iii
Acknowledgements…………………………………………………………..iv
Preface…………………………………………………………………………..vi
Table of Contents…………………………………………………………....vii
List of Figures…………………………………………………………………xi
List of Tables………………………………………………………………...xiii
List of Abbreviations……………………………………………………....xiv
Chapter 1 Introduction and Literature Review ………………………1
1.1 Epidermal growth factor receptors (EGFRs)………………………………2
1.1.1 Initial signaling events…………………………………………………...2
1.1.2 Endosome signaling……………………………………………………...6
1.1.3 Detergent-resistant membranes (DRMs) in signaling ………………….10
1.1.4 EGFRs in liver proliferation……………………………………………12
1.2 Vacuolar H+-ATPase (vATPase) …………………………………………..14
1.2.1 Structure of vATPase…………………………………………………...14
1.2.1.1 Molecular architecture of vATPase………………………………14
1.2.1.2 Multiple subunit isoforms of vATPase…………………………...17
1.2.2 Assembly and targeting of the vATPase………………………………...17
1.2.2.1 Assembly ………………………………………………………….17
1.2.2.2 Role of subunit ‘a’ isoforms in intracellular targeting ……………18
1.2.3 Regulation of vacuolar acidification…………………………………….19
1.2.3.1 Reversible assembly/disassembly of V1 and V0 domains …………19
1.2.3.2 Regulation of vATPase coupling efficiency between ATP hydrolysis
and proton pumping ……………………………………………….22
1.2.4 Functions of vATPase …………………………………………………..23
1.2.4.1 Functions of intracellular vATPase……………………………….23
1.2.4.2 Functions in the plasma membrane………………………………..28
vii
1.2.5 Modulations in vATPase function in disease ………………………….29
1.3 Mammalian target of rapamycin (mTOR)………………………………..30
1.3.1 Structure and organization of mTOR complexes……………………….30
1.3.1.1 mTOR domain structure…………………………………………..30
1.3.1.2 Organization of mTOR complexes……………………………….33
1.3.2 Downstream of mTOR complex 1(mTORC1) ………………………….35
1.3.2.1 Protein synthesis…………………………………………………..35
1.3.2.2 Ribosome biogenesis………………………………………………39
1.3.2.3 mTORC1 regulation of autophagy ………………………………..39
1.3.2.4 mTORC1 and metabolism………………………………………...40
1.3.3 Upstream regulation of mTORC1…………………………………….....43
1.3.3.1 mTORC1 activation by growth factors…………………………....43
1.3.3.2 mTORC1 regulation by energy……………………………………47
1.3.3.3 mTORC1 regulation by stress……………………………………..48
1.3.3.4 mTORC1 regulation by amino acids……………………………...48
1.4 Objectives of the current work…………………………………………...53
Chapter 2 Materials and Methods ……………………………………...54
2.1 Materials and animals……………………………………………………...55
2.2 Cell culture and liver fractionation………………………………………..56
2.2.1 Primary hepatocytes culture…………………………………………...56
2.2.2 HepG2 and FAO cell culture………………………………………….56
2.2.3 Preparation of microsomes and endosomes from rat liver……………56
2.2.4 Preparation of lysosomes from rat liver………………………………...57
2.2.5 Isolation of endosomal detergent resistant membranes (DRMs) from rat
liver…………………………………………………………………….57
2.3 Protein analysis…………………………………………………………….57
2.3.1 Preparation of cell lysates analysis …………………………………….57
2.3.2 Protein quantification…………………………………………………..58
2.3.3 Immunoprecipitation and Immunoblotting…………………………….58
2.3.4 Proteomic sample preparation and analysis…………………………....59
viii
2.4 Immunofluorescence and DAMP labelling………………………………..59
2.4.1 Immunofluorescence studies…………………………………………...59
2.4.2 DAMP labeling of vesicles in rat primary hepatocytes……………….61
2.5 [3H] Thymidine incorporation assay………………………………………62
2.6 In vitro S6 kinase assay…………………………………………………….62
2.7 7-Methyl-GTP (m7GTP) –Sepharose 4B pull-down assay ………………62
2.8 Amino acid analysis………………………………………………………...63
2.8.1 Leucine uptake assay…………………………………………………...63
2.8.2 Total intracellular amino acid analysis…………………………………63
2.8.3 Amino acid analysis by HPLC………………………………………….63
2.9 Statistical Analysis………………………………………………………….64
Chapter 3 Results …………………………………………………………. 65
3.1 EGF-induced recruitment of V1 subunits to the vacuolar system………66
3.1.1 Proteomic analysis of rat liver endosomes …………………………….66
3.1.2 EGF promotes recruitment of V1 subunits of vATPase to the vacuolar
system …………………………………………………………………..83
3.1.3 EGF increases the acidification of the vacuolar system………………..84
3.2 Effect of inhibiting vacuolar acidification on EGF action ……………91
3.2.1 Effect of bafilomycin on EGF-induced mitogenesis……………………91
3.2.2 Effect of bafilomycin on EGF-induced Akt and Erk signaling………...94
3.2.3 Bafilomycin inhibits EGF induced mTORC1 activation……………….94
3.2.4 Chloroquine mimics the effect of bafilomycin………………………..101
3.2.5 Effect of bafilomycin on insulin signaling ……………………………101
3.3 The role of acidification in mTORC1 activaiton………………………...107
3.3.1 Bafilomycin does not alter mTOR and Raptor association, energy status
of the cell and Akt effect on TSC2……………………………………107
3.3.2 Effect of bafilomycin on PRAS40…………………………………….111
3.3.3 Effect of bafilomycin on intracellular amino acid levels ……………111
ix
3.3.4 Effect of cycloheximide on mTORC1 activation and intracellular amino
acid levels……………………………………………………………118
3.3.5 MG132 mimics the effect of bafilomycin on mTORC1 activation…...126
3.3.6 Effect of in vivo chloroquine on mTOR signaling…………………….129
Chapter 4 Discussion and Summary…………………………………..133
Chapter 5 Contribution to Original Knowledge …………………..145
Chapter 6 References …………………………………………………….147
x
List of Figures
Figure 1.1 EGF cell surface signaling…………………………………………...5
Figure 1.2 EGF endosomes signaling.…………………………………………..9
Figure 1.3 Structural and mechanistic model of vATPase expressed in endosome
membrane.………………………………………………………….16
Figure 1.4 Schematic structure of mTOR.……………………………………..32
Figure 1.5 Regulating cap-dependent translation initiation.…………………...38
Figure 1.6 Model of mTOR signaling network.………………………………..45
Figure 1.7 Model for amino-acid induced mTORC1 activation.………………52
Figure 3.1 Proteomic analysis of EN-DRMs reveals a large number of
proteins……………………………………………………………...68
Figure 3.2 Proteomic analysis of EN-DRMs/rafts reveals an EGF-dependant
change in abundance of vATPase V1, but not V0 intrinsic subunits.81
Figure 3.3 EGF promotes recruitment of V1 subunits of vATPase to late
endosomes- lysosomes and increases their acidification.…………..86
Figure 3.4 Effect of bafilomycin on EGF- induced mitogenesis, and EGFR
content and tyrosine phosphorylation in rat hepatocytes…………...92
Figure 3.5 Effect of bafilomycin on EGF-induced Akt and Erk signaling…….95
Figure 3.6 Effect of bafilomycin on EGF-induced mTORC1 signaling……….98
Figure 3.7 Effect of chloroquine on EGF-stimulated mTORC1, Akt and Erk
activation…………………………………………………………..102
Figure 3.8 Similar effect of bafilomycin on Insulin and EGF-stimulated
mTORC1, Akt and Erk activation…………………………………105
Figure 3.9
Bafilomycin does not alter mTOR and Raptor association, energy
status of the cell and Akt effect on TSC2………………………...108
xi
Figure 3.10 Effect of bafilomycin on PRAS40 phosphorylation, intracellular
amino acid levels and leucine uptake of the cell…………………114
Figure 3.11 Effect of cycloheximide on mTORC1 activation and intracellular
amino acid levels…………………………………………………120
Figure 3.12 Effect of cycloheximide on p70S6K phosphorylation and REDD1
protein expression………………………………………………..127
Figure 3.13 Effect of in vivo chloroquine on mTOR signaling.………………130
Figure 4.1 Model of EGF induced mTORC1 activation…………………….144
xii
List of Tables
Table 3.1 Basic results of EN-DRM proteomic analysis………………………67
Table 3.2 Determination by proteomic analysis of proteins changing in ENDRMs following EGF………………………………………………..70
Table 3.3 Functional categorization of the proteins changing in EN-DRMs
following EGF……………………………………………………….79
Table 3.4 Concentrations of amino acids (AAs) in medium and primary
hepatocytes (nmol/ml) before and after treatment with EGF and
bafilomycin (Baf) ………………………………………………….117
Table 3.5 Concentrations of amino acids (AAs) in primary hepatocytes (nmol/ml)
after treatment with EGF with bafilomycin(Baf) and cycloheximide
(CHX) ……………………………………………………………...125
xiii
List of Abbreviations
4E-BP1
eukaryotic initiation factor 4E-binding 1
ACC
acetyl-CoA carboxylase
AMPK
AMP-activated protein kinase
AP-1
activator protein 1
ARF1
ADP-ribosylation factor 1
ARNO
ADP-ribosylation factor nucleotide site opener
bHLH-Zip
basic helix-loop-helix-leucine zipper
C/EBP-α
CCAAT/enhancer binding protein-α
CBP80
cap-binding protein of 80 kDa
DAMP
3-(2, 4-dinitroanilino)-3’-amino-N-methyldipropylamine
Deptor
DEP-domain-containing mTOR-interacting protein
DOPC
dioleoyl -phosphatidylcholine
DRMs
detergent resistant membranes
dRTA
distal renal tubular acidosis
ECM
extracellular matrix
ECV
endosomal carrier vesicles
EEA1
early endosome antigen 1
eEF2K
eukaryotic elongation factor 2- kinase
EGF
epidermal growth factor
EGFR
epidermal growth factor Receptor
eIF
eukaryotic initiation factor
ENs
endosomes
ER
endoplasmic reticulum
Erk
extracellular-signal-regulated kinase
FASN
fatty acid synthase
FAT
Frap, ATM, and TRAP PIKK-like
FAT/C
FAT domain C-terminal
F-ATPase
F-type ATP synthase
FIP200
200-kDa focal adhesion kinase family-interacting protein
xiv
FK506
Fujimycin or Tacrolimus, an immunosuppressive drug
FKBP12
FK506-binding protein 12
FRB
FKBP12-rapamycin binding
GAP
GTPase activating protein
Grb2
growth-factor-receptor-bound protein 2
GSK3
glycogen synthase kinase 3
HA
hemagglutinin
HIF1α
hypoxia-inducible Factors 1α
HM
hydrophobic motif
hVps34
human vacuolar protein-sorting associated protein 34
IGFR
insulin-like growth factor receptor
IGFs
insulin-like growth factors
IRS
insulin receptor substrate
JAK
Janus kinase
LAMP1
Lysosomal-associated membrane protein-1
LAT1
L-type amino acid transporter 1
m7GTP
7-Methyl-GTP
MAP4K3
mitogen-activated protein kinase kinase kinase kinase 3
MAPK
mitogen-activated protein kinase
MBCD
methyl-β-cyclodextrin
mLST8
mammalian lethal with Sec13 protein 8
MP1
MAPK scaffold protein 1
mRNA
messenger RNA
mSIN1
mammalian stress-activated protein kinase interacting protein 1
mTOR
mammalian target of rapamycin
mTORC
mammalian target of rapamycin complex
MVBs
multivesicular bodies
NRD
negative regulatory domain
PDCD4
programmed cell death 4
PDGFR
platelet-derived growth factor receptor
PDK1
3-phosphoinositide-dependent protein kinase 1
xv
PdtIns
phosphatidylinositol
PH
partial hepatectomy
PI3P
phosphatidylinositol 3 phosphate
PIKK
phosphoinositide 3-kinase -related protein kinases
PIP2
phosphatidylinositol-4,5-phosphate
PIP3
phosphatidylinositol-3,4,5-phosphate
PKC
protein kinase C
PLCγ
phospholipase C γ
PM
plasma membrane
Pol
RNA polymerase
PPAR-γ
peroxisome proliferator-activated receptor-γ
PRAS40
proline-rich AKT substrate 40 kDa
Protor-1
protein observed with Rictor-1
PTB
phosphotyrosine-binding
Raptor
regulatory-associated protein of mTOR
RAVE
regulator of the (H+)-ATPase of vacuolar and endosomal
membranes
REDD1
DNA damage response 1
Rheb
Ras homologue enriched in brain
Rictor
Raptor-independent companion of mTOR
RNAi
RNA interference
Rom2
Rho1 GDP–GTP exchange protein 2
RP
ribosomal protein
RPS6
ribosomal protein S6
rRNA
ribosomal RNA
RTKs
receptor tyrosine kinases
S6K
ribosomal S6 protein kinase
sAC
soluble adenylyl cyclase
Sc
Saccharomyces cerevisiae
SCD-1
stearoyl-CoA desaturase 1
SCF
Skp1-Cdc53- F-box
xvi
SGK
serum- and glucocorticoid-regulated kinase
SH2
Src homology 2
SKAR
S6K1 Aly/REF-like target
SLC1A5
system A amino acid transporter solute carrier family 1 member 5
SLC7A5
solute carrier family 7 member 5
SNARE
soluble N-ethylmaleimide-sensitive factor attachment protein
receptors
SOS
son-of-sevenless
SRE
sterol regulatory element
SREBPs
sterol regulatory element binding proteins
STAT
signal transducer and activator of transcription
STE20
sterile 20
TOP
terminal oligopyrimidine
TOS
mTOR signaling
TSC
tuberous sclerosis complex
UPS
ubiquitin proteasome system
ULK1
unc-51-like kinases 1
vATPase
vacuolar (H+)-ATPase
Vps15
vacuolar protein-sorting associated protein 15
xvii
Chapter 1
Introduction and Literature Review
1
1.1
Epidermal growth factor receptors (EGFRs)
Epidermal growth factor (EGF) was first detected in the 1960s in mouse
submaxillary gland as a protein that induced precocious eyelid opening and tooth
eruption of newborn mice [1]. It is a mitogen consisting of a single polypeptide
chain of 53 amino acid residues, including six Cys residues that form three intramolecular disulfide bonds [2]. EGF is widely distributed in many tissues, and has
been identified in most body fluids of mammalian species. It has been shown to
be important in mammalian development and function [2].
EGF binds to its cell surface receptor (EGFR), an approximately 170,000 Mr
transmembrane glycoprotein, that belongs to the receptor tyrosine kinase (RTK)
family [3]; and was the first RTK to be cloned [4]. EGFR mediates the biological
signals of a family of EGF-like growth factors of which EGF and transforming
growth factor-a (TGFα) are the best characterized family members [3]. Though
EGF and TGFα have similar actions, TGFα is more potent than EGF in
stimulating angiogenesis and in releasing calcium from bone [5, 6].
1.1.1
Initial signaling events
As with many other RTKs, EGFR-dependent signal transduction begins with
the stabilization of a receptor dimer through ligand binding, followed by the
activation of the intrinsic tyrosine kinase and tyrosine autophosphorylation at the
C-terminus of the receptor [7]. Phosphorylation of the EGFR C-terminus, effected
by transphosphorylation [8] provides specific docking sites for the Src homology
2
2 (SH2) or phosphotyrosine-binding (PTB) domains of intracellular signal
transducers and adaptors, leading to the assembly of signaling complexes [9]. The
interactions of the receptor with SH2 and PTB domains are essential steps in the
sequence of signaling activated by growth factors.
As with insulin signaling in which a key role for docking proteins (i.e. insulin
receptor substrates (IRS-1 -4) has been well documented [10], the Gab family
proteins constitute docking proteins involved in EGFR signaling. They have a
proline-rich domain (PRD) that binds adaptor protein growth-factor-receptorbound protein 2 (Grb2) and multiple tyrosine-based potential binding sites for
SH2-containing proteins, such as phosphatidylinositol 3' –OH kinase (PI3-kinase)
and protein-tyrosine phosphatase (PTPase) SHP2 [11-13]. Activated EGFR
effects phosphorylation of Gab-1 and Gab-2 on tyrosine residues, providing
binding sites for multiple proteins involved in signal transduction [10](Figure 1.1).
The cascade from EGFR activation to the stimulation of the proto-oncogene
Ras GTPase, and eventually of the extracellular-signal-regulated kinase (Erk)
42/44 [mitogen-activated protein kinase (MAPK)] has been examined [14].
Adaptor protein Grb2 is essential for EGF-dependent Ras activation [14]. The
SH2 domain of Grb2 binds to the tyrosine phosphorylated EGFR [15], whereas
the SH3 domain of Grb2 constitutively binds to the Ras guanidine-nucleotideexchange factor, son-of-sevenless 1(SOS1) [16]. Grb2 can also bind to the EGFR
indirectly, by binding to the EGFR-associated tyrosine phosphorylated Shc [17].
Binding of Grb2 to the EGFR recruits the Grb2–SOS complex to the membrane in
3
proximity to the membrane-anchored Ras, thus linking EGFRs to the Ras/MAPK
signaling pathway (Figure 1.1).
EGFR kinase triggers simultaneous activation of multiple downstream
pathways. These pathways include the Ras –MAPK cascade, the Janus kinase
(JAK), the signal transducer and activator of transcription (STAT) cascade, the
pathways involved in phospholipid metabolism (PLD, phospholipase C (PLCγ)
and its downstream calcium- and PKC-mediated cascades), as well as the
phospholipid-directed enzymes PI3-kinase cascade, and the non-receptor tyrosine
kinases - Src family kinases (reviewed in [7]).
.In this review, we focus on the MAPK, Pl3-kinase and mTOR pathways
which we have found to be important in the regulation of EGF-induced
mitogenesis.
4
Figure 1.1
Figure 1.1 EGF cell surface signaling.
Binding of ligand to each EGFR monomer promotes and stabilizes the dimer
configuration thus permitting tyrosine autophosphorylation. The tyrosinephosphorylated EGFR can phosphorylate substrates Gab-1 and Gab-2 and / or,
recruit adaptor molecules via their SH2 domains i.e. growth-factor-receptorbound protein 2 (Grb2). Both processes lead to enzyme activation and signaling
cascades.
5
1.1.2 Endosome signaling
The activated EGFR is rapidly internalized into early endosomes(ENs) [18],
and subsequently undergoes recycling to the plasma membrane or is sorted to late
endosomes-lysosomes [7, 19, 20]. The classical view considered that the receptor
was activated exclusively at the plasma membrane, and that cell signaling was
shut down by receptor endocytosis and degradation [21]. However, the concept
that endocytosis does not simply lead to receptor degradation, but is necessary to
initiate, extend and augment downstream signaling is now broadly accepted [2224]. Several studies have shown that signaling molecules are recruited to
endosomes following both EGF and insulin activation [25, 26], thus confirming
the endosome as a site of cellular signaling. These studies employed sucrose
gradient fractionation of rat liver, demonstrating an enrichment of signaling
molecules which associate with the endosomal fraction. Both signaling adaptor
proteins Grb2, SOS and Shc [25] and the p85 subunit of the PI3K) [26] were
enriched in the endosomal fraction in response to RTK activation.
Inhibiting internalisation of receptors and trapping them at the plasma
membrane has been used to study the role of endocytosis in signaling. By
exposing cells to low temperature(15 °C),to halt receptor trafficking , Chow et al
showed that insulin-like growth factor receptor (IGFR) signaling to Shc and Erk
required endocytosis of the receptor[27]. Dynamin is a molecular component
required for clathrin coated vesicle formation. By over-expressing the dominant
negative K44A dynamin mutant, Vieira et al. showed that clathrin-mediated
EGFR endocytosis is required for full activation of Erk signaling [28].
6
Similar
observations
on
changes
in
EGF-dependent
endosomal
phosphoproteins have been made by Stasyk et al [29]. All together 23 EGFregulated (phospho) proteins were identified as being differentially associated
with endosomal fractions by functional organellar proteomics. Among them, RRas, a small GTPase of the Ras family, associated with late endosomes in a
ligand-dependent manner.
Teis et al. have found that endosomal localization of the adaptor protein
p14/MP1-MAPK scaffold complex is crucial for EGFR signal transduction
(Figure 1.2). It is responsible for localization of Mek-Erk to the late endosomal
compartment [30, 31]. Subsequently, it was revealed that the p14-MP1-Mek
signaling complex is tethered to the cytoplasmic surface of late endosomes by p18,
which is anchored to lipid rafts of late endosomes through its N-terminal unique
region [32, 33]. Knocking down of MP1, P14 or P18 protein resulted in defective
signaling [30-32].
The above observations confirm that endocytosis allows for both temporal
and spatial organization of signaling from the cell surface throughout the
endosomal system thus providing an extended surface for interaction between
membrane bound receptors and cytosolic substrates; a concept first formulated in
studies on the endocytosis of insulin [34].
Besides sustaining and amplifying, there is also evidence that signals can be
uniquely generated within the endosomal compartment [35-37]. By treating cells
with EGF in the presence of AG-1478, a specific EGFR tyrosine kinase inhibitor,
7
and monensin which blocks the recycling of EGFR, Wang et al. established a
system to specifically activate EGFR when it is concentrated into endosomes.
Using this system, Wang et al. showed that endosomal signaling was sufficient for
activating the major signaling pathways of EGFR leading to cell proliferation and
survival [36].
Therefore, endosomes are a key site for mediating and modulating peptide
hormone and growth factor signaling. The endocytosis of activated receptors
provides temporal and spatial regulation in the signaling cascade.
8
Figure 1.2
Figure 1.2 EGF signaling in endosomes signaling.
Endosomal localization of the adaptor protein p18-p14-MAPK scaffold
protein 1(MP1)-MEK scaffold complex is crucial for EGFR signal transduction.
The p18-p14-MP1-MEK signaling complex is tethered to the cytoplasmic surface
of late endosomes by p18, which is anchored in lipid rafts of late endosomes
through its N-terminal unique region. EGFR is internalized and concentrated in
late endosomal rafts as well, where its proximity to the MEK–Erk complex could
lead to prolonged Erk activation.
9
1.1.3 Detergent-resistant membranes (DRMs) in signaling
Spatial control could be further modulated by activation of signaling
complexes in membrane subcompartments such as lipid domains. Membrane lipid
domains (lipid rafts) are heterogeneous, highly dynamic microdomains (10–200
nm) enriched in cholesterol, sphingolipids, and phospholipids with saturated acyl
chains [38]. They have lower buoyant density than bulk plasma membrane [39, 40]
and were initially proposed to play a role in sorting Golgi proteins to the apical
plasma membrane (PM) of polarized MDCK epithelial cells [41]
Lipid rafts are also characterized by their insolubility in non-ionic detergents
such as Triton X-100 or CHAPS at low temperatures. Extraction of cellular
membranes in cold 1% Triton X-100, followed by sucrose density gradient
centrifugation has been used to isolate detergent-insoluble, low-density
membranes (DRMs). Characterization of DRMs prepared from subcellular
fractions has been used to infer the biochemical characteristics of lipid rafts,
which can be viewed as sub-compartments of a given organelle, such as
endosomes [38]. Although DRMs do not represent intact rafts in vivo, it is
recognized that proteins co-purifying with DRM have a high affinity for these
lipid domains [42]. DRMs have been identified in a number of organelles
including plasma membrane (PM) [43], endoplasmic reticulum(ER) [44], Golgi
[45], and endosomes [46, 47].
In response to signaling, highly ordered lipid rafts may fuse into larger and
more stable structures resulting in the formation of efficient signaling platforms
[39]. They have been shown to be involved in modulating membrane trafficking
10
and signal transduction [39, 48, 49]. It has been reported that several transmembrane receptors were associated with lipid rafts [46, 50-54], including the
EGFR [46, 52]. The concentration of EGFR in lipid rafts is thought to have an
effect on the two principal functions of the EGFR: ligand binding and the
activation of signaling cascades. Adding cholesterol and phosphatidylinositol
(PdtIns) into dioleoyl -phosphatidylcholine (DOPC) liposome vesicles, into which
EGFR was also incorporated, resulted in an enhanced EGF-binding to EGFR [55].
Zhuang et al. reported that disruption of lipid rafts inhibited EGF receptor and Akt
signaling and reconstitution of the rafts with cholesterol restored EGFR-Akt axis
signaling both in vivo and in vitro [56, 57]. Puri et al. revealed that EGF
stimulation could induce the recruitment of EGFR, as well as signaling adaptor
proteins (i.e., Shc and Grb2) to DRMs [58]. Balbis et al. have also observed that
DRMs in endosomes and PM are greatly enriched in tyrosine phosphorylated
EGFR and downstream signaling molecules (i.e. Shc and Grb2) consistent with a
role for lipid rich membrane domains in EGF signaling [46, 59].
Conversely, several studies have reported that disruption of membrane rafts
by cholesterol depletion using methyl-β-cyclodextrin (MBCD) increased EGFbinding, and stimulated ligand-independent EGFR tyrosine kinase activity and
subsequent downstream signaling to Erk [60-63]. This argues that EGFR
signaling is suppressed in lipid rafts. It has been suggested that the conflicting
results from these groups were due to using MBCD at concentrations >5mM to
extract cholesterol from cells, since these concentrations of MBCD might not only
11
disrupt the membrane raft domains but also affect the integrity of the whole
membrane [64].
1.1.4 EGFRs in liver proliferation
The liver is well recognized as a biological system in which to study cell
proliferation and differentiation. EGFR is highly expressed in the adult liver and
EGFR-dependent signaling contributes to liver cell proliferation, representing an
important regulator of hepatic regeneration [65, 66].
Hepatocytes of normal adult liver are quiescent cells, arrested in G0. They
acquire a remarkable ability to proliferate after injury. Partial hepatectomy (PH) is
one of the models used to investigate liver regeneration in rodents [67]. During
liver regeneration after PH, normally quiescent hepatocytes undergo a transition
from G0 to G1, followed by one or two rounds of replication to restore liver mass
by a process of compensatory hyperplasia, before returning to the quiescent state.
The regeneration of liver results in the complete restoration of hepatic architecture
and tissue specific function [68].
The mechanisms underlying hepatic regeneration have been extensively
studied. In the acute-phase induced after PH, the earliest event observed is the
rapid and sequential induction of immediate early genes for c-fos, c-jun, and cmyc [69-71] mediated by different cytokines such as TNFα and IL-6 [67, 72],
which are undetectable in normal adult rat liver. Together with activator protein 1
(AP-1), STAT3, and NF-κB, they induce the expression of genes that encode cell
12
cycle regulators such as cyclin D [68, 73-75]. After replication hepatocytes leave
G1 and enter S phase, which is induced by up-regulation of a number of genes
such as cyclin E, cyclin A, and their kinases [72].
Growth factors that are highly expressed after PH such as TGFα, EGF,
heparin-binding EGF, and amphiregulin are important for inducing liver
regeneration [76-79]. Mice lacking amphiregulin were shown to have impaired
hepatocyte proliferation and delayed induction of cyclin D1 after PH [77]. In twothirds of PH mice, the expression of heparin-binding EGF-like growth factor (HBEGF) was shown to regulate the start of DNA replication, and thus a key factor
for progression through the G1/S transition during liver regeneration [80]. Mice
lacking hepatic EGFR display reduced hepatocyte proliferation with reduced and
delayed expression of cyclin D1 [81].
Band et al. have shown that in primary hepatocytes activation of the
mTORC1 pathway was critical to the induction of DNA synthesis by insulin and
EGF [82]. Inhibition of mTORC1 by rapamycin abrogated DNA replication [82,
83] as well as protein synthesis induced by growth factors. The expression of
cyclin D1 messenger RNA (mRNA) and protein levels were regulated by this
pathway [83].
13
1.2 Vacuolar H+-ATPase (vATPase)
Vacuolar H+-ATPase (vATPase) is a multisubunit enzyme that mediates
ATP-driven proton transport across membranes. It was discovered independently
in several laboratories working with animals, plants and fungi about 30 years ago.
It is now thought to be present in all eukaryotic cells as a regulator of acidification
[84]. vATPase resides within many intracellular compartments, including
endosomes, lysosomes and secretory vesicles, and has a function in processes
such as membrane trafficking, prohormone processing, receptor-mediated
endocytosis, protein degradation and the loading of synaptic vesicles [85].
vATPase is also present in the plasma membrane of certain cells, including renal
intercalated cells, osteoclasts and macrophages, where it plays an important role
in processes such as urine acidification, bone resorption and control of
cytoplasmic pH [86]. In accordance with its crucial roles in cellular function,
vATPase has been also implicated in various human diseases, including renal
tubular acidosis, osteopetrosis, and cancer (reviewed in [87]).
1.2.1 Structure of vATPase
1.2.1.1 Molecular architecture of vATPase
Yeast and mammalian vATPases share a high degree of similarity in their
subunit structure and biochemical mechanisms. The vATPase is a multimeric
complex divided into two distinct domains or sectors [84]- a peripheral catalytic
V1 domain (640 kDa), and a membrane-embedded V0 domain (240 kDa), together
forming a protein complex of ~900 kDa. The membrane-embedded V0 domain
14
carries out proton translocation and the peripheral V1 domain is responsible for
ATP hydrolysis (Figure 1.3). The V0 domain is composed of six different subunits
(ac4c′c″de) in which the hydrophobic subunits (c, c′, and c″) form a ring that sits
beside subunit ‘a’. The V1 domain is composed of eight different subunits with a
defined number of each subunit (A3B3CDEFG2H1-2). Functionally, three copies of
the A subunit alternate with three copies of the B subunit (A3B3) to form a
heterohexameric ring [88, 89], which is connected to the V0 domain by stalks.
Subunit A is responsible for ATP hydrolysis, while subunit B contains an ATP
binding site and plays a regulatory role. The D and F subunits form the central
stalk which interacts with the hydrophobic ring(c, c′, c″) through the ‘d’ subunit
and operates as a rotor [90]. The C, E, G, and H subunits together with the
cytosolic domain of the ‘a’ subunit form two peripheral stalks, which are attached
to the A3B3 hexamer and function as a stator (the stationary part of the rotor) [91,
92]. Hydrolysis of ATP by subunit A of the V1 domain drives the rotation of the
central stalk which is conveyed to the ring-like structure(c, c′, c″) in the V0
domain[93]. Part of the c-ring forms half of the proton channel which is
completed by a luminal (or extracellular) half-channel composed by the Cterminal domain of subunit ‘a’. The rotation of the c- ring against the stator drives
protons across the membrane [94] (Figure 1.3).
15
Figure 1.3
Figure 1.3 Structural and mechanistic model of vATPase expressed in
endosome membrane.
The peripheral V1 domain is composed of eight different subunits identified
with capital letters A-H. The integral membrane V0 domain is composed of six
different subunits identified with small letters (a, c, c′, c″, d, e). Subunit c and its
isoforms c′ and c″ form a H+-binding rotor ring. V0 and V1 domains are joined by
a central rotating stalk (subunits D, F) and two peripheral stationary stalks
(subunits C, E, G, H, a). The central stalk of the V1 domain interacts with the cring of the V0 domain through subunit ‘d’ to form the rotor (blue). The two
peripheral stalks attach to A3B3 hexamer to form the stator (red). Hydrolysis of
ATP by subunit A in the V1 domain drives clockwise rotation of the central stalk
together with the c-ring of the V0 complex. This rotation conveys H+ across the
membrane to acidify the lumen of the vacuole.
16
1.2.1.2 Multiple subunit isoforms of vATPase
Consistent with the presence of vATPase in diverse compartments, some of
the vATPase subunits are encoded by different genes and have various isoforms
which are tissue-specific and cell-specific in mammals. These include subunits B,
C, E, H, d which have two isoforms [95-97], subunit G with three isoforms and
subunit ‘a’ with four isoforms [96, 98]. Some subunit isoforms (i.e. B2, a1) are
found ubiquitously while the others are expressed in lung, kidney, osteoclasts,
epididymis or other tissues specifically [95, 97].
1.2.2 Assembly and targeting of the vATPase
1.2.2.1 Assembly
Assembly of the vATPase has been most extensively studied in yeast.
Deletion of individual vATPase subunits did not generally lead to loss of other
subunits [99]. However, loss of any subunit (except subunit H) led to the loss of
function and complete assembly; although vATPase can be assembled in the
absence of subunit H it is functionally inactive [84, 85]. V1 and V0 domains can be
assembled independently; the V0 domain can assemble and reach the vacuole in
the absence of the V1 subunits, and assembled V1 domains can be isolated from
mutants in absence of V0 subunits. In addition, a V1 sub-complex can be isolated
in the absence of V1 subunit C [100-102].
Assembly of the V0 domain is the early step in vATPase assembly. Several
assembly factors, including Vma12p, Vma21p and Vma22p are essential for
assembly of the V0. Both Vma12p and Vma21p are integral membrane proteins of
17
the ER, whereas Vma22p is a peripheral membrane protein that forms a complex
with Vma12p [103-105]. The Vma12p / Vma22p complex binds to the newly
synthesized V0 ‘a’ subunit in the ER, a step which seems necessary for exit of the
V0 domain from the ER [104, 105]. It has also been shown by coimmunoprecipitation experiments that Vma21p can bind directly to the ‘c′ subunit
in the hydrophobic ring of the V0 domain to form a complex that is able to recruit
subunit ‘d’ [106].
The association of V1 and V0 domains might occur before the V1 domain is
fully assembled. Complexes containing subunit ‘a’ bound to several V1 subunits
have been detected at an early stage of assembly [107]. It has also been shown
that the kinetics of association between V1 subunits A and B was slower than that
between V1 subunit A and V0 subunit ‘a’ [106]. These data suggest that parallel
pathways may be involved in the assembly of the vATPase.
1.2.2.2 Role of subunit ‘a’ isoforms in intracellular targeting the vATPase
Information for intracellular targeting of vATPases resides within the V0
subunit ‘a’. The largest subunit of the V0 domain, subunit ‘a’ is a 100-KDa
integral membrane protein containing a 50-KDa N-terminal cytosolic tail and a Cterminal hydrophobic domain with multiple putative membrane-spanning helices.
The targeting structure of vATPase is localized in the N-terminal domain [108]. In
yeast, subunit ‘a’ is the only V0 component that is encoded by more than one gene,
vph1 and stv1 [109]. In the worm, mice, and humans subunit ‘a’ is encoded by
four orthologous genes (a1-a4). The different isoforms of subunit ‘a’ have cell18
specific and intracellular compartment-specific distribution and thus appear to
target the vATPase complex to different intracellular compartments. In yeast, the
N-terminal cytosolic domain of subunit ‘a’ (Vph1p) targets vATPase to the
vacuolar compartment, and stv1p (another ‘a’ isoform) cycles between the Golgi
apparatus and prevacuolar endosome [109].
1.2.3 Regulation of vacuolar acidification
1.2.3.1 Reversible assembly/disassembly of V1 and V0 domains
Reversible dissociation of V1 and V0 domains is an important mechanism of
physiological regulation of vATPase [110] such as triggered by glucose depletion.
Dissociation of the V1 and V0 components of the holoenzyme was first observed
in insect tissue [111]. The reversible assembly/disassembly of V0 and V1
components was then documented in S.Cerevisiae [112] and renal epithelial cells
[113], and has been recognized as an important regulatory mechanism of
vATPase function [85, 114] widely conserved from yeast to mammalian cells
[112, 113, 115].
The reversible assembly/disassembly of the vATPase holoenzyme has been
mainly studied in yeast. Glucose deprivation resulted in disassembly of the
cytosolic V1 domain from the membrane bound V0 domain and inactivation of the
vATPase in yeast [115]. This process was rapid and did not require new protein
synthesis. None of the known glucose-induced signaling pathways were involved
in the disassembly. Restoration of extracellular glucose resulted in the rapid
19
reassembly of the free V1 complex with the V0 domain. These results established
that the activity of vATPase can be regulated at the level of assembly.
In yeast cells, glucose-induced reassembly of V1 and V0 requires the RAVE
complex (regulator of the (H+)-ATPase of vacuolar and endosomal membranes).
RAVE complex was first discovered in a proteomic analysis of the proteins
binding to Skp1p, which is known to be a component of SCF (Skp1-Cdc53- F-box)
ubiquitin ligases [116]. Skp1, Rav1 and Rav2 (Rav1 and Rav2 were
uncharacterized proteins and they were named on the basis of being the
components of RAVE complex) form the RAVE complex which associates with
the V1 domain of vATPase and promotes glucose-triggered assembly of the
vATPase holoenzyme. Subsequently it was shown that the RAVE complex could
also constitutively bind to cytosolic V1 in a mutant lacking V0 components; and
the association was not affected by changes in extracellular glucose. vATPase
complexes from cells lacking RAVE subunits showed serious structural and
functional defects even in glucose-grown cells [117]. It was also shown that
subunits E and G on the V1 peripheral stalk were critical for the binding of RAVE
to cytosolic V1 [117]. This suggests that RAVE may be important for docking of
the V1 peripheral stalk to V0. Further analysis found an interaction between the
RAVE complex and V1 subunit C, another subunit of the V1 peripheral stalk. In
the absence of the RAVE complex, subunit C was not able to stably assemble
with the vATPase [118]. Although the RAVE complex has, to date, only been
studied in yeast, the above data support a model where RAVE, through its
20
interaction with subunit C, docks the V1 peripheral stalk to V0 to facilitate
vATPase assembly.
In vATPase assembly, some of the vATPase subunits interact with enzymes
of the glycolytic pathway. It has been reported that the glycolytic enzyme aldolase
physically associates with the ‘a’, B, and E subunits of vATPase [119, 120]. The
association between aldolase and subunit B is important for proton pump function
[121]; and the binding of aldolase to vATPase in yeast cells increased
dramatically in the presence of glucose [120]. These data suggest that aldolase
may act as a glucose sensor and mediate vATPase assembly.
In mammalian cells there may be other mechanisms for triggering the assembly of
vATPase. In kidney epithelial cells, glucose activates vATPase activity through
the glycolytic pathway, and this activation requires PI3K activity [122]. In renal
epithelial cells, vATPase assembly and vATPase dependent acidification of
intracellular compartments is stimulated by glucose through PI3K dependent
signaling, but the factors linking glucose and PI3K are still unknown [113]. Carini
et al. also found a PI3K-dependent translocation and fusion of lysosomes with the
plasma membrane and the appearance of vATPase at the cell surface of isolated
hepatocytes. The inhibition of PI3K by wortmannin prevented the exocytosis of
lysosomes [123], further indicating that PI3K might be involved in the regulation
of the vATPase in mammalian cells. Recently, it has been shown that long-term
exposure of rat proximal tubules cells to angiotensin II (Ang II) caused upregulation of vATPase activity. This effect could be blocked by inhibition of PI3K
by wortmannin or of MAPK by SB 203580. Thus, the regulation of vATPase
21
assembly in mammalian cells seems not only dependent on mechanisms involving
PI3K activation but other kinase pathways as well
1.2.3.2 Regulation of vATPase coupling efficiency between ATP hydrolysis
and proton pumping
Role of specific subunits
Various subunits of the vATPase, including subunits a, d, A, and C, have been
implicated in regulating the activity of the vATPase in yeast by modulating the
coupling between ATP hydrolysis and proton pumping [97, 108, 124-127]. The
regulatory function of subunit H coupling ATP hydrolysis activity to proton
transport has been shown both in vivo and in vitro. Subunit H silences ATP
hydrolysis activity of the dissociated V1 domain, thereby minimizing
unproductive ATP hydrolysis in vivo [128]. Further studies have revealed that the
N-terminal domain of subunit H is required for the activation of the vATPase,
whereas the C-terminal domain is required for coupling ATP hydrolysis to proton
translocation [129-131]. This regulatory effect of subunit H was found not only in
yeast but also in mammalian cells, such as bovine brain vATPase [131]. Subunits
C, D, and A have also been shown to influence the coupling efficiency between
proton transport and ATP hydrolysis [125-127, 132].
Role of ATP
Early biochemical studies of vATPases suggested a loss of coupling
efficiency of vATPase at high ATP concentrations [133, 134]. Arai et al.
22
demonstrated that the bovine clathrin-coated vesicle vATPase had maximal rates
of proton pumping at ATP concentrations of approximately 0.3 mM, whereas
higher ATP concentrations decreased the pumping rate [133]. More recently,
Shao and Forgac provided further support demonstrating a decrease in coupling
efficiency observed at higher ATP concentrations for both wild type and subunit
A mutant vATPase [125].
There are likely to be other mechanisms modulating vATPase activity as well,
such as the roles of soluble adenylyl cyclase (cAMP) [135] and the actin
cytoskeleton [136] in vATPase recycling. Thus the regulation of vATPase is a
complex multilevel process.
1.2.4 Functions of vATPase
vATPase, as a specific proton pump of the cell, has a critical role in
controlling both intracellular and extracellular pH. vATPase is involved in
maintaining a relatively neutral intracellular pH, and an acidic luminal pH, as a
consequence of ATP-dependent proton transport from the cytoplasm into the
lumen of intracellular membrane-bound organelles and / or the extracellular
environment.
1.2.4.1 Functions of intracellular vATPase
vATPase plays an important role in both endocytosis and endosomal vesicular
trafficking. There are several sub-categories of endosomes, namely early
endosomes, recycling endosomes, and late endosomes/multivesicular bodies
23
(MVBs). The ratio of membrane associated V1 / V0 of vATPase varies along the
endocytic pathway and the relative abundance of V1 is higher in late endosomes
than in early endosomes. Notably, in a recent proteomic analysis, Lafourcade et al.
have found that all membrane-bound vATPase subunits are associated with
DRMs isolated from late endosomes. This raises the possibility that association
with lipid rafts plays a role in regulating the activity of the proton pump [137].
The constant activity of endosomal vATPase generates a pH gradient through
the endosomal system, ranging from pH
6.0 in early endosomes to pH 5.0–5.5
in lysosomes [138]. This is accomplished by a graded increase in the function of
vATPase. Thus during receptor-mediated endocytosis receptor-ligand complexes
are exposed to deceasing pH as they journey through the endosomal system [139].
The low pH within early endosomes initiates the dissociation of ligands from their
receptors, allowing those receptors to return to the cell surface through recycling
endosomes, while the ligands traffic to later intracellular compartments, such as
late endosomes and lysosomes where they are degraded.
Neutralization of endosomal compartments disturbs the dissociation of
internalized ligand–receptor complexes and recycling of unoccupied receptors to
the cell surface. There are three groups of pharmacological agents which have
been employed to inhibit vacuolar acidification. (1) specific inhibitors of vATPase
(such as bafilomycin and concanamycin), which bind to the V0 domain and
irreversibly inhibit pumping activity; (2) weak bases, which traverse membranes
and neutralize luminal protons, increasing vacuolar pH; and (3) ionophores (such
24
as monensin and nigericin) that can facilitate the movement of protons down the
electrochemical gradient.
Two of the most commonly used endosomal pH regulators are bafilomycin
and chloroquine. These inhibitors have different mechanisms of action, but both
result in a more neutral endosomal milieu. The specific inhibitor bafilomycin is a
plecomacrolide antibiotic which specifically and potently inhibits the vATPase
[140]. It has been shown to bind to both the V0a [141] and V0c [142, 143]
subunits, and is postulated to act by inhibiting rotation of the c-ring [142].
Inhibition of endosomal acidification by bafilomycin has been shown to modify
insulin signaling in adipose and liver cells [144, 145], and bafilomycin treatment
can also block EGFR degradation in NIH/3T3 Fibroblasts [146].
Chloroquine is a weak base which can cross biological membranes. It has
been used to treat malaria where its neutralizing effect on endosomal pH disrupts
the parasite’s life-cycle. Chloroquine was noted originally for its ability to inhibit
late-endosome/lysosomal proteolysis which occurs, not by directly inhibiting
proteases as initially thought, but rather by increasing the luminal pH rendering
the acidic proteases inactive [147]. Early studies using chloroquine showed that it
caused intracellular accumulation of
125
I-insulin in rat-liver [148]. The use of
isolated rat liver endosomes confirmed that low pH was responsible for
dissociation of insulin from its receptor, and that dissociation allowed the
degradation of insulin by endosomal proteases [149]. Later studies demonstrated
that there was also an accumulation of activated insulin receptors in rat liver
25
endosomes following chloroquine administration [150], as the receptor-ligand
interaction had not been disrupted.
In addition to activating ligand–receptor dissociation, vATPase is also
necessary for vesicular trafficking from early to late endosomes, which is another
step in the endocytic pathway and triggers the formation of endosomal carrier
vesicles (ECV). Formation of these vesicles requires the small GTP-binding
protein ADP-ribosylation factor 1(ARF1) which mediates the association of βCOPs to the membrane of the sorting endosomes - a process sensitive to
endosomal pH [151].
In proximal tubule epithelial cells, receptor-mediated endocytosis plays an
important role in protein homeostasis via re-absorption of albumin, hormones,
vitamin-binding proteins, etc. Budding of ECV from apical endosomes in these
cells requires the binding of the small GTPase, Arf6, and the ADP-ribosylation
factor nucleotide site opener (ARNO). The recruitment of these proteins from
cytosol to endosomal membranes is driven by vATPase-dependent intraendosomal acidification. In particular, the a2-isoform of vATPase is targeted to
early endosomes and interacts with ARNO in an intra-endosomal acidificationdependent manner. Inhibition of endosomal acidification abrogates protein
trafficking between early and late endosomal compartments. Thus vATPase plays
an essential role in the regulation of the endocytic degradative pathway and in
membrane trafficking [152].
26
The endocytic pathway is also exploited by certain enveloped viruses,
bacteria, and toxins to enter into cells. For example, The N-terminal domain of the
influenza hemagglutinin (HA), is the only portion of the molecule that enters
deeply into the membranes of cells to mediate the viral and host membrane-fusion.
The fusion between the viral and endosomal membrane is pH-activated [153, 154].
Emerging evidence also suggests a direct role for V0 in membrane fusion
during vesicular trafficking. Trans-SNARE (soluble N-ethylmaleimide-sensitive
factor attachment protein receptors) pairing mediates the attachment step in the
membrane fusion of vesicles in yeast. After SNARE mediated docking, V0
domains of vATPase on opposing membranes, form trans-complexes. The V0
trans-complexes form a continuous proteolipid-lined channel at the fusion site,
through a combination of the highly hydrophobic proteolipid subunits of two V0
domains thus promoting the association of the lipid bi-layers [155, 156]. It is not
yet clear whether participation of V0 is a general mechanism of membrane fusion.
Lysosomal vATPase provides a low pH environment, required for the
degradation of proteins and other macromolecules in this compartment. In
lysosomes and the vacuole of yeast, vATPase also generates the proton gradient
and/or the membrane potential to drive coupled transport of small molecules and
ions. For example, breakdown products of macromolecules such as amino acids
efflux from the lysosome into the cytoplasm by H+/amino acid cotransporters
driven by the vATPase generated proton gradient [84].
27
1.2.4.2 Functions in the plasma membrane
vATPase not only acidifies intracellular compartments, but also resides at the
plasma membrane of certain cells, such as renal cells, osteoclasts and insect
goblet cells where extracellular acidification is a critical function of these cells.
Functions in the kidney
Intercalated cells of the collecting duct express the highest levels of vATPase
among all acid-base transporting cells in the kidney. Acid-secreting type A renal
intercalated cells express vATPase and Na+/H+ exchanger at the apical membrane
and the Cl–/HCO3– exchanger at the basolateral membrane. This leads to the
extrusion of protons into the renal fluid, and HCO3- into the plasma thus playing
an important role in renal regulation of acid–base balance. In contrast, type B
renal intercalated cells express vATPase and the Na+/H+ exchanger on the
basolateral surface and the Cl–/HCO3– exchanger on the apical pole leading to the
secretion of bicarbonate under conditions of alkalosis [86].
Function in bone resorption
Osteoclasts are specialized macrophages involved in bone remodeling.
Osteoclasts attach to the bone’s matrix and create a sealed extracellular space,
where vATPases localized to the bone-facing apical plasma membrane of the
osteoclasts generate an acidic environment between the apical plasma membrane
and the bone surface. Acidification of this space dissolves the bone matrix and
increases the activity of acid hydrolases. Mutations in the gene encoding the 116kDa a3 subunit of vATPase in the osteoclast plasma membrane lead to defects in
28
bone resorption [157]. The vATPase is, therefore, essential for bone resorption
[158].
1.2.5 Modulations in vATPase function in disease
Mutations in the genes encoding the human kidney-specific isoforms B1 and
a4, which are highly expressed in renal intercalated cells, result in defective renal
acidification, and lead to distal renal tubular acidosis (dRTA) [95, 159]. Isoforms
of the ‘a’ subunit, expressed in the inner ear, are critical for maintaining the pH of
the fluid that surrounds the mechanosensory hair cell. Thus gene mutations in the
B1 and a4 subunit in the cochlea lead to sensorineural deafness [95, 160].
Mutations in the a3 subunit present in the osteoclast plasma membrane vATPase
lead to defects in bone resorption [161, 162].
vATPase is over expressed in many types of metastatic cancers and promotes
their invasion-potential and hence metastasis [87, 163]. Inhibition of vATPase
function by knockdown of subunit ‘c’ using RNA interference (RNAi) effectively
suppressed human hepatocellular carcinoma cell metastasis [164]. The low pH of
the tumor extracellular microenvironment promotes increased activation of
secreted lysosomal proteases which participate in the degradation and remodeling
of the extracellular matrix (ECM), thus contributing to cancer invasion and
metastasis. Both in vivo and in vitro findings provide evidence that, by preventing
the activation of ECM proteases, inhibition of vATPases may represent a strategy
for cancer therapy [165].
29
1.3 Mammalian target of rapamycin (mTOR)
Rapamycin, originally purified as a macrolide from a soil bacterium found on
Easter Island [166, 167], is currently used as an immunosuppressant and its
analogues are in clinical trials for their potential as anticancer drugs. Target of
Rapamycin (TOR) was first identified in the budding yeast Saccharomyces
cerevisiae (Sc) during a screening for resistance to the immunosuppressant drug
rapamycin in the early 1990s [168, 169]. Soon after, TOR was identified in many
organisms, from yeast to mammals. Mammals as well as other metazoans express
a single TOR gene, whereas yeast expresses two TOR genes. In all eukaryotes
TOR is found in two conserved complexes, TOR complex 1(TORC1) and TOR
complex 2(TORC2) [170]. TOR has been recognized as a major regulator of cell
growth and metabolism that integrates signals from nutrient, energy, and growth
factors. This review will focus on the structure and regulation of the mammalian
target of rapamycin (mTOR).
1.3.1 Structure and organization of mTOR complexes
1.3.1.1 mTOR domain structure
mTOR, a large protein of ~ 280KD, belongs to the phosphoinositide 3-kinase
(PI3K)-related protein kinases (PIKK) family, along with ATM, ATR, DNA-PK,
and hSMG1. mTOR consists of several distinct functional domains. Going from
the N-terminal to C-terminal domains one observes (Figure 1.4): 1) a number of
HEAT (Huntington, elongation factor 3, the PR65/A subunit of Protein
30
phosphatase 2A and the lipid kinase TOR1 heat-treatment) repeats, which are
likely involved in protein–protein interactions; 2) a large FAT (Frap, ATM, and
TRAP PIKK-like) domain, which is also present in other PIKK proteins; 3) an
FRB (FKBP12-rapamycin binding) domain; 4) a kinase domain; and 5) a FAT/C
(FAT domain C-terminal) regulatory domain (reviewed in [171]).
Rapamycin in association with its intracellular receptor, the FK506-binding
protein 12 (FKBP12), binds mTOR at its FRB domain and inhibits mTOR activity
[172, 173]. Although the sequence of the C-terminal kinase domain of mTOR is
similar to the catalytic domain of the PI3K, there is no evidence for mTOR lipid
kinase activity. Further research has shown that TOR is in fact a protein kinase,
belonging to the PIKK family, and that the FATC and FAT domains interact with
each other to form a configuration that exposes the catalytic domain [174, 175].
31
Figure 1.4
Figure 1.4 Schematic structure of mTOR.
See text for details.
32
1.3.1.2 Organization of mTOR complexes
The two distinct complexes of mTOR - mTORC1 and mTORC2 contain
shared and unique components (Figure 1.5). The principal distinct components
defining them are regulatory-associated protein of mTOR (Raptor) and Raptorindependent companion of mTOR (Rictor) respectively [176]. In early studies,
mTORC1 was characterized as rapamycin-sensitive while mTORC2 was
rapamycin-insensitive due to the binding of FKBP12-rapamycin directly to
mTORC1 but not mTORC2 [177, 178]. As one would predict the absence of
functional FKBP12 renders mTORC1 insensitive to rapamycin [179, 180]. To
complicate this apparently simple distinction it has been reported that prolonged
rapamycin treatment inhibits the assembly of mTORC2 thus reducing its activity
[181] In addition, recent reports suggest a rapamycin-resistant mTORC1 function
in regulating cap-dependent translation [182-184].
mTORC1 has four associated components [185, 186]: Raptor, mammalian
lethal with Sec13 protein 8 (mLST8, also known as GbL), proline-rich AKT
substrate 40 kDa (PRAS40), and DEP-domain-containing mTOR-interacting
protein (Deptor). Raptor functions as a scaffolding protein regulating the
assembly of mTORC1, and linking mTOR kinase with its downstream substrates
such as 4E-BP1, thus promoting mTORC1 signaling [187, 188]. Raptor has also
been reported to play a role in mTORC1 translocation in response to amino acids
[189]. mLST8 binds to the kinase domain of both complexes but it seems to have
a more critical function in mTORC2 assembly and signaling. Its function in
mTORC1 remains unclear, as ablation of mLST8 in mice doesn’t affect mTORC1
33
activity [190]. PRAS40 functions as a negative regulator as well as a competitive
substrate of mTOR. When PRAS40 is dephosphorylated it binds and represses
mTORC1 activity. Upon activation, mTORC1 phosphorylates PRAS40 which
then dissociates from mTORC1 further promoting mTORC1 activity [191-193].
Deptor binds to the FAT domain of mTOR and inhibits both mTORC1 and
mTORC2 activity [186].
Besides mLST8 and Deptor mTORC2 contains several unique components.
These are: Rictor, mammalian stress-activated protein kinase interacting protein
(mSIN1), and protein observed with Rictor-1 (Protor-1, also known as PRR5).
Rictor is required for mTORC2 catalytic activity and might recruit downstream
substrates for mTOC2. Rictor and mSIN1 stabilize each other to maintain
mTORC2 complex integrity [194, 195]. Protor-1 binds Rictor, but unlike other
mTORC2 components it is not required for mTORC2 integrity or kinase activity
[196, 197].
Compared to mTORC1, knowledge about upstream regulators and
downstream substrates of mTORC2 is more limited. It is known that TORC2
plays a role in organizing the actin cytoskeleton and in cell polarization in yeast
[170]. Below we focus on current understanding of the functions and regulation of
mTORC1.
34
1.3.2 Downstream targets of mTORC1
mTORC1 senses and integrates diverse signals to stimulate anabolic and
inhibit catabolic processes. It promotes cell growth and proliferation via the
regulation of protein synthesis and promotes cell survival by inhibiting autophagy.
mTORC1 also stimulates transcription from genes involved in ribosome
biogenesis. Below, among the multiple functions and substrates of mTORC1, we
place an emphasis on its best-known substrates in protein synthesis: ribosomal S6
protein kinase (S6K) and eukaryotic initiation factor 4E (eIF4E)-binding protein 1
(4E-BP1).
1.3.2.1 Protein Synthesis
By sensing the signal from growth factors or nutrients, mTORC1 is activated
and promotes protein synthesis mainly through regulating various components of
the translation initiation machinery, via direct or indirect phosphorylation events
(Figure 1.5). The best known substrates of mTOR in protein synthesis are 4E-BP1
and S6K. mTORC1 phosphorylates 4E-BP1 on multiple sites (including Thr37/46,
Thr70, and Ser65) [183, 184, 198-200]. Phosphorylation of 4E-BP1 controls the
activity of eIF4E, which is a translation factor that binds to the 5′-cap structure of
eukaryotic mRNAs to facilitate ribosome recruitment. 4E-BP1 competes with
eIF4G, a scaffold protein in the assembly of the translation pre-initiation complex,
for an overlapping binding site on eIF4E. Hypophosphorylated 4E-BP1 binds
tightly to eIF4E thereby preventing it from interacting with eIF4G and blocking
formation of a productive initiation factor complex. Upon mTORC1 activation,
35
hyperphosphorylated 4E-BP1 is released from eIF4E, allowing for the recruitment
of eIF4G and eIF4A to the 5' end of an mRNA to promote initiation complex
formation [171, 201, 202](Figure 1.5).
Raptor binds directly to mTOR signaling (TOS) motifs on 4E-BP1, S6K and
other known mTORC1 substrates (such as PRAS40 and Hif1α) to facilitate their
phosphorylation by mTORC1 [203]. It is noteworthy that not all output from
mTORC1 is inhibited by rapamycin. Among the four sites of 4E-BP1
phosphorylated by mTORC1, two (Thr70, and Ser65) are rapamycin sensitive and
two (Thr37/46) are rapamycin resistant [183, 184, 198-200].
Important targets of mTORC1 are the S6Ks, including S6K1 and S6K2.
While S6K1 and S6K2 have overlapping functions, studies in knockout mice and
in vitro analysis have revealed isoform-specific functions [204]. S6K1 seems to
play a more important role in mTORC1 regulation of cell growth [205, 206]. The
TOS motif is required for mTORC1/raptor-mediated phosphorylation of S6K1 on
its hydrophobic motif (HM) site (Thr389) [204]. Inactive S6K1 associates with
the eukaryotic initiation factor 3 (eIF3). Upon activation, mTORC1 is recruited to
the eIF3 complex and phosphorylates S6K1, resulting in the dissociation of S6K1
from eIF3, thus enabling the subsequent phosphorylation of its translational
targets, such as eukaryotic initiation factor 4B (eIF4B), which is then recruited
into the initiation complex [207]. Substrates of S6K1 involved in translational
control include the ribosomal protein S6 (RPS6), eIF4B, eukaryotic elongation
factor 2 kinase (eEF2K), programmed cell death 4(PDCD4), cap-binding protein
of 80 kDa (CBP80), and S6K1 Aly/REF-like target (SKAR) (reviewed in [208]).
36
RPS6, a component of the 40S ribosomal subunit, was the first S6K substrate
identified [209], and is considered a reliable readout for S6K activity.
Phosphorylation of RPS6 was previously assumed to promote translation of 5’
TOP (terminal oligopyrimidine) mRNAs, which encode ribosomal proteins and
translation elongation factors. However, it was later found that phosphorylation of
RPS6 is essential for regulating cell size, but is dispensable for translational
control of 5’ TOP mRNAs- its previously assigned targets[210].The role of RPS6
in promoting translation remains unclear.
The recruitment of the 40S ribosomal subunit to the 5’ end of mRNA is a
crucial and rate-limiting step during cap-dependent translation. A number of
translation initiation factors, including eukaryotic initiation factor 4A (eIF4A) and
eIF4B, are essential in this process. eIF4A has two major functions: first, it
promotes mRNA association with 40S ribosomal subunit; second, it is an RNA
helicase that unwinds double stranded RNA and therefore any secondary structure
at the 5’ end of mRNA thus allowing translation to proceed. eIF4B is a cofactor
which facilitates the RNA helicase function of eIF4A (Figure 1.5).
S6K1 promotes translation initiation by phosphorylating eIF4B on Ser422
which is located in an RNA-binding region required for increasing eIF4A helicase
activity [211] (Figure 1.5). PDCD4 binds to and inhibits the RNA helicase
activity of eIF4A [212]. S6K1 further promotes translation initiation by
phosphorylating PDCD4 at Ser67 which promotes its degradation thereby
preventing its inhibitory effect on eIF4A helicase function [213].
37
Figure 1.5
Figure 1.5 Regulating cap-dependent translation initiation.
(a) The recruitment of the 40S ribosomal subunit to the 5' end of mRNA is a
rate-limiting step during cap-dependent translation. Hypophosphorylated 4Ebinding proteins (4E-BPs) bind tightly to eIF4E, thereby preventing its interaction
with eIF4G and thus inhibiting the initiation of translation. mTORC1-mediated
phosphorylation of 4E-BP releases it from eIF4E, permitting the recruitment of
eIF4G to the 5' cap thereby allowing translation initiation to proceed. (b)
Following 40S ribosomal protein S6 kinase (S6K) or RSK-mediated
phosphorylation of eIF4B the latter is recruited to the translation pre-initiation
complex and enhances the RNA helicase activity of eIF4A. This is particularly
important for translating mRNAs that contain long and structured 5' untranslated
sequences, because the unwinding of these RNA structures is required for
efficient 40S ribosomal subunit scanning towards the initiation codon. GF,
growth factor. (from Ma, X. M. and Blenis, J., 2009, Nat Rev Mol Cell Biol)
38
1.3.2.2 Ribosome biogenesis
Consistent with the critical role of mTORC1 in cell growth via the modulation
of protein synthesis, mTORC1 positively regulates transcription of genes involved
in ribosome biogenesis [214]. It controls ribosome biogenesis by at least two
mechanisms: by promoting the translation of mRNAs for ribosomal proteins
(RPs), and by affecting ribosomal RNA (rRNA) synthesis [214]. mTORC1
coordinates transcription by all three classes of nuclear RNA polymerases. In both
yeast and mammals, rapamycin blocks transcription of rRNA genes by RNA
polymerase I (Pol I), transcription of ribosomal protein genes (RP genes) by RNA
polymerase II (Pol II) and transcription of tRNA and 5S genes by RNA
polymerase III (Pol III) [214]. Inhibition of mTORC1 signaling inhibits
translation more slowly and less pronouncedly than transcription of ribosomal
components [215, 216]. mTORC1 regulates Pol I by promoting the nuclear
translocation and activity of TIF-IA(RNA polymerase I transcription factor
RRN3), which is an essential initiation factor for Pol I-mediated transcription
[217, 218]. mTORC1 regulates the level of Pol III by interacting with the Pol III
specific transcription factor, TFIIIC. TFIIIC recruits mTORC1 to promoters
where it phosphorylates and inactivates the transcription repressor Maf1 thus
leading to the transcription of Pol III [219].
1.3.2.3 mTORC1 regulation of autophagy
Macroautophagy (referred to as autophagy below) is an evolutionarily
conserved homeostatic “self-eating” process which involves the digestion of
39
cytoplasmic proteins and organelles via the lysosomal pathway [220]. During
periods of starvation, stress or reduced availability of growth factors, stimulation
of autophagy mobilizes intracellular nutrient resources to maintain metabolism
and ATP levels. mTOR inhibits autophagy by influencing numerous proteins
required for the autophagic process.
The yeast serine/threonine kinase, autophagy-related 1 (Atg1), plays a key
role in initiating autophagosome formation.
Atg1 fulfills this role when
complexed with Atg13 and Atg17 [221-224]. TORC1 phosphorylates Atg13 at
multiple residues reducing its affinity for Atg1 and decreasing the Atg complex
resulting in repression of autophagy. During starvation or rapamycin treatment,
inactivation of TORC1 leads to dephosphorylation of Atg13, increasing Atg1–
Atg13–Atg17 complex formation and activating autophagy [221, 223, 224].
A recent study by Yu et al. demonstrated the role of mTORC1 in an
autophagy cycle governing lysosome homeostasis during starvation. Starvation
inhibits mTORC1 and induces autophagy which results in the release of amino
acids derived from protein degradation. This leads to reactivation of mTORC1
which attenuates autophagy and generates proto-lysosomal tubules and vesicles
which ultimately mature into functional lysosomes, thereby maintaining the
cellular lysosomal population [225].
1.3.2.4 mTORC1 and metabolism
The regulation of cell growth by mTORC1 has been studied mostly in regard
to protein synthesis. However, it has become clear that mTORC1 modulates
40
numerous metabolic pathways. Many lines of evidence indicate that mTORC1 can
control cell growth by promoting the synthesis of many classes of lipids
(unsaturated and saturated fatty acids, phosphatidylcholine, phosphatidylglycerol,
and sphingolipids) required for membrane biosynthesis and energy storage.
Recent work combining genomic, metabolomic, and bioinformatic approaches
established that mTORC1 regulates glycolysis and de novo lipid biosynthesis as
well as the oxidative arm of the pentose phosphate pathway [226].
1.3.2.4.1 mTORC1 promotes lipogenesis through SREBP-1
Sterol regulatory element binding proteins (SREBPs) are transcription factors
mediating the effect of sterols on expression of enzymes involved in lipid and
cholesterol homeostasis [227]. Akt has been shown to mediate the effect of insulin
on de novo lipogenesis by activating the transcription factor sterol regulatory
element-binding protein-1 (SREBP-1) [228].Work from different groups has
shown that expression of many SREBP-1 target genes, including acetyl-CoA
carboxylase (ACC) [229], fatty acid synthase (FASN) [230], and stearoyl-CoA
desaturase 1 (SCD-1) [231] were reduced by the mTORC1 inhibitor, rapamycin.
Porstmann et al. observed that rapamycin blocked all the metabolic effects of Akt
activation, including SREBP-1 nuclear localization and lipogenesis. This indicates
that mTORC1 is the major metabolic effector downstream of Akt. Knockdown of
Raptor by siRNA, but not Rictor, blocked the expression of SREBP-1
downstream targets, indicating Akt activates SREBP-1 via mTORC1 but not
mTORC2. Silencing SREBP-1 restricts mammalian cell growth and reduces cell
41
and organ size in Drosophila, suggesting that the PI3K–Akt–TORC1 pathway
regulates cell size by coordinating both protein and lipid biosynthesis [232].
Tuberous sclerosis complex (TSC) is a suppressor of mTORC1 signaling.
Duvel et al. demonstrated mTORC1 is activated in TSC1-/- or TSC2 -/- genetic
gain-of-function models leading to increased SREBP-1 and SREBP-2 activation,
produced at least partly by S6K1 [226]. Duvel et al. also showed that, in the
setting of TSC gene disruption, mTORC1 signaling drives glucose uptake and
glycolysis through up-regulation of the transcription factor Hypoxia-Inducible
Factor 1α (HIF1α) [226]. Consistent with previous findings [233, 234], mTORC1
activation alone is sufficient to increase HIF1α at translational levels under
normoxic conditions [226]. The regulation of HIF1α and SREBP-1 by mTORC1
suggests that mTORC1 coordinates carbohydrate and lipid metabolism and hence
the use of nutrient resources.
1.3.2.4.2 mTORC1 promotes adipogenesis through PPAR-γ
Peroxisome proliferator-activated receptor-γ (PPAR-γ) is a member of the
nuclear receptor superfamily of ligand-activated transcription factors which
controls the expression of various genes involved in fatty acid uptake, synthesis,
esterification, and storage. Activated mTORC1 induces the phosphorylation of
4E-BPs, which in turn releases eIF4E and increases the translation of
CCAAT/enhancer binding protein-α (C/EBP-α) and –δ, which are key
components required for the establishment of the adipogenic cascade. C/EBP-α
42
and C/EBP-δ augment the expression of PPAR-γ which promotes lipogenesis via
transcriptional regulation of lipogenic genes. mTORC1 stimulation of SREBP-1
also contributes to adipogenesis by promoting the production of endogenous
ligands for PPAR-γ(reviewed in [235]).
1.3.3 Upstream regulation of mTORC1
mTORC1 integrates signals from growth factors, nutrients, cellular energy
status and stress to promote cell growth (Figure 1.6). mTORC1 activity is
stimulated by the GTP-bound active form of the small G protein Ras homologue
enriched in brain (Rheb). Rheb binds directly to the kinase domain of mTORC1
and activates it in a GTP dependent manner [236]. Two isoforms of Rheb, Rheb1
and Rheb2 (of which Rheb1 has been extensively studied) have been identified in
mammalian cells.
As noted, mTORC1 is regulated by TSC1 and TSC2 (also known as tuberin).
TSC2 is a GTPase activating protein (GAP) which converts Rheb from its active
to its inactive GDP-bound form [237, 238]. Inactivation of TSC1 or TSC2 results
in an increase of GTP-bound Rheb and a constitutive activation of mTORC1
which is not further augmented by insulin or growth factors.
1.3.3.1 mTORC1 activation by growth factors
Growth factors stimulate mTORC1 through the PI3K pathway and the RasERK pathway. Binding of insulin or insulin-like growth factors (IGFs) to their
43
receptors leads to tyrosine phosphorylation of insulin receptor substrates (IRSs)
and recruitment of PI3K. PI3K binds to IRS and converts phosphatidylinositol4,5-phosphate(PIP2) into phosphatidylinositol-3,4,5-phos-phate (PIP3) which
localizes Akt to the membrane where it is phosphorylated by mTORC2 and 3phosphoinositide-dependent protein kinase 1 (PDK1). mTORC2 phosphorylates
Akt on ser473 located in the hydrophobic motif of Akt. This facilitates further Akt
phosphorylation by PDK1 at Thr308 in the catalytic domain. Phosphorylation at
these two sites together evoke full activation of Akt [239]. Activated Akt
phosphorylates TSC2 on several sites (Thr1462, Ser939 and Ser981), inducing its
binding to 14-3-3, which suppresses its GAP activity toward Rheb resulting in the
activation of mTORC1 [240]. Akt also promotes mTORC1 activation in a TSC1TSC2 independent manner by phosphorylating PRAS40, the inhibitory
component of mTORC1, leading to its dissociation from and subsequent
activation of mTORC1 [191-193]. Growth factors can signal to mTORC1 through
distinct pathways other than the PI3K-Akt axis. For example, Erk and ribosomal
protein S6 kinase (RSK) phosphorylate TSC2 at Ser664 and Ser1798 respectively,
leading to the suppression of TSC function [240].
Activated mTORC1 triggers a negative –feedback loop that inhibits the
insulin-PI3K-Akt pathway. Activation of mTORC1 and S6K1 stimulates the
Ser/Thr phosphorylation of IRS1 which uncouples IRS1 from PI3K leading to
reduced PI3K signaling. [241, 242]. This negative-feedback loop may play an
important role in the development of insulin-resistant states [243] and cancer
[244].
44
Figure 1.6
45
Figure 1.6 Model of mTOR signaling network.
mTOR is associated with two distinct complexes, mTORC1 and mTORC2.
mTORC1 integrates signals from growth factors, energy status of the cell,
nutrients (amino acids), and stress to promote cell growth. mTORC1 is stimulated
by the GTP-bound active form of Rheb which is regulated by an upstream tumor
suppressor complex TSC1–TSC2. TSC2 is a GTPase activating protein (GAP)
which converts Rheb from its active GTP-bound to its inactive GDP-bound form.
Several different kinases, including Akt, Erk and Rsk, phosphorylate TSC2
resulting in the inhibition of its GAP function towards Rheb. Conversely, AMPK
mediated phosphorylation of TSC2 augments the GAP activity of TSC2 thus
inhibiting mTORC1. Akt can also modulate mTORC1 activity in a TSC2independent manner by phosphorylating the mTORC1 inhibitory factor PRAS40.
The mTORC1 kinase is a master modulator of protein synthesis. In addition to its
direct phosphorylation of 4E-binding proteins (4E-BPs), activated mTORC1
promotes the activation of S6Ks which phosphorylate many translation initiation
factors. Besides protein synthesis, mTORC1 also controls metabolism, ribosome
biogenesis, and autophagy, pathways that collectively determine the mass (size)
of the cell. mTORC2 controls actin organization thereby determining cell shape.
Activating and inhibitory phosphates are red and green, respectively. Arrows
represent activation, whereas bars represent inhibition.
46
1.3.3.2 mTORC1 regulation by energy
mTORC1 senses cellular energy status through AMP-activated protein kinase
(AMPK). AMPK is activated under low cellular energy (high AMP levels or
AMP/ATP ratio). Activated AMPK phosphorylates TSC2 on Ser1345 and
enhances its GAP activity towards GTP-Rheb leading to inhibition of mTORC1
[245]. AMPK can also reduce mTORC1 activity in response to energy depletion
by directly phosphorylating Raptor thus inducing its binding by 14-3-3 resulting
in the inhibition of mTORC1 [246]. Moreover, phosphorylation of TSC2 by
AMPK at Ser1345 provides the priming phosphorylation for subsequent
phosphorylation of TSC2 at Ser1341 and Ser1337 sites by glycogen synthase
kinase 3(GSK3). AMPK and GSK3 coordinately phosphorylate and repress
mTOR signaling.
Besides directly phosphorylating TSC2 and PRAS40, Akt also activates
mTORC1 indirectly via maintaining high intracellular ATP levels which inhibit
AMPK and its
phosphorylation of TSC2 [247]. The effect of Akt on the
generation of ATP occurs via an increase in glycolysis and oxidative
phosphorylation [248]. Although the exact mechanisms by which Akt affects
these processes are not fully known, Akt can affect glycolysis through multiple
mechanisms, such as increasing glucose transporter expression and translocation
[249, 250], and increasing the expression and activity of glycolytic enzymes [248,
251].
47
1.3.3.3 mTORC1 regulation by stress
Hypoxia leads to a reduction of cellular ATP level and activates AMPK
mediated mTORC1 inhibition [252, 253]. In addition, hypoxia stabilizes the
transcription factor HIF1α, which drives the expression of DNA damage response
1(REDD1) [253]. REDD1 competes with TSC2 for 14-3-3 binding, thus
promoting the inhibitory effects of TSC2 on mTORC1 [254]. Other stresses such
as DNA damage also downregulate mTORC1 signaling. DNA damage activates
p53 which promotes transcription of the downstream target genes Sestrin1 and
Sestrin2 which activate AMPK thus inhibiting mTORC1 activity. [255, 256].
1.3.3.4 mTORC1 regulation by amino acids
Amino acid depletion of cells rapidly inhibits mTORC1 activation, even in
the presence of abundant growth factors. Readdition of amino acids quickly
restores mTORC1 signaling [257]. The branched chain amino acid leucine is a
primary regulator of mTORC1 activation. Withdrawal of leucine downregulates
mTORC1 signaling as effectively as the withdrawal of all amino acids [257].
mTORC1 remains sensitive to amino acid levels in TSC-null cells [258]whereas
overexpression of Rheb in Drosophila and mammalian cells is sufficient to
counteract the effect of amino acid starvation [259, 260]. Thus amino acidregulated signaling modulates mTORC1 downstream of TSC.
Amino acid transporters may play a role in amino acid-dependent mTOC1
activation (reviewed in [261]). System A amino acid transporter solute carrier
family 1 member 5(SLC1A5) transports glutamine into cells in a Na+ dependent
48
manner [262]; L-type amino acid transporter 1(LAT1) transports branch-chain
and neutral AAs into the cells in a Na+ independent manner [262, 263]. Coexpression of Systems A and L led to leucine accumulation, which was sufficient
to activate the TORC1 pathway in oocytes [264]. Recently, Nicklin et al. found
that L-glutamine uptake through SLC1A5 and its subsequent efflux by a
bidirectional transporter, solute carrier family 7 member 5 (SLC7A5)/ SLC3A2,
facilitated the uptake of leucine, leading to mTORC1 activation [265].
Several proteins have been implicated in the process of intracellular amino
acid sensing by mTORC1, including human vacuolar protein-sorting associated
protein 34(hVps34) which is a Class III PI3K [266, 267]. hVps34 may activate
mTORC1 signaling through the generation of phosphatidylinositol 3 phosphate
(PI3P). Deficiency of hVps34 suppressed leucine-responsive activation of
mTORC1 [267, 268]. However, Vps34 is not required for TORC1 signaling in
Drosophila [269]. Moreover, hVps34 together with its kinase-like partner
vacuolar protein-sorting associated protein 15(Vps15) is well recognized for its
requirement in the initiation of autophagy. This puts hVps34 downstream of
mTORC1 [270]. Further investigation is necessary to clarify the relation between
mTORC1 and hVps34 complexes.
Through a screen for protein kinases active in the TOR signaling pathway in
Drosophila, Findlay et al. identified a member of Ste20 serine/threonine kinases,
mitogen-activated protein kinase kinase kinase kinase 3(MAP4K3) as being
required for TORC1 activation. Knockdown of MAP4K3 in human HeLa cells
impaired mTORC1 activation by amino acids, whereas overexpression of
49
MAP4K3 was sufficient to stimulate S6K1 activity in a TSC1/2 independent
manner even under the condition of amino acid starvation [271]. These data
suggest MAP4K3 is involved in sensing amino acids in the control of mTORC1
signaling, but its precise role remains unclear.
Recent reports reveal that the Rag family of small GTPases may be important
elements linking amino acid availability to mTORC1 [189, 272-274]. Rag
GTPases are heterodimers consisting of RagA or RagB with RagC or RagD,
which constitutively reside on lysosomal membranes [189]. In the absence of
amino acids, the Rag GTPases are in an inactive form, where RagA or RagB is
loaded with GDP and RagC or RagD contains GTP. Amino acid availability
activates Rag GTPases to their active form. Now RagA or RagB is loaded with
GTP and RagC or RagD is loaded with GDP (Figure 1.7). Rag heterodimers
containing GTP-bound Rag A or B were shown to interact with the Raptor subunit
of mTORC1 and promote its translocation to lysosomes. This relocalization
enabled mTORC1 to interact with lysosome-localized Rheb GTP promoting
mTORC1 kinase activation [189, 274]. A lysosomal membrane-bound
heterotrimeric protein complex “Ragulator” consisting of p14, MAPK scaffold
protein 1 (MP1) and p18 may serve as the scaffolding apparatus that links Rag
heterodimers to the lysosomal surface (Figure 1.7). Knockdown of Ragulator
components led to the cytoplasmic redistribution of Rag GTPases and the failure
to recruit mTORC1 to lysosomes accompanied by the blocking of amino acid
signaling to mTORC1[274].
50
Taken together with other studies in late endosomes [275, 276], it would
appear that endomembranes play a key role in regulating the activity of mTORC1.
Moreover, since growth factor-dependent mTORC1 activation is strongly
dependent on the presence of amino acids, the subcellular targeting of mTORC1:
Rheb-GTP complex seems critical to integrate nutrient and growth factor
dependent pathways.
51
Figure 1.7
Figure 1.7 Model for amino-acid induced mTORC1 activation.
In this model of mTORC1 activation from the group of Sabatini et al [189,
274], Rag GTPases are tethered to the lysosomal surface by the “Ragulator”
complex consisting of p14, MAPK scaffold protein 1 (MP1) and p18. In the
absence of amino acids, the Rag GTPases are in an inactive form, where RagA or
RagB is loaded with GDP and RagC or RagD is loaded with GTP. mTORC1can
not associate with the endomembranes, and has no access to its activator Rheb. In
the presence of amino acids, RagA or RagB is loaded with GTP and RagC or
RagD is loaded with GDP and Rag GTPases becomes active. Activated Rag
GTPases serve as a docking site for mTORC1, allowing mTORC1 to associate
with endomembranes and thus bind and become activated by Rheb.
52
1.4 Objectives of the current work
Endosomes are critical sites for growth factor signaling. After internalization,
insulin and EGF signaling can be sustained and amplified [25, 26, 277] or
uniquely generated within the endosomal compartment [35, 36]. Further spatial
modulation of signaling within endosomes can be achieved by activating
signaling complexes in membrane subcompartments (i.e. lipid rafts). The
preparation of detergent-resistant membranes (DRMs) from cell lysates has been
used to characterize lipid rafts biochemically. Consistent with a role for lipid rich
membrane domains in EGF signaling, we have previously observed that DRMs in
endosomes are greatly enriched not only in tyrosine phosphorylated EGFR but
also downstream signaling molecules [46, 59]. The first objective of this project
was to identify the global protein changes in response to EGF in endosomal
DRMs by performing a proteomic analysis of rat liver endosomal DRMs before
and after EGF administration. We found EGF- induced recruitment of the
vacuolar ATPase to rat liver endosomes which was accompanied by a reduction in
vacuolar pH. Inhibiting vacuolar acidification blocked EGF induced mitogenesis
and mammalian target of rapamycin complex 1(mTORC1) activation. Therefore,
the second objective of this study was to explore the role of vacuolar acidification
in EGF induced mTORC1 signaling.
53
Chapter 2
Materials and Methods
54
2.1 Materials and animals
Porcine insulin was a gift from Eli Lilly and Co., (Indianapolis, IN, USA).
Epidermal growth factor (EGF) was from BD Biosciences (Mississauga, ON,
CA). Antibodies against EGFR (#sc-03, used for immunoblotting), and
phosphotyrosine proteins (PY99, #sc-7020) were from Santa Cruz Biotechnology
(Santa Cruz, CA, USA). Antibody against REDD1 was from Abnova (Walnut,
CA, USA). Antibodies against phospho-p70S6K(Thr389), p70S6K, 4E-BP1,
phospho-4E-BP1(Ser65), phospho-Akt(Ser473), Akt, phospho-Erk1/2
(Thr202/Tyr204), Erk1/2 , phospho-PRAS40(Thr246), PRAS40, phosphorAMPK(Thr172), AMPK, phospho-TSC2(Thr1462), TSC2 , mTOR and Raptor
were from Cell Signalling Technologies (Beverly, MA, USA). 3-(2, 4dinitroanilino)-3’-amino-N-methyldipropylamine (DAMP) and polyclonal antiDNP were from Oxford Biomedical Research (Oxford, MI, USA). The L-amino
acid quantitation kit was from Biovision (Mountain View, CA, USA). Antibodies
for immunofluorescence are described below. Bafilomycin A1, chloroquine,
cycloheximide and DMSO were from Sigma-Aldrich (ON, Canada).
Sprague-Dawley rats were purchased from Charles River Canada Ltd. (St.
Constant, Quebec), housed in an animal facility with 12 h light cycles at 25°C and
fed ad libitum on Purina normal chow. Animals were fasted overnight (16-18 h)
before preparation of liver subcellular fractions. All animal work protocols have
been approved by McGill University.
55
2.2 Cell culture and liver fractionation
2.2.1 Primary hepatocytes culture
Primary rat hepatocytes were prepared from male Sprague Dawley rats
(~130g) by collagenase perfusion, and maintained as described previously [278].
All studies were performed in a humidified 37C incubator with 5% CO2. For the
preparation of samples for immunoblotting cells were rinsed twice with ice-cold
PBS (pH 7.4), and solubilised in lysis buffer as described previously [278].
2.2.2 HepG2 and FAO cell culture
The human hepatocellular carcinoma cells (HepG2) were cultured at 37 °C in
a humidified atmosphere containing 5% CO2 in Eagle's minimum essential
medium supplemented with 100 units/ml penicillin G, 100 μg/ml streptomycin,
and heat-inactivated 10% fetal bovine serum.
Rat hepatoma cells (FAO) Cells were cultured at 37 °C in a humidified
atmosphere containing 5% CO2 in Dulbecco's modified Eagle medium
supplemented with 100 units/ml penicillin G, 100 μg/ml streptomycin, and heatinactivated 10% fetal bovine serum. All of the cell culture products were obtained
from Invitrogen.
2.2.3 Preparation of microsomes and endosomes from rat liver
Rats (160-180g female Sprague-Dawley) were anaesthetized and sacrificed by
decapitation following intra-jugular injections at the indicated times as described
in the appropriate figures and legends. Livers were exsanguinated, rapidly excised
56
and minced at scissor point in ice-cold buffer (5 mM Tris-HCl buffer, pH 7.4,
containing 0.25 M sucrose, 1 mM benzamidine, 1 mM PMSF, 1 mM MgCl2, 2
mM NaF, and 2 mM Na3VO4). Endosomes and microsomes were prepared as
previously described in our laboratory[46].
2.2.4 Preparation of lysosomes from rat liver
Lysosomes were prepared as described in detail by Wattiaux et al [279].
2.2.5 Isolation of endosomal detergent resistant membranes (DRMs) from rat
liver
DRMs were isolated as described in detail by Balbis et al [46].
2.3 Protein analysis
2.3.1 Preparation of cell lysates
After treatment, primary rat hepatocytes were rinsed twice with ice-cold
phosphate-buffered saline (pH 7.4) and solubilised with lysis buffer (50 mM
Hepes, pH 7.5, 150 mM NaCl, 10 mM sodium pyrophosphate, 100 mM sodium
fluoride, 1.5 mM MgCl2, 1 mM EGTA, 200 μM sodium orthovanadate, 1 mM
phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 10%
glycerol, and 1% Triton X-100). Cell lysates were clarified by centrifugation at
10,000 × g for 20 min at 4°C.
57
2.3.2 Protein quantification
Protein content of all samples was determined using a kit (BioRad, CA, USA)
with bovine serum albumin as an internal standard. The assays were performed as
described in the company manual.
2.3.3 Immunoprecipitation and Immunoblotting
Cell fractions were incubated in 1% (vol/vol) Triton X-100 and 0.5% (wt/vol)
sodium deoxycholate at 4ºC for 1 hour. Cell lysates, prepared from EGF-treated
or non-treated cells were pre-cleared with non-immune rabbit IgG (Sigma, St
Louis, MO, USA) and protein A/G plus agarose(Santa Cruz, CA, USA) for 1 hour
at 4ºC. After centrifugation, the resulting supernatants were incubated for 2 hours
at 4ºC with the antibody indicated in the figure legend. Protein A/G plus agarose
(40 μl) was added to each sample, and the incubation was continued for an
additional hour. Immune complexes were isolated by centrifugation, washed three
times in PBS, and boiled in Laemmli sample buffer. Immunoprecipitates or whole
lysates were subjected to SDS-PAGE, transferred to Immobilon-P membranes
(Millipore Ltd., Mississauga, Ontario, Canada), and immunoblotted with the
indicated first antibody for 90 minutes followed by 1 hour incubation with
horseradish peroxidase-, or [125I] - labeled goat anti-rabbit (GAR) or goat antimouse antibody (GAM) IgG. GAR and GAM immunoreactive proteins were
detected by autoradiography or by the ECL system (Amersham Biosciences)
respectively. Quantification of the signals was determined by densitometery
(Model GS-800, Bio-Rad Laboratories, Inc., Hercules, CA, USA).
58
2.3.4 Proteomic sample preparation and analysis
The methods used in
the proteomic studies were described in detail
previously [46]. Briefly, equal quantities of subcellular fractions from three
independent preparations of endosomes and endosomal DRMs were loaded on a
7-12% gradient gel and stained with Coomassie G. Each gel was sliced by hand
so as to yield approximately 86 equal bands. Each band was minced into ~ 1 mm3
pieces, and subjected to alkylation followed by in gel tryptic digestion, and
peptide extraction using a MassPrep Workstation (Micromass, Manchester, UK).
The extracts were then subjected to LC-MS in a Micro Q-TOF (Micromass, UK).
Equivalent bands from each sample were placed on the same 96-well tray, and
therefore subjected to all manipulations and MS as identically as possible.
Peptides were identified using Mascot, and subjected to clustering using an inhouse database (CellMapBase)[46] to produce a non-redundant list of proteins.
More detailed information about the ‘peptide counting’ has been previously
described [46].
2.4 Immunofluorescence and DAMP labelling
2.4.1 Immunofluorescence studies
Preparation of cells. Primary hepatocytes, harvested by collagenase in situperfusion (as described above) were grown on glass coverslips coated with
collagen, and starved for 2 days. Incubations with EGF (100 nM) were for 5
minutes at 37ºC; after which cells were washed rapidly with cold PBS and fixed
59
with pre-chilled methanol at -20ºC for 15 minutes. Cells were washed 3 times
with cold PBS and kept at 4C until the labeling procedure.
Labeling of the cells. Fixed cells were first blocked in 5% inactivated goat
serum (Gibco, Burlington, ON, Canada) in PBS for 30 minutes at room
temperature. Primary antibody incubation (in 2% inactivated goat serum) was for
45 minutes at 37ºC. Cells were then washed 3 times in PBS before a 30 minute
incubation with secondary antibody (diluted in 2% inactivated goat serum) at
room temperature in the dark.
For double labeling, the two primary or two
secondary antibodies were prepared in the same solution.
Following secondary
antibody incubation, cells were washed three times with PBS, then three times
with water, and mounted on slides using Prolong anti-fade mounting medium
(Molecular Probes, Burlington, ON, Canada).
Antibodies. The following summarizes the antibodies used and the dilutions
at which they were used. Anti-V1E chicken antibody, A22284F (GenWay
Biotechnologies, San Diego, CA, USA) was used at 1:2000. Anti-EEA1 rabbit
polyclonal, ab2900 (Abcam, Cambridge, UK) was used at 1:500. Anti-Rab5
rabbit polyclonal, sc-309 (Santa Cruz, CA, USA) was used at 1:50. Anti-LAMP1
rabbit polyclonal, ab24170 (Abcam) was used at 1:25.
Goat anti-chicken IgY-Alexa Fluor 488 (A11039) was used at 1:1000. Goat
anti-rabbit IgG-Alexa Fluor 594 (A11037) was used at 1:1000. Goat anti-rabbit
IgG-Alexa Fluor 750 (A21039) was used at 1:250. All the goat antibodies were
obtained from Molecular Probes (Burlington, ON, Canada).
60
Immunofluorescence microscopy. The cells were examined under an
epifluorescence Zeiss Axiovert 200 M inverted microscope, using a 100X
objective. Digital pictures were taken with a Roper Scientific CoolSNAP ES
digital camera and processed with a Perkin Elmer Metamorph Imaging system
(Santa Clara, CA, USA) for adding pseudo colors and merging the double
labelings. Images were finally saved in Tiff format. All immunofluorescence data
shown are representative of at least 3 independent experiments.
To assess the V1E distribution pattern, each coverslip was divided into 5
regions and at least 6 cells per region were analyzed (approximately 30 cells per
coverslip, from at least 10 different fields of view). The criteria for selecting the
cells were as follows: in each of the 5 regions, the first 6 cells seen which did not
display excessive amounts of secreted material masking the cell cytoplasm were
evaluated. Cells were double blindly assigned ‘vesicular’, ‘diffuse’ or ‘unclear’
labeling pattern.
The co-localisation of V1E with LAMP1, EEA1and Rab5 respectively was
examined in at least 10 EGF - treated cells per coverslip. The percentage overlap
between V1E and LAMP1, EEA1and Rab5 was estimated in 3 independent
experiments with each antibody. The cell selection criteria were the same as
described above.
2.4.2 DAMP labeling of vesicles in rat primary hepatocytes
The DAMP labeling of vesicles was performed using an Acidic Granule Kit
(D09, Oxford Biomedical Research). For DAMP labeling quantification, 10 cells
61
were selected randomly in each condition. For each selected cell a digital picture
was taken with a Roper Scientific CoolSNAP ES digital camera. Each picture
was analyzed with the Perkin Elmer Metamorph Imaging system. The
fluorescence intensity of all the vesicles labeled in the cell was measured. Total
fluorescence intensity for each cell was calculated using the Metamorph software.
The mean ± s.e.m. of 10 intensities was calculated for each condition.
2.5 [3H] Thymidine incorporation assay
[3H] thymidine incorporation was measured as previously described [278].
2.6 In vitro S6 kinase assay
The in vitro S6 kinase assay was performed using the S6 protein substrate as
previously described [280].
2.7 7-Methyl-GTP (m7GTP) –Sepharose 4B pull-down assay
The m7GTP –Sepharose 4B pull-down assay was performed as previously
described [281].
62
2.8 Amino acid analysis
2.8.1 Leucine uptake assay
Serum starved primary hepatocytes were incubated with either serum-free
medium or warm PBS, in the presence of DMSO or bafilomycin for 30 minutes.
Cells were then incubated with 10 nM EGF or insulin, and 1 µCi 3H-leucine
(Perkin Elmer) (humidified 37C, 5% CO2) for 15 minutes, then washed 3 times
with ice-cold PBS, and solubilised in 1 ml of 1 M NaOH. Subsequently 1 ml of 1
M HCl was added to neutralise the solubilised total cell extract which was
analysed by scintillation counting for 3H.
2.8.2 Total intracellular amino acid analysis
Total intracellular amino acid levels were measured using a chromogenic
technique (L-amino acid quantitation kit from Biovision).
2.8.3 Amino acid analysis by HPLC
Cells were washed three times with cold PBS, scraped into 1ml cold saline
and spun down by centrifugation. Pellets were re-suspended in 200 µl saline,
sonicated on ice, and then centrifuged to obtain the supernatant. From cell lysate
samples, proteins were precipitated with perchloric acid and supernatants
neutralized with potassium carbonate. Diluted samples (with water) were then
derivatized with O-phthaldialdehyde (OPA) reagent before injection into the
HPLC system (Beckman Coulter System Gold® 508 Autosampler, 126 Solvent
Module, 32 Karat 5.0 software). Amino acids were separated in reversed-phase
63
on a C18 silica column (4.6 mm  15 cm, 3 m particles, from Supelco) using a
sodium acetate buffer and increasing gradient of methanol, in about 52 minutes.
Detection was accomplished by fluorescence at excitation 340 nm and emission at
450 nm (Jasco FP 2020 Plus). Amino acid concentrations were quantified using
standard curves run in duplicate at the beginning, middle, and end of an overnight
sequence of injections. Using this approach, 21 individual amino acids were well
separated and quantified. Intra-assay coefficient of variation was <5.0% for all
amino acids, except ornithine and lysine (20%). Concentrations of amino acids
(AAs) (nmol/ml) in primary hepatocytes were calculated using an estimated cell
volume of 1/15 ml per 5x106 hepatocytes.
2.9 Statistical Analysis
All statistical analyses were carried out by unpaired Student’s t-test. The
number of replicates is indicated in the figure legends.
64
Chapter 3
Results
65
3.1 EGF-induced recruitment of V1 subunits to the vacuolar
system
3.1.1 Proteomic analysis of rat liver endosomes
Previous work in our lab has shown that an endosomal DRM subfraction is
greatly enriched in tyrosine phosphorylated EGF receptors and downstream
signaling molecules [46]. Therefore we subjected hepatic endosomal DRMs
derived from control and EGF-treated rats to a more detailed and complete
proteomic analysis.
Figure 3.1A shows a Coomassie-G stained gradient gel of hepatic endosomal
DRMs from biological triplicates of control and EGF-treated rats. Each lane was
cut into 86 horizontal bands, each of which was treated to yield tryptic peptide
fragments which were subjected to chromatographic resolution and onedimensional tandem mass spectrometry (1 D-MS-MS) as described in Materials
and Methods. The resulting spectra were analysed using Mascot™ in which the
peptides were identified by comparison to a frozen NCBI mammalian database.
The resulting identified proteins from each lane of the gel were clustered using
CellMapBase in order to minimize redundancy; and those in all 6 samples
(control and EGF-treated) were analysed using CD-HIT to facilitate the
identification of those affected by EGF treatment.
As a result, approximately 35% of the original MS-MS spectra matched
peptide sequences in the database and a total of 1472 proteins were identified. The
number of proteins identified in control and EGF-treated samples were 1127 ± 42
and 1192 ± 42 respectively and it can be seen that the data are very comparable in
66
terms of numbers of proteins identified across replicates for each condition (Table
3.1).
Table 3.1 Basic results of EN-DRM proteomic analysis.
Control
EGF
# of proteins identified 1127.3 ± 42.1 1192 ± 41.7
% of MS-MS assigned
to peptide sequences
39.4 ± 0.4
33.6 ± 0.6
Total
1472
35
The values in the table are means of 3 independent replicates ±s.d.
Figure 3.1B is a matrix depicting the number of proteins identified in 1, 2 or
all 3 of the biological replicates from control and EGF-treated rats. Of a total of
1,472 proteins, 503 proteins were identified in all 3 replicates from both control
and EGF-treated animals. 17 proteins were identified in all 3 control replicates but
never in EGF (unique to controls); and 33 proteins were identified in all 3 EGF
replicates but never in controls (unique to EGF).
In addition, we used peptide counting, as described in Materials and Methods,
to quantify identified proteins which were above the threshold of detection in both
control and EGF samples. We then determined that, of all 1472 identified proteins,
442 proteins reproducibly increased or decreased in abundance following EGF
(changing >1.5 fold in mean peptide counts) in the EN-DRM fraction. These are
listed in biological functional categories (Table 3.2).
67
Figure 3.1
A
B
Total =1472 proteins
68
Figure 3.1 Proteomic analysis of EN-DRMs reveals a large number of
proteins.
(A) Coomassie-G stained 7-12% gel of EN-DRMs from biological triplicates
of control and EGF-treated rats. M = protein molecular weight marker, ENs= total
liver endosome fraction, X=control EN-DRM fractions not cut for proteomic
analysis, C1-3=Control EN-DRM, E1-3 = EN-DRM from rats 5 min after EGF
administration. (B) Distribution of the proteins identified. Proteins identified are
placed in the matrix based on how many times they were identified in each
condition.
69
Table 3.2 Determination by proteomic analysis of proteins changing in EN-DRMs following EGF.
Database|Protein
Identifier
Protein Description
Fold
Change
C1 C2 C3
E1
E2 E3
Mean
Control
SD
Control
Mean
EGF
SD
Peptides (identifier Sequence)
EGF
1.8
1.8
49.5
0.6
1.2
0.7
2.6
0.6
2.1
0.7
4.6
1.8
2.8
1.7
6.2
0.6
6.0
0.1
2.1
2.5
1.0
0.6
2.0
23.2
1.7
0.6
1.0
6.3
1.0
8.0
4.3
4.0
2.5
2.0
15.6
22.9
126.1
6.3
6.0
7.9
10.9
7.6
7.3
2.0
18.7
21.8
3.3
6.7
21.9
4.0
46.6
1.0
2.3
3.7
2.0
0.7
1.3
35.2
0.3
0.3
0.0
3.5
0.0
2.0
1.5
3.0
0.5
1.0
6.4
6.1
28.2
1.9
1.0
1.3
0.6
4.9
1.2
1.0
5.7
1.3
1.5
3.2
7.3
0.0
1.6
1.0
0.6
0.7
1.0
0.0
0.6
2.9
3304382 NSELFDPFDLFDVR
828854 VMGPGVSYLVR
3127039 FNSLNELVDFYR
439344 FNSLNELVDYHR
168193 HFCPNVPIILVGNK
450797 EIFLSQPILLELEAPLK
218641 TAFDDAIAELDTLNEDSYK
66111 LTDFNFLMVLGK
235623 LLLLGAGESGK
1328372 SGEGFIDFIGQVHK
119653 ISEILLDHGAPIQAK
445529 VSAFENPYVDAIK
4162925 TALSDLFLEQLLR
567493 VPTTGIIEYPFDLENIIFR
305168 LIEDNEYTAR
235624 ILLLGAGESGK
2429404 NLEAVQTDFSTEPLQK
62808 GLLVEPAANSYLLAER
1354925 DGLEATALLHR
223848 VVVYSNTIQSIIAIIR
441210 VFDKDGBGYISAAELR
1033483 SQEHFTALGSFYFLHESLK
261381 VLEEHGEWWK
100412 ANVELDHATLVR
49947 IAQSDYIPTQQDVLR
45211 FADTHSMPLFETSAK
60452 VTAAIASNIWAAYDR
95115 FYGPAGPYGIFAGR
444625 NELHNLLDKPQLQGIPVLVLGNK
358325 YAGSNIVQLLIGNK
823455 LGGLDVLEAEFSK
92436 EGHVDTALALLEK
4625808 DLPLGASPR
172866 VELSDVQNPAISITDNVLHFK
EGF1-54
EGF1-39
EGF1-16
EGF1-14
Cont3-12
Cont1-25
Cont1-17
Cont1-52
Cont1-28
Cont1-50
Cont1-51
Cont1-29
Cont1-38
Cont2-28
Cont1-41
Cont1-28
Cont1-31
Cont2-49
Cont1-19
Cont1-16
Cont1-8
Cont1-31
Cont1-38
Cont1-32
Cont1-15
Cont1-16
Cont1-2
Cont1-10
Cont1-10
Cont1-15
Cont1-28
Cont1-51
Cont1-65
Cont1-75
IPR000219 (D) DH domain
IPR000980 (D) SH2 motif
IPR000980 (D) SH2 motif
IPR000980 (D) SH2 motif
IPR001806 (F) Ras GTPase
IPR004843 (D) MetalloIPR000308 (F) 14-3-3 protein
IPR000008 (D) C2 domain
IPR001019 (F) Guanine
IPR000242 (F) Tyrosine
IPR000488 (D) Death domain
IPR001019 (F) Guanine
1.3
2.0
1.7
1.7
2.0
3.0
2.3
2.0
1.7
11.0
64.7
34.7
1.5
1.0
126.0
5.3
30.3
0.6
1.7
0.6
0.6
0.0
1.7
2.3
1.0
0.6
4.0
10.1
6.1
0.7
0.0
10.3
1.2
4.9
1440726 ILVLDEATAAVDLETDDLIQSTVR
245322 LIMITDYLLLFR
4162679 GFELPDTPQGLIGEAR
3240171 FLTDQSYIDVLPEFR
109473 ERPQVGGTIK
177497 EHLALLAVPMVPLQIILPLLISK
45989 LLDLFSDNFR
484532 EENIANIVTYPDDGLIDLK
3198374 DVEGVEEVHELHVWQLAGSR
448800 LLEVSDDPQVLAVAAHDVGEYVR
246106 IPLENLQIIR
445671 VQENLLASGVDLVTYITR
54320 LTVNDFVR
3192904
237124 LAEMPADSGYPAYLGAR
57166 EGLQENVDGTENAK
305870 ARDDLITDLLNEAK
EGF1-69
EGF1-59
EGF1-52
EGF1-32
EGF1-11
Cont3-35
Cont2-41
Cont3-29
Cont1-47
Cont1-39
Cont1-64
Cont1-28
Cont1-45
Cont2-46
Cont1-2
Cont1-53
Cont1-20
IPR001140 (D) ABC
IPR000494 (D) Epidermal
Band
InterProScan
Predictions
Signaling (34 proteins)
Unique
1
1
1
Protein vav-2
UniRef100|Q60992
Unique
10
6
3
Src homology 2 domain-containing transforming
UniRef100|Q5M824 UniRef100|Q3U2Q7
Unique
1
1
1
GRB2-related adapter protein
UniRef100|Q13588
Unique
8
6
10
Bone marrow macrophage cDNA, RIKEN fullUniRef100|Q3U5I5
13.0
1
3
4
6
MGC140228 protein
UniRef100|Q1RMJ6 UniRef100|UPI000011035E
12.0
1
4
1
7
Hypothetical protein MGC129042
UniRef100|Q3SWW9
7.4
1
3
2.4
2
LOC495922 protein
UniRef100|Q5PQB4
6.0
1
3
2
1
protein kinase C alpha-polypeptide
nrdb|189979 UniRef100|P05696
4.9
4.2 4.2 1.1 22.9 11.2 13
Guanine nucleotide-binding
protein Gs alpha1
UniRef100|Q6X973 UniRef100|Q63803
UniRef100|Q12927
4.7
8.6
6
28.9 16.8 23
Protein-tyrosine
phosphatase delta precursor
UniRef100|P23468 UniRef100|Q5SPJ6
UniRef100|UPI0000506C96
IPI|IPI00375547.1
4.1
82 11.5 125 155 99
ankyrinUniRef100|UPI000013DF99
nrdb|178646 IPI|IPI00127163.1
3.8
1
2
2
4.3 8.1 6.5
signal-transducing G protein alpha q subunit
nrdb|260895 UniRef100|UPI0000167970
3.6
3
1
1
7
6
5
P55 protein
UniRef100|Q5BK33 UniRef100|Q6F6B2
3.4
4
3
8.7 6.4 8.5
Guanine nucleotide binding protein alpha 11
UniRef100|Q3HR09 UniRef100|Q9JID2
2.8
6
4.8
1
11 10.3 12
Viral oncogeneUniRef100|Q99PW1
yes-1 homolog 1
UniRef100|Q6AXQ3 UniRef100|Q3TJI7
2.2
3.2 4.2 3.2 2.1 9.3 12
Guanine nucleotide binding protein alpha 13
UniRef100|Q3HR08 UniRef100|Q6Q7Y5
2.0
3
6
2
6
8
8
Prolactin regulatory element-binding protein
UniRef100|Q6AYS1 UniRef100|UPI0000506E9B
2.0
1
2
2
1
3
Tissue-type transglutaminase
UniRef100|Q9WVJ6
1.7
15 12
6
14
25 17
PREDICTED: similar to KIAA0315
UniRef100|UPI00005071F1
1.7
11.2 15 13 UniRef100|Q3ZCA7
23
22 20
Guanine nucleotide binding
protein alpha
UniRef100|Q3HR12 UniRef100|UPI00001121B3
UniRef100|Q45QM8
IPI|IPI00231733.5
IPI|IPI00338854.1
1
5
3
2
5
C-like proteinnrdb|223872 UniRef100|Q9D6G4 1.7
nrdb|223036 nrdb|254772troponin
UniRef100|Q2KJE6
1.7
6
3
3
9
8
3
Inpp5a protein Activated spleen cDNA, RIKEN
UniRef100|Q7TNC9 UniRef100|Q8BNK3
1.7
19.4 13
7
25.7 26.4 14
lynnrdb|7434384
B protein tyrosine
kinase
nrdb|198943 nrdb|2105002
UniRef100|P25911
1.5
3
3
2
4
4
4
similar
to transducin (beta)-like 2
UniRef100|UPI00001D0815
UniRef100|Q9R099
1.5
28.1 39 28.8 46 45.4 49
Guanine nucleotide
binding protein alpha
UniRef100|Q45QN0 nrdb|1730227
nrdb|348273
2.1
2
2.1 2.1
2
0
1
Chain A, Gppnhp-Bound Rab33 Gtpase
nrdb|73535745 UniRef100|Q3TWI8
-1.6
6
3
2
3
2
2
Mitogen-activated protein-binding proteinUniRef100|Q9JHS3
-1.8
4
9
7
5
6
Membrane-associated progesterone receptor
UniRef100|Q80UU9 UniRef100|Q5XIU9
-2.0
3
4
5
3
2
1
unnamed protein
product [Mus musculus]
nrdb|74192952 UniRef100|Q96BM9
UniRef100|Q9CTB0
-2.1
1
2.1 1.1
1
1
1810048P08Rik protein
UniRef100|Q91Z34 UniRef100|Q8CG50
-2.3
1
3
5
2
1
1
REFSEQ:XP_873816
PREDICTED:
similar
to
IPI|IPI00732083.1 UniRef100|Q3UGL2
-3.0
125 80 110 34.9 38.2 33
PREDICTED:
similar to erythroid ankyrin
UniRef100|UPI0000507FE6
UniRef100|Q9N180
-6.0
4
1
1
1
PREDICTED: similar to neuronal RhoA GEF
nrdb|73956781
-13.0
4
5
4
1
B-IND1 protein
UniRef100|O09003
0.0
0.0
0.0
0.0
0.3
0.3
0.3
0.3
3.2
4.9
31.0
1.7
1.7
2.3
3.9
3.5
3.7
1.0
11.0
13.0
2.0
4.0
13.1
2.7
31.9
2.1
3.7
6.7
4.0
1.4
3.0
104.7
2.0
4.3
IPR001019 (F) Guanine
IPR000719 (F) Protein kinase
IPR000169 (F) Eukaryotic
IPR001019 (F) Guanine
IPR002048 (D) CalciumIPR000300 (F) Inositol
IPR000719 (F) Protein kinase
IPR001680 (R) G-protein beta
IPR001019 (F) Guanine
IPR001806 (F) Ras GTPase
IPR004942 (F)
IPR001199 (D) Cytochrome
IPR003579 (F) Ras small
IPR001806 (F) Ras GTPase
IPR000222 (F) Protein
IPR000906 (D) ZU5 domain
Receptor and Transporter (57 proteins)
Unique
Tax_Id=10116 Ensembl_locations(Chr-bp):None
IPI|IPI00411223.1
Unique
Insulin receptor precursor IR Insulin receptor
UniRef100|P15208
Unique
Endoglin
UniRef100|Q6Q3E8
Unique
Hepatocyte growth factor receptor precursor
UniRef100|Q63964
Unique
Tax_Id=10116 CytoChrome b-245, alpha
IPI|IPI00231970.3
9.0
Acetyl-coenzyme
A transporter 1 AT-1 AcetylUniRef100|Q99J27 UniRef100|O00400
UniRef100|UPI00004BDFB9
7.0
1
Cytochrome P450 monooxygenase CYP2T1
UniRef100|Q91Y29
6.0
Sodium/potassium-transporting ATPase subunit
UniRef100|Q63377
5.0
1
Zinc transporter 1 ZnT-1 Solute carrier family 30
UniRef100|Q62720 nrdb|67968089
4.1
2
3
PREDICTED:
similar to ATPase,
H+ transporting,
UniRef100|UPI000049270A
UniRef100|Q9UI12-2
UniRef100|UPI000016C681
UniRef100|Q8BVE3
3.7
12 19
Epidermal growth factor receptor
UniRef100|Q9QX70
3.4
8
12
ATPase, H+ transporting, V1 subunit C, isoform
UniRef100|Q5FVI6
3.3
0
0
PREDICTED:
similar to solute carrier family 25
nrdb|74004564 UniRef100|Q96AM8
UniRef100|Q8BH59
3.0
1
Ciliary neurotrophic factor receptor alpha
UniRef100|Q08406 UniRef100|UPI00000298D3
2.7
49 48
Vacuolar ATP synthaseUniRef100|UPI0000167BEF
catalytic subunit A,
UniRef100|P50516 UniRef100|UPI00001D0792
2.7
3
Solute carrier organic anion transporter family
UniRef100|O55224
2.5
20
Vacuolar ATP synthase subunit E V-ATPase E
UniRef100|P11019
1
1
3
21
11
1.4
41
3
16
1
4
2
1
2
2
1
1
1
11
71
36
2.3
1
138
6
36
2
1
1
1
2
1
2
2
2
2
2
5
5
1
2
3
2
2
7
15
53 70
40 28
1.2 1.1
1
1
120 120
4
6
27 28
0.0
0.0
0.0
0.0
0.0
0.3
0.3
0.3
0.3
2.7
17.3
10.3
0.5
0.3
46.0
2.0
12.0
70
0.6
4.7
2.1
0.8
4.4
0.0
2.8
IPR000719 (F) Protein kinase
IPR007732 (F) Cytochrome
IPR004752 (F) AmpG-related
IPR001128 (F) Cytochrome
IPR000402 (F) Na+/K+
IPR002524 (F) Cation efflux
IPR004908 (F) V-ATPase
IPR000345 (BS) Cytochrome
IPR004907 (F) V-ATPase
IPR001993 (F) Mitochondrial
IPR002996 (F) Cytokine
IPR000194 (D) H+IPR004156 (D) Organic anion
IPR002842 (F) H+-
Table 3.2 ___Continued
Database|Protein
Identifier
Protein Description
Fold
Change
C1 C2 C3
E1
E2 E3
2.5
2
2
2
1
Metaxin-1
UniRef100|Q13505
2.4
4
1
4
4
2
16
Solute carrier family 26 (Sulfate transporter),
UniRef100|Q5RKK1 UniRef100|P45380
2.4
49
29
27.6
99.6
78.7
72
5 days embryo UniRef100|Q3U791
whole body cDNA, RIKEN fullUniRef100|Q3TL62 UniRef100|Q71UA2
2.2
4
1
5
3
3
NADPH oxidase beta subunit gp91phox
UniRef100|Q9ERL1 UniRef100|Q9ER28
2.1
3
3
4
7
7
7
PREDICTED:
similar to ATPase, H+ transporting,
UniRef100|UPI0000493BF3
UniRef100|Q3U6X0
2.0
4
7
13
8
9
32
Na+ K+ ATPase alpha subunit
nrdb|179212 UniRef100|Q64609
2.0
46 51 24 108 68 70
Vacuolar ATP synthase subunit d V-ATPase d
UniRef100|Q9QWJ2
2.0
1
1
1
1
5
Endothelial differentiation sphingolipid G-proteinUniRef100|Q4V7F6
2.0
1
1
2
2
3
3
Vacuolar H+ ATPase
accessory subunit 1
UniRef100|Q8K567 UniRef100|Q3TWN7
UniRef100|Q6IRF8
2.0
2
3
1
4
2
6
Rieske
Iron-Sulfur Protein
UniRef100|UPI0000112C33
UniRef100|UPI0000110888
UniRef100|Q6QI13
1.9
17 24
35
30 11
Vacuolar ATP synthase 16 kDa proteolipid
UniRef100|Q3TD69
1.8
12
6
7
14
15 17
BWK4
UniRef100|Q5VLR5
4
5
1
5
8
5
L-type calcium channel
alpha2/delta subunit
UniRef100|Q8VHS9 UniRef100|Q5REF2
UniRef100|O08532-2
UniRef100|P542901.8
UniRef100|P54289
1.8
7
8
7.4 12.4 16.6 11
Voltage-dependent anion channel 2
UniRef100|Q5VWK3 UniRef100|Q9Y5I6
1.7
3
3
4
4
2
Protein disulfide-isomerase
A6 precursor
UniRef100|Q922R8 UniRef100|UPI00001CFB6B
nrdb|1710248
1.7
5
1
2
2
6
Solute carrier family 25, member 1,
UniRef100|Q498T8
1.7
1
3
2
1
5
4
Succinate dehydrogenase
Ip subunit
UniRef100|Q9Z1Z5 UniRef100|UPI00004A417D
UniRef100|O97650
1.7
3
1
5
2
2
11
Multidrug resistance protein 2 P-glycoprotein 2 PUniRef100|Q78E07 UniRef100|Q14813
1.6
3
4
4
6
5
7
Mitochondrial dicarboxylate carrier Solute carrier
UniRef100|O89035 UniRef100|UPI000016387A
1.6
8
8
6
10
14 11
ENSEMBL:ENSRNOP00000024423
IPI|IPI00734599.1 IPI|IPI00203106.3
1.5
1
1
1
1
1
Transferrin receptor P90, CD71
UniRef100|Q1HE24
1.5
10 13
9
16
13 18
PREDICTED: similar to ATPase, H+ transporting,
nrdb|73963311 UniRef100|Q3U861
1.5
7
9
10
13
14
11
unnamed
protein product [Rattus norvegicus]
nrdb|56905 UniRef100|Q9QV38
UniRef100|P11598
-1.6
28 24 31
19
11 22
PREDICTED: similar to golgi phosphoprotein 4
UniRef100|UPI00005064D7
-1.7
1
4
5
1
1
4
Chain D,UniRef100|P07309
Rat Transthyretin
nrdb|3212535 UniRef100|P02767
-1.7
6
4
2
2
2
Cytochrome
P450 17A1 (EC 1.14.99.9)
UniRef100|UPI000019C204
UniRef100|Q6LAE5
-1.8
4
9
5
4
4
2
oxidase IV,cytochrome
nrdb|223590 nrdb|40889867
-2.0
4
3
1
4
REFSEQ:XP_878579
PREDICTED: similar to
IPI|IPI00728779.1 UniRef100|Q9BTR6
UniRef100|Q9JKN2
-2.0
2
4
6
2
2
2
16
days
embryo
heart
cDNA,
RIKEN
full-length
UniRef100|Q3TF25 UniRef100|Q9DB20
-2.0
3
4
3
2
3
Low-density lipoprotein receptor precursor LDL
UniRef100|P35952
-2.0
39 55 13
31
11 11
PREDICTED: similar to Nucleoprotein TPR
UniRef100|UPI0000506C73
-2.3
2
4
1
3
2 days neonate
sympathetic ganglion cDNA,
UniRef100|Q3UF04 UniRef100|P19511
UniRef100|Q9CQQ7
4
4
4
2
3
nicotinamide
nucleotide transhydrogenase
nrdb|163397 UniRef100|Q9JK70
UniRef100|P11024
UniRef100|UPI000016A287 -2.4
-2.5
5
5
1
2
1
Tax_Id=10116 10 kDaUniRef100|Q54A32
protein
IPI|IPI00389152.2 UniRef100|UPI00005065E5
-2.5
63 47 33
21
11 25
Copper-transporting ATPase 2 Copper pump 2
UniRef100|Q63676 UniRef100|Q9QUG4
-2.6
5
13 29
1
4
13
ATP synthase beta subunit
nrdb|1374715 UniRef100|UPI000011257B
-3.0
1
1
1
1
10 day old male pancreas cDNA, RIKEN fullUniRef100|Q9CVJ6 IPI|IPI00692048.1
-4.0
1
2
1
1
Similar to CG6105-PA
UniRef100|Q6PDU7
-5.0
3
1
1
1
Hypothetical protein Dsm-1 D-serine modulator-1
UniRef100|Q497A2 UniRef100|Q9D1L5
-8.3
17.8
5
12
4.2
PREDICTED: similar to 1810073N04Rik protein
UniRef100|UPI0000250216
Mean
Control
SD
Control
Mean
EGF
SD
Peptides (identifier Sequence)
EGF
0.7
3.0
35.3
1.7
3.3
8.0
40.3
1.0
1.3
2.0
13.7
8.3
3.3
7.5
2.0
2.0
2.0
3.0
3.7
7.3
0.7
10.7
8.7
27.7
3.3
3.3
6.0
2.7
4.0
3.3
35.7
2.3
4.0
3.3
47.7
15.7
1.0
1.3
1.7
11.6
1.7
11.9
2.1
0.6
4.6
14.4
0.0
0.6
1.0
4.9
3.2
2.1
0.5
0.0
2.8
1.0
2.0
0.6
1.2
0.0
2.1
1.5
3.5
2.1
1.4
2.6
1.5
2.0
0.6
21.2
1.5
0.0
0.0
15.0
12.2
0.0
0.6
1.2
6.4
1.7
7.3
83.3
3.7
7.0
16.3
82.0
2.0
2.7
4.0
25.3
15.3
6.0
13.3
3.3
3.3
3.3
5.0
6.0
11.7
1.0
15.7
12.7
17.3
2.0
2.0
3.3
1.3
2.0
1.7
17.7
1.0
1.7
1.3
19.0
6.0
0.3
0.3
0.3
1.4
0.6
7.6
14.6
1.2
0.0
13.6
22.5
2.8
0.6
2.0
12.7
1.5
1.7
3.0
1.2
2.3
2.1
5.2
1.0
2.1
0.0
2.5
1.5
5.7
1.7
0.0
1.2
1.3
3.0
2.3
1.3
1.8
28.3
4.0
2.0
1.7
4.7
6.0
14.3
48.0
2.3
4.0
4.7
43.8
0.6
0.0
0.6
0.6
0.6
4.2
1.7
1.0
1.2
1.5
1.0
3.1
11.5
1.2
2.0
4.7
8.2
0.0
0.7
11.5
0.7
0.6
7.2
6.2
Band
InterProScan
Predictions
542630 EKYNADYDLSAR
77229 SLYSLTGLDAGYSATR
202306 TVSGVNGPLVILDHVK
3166384
349741 HPNFLVVEK
71224 SPDFTNENPLETR
191831 NIVWIAECIAQR
3081513 SQVSDYGNYDIIVR
446271 FDDHKGPTITLTQIV
91783 EIDQEAAVEVSQLR
212387 SGTGIAAMSVMRPELIMK
101949 TPADCPVIAIDSFR
386047 SGPGAYESGIMVSK
172030 LTFDTTFSPNTGK
46212 TGEAIVDAALSALR
263999 NTLDCGVQILK
193254 DLVPDLSNFYAQYK
80846 LATDAAQVQGATGTR
169146 VLLGGISGLTGGFVGTPADLVNVR
1397089 HSALGQHPTINDDLPNR
77241 SAFSNLFGGEPLSYTR
448978 FTAGDFSTTVIQNVNK
44365 FVMQEEFSR
1476149 SPYEEQLEQQR
128634 TAESGELHGLTTDEK
46196 TFTEGIVDATGDR
148122 ASGGGVPTDEEQATGLER
723071 DAGVNNLTIQVEK
158622 LVRPPVQVYGIEGR
899108 VYWTDVLNEAIFSANR
393743 VTSLEEELTDLR
162103 HYLFDVQR
57683 VALSPAGVQALVK
146831 SLCPVSWVSAWDDR
56791 HAGILSVLVALMSGK
77944 FTQAGSEVSALLGR
558339 LDFSTGNFNVLAVR
1377205 NIIHSAQTGNFK
4206075 APDEVPLAPR
221809 VVVDEGQDQEGPEEK
Cont1-24
Cont1-50
Cont1-16
Cont1-38
Cont1-2
Cont1-55
Cont1-11
Cont1-28
Cont1-31
Cont1-14
Cont2-3
Cont1-33
Cont1-62
Cont1-17
Cont2-37
Cont1-17
Cont1-16
Cont1-63
Cont1-16
Cont1-41
Cont2-54
Cont1-21
Cont1-40
Cont1-48
Cont1-5
Cont1-39
Cont1-3
Cont1-39
Cont1-12
Cont1-63
Cont1-54
Cont1-14
Cont1-57
Cont1-1
Cont1-63
Cont1-37
Cont1-23
Cont1-1
Cont1-24
Cont1-61
IPR004046 (D) Glutathione SIPR001902 (F) Sulphate
IPR000194 (D) H+IPR000778 (F) Cytochrome bIPR005772 (F) Eukaryotic
IPR001757 (F) ATPase, E1IPR002843 (F) H+IPR000276 (F) Rhodopsin-like
IPR008388 (F) Vacuolar ATP
IPR005805 (F) Rieske ironIPR000245 (F) Vacuolar H+IPR000886 (PTM)
IPR002035 (D) von
IPR001925 (F) Porin,
IPR000886 (PTM)
IPR001993 (F) Mitochondrial
IPR001450 (D) 4Fe-4S
IPR000504 (D) RNA-binding
IPR001993 (F) Mitochondrial
IPR000960 (F) FlavinIPR003137 (D) ProteaseIPR002699 (F) H+IPR005788 (D) Disulphide
3135122 AFEQFLGHLQAVPELR
150717 KNEGVNWLR
563725 ILYLIQAWAHAFR
459011 TALPTSGSSTGELELLAGEVPAR
189661 SSTNVEEAFFTLAR
918947 LNQELENLR
611379 TQYEQTLAELNR
672810 FTVPIPALNNSPQLK
540947 NHSIILSAPNPEGK
186515 ILFEITAGALGK
340484 LYGPTNFSPIINHVAR
217222 FEIPYFTTSGIQVR
452096
496849 EAAAAATAEFLQLHLESVEELKK
216866 LNVDEAFEQLVR
192612 YDVQHLQTALR
72085 LASQANIAQVLAELK
EGF1-18
Cont1-20
EGF1-56
EGF1-53
Cont2-13
Cont1-14
Cont1-37
Cont1-35
Cont3-21
Cont1-20
Cont1-41
Cont1-34
Cont1-14
Cont1-25
Cont1-15
Cont1-15
Cont1-24
IPR001683 (D) Phox-like
IPR005024 (F) Eukaryotic
IPR000306 (D) Zn-finger,
IPR001026 (D) Epsin NIPR001806 (F) Ras GTPase
IPR000895 (F) Transthyretin
IPR001128 (F) Cytochrome
IPR002124 (F) Cytochrome c
IPR002524 (F) Cation efflux
IPR000711 (F) H+IPR000033 (R) Low-density
IPR008688 (F) Mitochondrial
IPR004003 (F) NAD(P)
IPR003213 (F) Cytochrome
IPR001757 (F) ATPase, E1IPR000194 (D) H+IPR005547 (F) LongevityIPR006808 (F) Mitochondrial
Trafficking (73 proteins)
Sorting nexin-21 Sorting nexin L SNX-L
UniRef100|Q9BR16
Charged multivesicular body protein 1a
UniRef100|Q5R605
Hepatocyte growth factor-regulated tyrosine
UniRef100|Q5XIV8
Epsin 1
UniRef100|O88339
Ras-related protein Rab-8B
UniRef100|Q3U1Z3
DAMP-1 protein
UniRef100|Q811A2
PREDICTED:
protein kinase C and casein kinase
UniRef100|UPI0000506741
UniRef100|UPI000002A121
Sorting nexin-15
UniRef100|Q91WE1 UniRef100|Q4V896
Syntaxin-2 Epimorphin
UniRef100|Q08847
unknown [Homo
sapiens]
nrdb|62988824 UniRef100|Q3ZTS9
IPI|IPI00192445.1
PREDICTED:
similar toUniRef100|Q5CZX9
copine III
UniRef100|UPI0000506BFD
UniRef100|Q8IYA1
Adaptor-relatedUniRef100|UPI0000441F57
protein complex 1, mu 1 subunit
UniRef100|Q59EK3 UniRef100|Q3UG16
Similar to enthoprotin; epsin 4; clathrin interacting
UniRef100|Q6DGF2 UniRef100|Q5SUH7
Syntaxin-17
UniRef100|Q9Z158
B6-derived CD11 +ve dendritic cells cDNA,
UniRef100|Q3U1N3 UniRef100|P10301
Golgi SNAP receptor complex member 2 27 kDa
UniRef100|O08566
AP-1 complex UniRef100|UPI00004BE67D
subunit beta-1 Adapter-related
UniRef100|P52303 UniRef100|Q922E2
Unique
Unique
Unique
Unique
Unique
7.1
6.0
6.0
5.0
4.7
4.5
3.9
3.8
3.5
3.0
2.8
2.6
12
1
1
1
1
2
2
17
2
14.1
1
1
5
9
1
6
12
2
2
16
2
3
20
2
1
1
3
3
3
3
2
2
1
2
1
1.1 2.2 2.2
33
25 27
6
3
3
1
2
3
1
1
3
6
5
3
6
7
5
11
15 17
49
36 59
1
3
3
6
4
2
10
3
1
38.9 53.3 39
0.0
0.0
0.0
0.0
0.0
4.0
0.7
0.3
0.3
1.0
1.3
3.7
12.7
0.7
1.3
1.7
16.6
71
0.0
0.7
0.6
0.7
4.0
0.0
0.7
3.1
IPR001060 (D)
IPR001683 (D) Phox-like
IPR000727 (D) Target
IPR005024 (F) Eukaryotic
IPR000008 (D) C2 domain
IPR001392 (F) Clathrin
IPR001026 (D) Epsin NIPR000727 (D) Target
IPR001806 (F) Ras GTPase
IPR007705 (F) Vesicle
IPR002553 (D) Adaptin, N-
Table 3.2 ___Continued
Database|Protein
Identifier
Protein Description
Fold
Change
C1 C2 C3
E1
E2 E3
Mean
Control
2.6
4
7
14
11
4
3.7
Synaptosomal-associated protein 23 SNAP-23
UniRef100|O70377 UniRef100|O35620
2.3
3.4
4
5.2 7.1 4.9
2.5
Ras-related protein
Rab-5C
UniRef100|P51147 UniRef100|Q3TJ39
UniRef100|UPI000017ED6D
2.3
2
2
3
3
3
1.3
Adult male testis cDNA, RIKEN full-length
UniRef100|Q8C0W8
2.2
7
7
3
16
14
8
5.7
Charged multivesicular body protein 2a
UniRef100|Q9DB34
2.2
9.8 7.5 UniRef100|Q4W4Y1
1
14 14.2 13
6.1
2 days neonate UniRef100|Q5RB15
thymus thymic cells cDNA,
UniRef100|Q3TED2 UniRef100|Q9QZA2
IPI|IPI00694040.2 UniRef100|Q99LR3
nrdb|73989664
2.1
3
4
3
10
5
6
3.3
Syntaxin-8
UniRef100|Q9Z2Q7 UniRef100|O60712
2.0
8
12
6
21.9 14.5 16
8.7
ES cells cDNA, RIKEN full-length enriched
UniRef100|Q3TUW9 UniRef100|Q641Z6
2.0
2
2
1
1
0.7
Vacuolar protein sorting 33A
UniRef100|Q63615
2.0
2
1
2
1
0.7
LIN-7
homolog
C
LIN-7C
UniRef100|Q5RAA5
2.0
2
2
2
3
4
5
2.0
Chain A, Crystal Structures Of Ral-Gppnhp And
nrdb|56966231 UniRef100|P63320
2.0
1
1.1 1.1 2.1 1.1 3.2
1.1
Activated spleen cDNA, RIKEN full-length
UniRef100|Q3U0T9
2.0
12 10 10
22
23 18
10.7
Flot2 protein
UniRef100|Q5XIW9 UniRef100|Q5SS83
2.0
13 12
9
28 22.3 16
11.3
Annexin A11 Predicted
UniRef100|Q5XI77 UniRef100|P27214-2
1.9
7
6
3
16
7
8
5.3
Adapter-Related
Protein
Complex 1 Gamma 1 Su
UniRef100|UPI0000441F55
UniRef100|P22892
UniRef100|O43747
1.9
2.9 3.4 2.6 6.4 7.1 3.7
3.0
Small GTP-binding protein rab5
UniRef100|O88565
1.9
3
4
2
5
6
6
3.0
Caveolin 1
UniRef100|Q8R4A2 UniRef100|P33724
1.9
3.6 2.3
7
1.8 2.3
2.0
GES30
UniRef100|Q9Z2P7
1.8
2
3
1
3
6
2
2.0
Tax_Id=10090UniRef100|Q9UL25
Ras-related protein Rab-21
IPI|IPI00337980.3 UniRef100|Q6AXT5
1.8
5
6
7
15
6
11
6.0
Chain A, Mu2 Adaptin Subunit (Ap50) Of Ap2
nrdb|13399864 UniRef100|P20172
1.8
6
4.9 6.1 5.5 11.8 13
5.7
PREDICTED:
similar to Adapter-related protein
UniRef100|UPI000050609E
UniRef100|P17426
1.8
2
1
5
5
5
4
2.7
Synaptophysin-like protein
UniRef100|Q66H18
1.8
3
1
2
3
2
1.3
Adaptor-relatedUniRef100|Q9JKC8
protein complex 3, mu 1 subunit
UniRef100|Q6IRG9 UniRef100|Q5R478
1.8
4
10
6
12
16
7
6.7
PREDICTED:
similar to copine family member
nrdb|73996815 UniRef100|Q9CUZ8
UniRef100|UPI0000021F7D
1.7
9
6
9
13
4
5.0
PREDICTED: similar to Early endosome antigen
UniRef100|UPI00005070BF
1.7
16 16
8
24
26 19
13.3
Flotillin-1 Reggie-2
REG-2
UniRef100|Q9Z1E2 UniRef100|Q5TM70
UniRef100|Q767L6
UniRef100|UPI00004BBAD0
UniRef100|Q6P2A7
1.7
1
1
1
2
1
2
1.0
Hypothetical protein
UniRef100|Q2PFR5 UniRef100|Q53XZ1
1.6
5
8
7
13
10
9
6.7
MAL2A Mal, T-cell differentiation protein 2
UniRef100|Q7TPB7
1.6
3.3 2.2 3.2 2.2 3.2
1.8
Ras-related protein Rab-8
UniRef100|P24407
1.5
30
32
16.9
26.5
40.2
56
26.3
Ap2a2
protein
[Mus
musculus]
nrdb|14714884 UniRef100|Q8C2J5
1.5
6.2 5.4 5.4 8.2 8.4 9.4
5.7
Putative NFkB activating protein
UniRef100|Q7Z432 UniRef100|Q3TY55
1.5
1
1
1
1
1
0.7
dynein (Q13409) Isoform 2C of Q13409
UniRef100|Q13409-3
1.5
1
2
3
1
3
5
2.0
Vesicle-associated membrane protein 7
UniRef100|Q9JHW5
1.5
8
14
6
15
14 13
9.3
PREDICTED: similar to vesicle trafficking protein
RefSeq|XP_533025.1 UniRef100|O08595
1.5
1
1
1
1
1
0.7
SNAP-associated protein
UniRef100|Q4KM25 UniRef100|Q3U8V4
1.5
17 10 10
19
19 17
12.3
lipocortin I [Rattus sp.]
nrdb|235879 IPI|IPI00231615.3
-1.6
2
3
3
2
1
2
2.7
PREDICTED: similar to RAB17, member RAS
UniRef100|UPI0000507597
-1.6
27 34 34
24
11 23
31.7
Alpha-soluble NSF attachment protein SNAPUniRef100|P54921
-1.7
17
6
7
9
2
7
10.0
Hypothetical protein LOC498266
UniRef100|Q6AYB8 UniRef100|Q3TJC4
-1.7
4.3 1.4 1.6 1.7
2.6
2.4
PREDICTED:
similar to UniRef100|Q3SZN2
Protein transport protein
UniRef100|UPI00001806CD
UniRef100|Q503A9
UniRef100|Q9BS15
-1.7
3
4
5
3
2
2
4.0
Blocked early in transport 1 homolog (S.
nrdb|37589616 IPI|IPI00315437.5
-1.8
23 14 71
12
7
42
36.0
SM-11044 binding protein SMBP EP70-P-iso
UniRef100|Q5TB53 UniRef100|Q96JZ5
-1.8
3.2 2.5
1.8 1.4
1.9
PREDICTED: similar to Programmed cell death 6
UniRef100|UPI0000507416
-1.8
20 22 35
16
13 14
25.7
PREDICTED:
similar to Coatomer protein
UniRef100|UPI00002503B6
UniRef100|Q8CIE6
-1.8
18 19 18
14
3
13
18.3
18-day embryo whole body cDNA, RIKEN fullUniRef100|Q9D1J2 UniRef100|UPI0000507F48
-1.8
7
4
4
1
1
3.7
Vesicle associated protein
UniRef100|Q9Z2Q1
-1.8
4
3
4
3
3
3.7
(O75379) Isoform 2 of O75379
UniRef100|O75379-2
-2.0
51 36 69
35
29 14
52.0
Colon RCB-0549
Cle-H3 cDNA, RIKEN fullUniRef100|Q8C1Z9 UniRef100|P49020
UniRef100|Q3U9H7
-2.2
7
9
12
5
3
5
9.3
Endobrevin
UniRef100|UPI0000111953
UniRef100|UPI0000029ACA
-2.3
13 17 25
2
9
13
18.3
Coatomer subunit beta Beta-coat protein BetaUniRef100|P23514
-2.5
6
4
10
2
3
3
6.7
PREDICTED:
similar to archain; coatomer protein
UniRef100|UPI0000493215
UniRef100|P48444
-2.5
2
2
1
2
1.7
Sec22l2 protein
UniRef100|Q91Z54
-2.6
9
12 15.6
6
1
7
12.2
Coatomer protein
complex, subunit gamma
UniRef100|Q4AEF8 UniRef100|Q5U293
UniRef100|Q9Y678
-3.2
23 28 25
3
7
14
25.3
Coatomer protein complex, subunit beta 2 Beta
UniRef100|Q5M7X1 UniRef100|Q3U5Z9
-3.5
1
3
3
2
2.3
Phosphoinositide phosphatase SAC1
UniRef100|Q9ES21
-4.0
2
1
1
1
1.3
Adult male epididymis cDNA, RIKEN full-length
UniRef100|Q3TS10 UniRef100|UPI00000225EB
-5.0
2
2
1
1
1.7
TLOC1 protein
UniRef100|Q3KR08 UniRef100|O00682
SD
Control
Mean
EGF
SD
Peptides (identifier Sequence)
EGF
2.1
0.4
0.0
2.3
4.6
0.6
3.1
0.0
0.1
1.2
2.1
2.1
0.4
1.0
0.9
1.0
1.0
0.7
2.1
1.4
3.1
2.1
4.6
0.0
1.5
0.8
8.2
0.5
0.0
1.0
4.2
0.0
4.0
0.6
4.0
6.1
1.6
1.0
30.6
0.5
8.1
0.6
2.1
0.6
16.5
2.5
6.1
3.1
0.6
3.3
2.5
1.2
0.6
0.6
9.7
5.7
3.0
12.7
13.6
7.0
17.5
1.3
1.3
4.0
2.1
21.0
22.1
10.3
5.7
5.7
3.7
3.7
10.7
9.9
4.7
2.3
11.7
8.7
23.0
1.7
10.7
2.9
40.7
8.7
1.0
3.0
14.0
1.0
18.3
1.7
19.3
6.0
1.4
2.3
20.3
1.1
14.3
10.0
2.0
2.0
26.0
4.3
8.0
2.7
0.7
4.7
8.0
0.7
0.3
0.3
5.1
1.2
0.0
4.2
0.9
2.6
3.9
0.6
0.6
1.0
1.1
2.6
6.0
4.9
1.8
0.6
2.9
2.1
4.5
3.9
0.6
0.6
4.5
4.5
3.6
0.6
2.1
0.6
14.5
0.6
0.0
2.0
1.0
0.0
1.2
0.6
7.2
3.6
0.6
0.6
18.9
0.3
1.5
6.1
1.7
0.0
10.8
1.2
5.6
0.6
1.4
13.3
2.1
3.2
5.6
Band
574723 ILGLAIESQDAGIK
92312 GVDLQENSPASR
990888 NVVASLAQALQELSTSFR
3007409 ANIQAVSLK
57363 TPSNDLYKPLR
362570 IIQEQDAGLDALSSIISR
51311 VYIGSFWSHPLLIPDNR
362895 DKNFNAVGSVLSK
471382 RGDQLLSVNGVSVEGEHHEK
419610 ADQWNVNYVETSAK
45152 FAGQMGIQLFETSAK
610043 NVVLQTLEGHLR
49502 TPVLFDVYEIK
345264 IGYLGAMLLLDER
92656 TSMNVNEIFMAIAK
1029808 YVDSEGHLYTVPIR
180822 ALLLQGTESLNR
191459 HVSIQEAESYAESVGAK
62829 IPTPLNTSGVQVICMK
241673 VGGYILGEFGNLIAGDPR
928857 TITATFGYPFR
650291 VSSQNLVAIPVYVK
720422 TEVIDNTLNPDFVR
1357052 IQAGEGETAVLNQLQEK
382216 AQLIMQAEAEAESVR
441903 AAEEAFVNDIDESSPGTEWER
935455 ITLPAGPDILR
119590 ANINVENAFFTLAR
345267 SSPLIQFNLLHSK
192459 DKLTELQLR
1235775 SVSTPSEAGSQDSGDGAVGSR
446331 VTETQAQVDELK
156306 IMVANIEEVLQR
1305392 VVLVNNILQNAQER
567030 TPAQFDADELR
4203865 LNYQVSEIFNTIAQELLQR
441887 LLEAHEEQNVDSYTESVK
503726 VMADELTNFR
343487 IDMNLTDLLGELQR
719349 SLALDIDRDTEDQNR
788295
57363 TPSNDLYKPLR
73075 LVGQSIIAYLQK
491095 YGVVLDEIKPSSAPELQAVR
142755 DQTLSPTIISGLHSIAR
57026 HLNDDDVTGSVK
188933 HEQEYMEVR
196804 NKTEDLEATSEHFK
138291 VLQDLVMDILR
77073 NSNILEDLETLR
161921 INLSDMQMEIK
827883 ELAPAVSVLQLFCSSPK
105872 GSNNVALGYDEGSIIVK
48475 TQLGLVMDGFNSLLR
519946 ILDETQEAVEYQR
178609 KGEEALFTTR
Cont1-15
Cont1-15
Cont1-30
Cont1-21
Cont1-55
Cont1-14
Cont1-43
Cont2-45
Cont2-13
Cont1-15
Cont1-14
Cont1-33
Cont1-20
Cont1-54
Cont1-15
Cont1-12
Cont1-17
Cont1-15
Cont1-37
Cont1-52
Cont1-16
Cont1-33
Cont1-43
Cont1-65
Cont1-34
Cont1-23
Cont1-11
Cont1-14
Cont1-45
Cont1-14
Cont1-50
Cont1-14
Cont1-14
Cont1-6
Cont1-20
Cont1-14
Cont1-21
Cont1-16
Cont1-43
Cont1-3
Cont1-17
Cont1-55
Cont1-52
Cont1-21
Cont1-61
Cont1-7
Cont1-10
Cont1-3
Cont1-55
Cont1-43
Cont1-14
Cont1-54
Cont1-54
Cont1-42
Cont1-21
Cont1-6
1393740 SPTSSAIPFQSPR
Cont1-30
Transcription/Translation (16 proteins)
MTSG1 346aa isoform
UniRef100|Q6XUU5 UniRef100|Q80Z99
5.0
3
5
14
11
15
2.7
72
InterProScan
Predictions
IPR000727 (D) Target
IPR001806 (F) Ras GTPase
IPR000727 (D) Target
IPR005024 (F) Eukaryotic
IPR004328 (D) BRO1
IPR000727 (D) Target
IPR000261 (D) EPS15
IPR001619 (F) Sec1-like
IPR001478 (D)
IPR001806 (F) Ras GTPase
IPR001806 (F) Ras GTPase
IPR001107 (F) Band 7 protein
IPR001464 (F) Annexin
IPR002553 (D) Adaptin, NIPR001806 (F) Ras GTPase
IPR001612 (F) Caveolin
IPR000727 (D) Target
IPR001806 (F) Ras GTPase
IPR001392 (F) Clathrin
IPR002553 (D) Adaptin, NIPR001392 (F) Clathrin
IPR000008 (D) C2 domain
IPR001107 (F) Band 7 protein
IPR000996 (F) Clathrin light
IPR008253 (D) Marvel
IPR001806 (F) Ras GTPase
IPR002553 (D) Adaptin, NIPR000348 (F)
IPR001680 (R) G-protein beta
IPR001388 (F) Synaptobrevin
IPR001388 (F) Synaptobrevin
IPR001464 (F) Annexin
IPR000744 (F) NSF
IPR006895 (D) Sec23/Sec24
IPR000727 (D) Target
IPR001064 (D) Beta and
IPR001680 (R) G-protein beta
IPR006822 (F) Coatomer
IPR001680 (R) G-protein beta
IPR001388 (F) Synaptobrevin
IPR000348 (F)
IPR001388 (F) Synaptobrevin
IPR002553 (D) Adaptin, NIPR001392 (F) Clathrin
IPR001388 (F) Synaptobrevin
IPR002553 (D) Adaptin, NIPR001680 (R) G-protein beta
IPR002013 (D) Synaptojanin,
IPR005024 (F) Eukaryotic
IPR004728 (F) Translocation
Table 3.2 ___Continued
Database|Protein
Identifier
Protein Description
Fold
Change
60S acidic ribosomal
protein P0 L10E
UniRef100|P19945 IPI|IPI00457715.2
UniRef100|Q9BVK4
PREDICTED: similar to eukaryotic translation
nrdb|73947646 UniRef100|UPI00000E75B7
Ribosomal protein S7 2 days neonate thymus
UniRef100|Q4FZE6
Translation initiation factor-3 subunit 5
UniRef100|Q8VH52 UniRef100|Q8N978
PURA protein
UniRef100|Q2NLD4 UniRef100|P42669
40S ribosomal protein S18
UniRef100|Q3T0R1
REFSEQ:XP_875759 PREDICTED: similar to
IPI|IPI00728322.1 UniRef100|Q3T0A5
5 nucleotidase
UniRef100|Q4G083 nrdb|539794
UniRef100|P21588 UniRef100|Q3U3S1
ribosomal protein S10 [Homo sapiens]
nrdb|3088338 UniRef100|Q3T0F4
40S ribosomal protein S5
UniRef100|P46782
Prohibitin-2 B-cell receptor-associated protein
UniRef100|Q5XIH7 UniRef100|Q2YDA4
ribosomal protein L11 [Homo sapiens]
nrdb|4432750 UniRef100|Q4V8I6
RPL23 protein
UniRef100|Q8N4F9 UniRef100|Q3SWV7
SWISS-PROT:Q71UM5 TREMBL:Q49A11
IPI|IPI00420117.5 IPI|IPI00124709.1
Ribosomal protein S2
UniRef100|O55211 nrdb|3122811
3.0
2.6
2.2
2.0
2.0
2.0
1.7
1.6
Unique
-1.7
-1.9
-2.0
-3.0
-3.0
-3.0
C1 C2 C3
12.3
1
1
2
20
3
1
1
2
57
1
2
6
1
1
1
1
54
2
1
13
2
1
1
1
4
1
7
2
3
2
1
4
3
2
3
E1
E2 E3
Mean
Control
SD
Control
Mean
EGF
1.0
14.3
1.7
0.7
0.7
0.3
1.0
52.7
1.3
1.7
9.0
1.3
1.0
1.0
1.0
0.7
4.9
0.7
0.0
0.0
3.0
36.9
3.7
1.3
1.3
0.7
1.7
83.3
0.0
1.0
4.7
0.7
0.3
0.3
0.3
1.0
6.8
2.5
0.6
0.6
0.0
0.6
13.1
2.1
1.0
0.6
1.0
1.3
11.7
1.3
1.3
13.0
3.7
1.3
1.0
3.2
0.7
0.0
2.1
1.2
1.5
0.5
1.5
1.0
1.2
12.1
5.9
1.2
3.6
0.6
1
2
4
10.8 31.5 44.5
2
4
6
2
1
1
1
1
1
1
2
1
47
98
79
1
2
1
1
8
3
3
1
1
1
1
1
2
1
3
35
1
1
2
1
17
2
1
23
4
1
2
1
7
1
1
6
5
2
1
0.0
2.0
0.3
0.3
3.7
2.0
2.3
3.0
2
1
3
1
3
4
1
13
8
2
50
8
16
5
8
5
5
19
4
1
11
0.0
0.0
0.0
0.0
0.0
0.0
0.3
3.7
3.7
0.7
24.6
4.7
6.3
2.7
6.7
2.0
3.3
17.7
18.7
2.3
53.9
2.7
2
2
1
1
1
4
1
6
0.0
0.0
0.3
0.3
0.3
1.7
0.3
1.3
2
73
1
8
1
1
0.7
5.1
0.6
0.6
3.6
0.6
0.0
0.0
0.7
SD
Peptides (identifier Sequence)
EGF
Band
InterProScan
Predictions
90291 GHLENNPALEK
85453 STTTGHLIYK
162543 TLTAVHDAILEDLVFPSEIVGK
924071 VIGLSSDLQQVGGASAR
274070 GPGLGSTQGQTIALPAQGLIEFR
144321 IPDWFLNR
95995 HPGSFDVVHVK
139343 VVLPSYLVNGGDGFQMIKDELLK
90066 IAIYELLFK
90700 TIAECLADELINAAK
119012 IVQAEGEAEAAK
89363 VLEQLTGQTPVFSK
147146 ISLGLPVGAVINCADNTGAK
146744 DLLHPSLEEEK
95103 SPYQEFTDHLVK
Cont2-24
Cont1-17
Cont1-11
Cont1-36
Cont1-30
Cont1-7
Cont1-18
Cont1-17
Cont1-8
Cont1-11
Cont1-22
Cont1-9
Cont1-5
Cont1-2
Cont2-20
IPR001790 (F) Ribosomal
IPR000795 (F) Elongation
IPR000554 (F) Ribosomal
IPR000555 (F) Mov34
IPR006628 (F) PURIPR001892 (F) Ribosomal
IPR000876 (F) Ribosomal
IPR004843 (D) MetalloIPR005326 (D) Plectin/S10, NIPR000235 (F) Ribosomal
IPR001107 (F) Band 7 protein
IPR002132 (F) Ribosomal
IPR000218 (F) Ribosomal
IPR000592 (F) Ribosomal
IPR000851 (F) Ribosomal
0.6
5.0
0.6
0.6
8.9
1.5
0.6
0.7
964924 AFLDGFNEVAPLEWLR
3133060 NEFTITHVLIPR
182907 LPPTPLLLFPEEEATNGR
3159588 IHQESELHSYLTR
1186714 TFEAFMYLSLPLASTSK
478454 KMPETFSNLPR
408110 NYSMIVNNLLKPISVESSSK
200886 PLENLEEEGLPK
EGF1-56
Cont1-20
Cont3-32
Cont2-23
Cont1-60
Cont1-20
Cont1-17
Cont1-31
IPR000569 (D) HECT domain
IPR000555 (F) Mov34
IPR003892 (D) Ubiquitin
IPR001440 (R) TPR repeat
1.7
1.3
4.0
1.7
2.7
3.3
1.0
10.9
9.3
1.7
59.3
10.7
14.3
5.7
12.3
3.7
6.0
26.7
9.3
1.0
10.3
0.0
0.6
0.6
1.7
1.2
0.6
1.2
0.0
2.1
1.5
0.6
10.7
2.5
2.1
0.6
4.0
1.2
5.7
6.8
5.5
0.0
2.1
466314 SLLAELDEVNKELSR
104271 DITYFIQQLLR
81362 TLFSNIVLSGGSTLFK
1334291 YRDLDEDEILGALTEEELR
761361 EQSPPLIGQQSTVSDVPR
275627 KDEGSYSLEEPK
620910 IVEANPLLEAFGNAK
69274 LALDIEIATYR
83786 NFHVFYQLLSGASEELLHK
837280 KVESLQEEIAFLK
71015 MSLLQLVEILR
166383 SGSGTMNLGGSLTR
1279481 NQNINLENNLGEVEAR
241964 IGELVGVLVNHFK
64032 TLVTQNSGVEALIHAILR
321395 LLEVQSQVEELQK
71087 HVNPVQALSEFK
470144 LLSEQDGSLKDILR
834499 ETEVIDPQDLLEGR
214454 LNGTDPEDVIR
88384 NTFAEVTGLSPGVTYLFK
275627 KDEGSYSLEEPK
EGF1-43
EGF1-35
EGF1-32
EGF1-31
EGF1-74
Cont1-80
Cont3-62
Cont1-1
Cont1-59
Cont1-41
Cont1-52
Cont1-19
Cont1-31
Cont1-56
Cont1-52
Cont1-52
Cont1-56
Cont1-14
Cont1-72
Cont1-8
Cont1-67
Cont1-80
IPR008253 (D) Marvel
IPR004000 (F) Actin/actin-like
IPR004000 (F) Actin/actin-like
IPR002016 (F) Haem
3.7
1.3
1.7
1.3
1.3
5.7
1.0
4.0
1.5
0.6
1.2
0.6
0.6
2.1
0.0
1.7
153708 GGDFLEGSIITGAQLSQVNAR
3263739 NPSASTFLHLSPSSFR
549333 VLNVGDIGGNETVTLR
46779 ALQSDYITYIDDLLTSINAKPDLR
2654826 VEEVDNEVLLAAFQEK
1099703 AVGAFLFGASASQSLTDIAK
259444 GIVSLSDILQALVLTGGEK
121777 KEEEVNNLVK
EGF1-12
EGF1-39
Cont1-53
Cont3-39
Cont2-53
Cont1-18
Cont1-25
Cont1-24
IPR000634 (BS)
IPR001968 (F) Glycoside
IPR001102 (F) ProteinIPR000566 (F) Lipocalin-
0.0
2.9
0.0
Ubiquitination/Proteasome (8 proteins)
Nedd-4-like ubiquitin-protein ligase WWP2
UniRef100|Q9DBH0
STAM-binding protein Associated molecule with
UniRef100|Q8R424 UniRef100|Q3UTI9
AUP1 protein
UniRef100|Q5XKR6
STIP1 homology and U box-containing protein 1
UniRef100|Q9DCJ0
PREDICTED: similar to mKIAA0055 protein (94%
UniRef100|UPI00005067FB
hypothetical protein [Homo sapiens]
nrdb|52545759 UniRef100|Q9BT67
Calcyclin binding protein
UniRef100|Q6AYK6 UniRef100|Q9CXW3
PREDICTED: similar to 26S proteasome nonnrdb|73985099 UniRef100|Q15008
Unique
5.8
4.0
4.0
3.5
1.8
-1.8
-3.0
1
1
1
2
4
2
11
1
2
10
2
1
1.7
IPR007699 (D) SGS
IPR000717 (D) Proteasome
Structural/Cytoskeleton (22 proteins)
Unique
Tax_Id=10116 Ensembl_locations(Chr-bp):2IPI|IPI00210150.1
Unique
Actin-related protein Arp11
UniRef100|Q9C0K3
Unique
Alpha-centractin
Centractin CentrosomeUniRef100|Q3TJF9 UniRef100|Q8R5C5
UniRef100|P42025
Unique
Tropomodulin-1 Erythrocyte tropomodulin EUniRef100|Q9ERR9
Unique
Fibronectin
UniRef100|Q28692
Unique
Syndecan-1 precursor SYND1 CD138 antigen
UniRef100|Q96HB7
3.0
Myosin
UniRef100|Q14784 UniRef100|Q9UEG2
2.9
5.8
0
cytokeratin 8 polypeptide
nrdb|203734 UniRef100|UPI00001C3B6A
UniRef100|Q5U2M3 UniRef100|Q10758
2.5
6
Myosin Ib Myosin I alpha MMI-alpha MMIa
UniRef100|Q05096
2.5
1
Vimentin
UniRef100|Q6S5G2 UniRef100|P48670
2.4
27 23
Myosin I heavy chain
UniRef100|Q63355
2.3
4
4
CAPZB protein [Homo sapiens]
nrdb|19352984 UniRef100|Q3T012
2.3
6
5
TREMBL:Q5BJY9;Q63278
IPI|IPI00480679.2
2.1
3
2.1
Myo1d protein UniRef100|Q5SSK7
UniRef100|Q8K063 UniRef100|Q8NHP9
1.8
7
5
Junction UniRef100|P70565
plakoglobin (Desmoplakin III)
nrdb|1709649 UniRef100|Q15093
1.8
3
2
HOOK3 protein [Homo sapiens]
nrdb|29387173 UniRef100|Q8NBH0
1.8
2
4
Hypothetical protein
MGC128363
UniRef100|Q3MHM6 UniRef100|Q5U302
UniRef100|UPI000011236B
UniRef100|P35221
1.5
27 22
PREDICTED:
similar toUniRef100|Q6WGK6
connexin 32
UniRef100|UPI00004A7732
UniRef100|P08034
-2.0
21
23
Syndecan-4
precursor
SYND4
Ryudocan
core
UniRef100|P34901
-2.3
3
1
unnamed protein product [Rattus norvegicus]
nrdb|4490977 UniRef100|O14950
-5.2
54.8 57
Fibronectin precursor FN
UniRef100|Q6LDX9
Unique
3
3
syndecan [Rattus norvegicus]
nrdb|57324 UniRef100|P26260
1
5.4
5
1
24
6
8
3
8
1
4
4
12
3
50
2
2
1
1
2
3
6
3
1
3
2
2
4
1
1
8.6 11.7
9
11
2
1
57
71
13
11
15
12
6
6
15.8 13
3
3
13
32
29
9
15
1
1
12
8
IPR001050 (F) Syndecan
IPR001609 (D) Myosin head
IPR001664 (F) Intermediate
IPR000048 (D) IQ calmodulinIPR001664 (F) Intermediate
IPR000048 (D) IQ calmodulinIPR001698 (F) F-actin
IPR000048 (D) IQ calmodulinIPR000225 (R) Armadillo
IPR008636 (F) HOOK
IPR001033 (F) Alpha-catenin
IPR000500 (F) Connexins
IPR001050 (F) Syndecan
IPR002048 (D) CalciumIPR000083 (D) Fibronectin,
IPR001050 (F) Syndecan
Metabolism (57 proteins)
Acyl-coenzyme A oxidase 1, peroxisomal
UniRef100|P11354
Hyaluronidase-2 precursor Hyal-2
UniRef100|Q9Z2Q3
Protein-glutamine gamma-glutamyltransferase K
UniRef100|Q9JLF6 UniRef100|Q4QRA6
Dimethylaniline monooxygenase [N-oxideUniRef100|Q6P7Q5
PREDICTED: similar to B aggressive lymphoma
UniRef100|UPI00005078DA
Phosphatidic acid phosphatase 2a
UniRef100|Q6P766 UniRef100|O08564
PREDICTED: similar to 5-AMP-activated protein
nrdb|73996585 UniRef100|P80385
Peroxisomal trans-2-enoyl-CoA reductase RLF98
UniRef100|Q9WVK3
Unique
Unique
5.0
4.0
4.0
3.4
3.0
3.0
1
1
2
1
1
1
2
1
3
4
1
3
1
1
5
1
3
5
1
1
2
2
8
1
3
73
0.6
1.4
IPR000326 (F) PAIPR000644 (D) CBS domain
IPR002198 (F) Short-chain
Table 3.2 ___Continued
Database|Protein
Identifier
Protein Description
Fold
Change
C1 C2 C3
E1
E2 E3
2.9
3
6
8
9
9
Fatty-acid amide hydrolase Oleamide hydrolase
UniRef100|P97612
2.5
1
2
1
3
4
3
Chain A, Crystal Structure Of The Inhibitory Form
nrdb|55670739 UniRef100|P22288
2.3
2
4
5
8
1
B6-derived CD11 +ve dendritic cells cDNA,
UniRef100|Q3TB74 UniRef100|P07824
2.3
3
4
3
7
9
7
Argininosuccinate synthase Citrulline--aspartate
UniRef100|P09034
2.3
23 12
9
48
28 24
Membrane-bound aminopeptidase P X-prolyl
UniRef100|Q99MA2
2.1
8
17
10
20
31
24
Bile acyl-CoA synthetase BACS Bile acid CoA
UniRef100|Q9ES38
2.1
1
5
5
10
8
5
Dehydrogenase/reductase SDR family member 8
UniRef100|Q6AYS8
2.0
2
2
1
1
Very-long-chain specific acyl-CoA
UniRef100|P45953
2.0
6
5
6
1
Fructose-bisphosphate
aldolase
B
UniRef100|P00884
2.0
1
2
2
2
2
hydroxyacyl-Coenzyme
UniRef100|UPI000019614C
UniRef100|Q60587 A dehydrogenase/31.9
19 23 27
52 49.4 33
Aminopeptidase N
UniRef100|P15684
1.8
4
2
5
4
2
S-adenosylmethionine synthetase isoform type-1
UniRef100|Q5FVU2
1.8
3
4
2
4
9
3
Dehydrogenase/reductase SDR family member 4
UniRef100|Q8VID1
1.8
2
2
2
4
1
Acyl-coenzyme A oxidase 2, peroxisomal 3UniRef100|P97562
1.8
1
1
2
3
3
1
Adult retina cDNA, RIKEN full-length enriched
UniRef100|Q3UEY0 UniRef100|Q3UVJ7
1.7
3
8
4
10
5
LRRGT00111
UniRef100|Q6TUD3
1.7
1
3
3
4
4
4
PREDICTED: similar to Amine oxidase [flavinnrdb|109139486 UniRef100|Q5EBB5
1.7
8
15
7
18
19 14
PREDICTED: similar to NADH-ubiquinone
UniRef100|UPI0000180F4C
1.7
3
1
1
3
4F2 heavy chain 4F2hc Slc3a2 protein Type II
UniRef100|Q794F9
1.7
10 11
3
14
15 11
13 days embryo
head cDNA, RIKEN full-length
UniRef100|Q3TUI2 UniRef100|Q569X5
UniRef100|P51640
1.6
5
7
3
2
9
13
Lpgat1 protein UniRef100|Q92604
UniRef100|Q8R1E1 UniRef100|Q91YX5
1.5
5
10
4
6
13 10
Alcohol dehydrogenase A chain
UniRef100|P06757 UniRef100|Q8K571
1.5
27 23 17 40.6 33 27
Dipeptidase 1 precursor Microsomal dipeptidase
UniRef100|P31430 UniRef100|Q6IN35
1.5
2
1
1
1
Adult male liver tumor cDNA, RIKEN full-length
UniRef100|Q3UEF5
1.5
3
4
5
6
6
6
Squalene synthetase SQS SS FarnesylUniRef100|Q02769
-1.6
3
6
7
3
5
2
17-beta hydroxysteroid dehydrogenase 13
UniRef100|Q5M875
-1.7
18 15 13
8
5
14
Calcium-transporting ATPase 2C1
UniRef100|Q5PQW5 UniRef100|Q64567
-1.7
35 40 33
36
17
9
Tax_Id=10116 35 kDa protein
IPI|IPI00551713.1 UniRef100|Q543J0
-1.8
20 13 11
9
7
9
UDP-Gal:betaGlcNAc beta 1,3UniRef100|Q920V5 UniRef100|UPI0000028327
-1.8
10.3 15 47.7 12.1 12 17
Aa2-111
UniRef100|Q7TP82
-1.8
23 17 32
8
9
23
PREDICTED:
similar toUniRef100|Q922K5
UDP-N-acetyl-alpha-DUniRef100|UPI00005083B6
UniRef100|Q3UA32
-1.8
11
4
14
3
4
9
Alpha-1,6-mannosyl-glycoprotein 2-beta-NUniRef100|Q09326
-1.9
12 16
9
11
9
0
Nucleoside diphosphate kinase B
UniRef100|P19804
-1.9
19 14 46
14
10 17
CMP-N-acetylneuraminate-beta-galactosamideUniRef100|P13721
-2.0
2.5
1
4.5
0
3
1.1
UDP-glucuronosyltransferase
1-1 precursor
UniRef100|Q63886 UniRef100|Q561M6
UniRef100|Q64635
-2.0
13 14
7
7
3
7
PREDICTED:
similar to FLJ16237 protein
UniRef100|UPI0000506EBA
UniRef100|Q8C7H5
-2.1
10 13 11
4
7
5
glucose-6-phosphatase, catalytic [Rattus
RefSeq|NP_037230.1 UniRef100|P43428
-2.2
15
9
29
9
3
12
PREDICTED:
similar to mannosylUniRef100|UPI000024FEB9
UniRef100|P45700
-2.5
1
1
3
1
1
N-acetylglucosamine galactosyltransferase
UniRef100|Q9WVK1
-2.6
0
13.9 2.2
1
2.2
UDP-glucuronosyltransferase 2B2 precursor,
UniRef100|P08541 UniRef100|P08541
-2.6
4
9
18
3
3
6
Mannoside acetylglucosaminyltransferase 1
UniRef100|Q8CIC9 UniRef100|Q09325
-2.6
24 26 12
24
Ab1-219
UniRef100|Q7TP88
-2.7
4
3
9
2
1
3
UDP-GalNAc:polypeptide,
Nnrdb|304259 UniRef100|UPI00004523DC
UniRef100|Q10473
UniRef100|Q07537 UniRef100|UPI00004C0C7A
-2.8
7
7
1
3
1
NAD(P) dependent steroid dehydrogenase-like
UniRef100|Q5PPL3
-3.3
39.7 55 131 18.9 16 33
Alpha-mannosidase
2 Alpha-mannosidase II
UniRef100|P27046 nrdb|807679
UniRef100|P28494
-3.3
5
3
2
2
1
Lanosterol synthase Oxidosqualene--lanosterol
UniRef100|P48450 UniRef100|UPI00000E8107
-4.5
28 33 47
1
11 12
(P16615) Isoform SERCA2A of P16615
UniRef100|P16615-2 UniRef100|P11507-2
-4.7
7
2
5
1
1
1
Adult male medulla oblongata cDNA, RIKEN fullUniRef100|Q3UYN7 UniRef100|Q921U1
-5.8
17
8
33
10
PREDICTED: similar to Endoplasmic reticulum
UniRef100|UPI000050667A
Mean
Control
SD
Control
Mean
EGF
3.0
1.3
2.0
3.3
14.7
11.7
3.7
0.7
2.0
1.0
23.0
2.0
3.0
1.3
1.3
3.7
2.3
10.0
1.0
8.0
5.0
6.3
22.3
0.7
4.0
5.3
15.3
36.0
14.7
24.5
24.0
9.7
12.3
26.3
2.7
11.3
11.3
17.7
1.7
4.6
10.3
20.7
5.3
4.7
75.2
3.3
36.0
4.7
19.3
2.1
0.6
1.4
0.6
7.4
4.7
2.3
8.7
3.3
4.7
7.7
33.3
25.0
7.7
1.3
4.0
2.0
44.8
3.7
5.3
2.3
2.3
6.3
4.0
17.0
1.7
13.3
8.0
9.7
33.5
1.0
6.0
3.3
9.0
20.7
8.3
13.6
13.3
5.3
6.7
13.7
1.4
5.7
5.3
8.0
0.7
1.8
4.0
8.0
2.0
1.7
22.7
1.0
8.0
1.0
3.3
0.6
0.6
3.5
1.2
12.9
5.6
2.5
0.6
2.6
0.0
10.3
1.5
3.2
1.5
1.2
3.2
0.0
2.6
1.2
2.1
5.6
3.5
6.8
0.0
0.0
1.5
4.6
13.9
1.2
2.7
8.4
3.2
5.9
3.5
1.5
2.3
1.5
4.6
0.0
0.7
1.7
1.0
2.4
1.3
1.3
1.0
1.0
1.7
1.7
0.0
1.4
0.6
0.6
0.0
0.0
0.6
0.6
0.7
4.0
1.4
1.0
0.0
0.6
3.5
1.2
4.4
4.4
2.0
3.2
5.0
1.0
2.1
2.5
3.6
4.7
20.3
7.5
5.1
3.5
17.2
1.8
3.8
1.5
10.3
1.2
9.8
7.1
7.6
3.2
0.0
49.2
1.5
9.8
2.5
12.7
SD
Peptides (identifier Sequence)
EGF
1.0
1.2
9.3
0.7
6.1
0.0
Band
45522 LQNPDLDSEALLTLPLLQLVQK
3135525 SEEDNELNLPNLAAAYSSILR
82227 VMEETFSYLLGR
89326 APNTPDVLEIEFK
814542 GTVDEFSGAEHIDQLR
61217 IQDSLEITNTYK
1388565 MIFVPGSIALLTVLER
53813 VPAENVLGEVGDGFK
99425 GILAADESVGTMGNR
210062 IPFLLSGTSYK
73004 LPASVSTIMDR
228304 FVIGGPQGDAGVTGR
166023 LAEDGAHVVISSR
130849 LTNILDGGLPNTVLR
166875 VNSLAPGAISGTEGLR
1429146 GELVASEIQATTGNSQVLVR
45143 YVDLGGSYVGPTQNR
168744 DFPLTGYVELR
278967 IGDLQAFVGPEAR
124372 VPTPNVSVVDLTCR
271171 AEPIDIQTWILGYR
144648 AAVLWEPHKPFTIEDIEVAPPK
611184 VASLIGVEGGHLIDSSLGVLR
58829 ELLQSVEEQYK
80372 NFHTFLYEPEWR
1389210 LWPVLEPDEVAR
529735 MITGDSQETAIAIASR
121994 AHVYVEEVPWK
961389 YFTGYVINGGPIR
84537 IIGDSAFLLILK
774938 HFYYAAVPSAR
316274 KNDALAPPLLDSEPLR
188395 VMLGETNPADSKPGTIR
61322 FNGAPTDNFQQDVGSK
46308 WLPQNDLIGHPK
1391093 LDDILTSMSAGVVSR
172112 MNVLHDFGIQSTR
572980 YAWGLNELKPISK
773999 LDGLNSLTYQVLDIQR
46309 WLPQNDILGHPK
496963 EQMVDSSKPELLYR
1510706 EHYDFLAELADSLK
47852 HYFSLGEIR
76720 ILTGLNYEAPK
61643 LLAENNEIISNIR
66074 ILGIGPDDPDLVR
57721 NMLFSGTNIAAGK
337987 HADEILLDLGHHER
488042 NVDVNLFESTIR
Cont2-41
Cont1-19
Cont1-28
Cont1-31
Cont1-45
Cont1-46
Cont1-20
Cont2-45
Cont2-27
Cont1-35
Cont1-59
Cont1-36
Cont1-16
Cont1-46
Cont1-19
Cont1-19
Cont1-41
Cont1-16
Cont3-51
Cont1-24
Cont1-24
Cont1-28
Cont1-35
Cont1-19
Cont1-33
Cont1-22
Cont1-28
Cont1-22
Cont1-29
Cont1-52
Cont1-30
Cont1-38
Cont1-7
Cont1-25
Cont1-38
Cont1-30
Cont1-22
Cont1-45
Cont1-38
Cont1-38
Cont1-36
Cont1-31
Cont1-46
Cont2-24
Cont1-52
Cont1-50
Cont1-45
Cont1-44
Cont1-48
3163854 LLADDAEALSLLALNPFEGR
45503 LAMDEIFQKPFQTLMFLVR
3260048 ALQGPLFAYIQEFR
434827 VQAVQPTLILQDGDVINLGDR
1459910 LLPPQDNPLWQYLLSR
958934 SIASADMDFNQLEAFLTAQTK
3301756 VPASEAFRDPIWSTWALYGR
1448340 ISTTLIGLEEHLNALDR
EGF1-37
Cont1-40
EGF1-38
EGF1-19
EGF1-17
Cont2-10
EGF1-53
EGF1-44
InterProScan
Predictions
IPR000120 (F) Amidase
IPR001474 (F) GTP
IPR005924 (F) Arginase
IPR001518 (F)
IPR000994 (F)
IPR000873 (F) AMPIPR002198 (F) Short-chain
IPR006089 (F) Acyl-CoA
IPR000741 (F) FructoseIPR000408 (F) Regulator of
IPR001930 (F) Peptidase M1,
IPR002133 (F) SIPR001092 (D) Basic helixIPR002655 (F) Acyl-CoA
IPR002198 (F) Short-chain
IPR002198 (F) Short-chain
IPR001268 (F) NADH
IPR006047 (D) Alpha
IPR000173 (F)
IPR002123 (F)
IPR002085 (F) ZincIPR000180 (AS) Peptidase
IPR002181 (D) Fibrinogen,
IPR002060 (F)
IPR002198 (F) Short-chain
IPR000695 (F) H+
IPR002042 (F) Uricase
IPR002659 (F) Glycosyl
IPR000602 (F) Glycoside
IPR000772 (D) Ricin B lectin
IPR007754 (F) NIPR001564 (F) Nucleoside
IPR001675 (F) Glycosyl
IPR002213 (F) UDPIPR006087 (D) SUR2-type
IPR000326 (F) PAIPR001382 (F) Glycoside
IPR003859 (F) Metazoa
IPR002213 (F) UDPIPR004139 (F) Glycosyl
IPR004455 (F) NADP
IPR000772 (D) Ricin B lectin
IPR002225 (F) 3-beta
IPR000602 (F) Glycoside
IPR000923 (D) Blue (type 1)
IPR001757 (F) ATPase, E1IPR001452 (D) SH3 domain
Unknown Function (86 proteins)
PREDICTED: similar to RIKEN cDNA
UniRef100|UPI0000507623
PREDICTED: similar to RIKEN cDNA
UniRef100|UPI0000506262
Tax_Id=9606 KIAA1376 protein
IPI|IPI00002205.1
PREDICTED: RIKEN cDNA 2900024O10
UniRef100|UPI00001C527E
PREDICTED: similar to catechol-OUniRef100|UPI0000180920
COMM domain-containing protein 1 Protein
UniRef100|O97832
ENSEMBL:ENSRNOP00000033466
IPI|IPI00369029.1 UniRef100|Q9ULQ9
similar to cDNA sequence BC021917
UniRef100|UPI00001CEB5D
Unique
Unique
Unique
Unique
Unique
Unique
Unique
Unique
0
1
4
1
1
1
1
2
2
1
2
1
2
1
1
2
2
1
1.3
2
1
1
1
1
1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
74
IPR000698 (F) Arrestin
IPR001279 (D) BetaIPR000322 (F) Glycoside
IPR004006 (D) Dak kinase
Table 3.2 ___Continued
Database|Protein
Identifier
Protein Description
Fold
Change
Unique
MKIAA0247 protein
UniRef100|Q6ZQF4
Unique
Charged multivesicular body protein 2b
UniRef100|Q53HC7
Unique
Tex261 protein Bone marrow macrophage cDNA,
UniRef100|Q62302
15.0
PREDICTED: similar to desmoglein 2
UniRef100|UPI00005081C7
12.5
PREDICTED: similar to RIKEN cDNA
UniRef100|UPI0000508392
12.0
PREDICTED: similar to 5730405I09Rik protein
UniRef100|UPI000017EE3C
10.0
RIKEN cDNA C920006C10
UniRef100|Q922I2 UniRef100|Q14156
8.0
WD repeat and FYVE domain-containing protein
UniRef100|Q9H9D5
7.0
LOC495763
protein
UniRef100|Q5RKA2
6.5
PREDICTED: similar to mouse Rp2 protein
UniRef100|UPI00001D16BA
5.4
spastic paraplegia 4 homolog
UniRef100|UPI000000B99B
5.2
Chmp5 protein
[Rattus norvegicus]
nrdb|55778532 UniRef100|Q5RBR3
UniRef100|Q3UI64
4.6
similar to cDNA sequence BC029169
UniRef100|UPI00001812BB
3.5
Tyrosine-protein phosphatase
non-receptor type
UniRef100|O88902 UniRef100|UPI00000E5DAD
UniRef100|Q6PB44
3.3
mKIAA0174
protein [Mus musculus]
nrdb|28972087 UniRef100|Q9BQ81
UniRef100|Q568Z6
3.3
(Q9D024) Isoform 2 of Q9D024
UniRef100|Q9D024-2 RefSeq|NP_080285.1
3.2
PREDICTED:
similar to RAB, member of RAS
UniRef100|UPI0000251426
UniRef100|Q9D4V7
3.0
MIPP65
UniRef100|O54755
3.0
Hypothetical protein
UniRef100|Q6AY64
3.0
10 days embryo whole body cDNA, RIKEN fullUniRef100|Q9CZX9
3.0
Chromosome 6 open reading frame 55
UniRef100|Q5TGM0 UniRef100|Q9P0Q0
2.8
Ac2-233
UniRef100|Q7TPJ1
2.8
Hypothetical LOC297077
UniRef100|Q4G068
2.7
PREDICTED: similar to lemur tyrosine kinase 2
UniRef100|UPI0000507992
2.7
(Q8NBJ9) Isoform 2 of Q8NBJ9
UniRef100|Q8NBJ9-2
2.6
SPFH
domain family, member
1
UniRef100|UPI00000E8C5F
UniRef100|O75477
UniRef100|Q91X78
IPI|IPI00706483.2
2.5
PREDICTED: similar to deltex 3-like
UniRef100|UPI00005078DB
2.5
Hypothetical protein RGD1307700
UniRef100|Q5I0J5
2.5
Charged
multivesicular
body
protein
4c
UniRef100|Q569C1 UniRef100|Q3TVD7
2.5
RIKEN cDNA 5730439E10
UniRef100|Q80XL6
2.3
PREDICTED: similar to family 4 cytochrome
UniRef100|UPI0000507FA7
2.2
PREDICTED:
similar to Expressed sequence
UniRef100|UPI00001CBEDF
UniRef100|UPI0000507C3C
2.2
PREDICTED: similar to chromosome 10 open
UniRef100|UPI0000508784
2.2
PREDICTED:
similar to COMM domain
UniRef100|UPI00001CFFDA
UniRef100|Q9CQ02
2.2
55 kDa type II phosphatidylinositol 4-kinase
UniRef100|Q99M64 UniRef100|Q9NSG8
2.1
Adult male liver cDNA, RIKEN full-length
UniRef100|Q9CW42
2.1
Leucine-rich repeat-containing protein 59
UniRef100|Q3TJ35 UniRef100|Q5RJR8
2.0
BAI1-associated protein 2-like 1
UniRef100|Q3KR97
2.0
Tax_Id=10090 1300006C19Rik protein
IPI|IPI00515450.1 IPI|IPI00515535.1
2.0
Hypothetical protein
UniRef100|Q9BGQ2
2.0
MOCO sulphurase C-terminal domain containing
UniRef100|Q922Q1 UniRef100|O88994
2.0
Interferon-induced protein 35 Predicted
UniRef100|Q5M849
2.0
N-myc (And STAT) interactor
UniRef100|Q498S7
2.0
WD repeat protein 48 WD repeat endosomal
UniRef100|Q8TAF3
1.7
Hypothetical protein
UniRef100|Q5I0K9
1.7
Hypothetical protein RGD1309362
UniRef100|Q4V797 UniRef100|Q5FVQ6
1.7
PREDICTED: similar to Thymic dendritic cellUniRef100|UPI000018149F
1.7
Commd2 protein 6 days neonate spleen cDNA,
UniRef100|Q497T6 UniRef100|UPI00000EB6C7
1.6
PREDICTED:
similar
to
hypothetical
protein
UniRef100|UPI000050462E
1.6
30 kDa protein variant [Homo sapiens]
nrdb|62897465 IPI|IPI00116077.1
1.6
COMM domain-containing protein 3
UniRef100|Q6P9U3
1.5
PREDICTED: complement component 5
UniRef100|UPI00005066DB
1.5
D15Ertd621e protein
UniRef100|Q8K077 UniRef100|Q80TZ0
1.5
Hypothetical protein FLJ21040
UniRef100|Q9H7C8
1.5
PREDICTED: similar to RIKEN cDNA
UniRef100|UPI0000508788
1.5
Chronic lymphocytic
leukemia deletion region
UniRef100|Q2T9X3 IPI|IPI00030959.1
nrdb|12857585
1.5
NOD-derived CD11c +ve dendritic cells cDNA,
UniRef100|Q3TDR2 UniRef100|Q8K1X1
1.5
Stomatin (Epb7.2)-like 2
UniRef100|Q4FZT0 UniRef100|Q9DCG8
C1 C2 C3
1
2
1
2
1.3
4
9
4
2
1
1
1
4
5
11
9
1
2
1
2
4
5
1
1
8
1
5
1
5
2
2
1
2
1
8
1
1
1
1
1
1
7
2
1
7
1
2
2
1
1
4
2
1
1
16
2
4
2
1
2
1
1
3
3
7
7
3
1
3
2
1
4
2
19
3
5
5
4
2
9
2
2
6
9
1
9
1
1
1
1
1
2
1
1
5
3
2
9.8
3
1
1
1
2.3
2
1
1
1
3
1
1
2
1
7
6
3
3
2
4
3
1
4
1
3
1
E1
E2 E3
1
1
1
3
2
1
4
2
2
7
4
4
10
6
9
5
4
3
8
7
5
4
2
2
2
3
2
6
4
3
22
5.7 10
11
7
8
2
1
3
14
5
2
21
15 20
5
6
12
5
6
5
1
1
1
4
3
2
1
2
3
1
4
1
10
7
8
11
6
8
8
4
4
2
3
3
30.9 35.6 29
1
2
2
15
13 12
6
3
1
2
2
1
1
4
2
2.2
4
1.1
3
6
2
2
3
6
7
4
2
4
10
3
8
10
3
6
3
5
1
13
4
5
1
1
2
1
1
3
2
4
4
4
1
2
1
19
24 15
11
2
4
6
7
9
5
3
7
12
2
4
4
5
4
6
8
8
1
2
3
3
2
1
4
7
4
1
1
1
12
9
9
1
1
1
3
7
6
Mean
Control
0.0
0.0
0.0
0.3
0.7
0.3
0.7
0.3
0.3
0.7
2.3
1.7
0.4
2.0
5.7
2.3
1.7
0.3
1.0
0.7
0.7
3.0
3.0
2.0
1.0
12.2
0.7
5.3
1.3
0.7
1.0
1.1
1.7
1.7
2.0
2.7
3.3
2.3
2.3
1.7
0.7
1.0
2.0
0.7
11.3
3.3
4.3
3.0
3.7
2.7
4.7
1.3
1.3
3.3
0.7
6.7
0.7
3.7
75
SD
Control
0.0
0.0
0.6
1.7
4.2
1.5
0.6
0.7
0.0
0.0
0.7
1.0
2.8
0.7
3.2
3.2
0.6
0.0
0.7
0.9
0.6
1.2
1.0
4.2
2.8
0.7
2.3
1.2
0.7
1.7
6.7
2.5
1.2
2.0
1.5
1.2
3.8
0.6
0.0
1.4
0.0
3.2
0.0
4.6
Mean
EGF
1.0
2.0
2.7
5.0
8.3
4.0
6.7
2.7
2.3
4.3
12.6
8.7
2.0
7.0
18.7
7.7
5.3
1.0
3.0
2.0
2.0
8.3
8.3
5.3
2.7
32.0
1.7
13.3
3.3
1.7
2.3
2.4
3.7
3.7
4.3
5.7
7.0
4.7
4.7
3.3
1.3
2.0
4.0
1.3
19.3
5.7
7.3
5.0
6.0
4.3
7.3
2.0
2.0
5.0
1.0
10.0
1.0
5.3
SD
Peptides (identifier Sequence)
EGF
0.0
1.0
1.2
1.7
2.1
1.0
1.5
1.2
0.6
1.5
8.4
2.1
1.0
6.2
3.2
3.8
0.6
0.0
1.0
1.0
1.7
1.5
2.5
2.3
0.6
3.2
0.6
1.5
2.5
0.6
1.5
1.5
2.1
2.1
2.5
3.8
3.6
1.5
8.5
2.1
0.6
1.0
0.0
0.6
4.5
4.7
1.5
2.0
5.3
0.6
1.2
1.0
1.0
1.7
0.0
1.7
0.0
2.1
3021788 VQIVLSEGSAPSGR
623784 TVDDVIKEQNR
598749 LGILVVFSFIK
3242172
2941482 FTEDLVASVVHVLTHR
1373769 SLAEANSLSFPLEPLSR
193257 GSIGPTVLEVFNTLLK
409647 IEGHQDAVTAALLIPK
117234 GSLLWNQQDGTLSATQR
3168218 DYTFSGLKDETVGR
674960 MTDGYSGSDLTALAK
980136 NKDGVLVDEFGLPQIPAS
1513454 ESLLEDQLTPVLTEPHLLALDR
1094260 LQETLGQAGAGPGPSVTK
202491 EIADYLAAGKDER
47960 RQDLLNVLAR
192793 EQFADNQIPLLVIGTK
3186369
4203866 TVDQYPFGEAAYAADQTGTSQK
188331 GSGQGDSLYPVGYLDK
1214869 YAGSALQYEDVSTAVQNLQK
1449501 LIGHEEEDSAPTGVVR
3130717 EFNNYWYYLR
4201542 VLLGETYTGTSVTR
1085712 EAVVSFQVPLILR
98174 ISEIEDAAFLAR
4206012 MLTLLGSAADIAAAAEK
374191 RAVELDAESR
4206362 MTSMELPNVPSSSLPAQPSR
1621270 VPASNLILGEGR
1667819 SLHTFTNNVIAER
4211366 LDFLTMSSTDLLTALR
3138883 TLVQQLYTTLCIEQHQLNK
3111766 HSVDSDSLSSELQQLGLPK
962606
270620 LQQVGTVAQLWIYPIK
95021 LVTLPVSFAQLK
3180720 NVVEQFNPGLR
57448 EGDYFTQQGEFR
612406 ETLLNSATTSLNSK
553214 LGLPGQPR
1359518 VSPYVSGEIQEAEIK
1401713 KLEAELQSNAR
97243 DKELVASAGLDR
4205195
1444943 TVFGVDDAALQSLAK
1018913 YPAPSLVVVR
3007056
467631 YVFLDPLAGAVTK
120933 LTQEQVSDSQVLIR
3001652 AAFQSLLDAQADEAALDHPDLK
1403683 GLLIGEFLSTVLSK
760786 LSDESLDSFLIELEK
457165 ELLNLTQQDYVNR
4204776 HSELVQHFR
212274 IQSTGIWGIGVATQK
899129 FLLTGLLSGLPSPQFAIR
127814 ILEPGLNVLIPVLDR
Band
EGF1-24
EGF1-20
EGF1-68
Cont3-62
Cont1-34
Cont3-26
Cont2-53
Cont2-32
Cont2-13
Cont1-29
Cont1-41
Cont1-21
Cont1-50
Cont1-68
Cont1-31
Cont1-43
Cont1-16
Cont1-43
Cont1-14
Cont1-8
Cont1-29
Cont1-34
Cont1-16
Cont1-50
Cont1-76
Cont1-28
Cont2-53
Cont1-17
Cont1-23
Cont2-52
Cont2-41
Cont1-38
Cont1-20
Cont1-13
Cont1-38
Cont1-24
Cont1-22
Cont1-21
Cont1-45
Cont1-41
Cont1-24
Cont1-21
Cont1-25
Cont2-51
Cont1-19
Cont1-35
Cont1-29
Cont1-12
Cont1-49
Cont1-19
Cont1-12
Cont1-68
Cont1-54
Cont1-53
Cont1-47
Cont1-14
Cont1-60
Cont1-28
InterProScan
Predictions
IPR000436 (D) Sushi
IPR005024 (F) Eukaryotic
IPR007277 (F) Protein of
IPR000306 (D) Zn-finger,
IPR002088 (R) Protein
IPR003593 (D) AAA ATPase
IPR005024 (F) Eukaryotic
IPR000242 (F) Tyrosine
IPR005061 (D) Eukaryotic
IPR001806 (F) Ras GTPase
IPR006745 (F) Protein of
IPR001687 (BS) ATP/GTP-
IPR001107 (F) Band 7 protein
IPR005024 (F) Eukaryotic
IPR000169 (F) Eukaryotic
IPR002213 (F) UDPIPR000403 (D)
IPR005302 (D) MOSC domain
IPR001611 (R) Leucine-rich
IPR003674 (F) Oligosaccharyl
IPR001844 (F) Chaperonin
IPR005302 (D) MOSC domain
IPR001680 (R) G-protein beta
IPR001687 (BS) ATP/GTP-
IPR008568 (F) Eukaryotic
IPR001032 (F)
IPR003877 (D)
IPR001680 (R) G-protein beta
IPR001107 (F) Band 7 protein
Table 3.2 ___Continued
Database|Protein
Identifier
Protein Description
Fold
Change
PREDICTED:
similar toUniRef100|Q2M389
KIAA1033 protein
UniRef100|UPI0000507081
UniRef100|Q8CAK8
PREDICTED: similar to novel protein of unknown
UniRef100|UPI0000181FBE
Hypothetical protein RGD1308697
UniRef100|Q66H15 UniRef100|Q7TNF2
Hypothetical protein LOC203547
UniRef100|Q3ZAQ7 UniRef100|Q78T54
PREDICTED: similar to ATP-binding cassette
UniRef100|UPI00005077EC
PREDICTED: hypothetical protein XP_217094
UniRef100|UPI000017E10A
PREDICTED: similar to expressed sequence
UniRef100|UPI00005066BB
Hypothetical protein LOC90693
UniRef100|Q75MQ6
family with sequence similarity 20, member C
UniRef100|UPI00001D08A6
RIKEN cDNA 9130011E15
UniRef100|Q6PD19 UniRef100|Q80V50
PREDICTED: similar to RIKEN cDNA
UniRef100|UPI00001805B1
Transmembrane 9 superfamily protein member 1
UniRef100|Q9DBU0 UniRef100|Q3U7S4
Bone marrow macrophage cDNA, RIKEN fullUniRef100|Q3U649 UniRef100|Q9DCN7
PREDICTED: similar to RIKEN cDNA
UniRef100|UPI0000508057
PREDICTED: similar to Golgi-specific brefeldin AUniRef100|UPI0000506323
Transmembrane 9 superfamily
protein member 4
UniRef100|Q2TBF8 UniRef100|UPI0000506855
UniRef100|Q8CHH4
In vitro fertilized eggs cDNA, RIKEN full-length
UniRef100|Q3UX01 UniRef100|Q95LN3
Hypothetical protein RGD1305703
UniRef100|Q68FV1
PREDICTED:
similar to mKIAA1354 protein
UniRef100|UPI000050363A
UniRef100|Q8C7P1
PREDICTED: similar to PL6 protein
UniRef100|UPI00001D00B7
1.5
-1.7
-1.8
-2.0
-2.2
-2.3
-2.3
-2.3
-2.6
-3.0
-3.0
-3.2
-4.0
-4.8
-5.0
-5.3
-5.5
-8.0
Unique
Unique
C1 C2 C3
E1
E2 E3
17
2
8
3
82
5
6
7
2.7
2
1
9
3
8
13
16
3
2
6
2
30
1
4
1
28
15
1
8
1
18
1
19
2
8
1
70
8
1
8
4
1
1
3
1
7
4
4
2
6
1
6
1
5
2
102
14
2
13
5
3
1
28
2
10
5
59
4
4
2
4
3
1
0
1
1
1
2
3
2
1
1
1
16
1
1
68
11
4
9
1
3
1
11
2
2
12
1
1
Mean
Control
SD
Control
Mean
EGF
SD
Peptides (identifier Sequence)
EGF
14.0
1.7
7.0
2.0
84.7
9.0
3.0
9.3
2.6
3.0
1.0
12.7
2.7
6.3
8.3
26.3
3.7
2.7
4.7
2.3
7.0
0.6
1.7
1.0
16.5
4.6
2.6
3.2
1.6
1.0
0.0
13.9
0.6
4.7
4.2
28.9
0.6
1.2
2.3
1.5
20.3
1.0
4.0
1.0
38.0
4.0
1.3
4.0
1.0
1.0
0.3
4.0
0.7
1.3
1.7
5.0
0.7
0.3
0.0
0.0
8.4
0.0
2.8
0.0
26.5
7.1
1.8
2.0
4.0
5.3
3.0
3.7
3.7
4.3
9.7
11.3
2.7
2.7
1.7
5.0
2.8
23.7
4.2
10.0
3.0
2.7
1.3
1.3
2.0
4.7
8.5
10.7
12.0
1.7
3.3
1.7
1.7
31.7
2.7
7.9
4.7
9.0
2.0
1.3
1.0
1.0
2.1
1.0
2.5
1.5
1.2
1.2
1.5
1.5
0.6
0.6
1.0
1.9
8.1
2.0
5.6
1.0
0.6
0.6
0.6
0.0
0.6
2.4
2.1
0.1
0.6
0.6
0.6
1.2
17.3
0.6
1.7
1.5
1.7
1.4
4.2
0.0
2.1
7.1
0.6
0.6
6.4
0.0
Band
450789 LGITPEGQSYLDQFR
2060434
1486226 SLQGLAGEIVGEVR
144179 AALNALQPPEFR
1476395 ILLLDEPTAGLDPFSR
1379642 GLGTEVPGSLQGPDPYR
444160 NPDSYLSAGEIPLPK
770322 TIAVLLDDILQR
1465453 LPPAAEPVDHAPR
482403 LQDGLDQYER
1378123 VVNEINIEDLNLTK
769060 SDELLGLTHTYSVR
3052235 STEGSLHPGDVHIQINSGPK
317423 IQSSHSFQLENVNK
4200971
1011121 ITEEYYVHLIADNLPVATR
59288 SMLQATAEANNLAAVAGAR
1386210 DAAVTLTPFEDTLTR
185270 SAAGELATVECYNPR
823244 YDVGAPSSITISLPGTDPQDAER
Cont1-60
Cont1-1
Cont1-18
Cont1-3
Cont1-63
Cont1-69
Cont1-40
Cont1-14
Cont1-53
Cont1-47
Cont1-5
Cont1-34
Cont1-25
Cont1-49
Cont1-14
Cont1-38
Cont1-41
Cont1-15
Cont1-44
Cont1-24
822454 SQVLFAVVFTAR
958622 FQETFEDVFSDR
725512 NNDGNLVIDSLLQYINQR
561067 DIALHLNPR
963174 EPGEGAITYLVTSVLR
69851 MVVDAVMMLDELLQLK
1398326 FQYNTDVVFDSR
363497 LYGPSSVSFADDFVR
803749 ILDYEVVLTQSK
349849 SGFTTEPVTVEAK
66026 AQFEGIVTDLIK
318760 HVINFDLPSDIEEYVHR
3186892 TTLPGVVDGANNPAIR
452858 EFEGEEEYLEILGITR
565440 YANVIAYDHSR
178316
132825 AHVTLHSRPEGDVTLR
3158159 RVDTEANLGQYTDIIR
393916 EGVNDNEEGFFSAR
470481 GPVYIGELPQDFLR
1387792 GLFQVLAGGTVLQLQR
756115 LQEEHSLQDVIFK
62730 LVSSSGGLQNAQFGIR
1481803 IASILNASLDEK
544604 EIEIDYTGTEPSSPCNK
373654 ITMEVYDLVSKPAIK
141925 LQVNILVKPAR
125210 GTEDFIVESLDASFR
3008062 IIPSPEDPNEDIVER
3111438
78122 ADTGTTSEFIDEGAGIR
46447 FLEQQNQVLQTK
751031 KGVQLLLSER
226485 LQTEGDGIYTLNSEK
406143 AGVNFSEFTGVWK
196088 VLAQQGEYSEAIPILR
1467797 VLTTALLLQAASALANYIHFSR
Cont1-10
Cont1-6
Cont1-24
Cont1-21
Cont2-41
Cont1-40
Cont1-45
Cont1-33
Cont1-63
Cont1-48
Cont1-48
Cont2-49
Cont1-44
Cont1-42
Cont1-50
Cont1-33
Cont1-33
Cont1-36
Cont1-1
Cont2-19
Cont1-18
Cont2-34
Cont1-53
Cont1-17
Cont1-40
Cont1-28
Cont1-51
Cont1-23
Cont1-12
Cont2-12
Cont1-42
Cont1-1
Cont1-30
Cont1-24
Cont1-10
Cont1-35
Cont1-35
InterProScan
Predictions
IPR006696 (F) Protein of
IPR001064 (D) Beta and
IPR006622 (D) Zn-finger,
IPR004240 (F) Nonaspanin
IPR001841 (D) Zn-finger,
IPR004240 (F) Nonaspanin
IPR003191 (F) GuanylateIPR000210 (D) BTB/POZ
Miscellaneous (89 proteins)
PREDICTED: similar to KDEL (Lys-Asp-Glu-Leu)
nrdb|73946384
Tartrate-resistant acid phosphatase type 5
UniRef100|O97860
MEST
UniRef100|Q5MBK5 UniRef100|Q92571
Galectin 8
UniRef100|Q6IN24 UniRef100|Q62665
BWK3
UniRef100|Q5VLR6
Bone marrow macrophage
cDNA, RIKEN fullUniRef100|Q3UDB1 UniRef100|Q3TIJ7
UniRef100|Q3THH8
PREDICTED: vanin 1 (predicted)
UniRef100|UPI00001824E8
Rheumatoid arthritis
related antigen RA-A47
UniRef100|Q9NPA9 UniRef100|Q9NP88
UniRef100|P19324
Interleukin-6 receptor beta chain precursor IL-6RUniRef100|P40190
Ig mu chain
C region - rat (fragment)
nrdb|111977 UniRef100|Q5RK07
UniRef100|Q5I0L9
12 days embryo embryonic body between
UniRef100|Q3UVN1 UniRef100|UPI0000170CC4
DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, XUniRef100|Q5JSI3 UniRef100|Q3TQX5
Sphingomyelin phosphodiesterase, acid-like 3B
UniRef100|Q4V7D9
Limbic system-associated membrane protein
UniRef100|Q13449 UniRef100|Q6VUH9
Leukocyte common antigen-related phosphatase
UniRef100|Q63294 UniRef100|P10586
Bone marrow macrophage
cDNA, RIKEN fullUniRef100|Q3UC51 UniRef100|O54734
UniRef100|Q641Y0
MHC class I RT1.E precursor MHC class I
UniRef100|Q31267 UniRef100|Q31265
Monocyte differentiation antigen CD14
UniRef100|O88955 UniRef100|Q6GT04
fibrinopeptide B
nrdb|229334
Toll-interacting protein
UniRef100|Q9D5P5
Complement C1q subcomponent subunit A
UniRef100|Q5RJK1
PTX1 protein [Homo sapiens]
nrdb|15010925 IPI|IPI00319058.1
NOD-derived CD11c +ve dendritic cells cDNA,
UniRef100|Q3U273 UniRef100|UPI00000EB740
PREDICTED: similar to 5830458K16Rik protein
UniRef100|UPI00001D07A4
unnamed protein product [Mus musculus]
nrdb|74217938 UniRef100|Q3UDH8
CD48 antigen precursor MRC OX-45 surface
UniRef100|P10252
Cd36 protein
UniRef100|Q5BKE5 UniRef100|Q925W0
Translocon-associated protein subunit alpha
UniRef100|P16967
Igj protein
UniRef100|Q4QQV9
Glycophorin C Gerbich blood group Predicted
UniRef100|Q6XFR6
fibrinopeptide A
nrdb|229210
Keratin 2A Epidermal ichthyosis bullosa of
UniRef100|Q4VAQ2 UniRef100|P35908
(Q8BUE4) Isoform 2 of Q8BUE4
UniRef100|Q8BUE4-2 UniRef100|UPI000021EF7B
Ba1-647
UniRef100|Q7TP23
Programmed cell death protein 6
UniRef100|P12815 UniRef100|UPI0000181133
FK506-bindingUniRef100|Q3B7U9
protein 8
UniRef100|Q811R4 UniRef100|Q99L93
Intimal
thickness-related receptor
UniRef100|Q5XIJ2
Unique
6.0
6.0
5.3
4.5
3.7
3.7
3.3
3.2
3.1
2.7
2.7
2.5
2.5
2.5
2.4
2.3
2.3
2.3
2.0
2.0
2.0
2.0
2.0
2.0
1.9
1.7
1.7
1.7
1.7
1.7
1.6
1.6
1.6
1.6
1.5
1.5
1.2
1
3.3
2
1
3
4
5
3
6
3
7
1
1
3
2
4
1
6
4
1
1
5
4
2
1
2
5
3
5
4
4
9
11
9
3
4
10
13 11
3
4
3
1
2
1
3
2
3
1
1
2
2
4
2
5
6
4
2.4
1
1.2 4.9 2.3
11
8
20
18 33
1.4 1.5
2
4.5
6
5
5
15
11
4
1
2
3
4
2
1
3
3
2
3
1
1
1
2
1
1
2
1
1
1
1
2
2
2
2
5
4
5
3.8
5
10.7 8.8
6
9
1
13
10
9
9
7
11.9 12 12
1
2
2
2
1
2
3
3
3
4
2
1
1
2
2
1
3
1
35 15.3 17.2 50.9 27
3
1
3
2
3
8
7
7
9.9 6.9
3
2
6
3
5
8
4
11
8
8
1
2
2
4
1
1
3
2
2
1
1
4
0
1
10
2.5
3
1
1
1
5
4
7
5
1
3
8.1
1
4
6
1
1
0.0
0.3
0.7
1.0
0.7
1.0
1.0
1.3
3.0
3.7
1.0
1.0
0.7
2.0
1.1
9.7
1.8
4.3
1.3
1.3
0.7
0.7
1.0
2.3
4.3
5.7
7.0
1.0
2.0
1.0
1.0
19.4
1.7
5.0
3.0
6.0
1.3
76
0.0
0.0
0.7
0.7
0.6
1.7
0.6
2.1
0.7
0.0
1.4
1.0
1.5
0.6
1.2
0.6
1.4
0.0
0.0
0.0
2.1
0.6
4.2
2.0
0.7
1.0
0.7
13.8
1.2
0.7
1.0
2.0
0.6
IPR004843 (D) MetalloIPR000073 (D) Alpha/beta
IPR001079 (F) Galectin,
IPR002194 (F) Chaperonin
IPR000215 (F) Serpin
IPR002996 (F) Cytokine
IPR003006 (F)
IPR001023 (F) Heat shock
IPR000629 (D) ATPIPR004843 (D) MetalloIPR003598 (D)
IPR000242 (F) Tyrosine
IPR005013 (F) DolichylIPR001039 (D) Major
IPR001611 (R) Leucine-rich
IPR000008 (D) C2 domain
IPR006209 (D) EGF-like
IPR005045 (F) Eukaryotic
IPR003599 (D)
IPR002159 (F) CD36 antigen
IPR005595 (F) Translocon-
IPR001664 (F) Intermediate
IPR000103 (F) Pyridine
IPR000436 (D) Sushi
IPR002048 (D) CalciumIPR001179 (F) Peptidylprolyl
Table 3.2 ___Continued
Database|Protein
Identifier
Protein Description
Fold
Change
C1 C2 C3
E1
1.5
1
1
1
Golgin subfamily A member 7
UniRef100|Q5EA55
1.5
8
9
2
8
Transmembrane 7 superfamily member 2
UniRef100|Q5BK21
-1.6
4
13
5
3
Complement component 4, gene 2
UniRef100|Q6MG90 UniRef100|Q6MG79
-1.6
2
6
2
Complement C3 precursor Complement C3 beta
UniRef100|P01026
-1.7
7
14 11
3
(P24008) Isoform Short of P24008
UniRef100|P24008-2
-1.7
9
4
9
6
DMT1-associated protein
UniRef100|Q5XI18 UniRef100|UPI0000029630
-1.7
67 46 28
31
PREDICTED:
similar
to DnaJ homologUniRef100|Q3L0T1
subfamily
UniRef100|UPI00005073C8
nrdb|73990575
IPI|IPI00458074.2
-1.8
3
1
3
1
PREDICTED:
similar to Exostosin 2
UniRef100|UPI000050676F
UniRef100|P70395
-1.8
8
10 33
8.1
PREDICTED: similar to alpha-mannosidase
UniRef100|UPI0000506586
-1.8
30.9 38 23.9 14.9
Inter-alpha-inhibitor H4 heavy chain
UniRef100|Q5EBC0
-1.8
7.3 7.8 19.6 9.1
Serum albumin precursor
UniRef100|P11382 UniRef100|Q5U3X3
-1.8
16 22 26
15
Ferritin light chain 1
UniRef100|Q6P7T1 UniRef100|P02793
-1.9
8
6
18
3
Ectonucleoside triphosphate diphosphohydrolase
UniRef100|Q4V8N9
-1.9
12 10 13
7
Cyclophilin B
UniRef100|O88541
-2.0
6
7
5
4
Secreted acidic cysteine rich glycoprotein
UniRef100|Q5NBV5 UniRef100|P16975
-2.0
2
1
1
2
Claudin 14 Predicted
UniRef100|Q5BJQ1 UniRef100|Q9Z0S3
-2.0
7
5
8
2
PREDICTED:
similar to D-glucuronyl C5UniRef100|UPI0000504243
UniRef100|O18756
-2.0
1
2
1
1
PREDICTED: similar to chaperonin containing
nrdb|73960904 UniRef100|Q6P502
-2.0
1
2
1
1
Ctdsp1 protein
UniRef100|Q3B8P1 UniRef100|UPI0000181A5D
UniRef100|Q9GZU7
-2.1
65 66 82
30
Golgi apparatusUniRef100|Q9Z1E9
protein 1 precursor Golgi
UniRef100|Q9QZ40 UniRef100|Q13221
-2.1
83.7 72 142 42.9
albumin
UniRef100|UPI000016C268
UniRef100|P02769 UniRef100|Q3SZR2
-2.2
10
8
8
3
PREDICTED: similar to beta-1,3UniRef100|UPI0000180178
-2.3
1
1
5
Tax_Id=10116 CD36 antigen (collagen type I
IPI|IPI00231478.3
-2.4
1
2
16
3
REFSEQ:XP_892340 PREDICTED: similar to
IPI|IPI00677435.1 UniRef100|Q6AY20
-2.4
2
10
3
Alpha-1-macroglobulin precursor
UniRef100|Q63332 UniRef100|Q63041
-2.5
27
16
36
9
Aa1064 Ac1-060
UniRef100|Q7TMA5
-2.5
2
2
1
PREDICTED:
collagen, type XVIII, alpha 1
UniRef100|UPI0000508428
UniRef100|Q9QZD2
-2.5
6
3
6
1
Xylosyltransferase
II
UniRef100|Q3KRD6 UniRef100|Q9EPI0
UniRef100|Q9H1B5
-2.6
2
4
7
2
Lipoprotein lipase
UniRef100|Q8R4V8 UniRef100|UPI0000161D95
UniRef100|O46647
-2.7
4
6
6
3
Ferritin heavyUniRef100|UPI0000163C2E
chain Ferritin H subunit
UniRef100|P25915 UniRef100|P02794
-2.8
14 12
5
Histidine-rich glycoprotein 1
UniRef100|Q99PS8 UniRef100|Q99PS7
-3.0
7.7 3.6 17.4 4.9
Keratin,
type II cytoskeletal
6B (Cytokeratin 6B)
UniRef100|UPI000013CD50
UniRef100|Q2TAZ9
UniRef100|UPI0000167E31
UniRef100|P04259
-3.0
1
1
1
1
5 days embryo whole body cDNA, RIKEN fullUniRef100|Q3TL79 UniRef100|Q8R3E6
-3.0
2
1
1
ENSEMBL:ENSRNOP00000011183
IPI|IPI00365897.1
-3.0
1
1
1
Tax_Id=10116 Similar to Cox7a2l protein
IPI|IPI00365505.1 UniRef100|UPI0000506E36
-3.1
151 204 263 75
PREDICTED: similar to lipoprotein receptorUniRef100|UPI0000504CBE
-3.9
7
2.5 17.2 4.8
PREDICTED:
similar to Keratin, type I
nrdb|73965817 UniRef100|P08779
UniRef100|P08779
-4.0
1
2
1
Three prime repair exonuclease 1
UniRef100|Q5BK16
-4.3
9
1
37
1
PREDICTED:
similar to macrophage mannose
UniRef100|UPI0000508181
UniRef100|Q8C502
-4.5
25 24 45
3
PREDICTED: similar to ATP-binding cassette
UniRef100|UPI00005077EA
-6.0
2
1
3
1
TIB-55 BB88 cDNA,
RIKEN full-length enriched
UniRef100|Q3UJR4 UniRef100|Q9D0T9
UniRef100|O88824
-6.4
7.2 3.7 9.7 3.2
Keratin, type IIPI|IPI00646190.1
cytoskeletal 14 Cytokeratin-14 CKUniRef100|Q91VQ4 IPI|IPI00384444.3
-9.9
10
8
11.8
2
Stabilin-2 precursor Hyaluronan receptor for
UniRef100|Q8CFM6
-12.2
118 120 116 20
Rat GCP360
UniRef100|Q63714
-12.2
1.7
9
26
alpha-1-inhibitorUniRef100|Q63018
III precursor
nrdb|554401 UniRef100|UPI0000506B73
UniRef100|Q6LDP2
UniRef100|UPI000019B93C
-14.2
5
6
3.2
1
PREDICTED: similar to Stabilin 2 precursor
UniRef100|UPI0000507087
-20.0
5
8
7
Inter-alpha-trypsin inhibitor heavy chain H3
UniRef100|Q63416 UniRef100|Q61704
Unique
1
2
3
apolipoprotein
A-IV
UniRef100|UPI0000167943
UniRef100|P02651
Unique
5
2
3
Interleukin 3
UniRef100|Q8HZ84 UniRef100|Q6GS87
Unique
1
1
1
PREDICTED: similar to stabilin-1
UniRef100|UPI0000507EED
Unique
1
1
1
Serum amyloid A 4 Predicted
UniRef100|Q5M878
Unique
1
1
1
Atp5d protein Lung RCB-0558 LLC cDNA,
UniRef100|Q4FK74 UniRef100|Q9DCZ0
E2 E3
1
8
6
1
7
3
25
6
9.9
7
5
6
2
2
1
23
59
4
1
1
1
12
1
1
1
8
4.7
54
2
1
12
5
2
9
4
27
3
15
28
9.9
13
9
5
3
6
1
47
37
5
2
4
1
11
1
4
3
2
3
0
1
72
13
1
10
5
5
2
1
4
1
1
Mean
Control
SD
Control
Mean
EGF
0.7
6.3
7.3
2.7
10.7
7.3
47.0
2.3
17.0
30.9
11.6
21.3
10.7
11.7
6.0
1.3
6.7
1.3
1.3
71.0
99.4
8.7
2.3
6.3
4.0
26.3
1.7
5.0
4.3
5.3
10.3
9.6
1.0
1.0
1.0
206.0
8.9
1.3
15.7
31.3
2.0
6.9
9.9
118.0
12.2
4.7
6.7
2.0
3.3
1.0
1.0
1.0
0.0
3.8
4.9
2.8
3.5
2.9
19.5
1.2
13.9
7.0
7.0
5.0
6.4
1.5
1.0
0.6
1.5
0.6
0.6
9.5
37.7
1.2
2.3
8.4
5.7
10.0
0.6
1.7
2.5
1.2
4.7
7.1
0.0
0.7
0.0
56.0
7.5
0.6
18.9
11.8
1.0
3.0
1.9
2.0
12.5
1.4
1.5
1.0
1.5
0.0
0.0
0.0
1.0
9.3
4.7
1.7
6.3
4.3
27.7
1.3
9.7
17.5
6.3
11.7
5.7
6.0
3.0
0.7
3.3
0.7
0.7
33.3
46.3
4.0
1.0
2.7
1.7
10.7
0.7
2.0
1.7
2.0
3.7
3.2
0.3
0.3
0.3
67.0
2.3
0.3
3.7
7.0
0.3
1.1
1.0
9.7
1.0
0.3
0.3
0.0
0.0
0.0
0.0
0.0
77
SD
Peptides (identifier Sequence)
EGF
0.0
2.3
1.5
0.6
3.1
1.5
3.1
1.4
4.7
9.1
0.6
4.2
3.1
1.0
1.0
2.3
0.0
0.0
12.3
11.3
1.0
0.7
1.5
1.2
1.5
0.0
1.7
0.7
1.0
3.5
2.8
11.4
2.0
6.4
5.3
0.7
9.0
0.7
289336 IYAPQGLLLTDPIER
77274 NPSDPSVAGLETIPTATGR
1443021 ILSLAQEQIGDSPEK
60281 IFTVDNNLLPVGK
53331 YSPQWPGIR
1000802 EPGEAAAEGAAEEAR
989633 NEETNQQEVANSLAK
762521 YVDDAGVPVSSAISR
759087 MYDDAVEAIEK
243787 TTFELIYQELLQR
67590 FPNAEFAEITK
81660 TLEAMEAALALEK
772701 AQEENTWFSYLK
89262 TVDNFVALATGEK
961654 LEAGDHPVELLAR
773689 APSVTSAAHSGYR
812479 AMLPLYDTGSGTIYDLR
56190 DMMLNIINSSITTK
3148072 RPHVDEFLQR
531124 IIIQESALDYR
66220 LGEYGFQNALIVR
1466664 FAVGTSGLGAEER
63644 KLDDFVETGNIR
444233 SFESTVGQGSDTYSYIFR
81370 YNILPEAEGEAPFTLK
995753 IAQDGVSTSATTNLK
1372268
439086 SFVEYVVYTEDPLVAQLR
969746 LSPDDADFVDVLHTFTR
81597 SVNQSLLELHK
760835 DSPVLVDSFEDSEPYRK
47177 NLDLDSIIAEVK
2352771 VFTTQELVQAFTHAPAALEADR
1378728 ELNGLPIMESNYFDPSK
1445994 LTSSVTAYDYAGK
572094 NGDTCVTLLDLELYNPK
170287 ALEEANTELEVK
2948735 TLEQASSPSEHGPR
80089 AYLTTVEDRYEQAFLTSLVGLRPEK
996942 GQITAILGHSGAGK
447988 TTPECGSTGYVEK
50469 VTMQNLNDR
994821 ELAGPGPFTVFAPLSSSFNHEPR
102875 ILLDDTQSEAAR
248075 ESVVFVQTDKPVYKPGQSVK
4200733 TMLGSQLLITSSQDQLHQETR
381950 SMTNINDGLLR
128627 LGNINTYADDLQNK
539618 NLLPCLPLATAAPTR
2796121 NIEASASDLPNLGQLR
1460870 DHGLESLQSTQK
157987 AQSELSGAADEAAR
Band
Cont2-4
Cont1-25
Cont1-37
Cont1-44
Cont1-12
Cont1-45
Cont1-64
Cont1-52
Cont1-47
Cont1-42
Cont1-1
Cont1-10
Cont1-29
Cont1-10
Cont1-30
Cont1-10
Cont1-49
Cont1-44
Cont1-21
Cont1-24
Cont1-1
Cont1-24
Cont1-52
Cont1-30
Cont2-26
Cont1-69
Cont1-77
Cont1-56
Cont1-24
Cont1-10
Cont1-50
Cont1-1
Cont1-31
Cont2-7
Cont1-3
Cont1-54
Cont1-11
Cont1-22
Cont1-67
Cont1-74
Cont1-11
Cont1-1
Cont1-69
Cont1-52
Cont1-68
Cont1-64
Cont1-51
Cont1-30
Cont1-3
Cont1-75
Cont1-3
Cont1-6
InterProScan
Predictions
IPR000020 (D)
IPR000020 (D)
IPR001104 (D) 3-oxo-5-alphaIPR000582 (F) Acyl-coAIPR004263 (F) Exostosin-like
IPR000264 (F) Serum
IPR001519 (F) Ferritin
IPR002130 (D) Peptidyl-prolyl
IPR001999 (D) OsteonectinIPR004031 (F) PMPIPR002194 (F) Chaperonin
IPR004274 (D) NLI interacting
IPR001893 (R) Cysteine rich
IPR000264 (F) Serum
IPR002659 (F) Glycosyl
IPR002159 (F) CD36 antigen
IPR000296 (F) CationIPR001599 (F) Alpha-2IPR003406 (F) Glycosyl
IPR000379 (D)
IPR001519 (F) Ferritin
IPR000010 (F) Cysteine
IPR001664 (F) Intermediate
IPR007821 (D) Protein of
IPR003177 (F) Cytochrome c
IPR001664 (F) Intermediate
IPR006055 (F) Exonuclease
IPR000562 (D) Type II
IPR008657 (F) Jumping
IPR001664 (F) Intermediate
IPR000538 (D) Link
IPR003345 (R) M protein
IPR000531 (D) TonBIPR001117 (F) Multicopper
IPR000074 (F) Apolipoprotein
IPR002183 (F) Interleukin-3
IPR000096 (F) Serum amyloid
IPR001469 (F) H+-
Table 3.2 ___Continued
Proteins identified by proteomics as changing >1.5 fold (increase or decrease in mean peptide counts) in the EN-DRM fraction
were grouped according to biological function into the following categories: Signalling, Receptor and Transporter, Trafficking,
Transcription and Translation, Ubiquitination/Proteasome, Structural/Cytoskeleton, Metabolism, Miscellaneous, and Unknown
Function. From left to right, the first column gives the database name and unique identifiers for the proteins. "Protein
Description" gives the name/description associated with the entry. "Fold change" indicates the mean fold change, and the
following 6 columns give the 'peptide counts' for each sample from control1 to EGF3 as indicated. The following 4 columns give
the means and standard deviations (SD) for the peptide counts. "Peptides" gives the peptide unique identifier (within our in-house
database [46]) followed by the amino acid sequence of the peptide identified. "Band" lists the sample number and band in which
peptides for this protein were identified. "Interproscan Predictions" lists the protein family (F), domains (D), active sites (AS),
repeat motifs (R) and binding sites (BS) as predicted by Interproscan.
78
Table 3.3 summarizes the number of these proteins by function. Of interest is
the large number of signaling and trafficking proteins which change in response to
EGF stimulation, consistent with the dual signaling and trafficking roles of the
endosomal system [59, 282, 283]. Also noteworthy are 8 ubiquitination-related
proteins, whose presence emphasizes the important role of ubiquitin-related
processes in trafficking events [284].
Table 3.3 Functional categorization of the proteins changing in ENDRMs following EGF.
# of
Protein Category
proteins
% of total
Signaling
34
7.7
Receptor and Transporter
57
12.9
Trafficking
73
16.5
Transcription/Translation
16
3.6
Ubiquitination/ Proteasome
8
1.8
Structural/Cytoskeleton
22
5.0
Metabolism
57
12.9
Unknown Function
86
19.5
Miscellaneous
89
20.1
Total
442
100
↑, increased post EGF; ↓, decreased post EGF.
# of
proteins↑
# of
proteins↓
26
41
53
9
6
18
33
67
39
292
8
16
20
7
2
4
24
19
50
150
The vATPase is a multimeric structure (Figure 1.3), responsible for the
acidification of intracellular organelles such as endosomes and lysosomes [139]. It
is structurally similar to the F-ATPase, the H+ dependant ATP synthase of
mitochondria and chloroplast (reviewed in ref. [139]), and is composed of a V1
79
(extrinsic) domain which consists of 8 different subunits involved in the binding
and hydrolysis of ATP; as well as a V0 domain consisting of multiple intrinsic
membrane components responsible for H+ transport across the membrane. A
striking observation was that, following EGF treatment, almost all the extrinsic V1
subunits of the vATPase were observed to increase 1.5 to 4.3 fold in endosomal
DRMs whereas the intrinsic V0 subunits, did not change except subunit ‘d’, which
is the only V0 component that is not an integral membrane protein(Figure 3.2).
The proteomic studies suggest that EGF stimulates the rapid assembly of the
vATPase holo-enzyme at the endosomal membrane.
80
Figure 3.2
81
Figure 3.2 Proteomic analysis of EN-DRMs/rafts reveals an EGF-dependant
change in abundance of vATPase V1, but not V0 intrinsic subunits.
Proteomic results for the identification of vATPase subunits in hepatic
endosomal rafts. Endosomal rafts were prepared from rat livers at 5 minutes after
EGF (1.0 µg/100 g BW) or vehicle administration and subjected to proteomic
analyses as described in Materials and Methods. The Mascot database identifiers
for each subunit are noted. The mean peptide ‘count’ for each subunit was
calculated by summing the results of all the subunit isoforms; and is listed for 3
replicate studies of controls (C ± s.d.) and EGF-treated (E ± s.d.) rats. The fold
change in peptide ‘count’ for EGF-treated vs control endosomal rafts
demonstrates an EGF-dependent augmentation of V1 but not V0 subunits as
shown in the upper and lower panels respectively.
82
3.1.2 EGF promotes recruitment of V1 subunits of vATPase to the vacuolar
system
The proteomic results, indicating V1 subunit recruitment, were confirmed by
immunological methods. Immunoblotting of rat liver combined endosomal
fractions and endosomal DRMs for subunit V1E and V0a demonstrated
recruitment of the former but not the latter following EGF administration (Figure
3.3A and B). It should be noted that this endosomal fraction is depleted of
lysosomes [285] which are known to contain a high concentration of vATPase.
In addition we employed immunofluorescence microscopy to localize V1E in
primary rat hepatocytes (Figure 3.3C and D). Following EGF stimulation for 5
minutes, V1E labeling can be seen to shift from a primarily diffuse basal pattern to
a more vesicular pattern consistent with vacuolar labeling. We did observe V1E
labeled vesicles in the basal state, but this population of structures, which most
likely consists of lysosomes and late endosomes, increased markedly following
EGF treatment. A negative control antibody (non-immune chicken IgY) was used
to demonstrate the specificity of the V1E labeling (Figure 3.3C). Double-blind
quantification of multiple independent experiments confirmed differential
localization of V1E following EGF treatment (Figure 3.3D). Comparable results
were obtained using an antibody against V1B (data not shown); however detailed
studies were pursued with antibody to V1E as this proved of superior quality in
our hands.
The rapid effect of EGF on V1E localization to vacuolar structures prompted
us to identify them more fully. Figure 3.3E shows that following EGF stimulation,
V1E co-localizes with lysosomal-associated membrane protein-1(LAMP1), which
83
is a late endosomal and lysosomal marker. Notably V1E did not co-localize with
early endosome antigen1(EEA1) or Rab5, markers for the early endosomal
components. These data, taken together with the recruitment of V1E to lysosomefree endosomal fractions leads us to conclude that EGF stimulates the rapid
recruitment of V1E to late endosomes as well as lysosomal structures.
In the course of these studies we frequently encountered cells in which there
was significant autofluorescence as previously observed by others [286].
Autofluorescence is visible in all channels, but is much weaker in the infra-red
channel (Figure 3.3G). Therefore, we could falsely interpret autofluorescence as
co-localization, as it would appear yellow in a merged image. We employed an
antibody coupled to a fluorophore which emits in the infra-red range, to limit the
impact of autofluorescent material appearing in the co-localization studies (Figure
3.3G).
Comparison of the specific infra-red fluorescence to channels in the
visible light range (red, in Figure 3.3G) allowed identification of interfering
autofluorescent material. Thus, use of the infra-red channel allowed us to verify
that our co-localization was true V1E-LAMP1 co-localization, and not
confounding autofluorescent material.
3.1.3 EGF increases the acidification of the vacuolar system
Since the vATPase is responsible for the acidification of intracellular
organelles, we sought to determine if EGF induced- recruitment of V1 vATPase
subunits to the vacuolar system could cause a decrease in the pH of these
structures. We performed 3-(2, 4-dinitroanilino)-3’-amino-N-methyl-dipropy-
84
lamine (DAMP) labeling in primary hepatocytes. DAMP, when incubated with
viable cell, accumulates in acidic compartments. The location of the acidic
compartments may then be visualized by immunofluorescence after fixation by
treatment with polyclonal anti-DNP, followed by a fluorophore-labeled,
secondary antibody. DAMP labeling has been successfully used to study low pH
compartments in a number of cell types [287, 288]. We found that EGF induced
acidification of vacuolar structures was sustained for as long as 20 minutes
(Figure 3.3F).
85
Figure 3.3
A
B
86
Figure 3.3 __Continued.
C
D
87
Figure 3.3 __Continued
E
F
88
Figure 3.3 __Continued
G
89
Figure 3.3 EGF promotes recruitment of V1 subunits of vATPase to late
endosomes- lysosomes and increases their acidification.
(A) Rat liver subcellular fractions were prepared as described in Materials and
Methods. Immunoblotting of V1E and V0a1 in rat liver microsomes (M),
endosomes (EN) and endosomal DRMs (EN-DRMs) from control and EGFtreated rats is shown. The results are from a single exposed gel which was sliced
to allow side-by-side comparison of the data. (B) Densitometric quantification of
the immunoblotting data was normalized to controls in each of 3 independent
studies. *p<0.05; bars indicate s.e.m. (C) Immunofluorescence microscopy of
primary rat liver hepatocytes stained with antibodies against V1E, or non-immune
IgY, +/- 5 minutes EGF. (D) Quantification of double-blind assigned V1E labeling
pattern was determined in 6 different experiments in each of which ~30 cells were
counted per condition (i.e. ~ 60 cells per experiment). EGF induced a vesicular vs
diffuse labeling pattern at *p=0.008; bars indicate s.e.m. (E) Immunofluorescence
microscopy shows double immunolabeling of endogenous V1E (green) with
EEA1 (red), Rab5 (red) and LAMP1 (red) in EGF-stimulated (100 nM, 5 minutes)
primary hepatocytes. All exposure times are equal. Bar is 5 m. (F) Rat primary
hepatocytes were incubated with 30 μM DAMP for 30 minutes and treated with
EGF for different times as noted in the figure. Indirect immunofluorescence was
performed and mean vesicle fluorescence intensity per cell was quantified as
described in Material and Methods. For each condition 10 cells were selected
randomly. *p<0.03, EGF treatment vs basal; bars indicate s.e.m. (G) Use of the
infra-red filter confirms observed co-localisation of V1E and LAMP1.
Immunofluorescence microscopy of primary hepatocytes labeled for V1E (green),
LAMP1 (infra-red), and visible interfering autofluorescence in the red channel.
The pseudocolour merge shows V1E as green, LAMP1 as red, and the
autofluorescence as blue. Autofluorescent material therefore appears in the merge
as cyan or white, whereas true co-localization appears yellow.
90
3.2 Effect of inhibiting vacuolar acidification on EGF action
3.2.1 Effect of bafilomycin on EGF-induced mitogenesis
Bafilomycin, a plecomacrolide antibiotic which specifically and potently
inhibits the vacuolar ATPase [140], has been shown to bind to both the V0c
[289]and V0a [141] subunits. Bafilomycin was previously observed to inhibit
insulin stimulated mitogenesis in primary hepatocytes [145], Swiss 3T3 cells
[290], BNL CL.2 (murine embryonic liver) cells [291], and eight different human
cancer cell lines [292]. In this study we showed that EGF-stimulated DNA
synthesis in primary rat hepatocytes was also inhibited by bafilomycin using both
10 nM and 100 nM EGF (Figure 3.4A). Notably bafilomycin maintained the
cellular content of the EGFR for 6 hours, during which time EGFR remained
tyrosine phosphorylated (Figure 3.4B and C).
Thus bafilomycin treatment
decoupled the relationship between EGFR tyrosine phosphorylation status and its
mitogenic potential.
91
Figure 3.4
A
B
C
92
Figure 3.4 Effect of bafilomycin on EGF- induced mitogenesis, and EGFR
content and tyrosine phosphorylation in rat hepatocytes.
(A) Hepatocytes were incubated for 30 minutes with100 nM bafilomycin or
DMSO before incubating for 18 hours in serum-free medium containing 5 µCi
3
H-methylthymidine with 0, 10 nM or 100 nM EGF. Incorporation of 3H-
thymidine into DNA was determined as described in Materials and Methods.
Results are expressed as fold over 10 nM EGF in each of three independent
studies. *p<0.02; bars indicate s.e.m. (B) Hepatocytes were pre-treated with either
DMSO or 100 nM bafilomycin, and then stimulated with 10 nM EGF for the
times shown. Cell lysates were subjected to SDS-PAGE followed by
immunoblotting with anti-EGFR and PY99 antibody (an antibody specifically
directed at phosphorylated tyrosine residues). SE: short exposure; LE: long
exposure. (C) Densitometric quantification of the immunoblotting data. Results
are means ± s.e.m. of at least 3 independent experiments.*p< 0.05.
93
3.2.2 Effect of bafilomycin on EGF-induced Akt and Erk signaling
To determine the basis by which bafilomycin inhibited EGF-induced DNA
synthesis we extended our studies on EGF signaling. Activation of both the
PI3K/Akt [278] and Erk pathways (Figure 3.5A) are important for EGFdependent mitogenesis in primary rat hepatocytes. An assessment of the time
course of EGF-induced phosphorylation of Akt and Erk clearly demonstrated no
inhibitory effect of bafilomycin on these processes (Figure 3.5B and C).
3.2.3 Bafilomycin inhibits EGF induced mTORC1 activation
However, bafilomycin markedly inhibited EGF-stimulated activation of
p70S6K as measured by pThr389 (Figure 3.6A and B) and p70S6 kinase activity
(Figure 3.6C), as well as EGF-stimulated phosphorylation of 4E-BP1 (Figure
3.6D). Both p70S6K and 4E-BP1 are downstream substrates of the mammalian
target of rapamycin (mTOR) and generally reflect mTORC1 activity [171]. The
inhibitory effect of bafilomycin on p70S6K and 4E-BP1 phosphorylation is
compatible with previous data showing that rapamycin inhibited EGF-dependent
DNA synthesis in hepatocytes [82].
Further investigation in HepG2 and FAO cell lines confirmed that
bafilomycin indeed inhibited EGF -stimulated activation of p70S6K as measured
by pThr389 in these cell lines (Figure 3.6E). Since most cell types respond to EGF,
we propose that this phenomenon might be widespread.
94
Figure 3.5
A
B
95
Figure 3.5 __Continued
C
96
Figure 3.5 Effect of bafilomycin on EGF-induced Akt and Erk signaling.
(A) Primary rat hepatocytes were incubated with the MEK1 inhibitor
U0126/DMSO (or DMSO control) for 30 minutes and subsequently stimulated
with EGF in the presence of
3
H-thymidine as described in the methods.
Quantitation of 3 independent experiments is shown. *p< 0.05; bars indicate s.e.m.
(B) Hepatocytes were treated as described in the legend of Fig. 3.4B. Cell lysates
were subjected to SDS-PAGE followed by immunoblotting with anti-phosphoAkt (Ser473) and anti-Akt antibody (top). Densitometric quantification was
performed on immunoblotting data from at least 3 independent experiments
(bottom). Bars indicate s.e.m. (C) Cell lysates were subjected to SDS-PAGE
followed by immunoblotting with anti-phospho-Erk1/2 (Thr202/Tyr204) and antiErk1/2 antibody (top). Densitometric quantification was performed on
immunoblotting data of at least 3 independent experiments (bottom). Bars indicate
s.e.m.
97
Figure 3.6
A
B
C
98
Figure 3.6 __Continued
D
E
99
Figure 3.6 Effect of bafilomycin on EGF-induced mTORC1 signaling.
(A) Hepatocytes were treated as described in Figure 3.4B. Cell lysates were
subjected to SDS-PAGE followed by immunoblotting with anti-phospho-p70S6K
(Thr389) and anti-p70S6K antibody. (B) Densitometric quantification was
performed on immunoblotting data of at least 3 independent experiments. *p<
0.005, **p<0.001; bars indicate s.e.m. (C) The p70S6K kinase assay was
performed using the S6 peptide substrate as described in Materials and Methods.
Data are representative of 2 experiments performed with internal duplicates. (D)
Cell lysates were subjected to SDS-PAGE followed by immunoblotting with antiphospho-4E-BP1 (Ser65) and anti-4E-BP1antibody. (E) HepG2 or FAO cells were
pre-treated with either DMSO or 100 nM bafilomycin, and then stimulated with
10 nM EGF for the times shown. Cell lysates of HepG2 cells were subjected to
SDS-PAGE followed by immunoblotting with anti- phospho-p70S6K (Thr389),
anti-p70S6K, anti-phospho-PRAS40 (Thr246) and anti-PRAS40 antibody (top);
cell lysates of FAO cells were subjected to SDS-PAGE followed by
immunoblotting with anti-phospho-p70S6K (Thr389) and p70S6K antibody
(bottom).
100
3.2.4 Chloroquine mimics the effect of bafilomycin
To confirm that the bafilomycin effect was due to its inhibition of endosomal
acidification we investigated the effect of chloroquine, an acidotropic inhibitor of
vacuolar acidification [293]. As shown in Figure 3.7, chloroquine also inhibited
activation of p70S6K and 4E-BP1, but had no inhibitory effect on Akt and Erk
signaling.
3.2.5 Effect of bafilomycin on insulin signaling
We also assessed the effect of bafilomycin on insulin signaling; and found
that here as well there was no effect on Akt and Erk activation by insulin but a
noticeable reduction in TORC1 activation as inferred from the inhibition of
p70S6K and 4E-BP1 phosphorylation (Figure 3.8). Since chloroquine, unlike
bafilomycin, has minimal or no inhibitory effect on vATPase [125], it appears that
the acidic pH of the endosomal milieu is required for hormone and growth-factor
activation of mTORC1-dependent signaling.
101
Figure 3.7
A
102
Figure 3.7 __Continued
B
Time after EGF (min)
Time after EGF (min)
103
Figure 3.7 Effect of chloroquine on EGF-stimulated mTORC1, Akt and Erk
activation.
(A) Hepatocytes were pre-treated with either vehicle or 100 µM chloroquine
for 30 minutes, and then stimulated with 10 nM EGF for the times shown. Cell
lysates were subjected to SDS-PAGE followed by immunoblotting with
antibodies as indicated. Data shown are representative of at least 3 independent
experiments. (B) Densitometric quantitation of selected results from Figure 3.7A
is shown. Results are means ± s.e.m. of at least 3 independent experiments. *p<
0.05.
104
Figure 3.8
105
Figure 3.8 Similar effect of bafilomycin on Insulin and EGF-stimulated
mTORC1, Akt and Erk activation.
Hepatocytes were pre-treated with either DMSO or 100 nM bafilomycin, and
then stimulated with 10 nM EGF or insulin for the times shown. Cell lysates were
subjected to SDS-PAGE followed by immunoblotting with the indicated
antibodies. Data shown are representative of at least 3 independent experiments.
106
3.3 The role of acidification in mTORC1 activation
3.3.1 Bafilomycin does not alter mTOR and Raptor association ， energy
status of the cell and Akt effect on TSC2
Mammalian TORC1 is composed of mTOR, Raptor, mLST8 and PRAS40
and sensitive to the inhibition of macrolide antibiotic rapamycin. Its activation
and downstream events can be influenced by many factors, including amino acids,
glucose (reviewed in [171]) and growth factors (Figure 1.6). We demonstrated that
the mTOR and Raptor association status was unchanged by bafilomycin (Figure
3.9A and B), so the inhibitory effect of bafilomycin on mTORC1 is not a
‘rapamycin-like’ effect.
Mammalian TORC1 activity is linked to energy status in a manner
independent from nutrient status [294]. AMPK is the sensor of ATP levels in cells
[295], and AMPK has been shown to inhibit mTORC1 by phosphorylation and
activation of TSC2, an upstream GAP which inhibits Rheb activity [245]. We
therefore assessed AMPK status by immunoblotting for pT172 of AMPK, the
target of upstream LKB1 [296] and a readout of AMPK activity status [297].
Figure 3.9C shows that EGF and insulin gradually de-activated AMPK in primary
hepatocytes, and bafilomycin did not interfere with this process.
107
Figure 3.9
A
B
C
108
Figure 3.9 __Continued
D
109
Figure 3.9 Bafilomycin does not alter mTOR and Raptor association, energy
status of the cell and Akt effect on TSC2.
(A) Serum starved primary hepatocytes were pre-incubated with either serumfree medium or warm PBS, in the presence of DMSO or 100 nM bafilomycin for
30 minutes. Cells were then stimulated with 10 nM EGF for the times shown. Cell
lysates were incubated with anti-mTOR antibody, and the immunoprecipitates
were subjected to SDS-PAGE followed by immunoblotting with anti-mTOR and
raptor antibodies. (B) Densitometric quantification of the immunoblotting data.
Raptor- mTOR association is expressed as fold over basal in each of three
independent studies. Bars indicate s.e.m. (C) Serum starved primary hepatocytes
were pre-incubated with either serum-free medium or warm PBS, in the presence
of DMSO or 100 nM bafilomycin for 30 minutes. Cells were then stimulated with
10 nM EGF or insulin for the times shown. Cell lysates were subjected to SDSPAGE followed by immunoblotting with anti-phospho-AMPK (Thr172) and
AMPK antibody. Data are representative of 3 independent experiments. (D) Cells
were then stimulated with 10 nM EGF for the times shown. Cell lysates were
subjected to SDS-PAGE followed by immunoblotting with anti-phospho-TSC2
(Thr1462) and TSC2 antibody (top). Densitometric quantification was performed
on immunoblotting data of at least 3 independent studies (bottom). Bars indicate
s.e.m.
110
3.3.2 Effect of bafilomycin on PRAS40
The interaction of the proline-rich Akt substrate (PRAS40) with mTORC1
inhibits the activity of mTORC1 [298]. Phosphorylation of PRAS40 reduces its
binding to mTOR thus relieving its inhibitory constraint on mTORC1 activity
[192]. Growth factor dependent phosphorylation of PRAS40 is effected by Akt
activation and modulated by intracellular amino acid levels [299]. We found that
EGF induced phosphorylation of PRAS40 at 15 minutes was decreased by both
bafilomycin (by ~20%) and chloroquine (by ~30%) (Figure 3.10A). This was not
due to an inhibition of PIM1 kinase (data not shown) which was recently reported
to regulate mTOR activity by phosphorylation of PRAS40 at Thr246 [300]. We
checked both FAO and HepG2 cell lines and found that EGF-induced
phosphorylation of PRAS40 was also decreased by bafilomycin in these cell lines
(Figure 3.6E). Since bafilomycin had no effect on EGF-induced Akt activation
(Figure 3.4A) as further confirmed by lack of change in Akt-dependent
phosphorylation of TSC2 at Thr1462 site (Figure 3.9D), we deduced that
bafilomycin might inhibit EGF induced mTORC1 activity through influencing
amino acid sufficiency.
3.3.3 Effect of bafilomycin on intracellular amino acid levels
Using a chromogenic technique to measure amino acids (L-amino acid
quantitation kit from Biovision), we found that bafilomycin had no effect on total
free intracellular amino acid levels in primary hepatocytes (Figure 3.10B). This
prompted us to do further individual intracellular amino acid analysis by HPLC.
111
Consistent with our data using the chromogenic method we found total
intracellular amino acid levels to be unchanged following bafilomycin. However,
interestingly, the total concentration of essential amino acids decreased
dramatically with bafilomycin treatment (Figure 3.10 C top) while that of nonessential amino acids remained unchanged in EGF-treated primary hepatocytes.
Notably, the intracellular concentration of the amino acids leucine, isoleucine and
arginine whose sufficiency are critical for mTORC1 activation [301], decreased
markedly with bafilomycin in EGF-treated primary hepatocytes (Figure 3.10C
bottom and Table 3.4). It is also noteworthy that the concentrations of essential
and non-essential amino acids in the medium were 2,764 and 4,259 nmol/ml
respectively, which was ~10 times higher than the corresponding intracellular
amino acid levels (Table 3.4). Thus Intracellular amino acid levels in cultured
hepatocytes appear to reflect primarily the balance between the intracellular
processes of protein synthesis and proteolysis.
To complement these observations we sought to investigate whether
neutralising endosomal pH could affect amino acid transport into cultured
hepatocytes. This is relevant in view of the report that in skeletal muscle
chloroquine can inhibit the movement of amino acid transporters to the plasma
membrane, thus impairing amino-acid uptake [302]. The branched chain amino
acid leucine is sensed by cells as a reporter of external nutrient status. We thus
determined leucine uptake in the absence and presence of bafilomycin. Neither
EGF nor insulin stimulated an increase in leucine uptake (Figure 3.10 D), nor did
bafilomycin inhibit uptake. To further eliminate the possibility that bafilomycin
112
inhibited leucine uptake, we starved cells for 30 min in warm PBS.
The
accelerated uptake of 3H-Leucine by these starved cells was also unaffected by
bafilomycin (Figure 3.10D).
113
Figure 3.10
A
B
114
Figure 3.10 __Continued
C
D
115
Figure 3.10 Effect of bafilomycin on PRAS40 phosphorylation and
intracellular amino acid levels and leucine uptake of the cell.
(A) Hepatocytes were pre-treated with DMSO, 100 nM bafilomycin or 100
µM chloroquine, and then stimulated with 10nM EGF for the times shown. Cell
lysates were subjected to SDS-PAGE followed by immunoblotting with antiphospho-PRAS40 (Thr246) and PRAS40 antibody (top). The results are from a
single exposed gel which was sliced to allow side-by-side comparison of the data.
Densitometric quantification was performed on immunoblotting data of at least 3
independent experiments. *p< 0.05; bars indicate s.e.m. (bottom). (B)
Hepatocytes were pre-treated with either vehicle or 100 nM bafilomycin, and then
stimulated with 10 nM EGF for the times shown. Total amino acid analysis was
performed by using the L-amino acid quantitation kit from Biovision. (C) Serum
starved primary hepatocytes were pre-incubated with either serum-free medium or
warm PBS, in the presence of DMSO or 100 nM bafilomycin for 30 minutes.
Cells were then stimulated with 10 nM EGF for another 30 minutes. Individual
intracellular amino acids were determined by HPLC analysis as described in
Materials and Methods. Total concentration of essential amino acids and nonessential amino acids were calculated by adding the respective individual amino
acids concentration together. Quantification was performed on 3 independent
experiments. *p<0.001; bars indicate s.e.m. (top).
*p<0.05, **p<0.005; bars
indicate s.e.m. (bottom). (D) Serum starved primary hepatocytes were preincubated with either serum-free medium or warm PBS, in the presence of DMSO
or 100nM bafilomycin for 30 minutes. Cells were then stimulated with EGF or
insulin in the presence of 3H-leucine for 15 minutes. Leucine uptake was
measured by scintillation counting. Results are means ± s.e.m. of 3 independent
experiments.
116
Table 3.4 Concentrations of amino acids (AAs) in medium and primary
hepatocytes (nmol/ml) before and after treatment with EGF and
bafilomycin (Baf).
Hepatocytesa
EGF
-Baf
+Baf
Medium
Basal
AA
Essential
MET
VAL
PHE
ILE
LEU
LYS
THR
TRP
ARGb
TYRb
HISb
SUM
Non-essential
ORN
ASN
ASP
TAU
GLU
SER
GLN
GLY
ALA
CIT
SUM
TOTAL
449.8
115.6
624.2
451.1
214.9
415.5
449.1
44.1
700.2
212.4
150.3
2764.3
%change
49.5±1.3
18.8±0.5
37.5±0.8
30.1±0.5
27.2±0.8
14.4±0.2
35.8±0.5
4.8±0.1
28.6±1.0
26.7±0.5
12.7±0.8
267.1±6.5
53.8±6.4
20.3±2.6
44.8±3.1
33.3±3.5
29.2±3.1
16.0±1.6
41.2±4.2
5.1±0.6
33.7±2.8
29.0±3.0
13.4±2.3
299.6±30.6
19.0±1.8
7.8±1.4
21.4±2.3
17.0±2.0
15.3±1.3
9.0±1.0
37.5±3.5
3.8±0.6
13.8±2.5
15.9±1.7
14.3±3.4
167.1±19.9
-65%**
-62%**
-52%**
-49%**
-48%**
-44%**
n/s
n/s
-59%**
-45%**
n/s
-46%**
4259.0
29.0±1.2
13.9±0.1
23.9±0.4
6.5±0.3
53.5±2.6
54.6±1.0
73.6±0.6
47.6±4.4
55.1±0.6
2.7±0.3
360.2±11.5
32.3±0.9
15.1±1.5
24.0±1.8
7.0±0.8
62.1±5.1
60.3±6.0
80.2±8.0
47.6±5.2
58.5±3.3
3.0±0.2
390.1±32.7
25.4±1.8
9.9±1.2
38.6±3.9
12.6±1.0
74.8±3.1
41.3±3.7
80.9±5.4
54.4±5.7
48.9±4.3
3.8±0.6
390.5±30.6
-21%*
-35%*
+61%*
+80%**
n/s
n/s
n/s
n/s
n/s
n/s
n/s
7023.3
627.3±18.0 689.7±63.3 557.5±50.5
99.2
50.0
249.8
2497.5
249.8
50.0
a
n/s
The values in the table are means of 3 independent replicates ±SEM. *p<0.05,
**p<0.01.
b
Essential only in certain cases.
117
3.3.4 Effect of cycloheximide on mTORC1 activation and intracellular amino
acid levels
To confirm that the inhibition of mTORC1 activation was due to a
bafilomycin dependent decrease in amino acid levels, we inhibited this decrease
by co-incubating cells with cycloheximide, a translation elongation inhibitor,
known to augment intracellular amino acid levels [281]. As illustrated in Figure
3.11A and Table 3.5 cycloheximide significantly blunted the bafilomycin-induced
decrease in intracellular essential amino acid levels, including the amino acids
critical for mTORC1 activation- leucine, isoleucine and arginine. At the same time
cycloheximide completely reversed the inhibition by bafilomycin of 4E-BP1
phosphorylation in EGF-treated hepatocytes (Figure 3.11B). It also increased the
phosphorylation of p70S6K over the basal state (Figure 3.11D).
Hypophosphorylated 4E-BP1 functions as a translational repressor by binding
to eukaryotic translation initiation factor 4E (eIF-4E), thereby preventing its
interaction with eIF-4G and thus inhibiting translation [171]. We examined the
binding of 4E-BP1 to eIF4E by affinity chromatography using m7GTP-Sepharose,
which retained eIF4E and associated proteins. Consistent with the 4E-BP1
phosphorylation data, cycloheximide treatment led to a decrease in the amount of
4E-BP1 associated with eIF-4E and a corresponding increase of eIF-4G binding
with eIF-4E (Figure 3.11 C).
Due
to
the
extent
to
which
cycloheximide
augmented
p70S6K
phosphorylation we examined the possibility that the mTORC1 repressor, REDD1
[303] was affected by the inhibition of protein synthesis as previously described
118
[304]. However in rat hepatocytes the expression level of REDD1 was not
downregulated by cycloheximide (Figure 3.11E).
Besides cell-surface amino acid transporters, a different class of amino acid
transporter resides in late endosomes and lysosomes, known as PAT (protonassisted amino acid transporter),
which has been shown to be required for
mTORC1 activation by amino acids in both Drosophilia [305]and human cell
lines [306]. Since EGF induces vacuolar acidification in late endosomal
compartments (Figure 3.3F), we sought to determine if the effect of EGF and
bafilomycin on mTORC1 through amino acids included modulation of PAT1
function. However, we did not detect the PAT1 protein in rat liver endosomes by
western blot while the expression level of PAT1 in rat brain was readily observed
(Figure 3.11F).
119
Figure 3.11
A
B
120
Figure 3.11 __Continued
C
121
Figure 3.11 __Continued
D
122
Figure 3.11 __Continued
E
F
123
Figure 3.11 Effect of cycloheximide on mTORC1 activation and intracellular
amino acid levels.
(A) Hepatocytes were pre-treated with either DMSO, 100nM bafilomycin (Baf),
25 µg/ml cycloheximide (CHX) or Baf and CHX together (Baf+CHX) for 30
minutes, and then stimulated with 10nM EGF for another 30 minutes. Individual
intracellular amino acids were determined by HPLC analyses as described in
Materials and Methods. Total concentrations of essential and non-essential amino
acids were calculated by adding the respective individual amino acids
concentration together. Quantification was performed from 3 independent
experiments. *p<0.005, **p<0.001; bars indicate s.e.m. (top). *p<0.05,
**p<0.005; bars indicate s.e.m. (bottom). (B) Cell lysates were subjected to SDSPAGE followed by immunoblotting with anti-phospho-4E-BP1 (Ser65) and 4EBP1 antibody. Quantification was performed in 3 independent experiments. *
p<0.05; bars indicate s.e.m. (bottom). (C) Hepatocytes were treated as indicated
and samples of cell lysates were subjected to affinity chromatography on m 7GTPSepharose and bound materials were subjected to SDS-PAGE followed by
immunoblotting with anti-eIF4G, eIF4E and 4E-BP1 antibody. The signals of 4EBP1 and eIF4E were quantified and presented as a ratio. Quantification was
performed in 3 independent experiments. * p<0.005; bars indicate s.e.m. (D) Cell
lysates were subjected to SDS-PAGE followed by immunoblotting with antiphospho-p70S6K (Thr389) and p70S6K antibody (top). Quantification was
performed in 3 independent experiments. * p<0.05, ** p<0.005; bars indicate
s.e.m. (bottom). (E) Cell lysates were subjected to SDS-PAGE followed by
immunoblotting with anti-REDD1 antibody (top). Quantification was performed
on 6 independent replicates. Data are normalized to control values. *p<0.05,
**p<0.01; bars indicate s.e.m. (bottom). The right bottom inserted figure shows
induction of REDD1 by CoCl2 as a positive control. (F) Rat liver lysosomes were
prepared. Immunoblotting of PAT1 is shown. A rat brain homogenate was used as
a positive control for PAT1 protein.
124
Table 3.5 Concentrations of amino acids (AAs) in primary hepatocytes
(nmol/ml) after treatment with EGF with bafilomycin (Baf) and
cycloheximide (CHX).
AA
Essential
LEU
MET
LYS
VAL
PHE
ILE
THR
TRP
ARGa
TYRa
HISa
SUM
Non-Essential
ORN
ASN
ASP
TAU
GLU
SER
GLN
GLY
ALA
CIT
SUM
TOTAL
Control
Baf
CHX
Baf+CHX
53.8±4.5
22.6±1.7
41.1±3.5
37.3±3.2
30.2±2.0
16.8±1.4
47.1±3.1
6.5±0.3
26.9±1.7
27.4±1.5
16.9±1.2
326.6±22.6
26.5±1.7
14.3±0.9
25.1±1.5
23.6±1.4
18.0±0.9
10.6±0.6
43.4±2.5
5.0±0.2
15.5±0.5
16.4±0.8
13.2±1.2
211.6±11.2
67.2±5.1
25.2±2.1
51.6±3.9
45.0±2.8
32.0±2.6
19.8±1.3
60.8±2.8
6.7±0.5
33.0±3.4
29.6±2.7
17.0±1.1
387.9±27.5
41.4±1.1
19.8±0.5
38.3±0.9
33.7±0.7
24.3±0.8
14.4±0.3
57.7±1.3
6.1±0.2
22.7±1.3
22.2±0.4
16.0±0.3
296.4±6.8
30.9±1.8
16.6±1.2
25.1±1.5
12.4±0.8
71.3±1.7
79.8±4.8
160.0±3.0
45.6±2.4
58.9±1.2
5.0±0.2
505.6±18.3
27.1±1.9
12.2±0.7
36.6±2.3
14.0±1.4
80.0±3.0
65.7±3.2
165.5±6.3
46.4±2.6
52.4±1.9
6.8±0.1
506.6±21.8
37.8±2.5
20.3±1.2
27.1±1.4
12.2±0.6
73.2±4.4
95.3±5.2
163.7±9.6
59.4±7.5
63.0±4.0
5.7±0.4
557.8±35.8
36.3±0.6
16.1±0.2
40.1±0.6
14.2±0.3
86.6±2.9
87.1±2.1
178.5±4.3
66.1±12.0
62.4±1.7
7.5±0.4
594.7±10.6
832.1±37.8 718.1±32.9 945.7±62.9 891.1±11.2
a
Essential only in certain cases
The values in the table are means of 3 independent replicates ±s.e.m.
125
3.3.5 MG132 mimics the effect of bafilomycin on mTORC1 activation
Since bafilomycin inhibited mTORC1 activation by decreasing amino acid
levels in the cells, we checked if blocking proteasome-dependent proteolysis
would mimic the effect of bafilomycin. MG132 is a specific, reversible, and cellpermeable proteasome inhibitor which reduces the degradation of ubiquitinconjugated proteins in mammalian cells and permeable strains of yeast by the 26S
complex without affecting its ATPase or isopeptidase activities. Like bafilomycin,
MG132 markedly inhibited EGF -stimulated activation of p70S6K as measured
by pThr389. At the same time, cycloheximide dramatically reversed the inhibition
of MG132 by restoring the phosphorylation of p70S6K in EGF-treated
hepatocytes (Figure 3.12).
126
Figure 3.12
127
Figure 3.12 Effect of MG132 on EGF-induced mTORC1 signaling.
Hepatocytes were pre-treated with 50 µM MG132 for 5 hours or /and 25
µg/ml cycloheximide (CHX) for 30 minutes, and then stimulated with 10nM EGF
for the times shown. Cell lysates were subjected to SDS-PAGE followed by
immunoblotting with anti-phospho-p70S6K (Thr389) and p70S6K antibody (top).
Quantification was performed from 3 independent experiments. * p<0.05, **
p<0.005; bars indicate s.e.m. (bottom).
128
3.3.6 Effect of in vivo chloroquine on mTOR signaling
We then sought to evaluate in vivo the effect of inhibiting vacuolar
acidification on mTOR signaling. Fasted rats were treated with chloroquine or
normal saline and EGF was then administered. At 15 minutes after EGF
administration, rat liver endosomes and cytosols were prepared and subjected to
western blot analysis. As shown in Figure 3.13A, chloroquine administration
markedly inhibited EGF -stimulated activation of p70S6K as measured by
pThr389; during which time there was augmented accumulation of EGFR and
tyrosine phosphorylated EGFR in endosomes following chloroquine (Figure
3.13B). The activity of mTORC1 is stimulated by the GTP-bound (active) form of
the small G protein Ras homologue enriched in brain (Rheb). Rheb binds directly
to the kinase domain of mTOR and activates mTOR [236]. Recent reports have
suggested a movement of mTOR to subcellular structures where Rheb resides in
response to amino acids [189, 274]. Intriguingly, in our study EGF induced a
marked recruitment of Rheb into endosomes while chloroquine significantly
inhibited this recruitment. The endosomal content of mTOR was unaffected by
either EGF or chloroquine treatment. EGF also induced an increase of Rheb in rat
liver lysosomal fractions while the content of mTOR was unchanged (Figure
3.13C).
129
Figure 3.13
A
130
Figure 3.13 __Continued
B
C
131
Figure 3.13 Effect of in vivo chloroquine on mTOR signaling.
(A) Animals received 10 mg/200g body weight of chloroquine by intraperitoneal
injection, 2 hours and 1 hour prior to EGF stimulation. Control animals received a
comparable volume of normal saline. Rat liver subcellular fractions were prepared
as described in Materials and Methods from rat livers at 15 minutes after either
EGF (1.0 µg/100 g BW) or vehicle administration. Immunoblotting of mTOR,
Rheb, p70S6K and phospho-p70S6K (Thr389) in rat liver cytosol (CY) is shown
(top). Densitometric quantification of the immunoblotting data was normalized to
controls in each of 3 independent studies. *p<0.01; bars indicate s.e.m. (bottom).
(B) Rat liver endosomes were prepared as described in Fig. 8A.Immunoblotting of
mTOR, Rheb, RagA, RagC, EGFR and PY-EGFR in rat liver endosomes (EN) is
shown (top). Densitometric quantification of the immunoblotting data was
normalized to controls in each of 3 independent studies. *p<0.002; bars indicate
s.e.m. (bottom). (C) Rat liver lysosomes were prepared as described in Materials
and Methods from rat livers at 5 and 30 minutes after EGF (1.0 µg/100 g BW) or
vehicle administration. Immunoblotting of mTOR and Rheb in rat liver lysosomes
is shown.
132
Chapter 4
Discussion and Summary
133
In previous work it was shown that insulin and EGF treatment effected the
rapid internalization of their respective receptors into endosomes with the
concomitant recruitment of both signaling and trafficking molecules [22, 26, 46].
Similar observations on changes in endosomal EGF-dependent phosphoproteins
have been made by Stasyk et al [29]. In the present study we extended these
earlier observations on endosomal events following EGF treatment and have
demonstrated that a large number of molecules involved in signaling and
trafficking are significantly augmented or decreased in endosomes following EGF
treatment.
The vATPase is the principal agent responsible for endosomal acidification
accomplished by the translocation of protons across the endosomal membrane
[139]. The ongoing activity of endosomal vATPases generates a pH gradient
within the endosomal system from pH
6.0 in early endosomes to pH 5.0–5.5 in
lysosomes [138]. Of particular interest was our novel finding that EGF treatment
induced the accumulation of V1 components of the vacuolar (H+)- ATPase
(vATPase) in endosomal DRMs/rafts while the V0 components showed little or no
change in this fraction. The increase in subunit ‘d’ association is interesting as,
unlike the other V0 subunits, it is a peripheral membrane protein [307]. The
augmentation of V1 components in DRMs/rafts did not reflect translocation of V1
components from non-hydrophobic to hydrophobic membrane domains since we
could demonstrate that there was net recruitment of V1 subunits to intact
endosomes.
134
Dissociation of the V1 and V0 components of the holoenzyme was first
observed in insect tissue [111]. The reversible assembly/disassembly of V0 and V1
components was then documented in S.Cerevisiae [112] and renal epithelial
cells[113], and has been recognized as an important regulatory mechanism of
vATPase function [85, 114]. In renal epithelial cells, vATPase assembly and
vATPase dependent acidification of intracellular compartments is stimulated by
glucose through PI3K signaling, but the factors linking glucose level and PI3K
remain unknown [113]. It is possible that EGF- induced V1/V0 assembly in our
study follows activation of PI3K in analogy with the demonstrated mechanism by
which glucose promotes the assembly of V1 and V0 [113].
Using DAMP fluorescence to measure vacuolar pH we found that the
recruitment of V1 components was accompanied by augmented vacuolar
acidification. This represents the first demonstration of a growth factor inducing
the rapid assembly of the vATPase as a functional holoenzyme leading to
vacuolar acidification.
EGF has fundamental roles in mammalian embryonic development, where it
regulates diverse processes such as eyelid opening, tooth growth, hair follicle and
mammary gland development. EGFR knockout mice often suffer from early and
lethal defects associated with neural and myocyte development. The survival of
EGFR knockout mice depends on their genetic background-MF1 and CD1 mice
can live until postnatal day 20. However, all the mice suffer neurodegeneration
and defective development of surplus neurons in the hippocampus [308]. EGF
also strongly influences the synthesis and turn-over of proteins of the extracellular
135
matrix, increases bone resorption through the release of calcium from bone and
regulates wound healing. On the cellular level, it is a strong mitogen and
stimulates the proliferation of fibroblasts, epidermal and epithelial cells, including
kidney epithelial cells, human glial cells, hepatocytes, and thyroid cells [2].
This thesis focuses on the biological role of EGF in liver. EGFR-dependent
signaling contributes to liver cell proliferation, and represents an important
regulator of hepatic regeneration [66]. Mice lacking hepatic EGFR display
reduced hepatocyte proliferation with reduced and delayed expression of cyclin
D1 [81]. In previous work we showed that mammalian TOR is the critical
regulator of EGF-induced cell growth and DNA synthesis in primary hepatocytes
[82]. Inhibition of mTORC1 by rapamycin abrogated both DNA replication and
protein synthesis induced by growth factors [82, 83]. Rapamycin also decreased
messenger RNA (mRNA) and protein levels of cyclin D1 [83].
The mTORC1 molecular complex responds to a range of signals relating to
the energy and nutrient status of the cell [171]. It is known that growth factors can
stimulate mTORC1 through the Akt pathway and the Erk pathway [309]. In the
present study we examined the role of EGF-induced vacuolar acidification on
EGF action by neutralizing vacuolar pH during EGF treatment. Bafilomycin, a
specific inhibitor of vATPase, known to bind both V0c [142] and V0a [141]
subunits, has been proposed to block vacuolar acidification by interfering with
rotation of the proteolipid ring [142]. We found that bafilomycin inhibited EGFinduced mTORC1 activation. However bafilomycin did not interfere with EGF
induced Akt activation nor with Akt –dependent TSC2 phosphorylation; nor did
136
bafilomycin inhibit EGF-dependent Erk activation or alter AMPK activity. That
this effect derived from the inhibition of vacuolar acidification and not other
potential effects of bafilomycin (viz. on vacuolar coat assembly) was supported
by the use of the acidotropic agent chloroquine [147] which, unlike bafilomycin
has minimal or no effect on vATPase activity [125] but nevertheless, selectively
inhibited mTORC1 activation.
In our study the inhibition of EGF-induced phosphorylation of PRAS40 at
Thr246 was decreased by both bafilomycin and chloroquine. Amino acid
deficiency has been observed to decrease PRAS40 phosphorylation [299]. A
number of observations have suggested that cell surface amino acid transporters
may play a role in amino acid-dependent mTORC1 activation [261, 263, 264].
These include System A amino acid transporters (i.e. solute carrier family 1
member 5(SLC1A5)) which transport glutamine into cells in a sodium (Na+)
dependent manner[262]; and System L amino acid transporters (i.e. LAT1) [262,
263], which transport branch-chain amino acids and neutral amino acids into the
cells in a sodium (Na+) independent manner. Co-expression of Systems A and L
led to leucine accumulation and activation of TORC1 in oocytes [264]. However,
in our studies preincubation of primary cultured hepatocytes with bafilomycin for
30 minutes resulted in a decrease of the intracellular concentration of essential
amino acids despite concentrations of amino acids in the incubation medium that
were ~10 times higher than the corresponding intracellular amino acid levels.
There was no corresponding decrease in the intracellular level of non-essential
amino acids. Combined with the fact that EGF did not stimulate an increase in
137
leucine uptake, nor did bafilomycin inhibit uptake, we propose that the inhibition
of vATPase by bafilomycin affects intracellular essential amino acid levels
primarily by an intracellular process rather than by the modulation of amino acid
transporter function at the plasma membrane.
It has been previously noted that the intracellular level of amino acids can be
refractory to changes in the level of extracellular amino acids [310]. Studies in
rats [311, 312] have shown that ethanol and fasting can produce significant
changes in the levels of amino acids and related compounds in plasma and various
tissues. However, the changes in tissue amino acid levels were found to be highly
tissue specific and generally unrelated to changes seen in the plasma. These
observations suggest that individual tissues have strong regulatory mechanisms
essential to maintaining intracellular amino acid homeostasis. By using inhibitors
of protein synthesis and autophagy, Beugnet et al. showed that the regulation of
mTOR in mammalian cells was responsive to the intracellular amino acid pool;
and that the levels of intracellular amino acids were critically determined by the
intracellular rates of protein synthesis and degradation [281]. In our study the
reduction in essential amino acid levels, following the inhibition of vacuolar
acidification, occurred despite a large excess of extracellular amino acids. Thus, in
cultured hepatocytes, intracellular amino acid levels largely reflected a balance
between the intracellular processes of protein synthesis and protein degradation.
We therefore suggest that bafilomycin/chloroquine, blocked EGF-induced
mTORC1 activation by inhibiting lysosomal proteolysis leading to reduced
intracellular levels of essential amino acids. The fact that only the levels of
138
essential amino acids decreased may reflect the total dependence of intracellular
essential amino acid levels on recycling in the absence of inflow from the
extracellular space. This possibility is supported by the observation that
cycloheximide prevented the bafilomycin-induced decrease in intracellular levels
of essential amino acids, and correspondingly prevented the inhibition by
bafilomycin of mTORC1 activation by EGF. A recent study in C. elegans showed
that EGF signaling altered protein homoeostasis by increasing the activity of the
ubiquitin proteasome system (UPS) and polyubiquitination, while decreasing
protein aggregation [313]. Interestingly inhibition of proteasome-dependent
proteolysis by MG132 in our study inhibited EGF -stimulated activation of
p70S6K; and this effect was reversed by cycloheximide, again emphasizing the
role of proteolysis in maintaining intracellular amino acid levels thus sustaining
mTOR activation.
Notably, besides cell-surface amino acid transporters, there is a different class
of amino acid transporter, mammalian proton-coupled SLC36 amino acid
transporters known as PAT (proton-assisted amino acid transporter) shown to be
required for mTORC1 activation by amino acids in both Drosophilia [305] and
human cell lines[306]. PAT1 resides in intracellular compartments, such as late
endosomes and lysosomes rather than at the cell surface [306] and regulates
mTORC1 activity without requiring bulk transport of amino acids [305]. Heublein
et al. proposed a model in which intracellular PATs alter the sensitivity of
mTORC1 to amino acids in late endosomal compartments. However, we did not
139
find detectable PAT1 protein level in rat liver lysosomes, which suggests that
PAT1 modulation of mTORC1 might be tissue-specific.
In previous studies it was observed that the Rag family of small GTPases are
critical elements that link amino acid availability to mTORC1 [189, 272-274].
Rag GTPases are heterodimers, consisting of RagA or RagB with RagC or RagD,
which constitutively reside on lysosomal membranes. Stimulation by amino acids
is proposed to convert Rag GTPases to their active form, in which RagA or RagB
is loaded with GTP and RagC or RagD is loaded with GDP. Activated Rag
heterodimers interact with the Raptor subunit of mTORC1 and promote the
translocation of mTORC1 to lysosomes. This relocalization enables mTORC1 to
interact with the lysosome-localized Rheb (in its GTP-bound state), promoting
mTORC1 kinase activation [189, 274]. A lysosomal membrane-bound
heterotrimeric protein complex “Ragulator”, consisting of adaptor protein p14,
MAPK scaffold protein 1 (MP1) and p18, serves as the apparatus linking Rag
GTPases to the lysosomal surface [274]. This likely involves late endosomes as
well since blocking early to late endosomal trafficking prevented the interaction
between mTOR and Rheb in the latter compartment [276].
In our work we did not observe mTOR recruitment to endosomes. We
speculate that this might reflect a difference between the significant amino acid
deprivation in the studies of Sancak et al [274] compared to a relatively milder
level of intracellular amino acid deprivation in our studies. The localization of
endogenous Rheb was not demonstrated by Sancak et al. However, using rat liver
fractionation, we showed that EGF induced the translocation of endogenous Rheb,
140
but not mTOR, to late endosomes/lysosomes. Notably, preventing vacuolar
acidification with chloroquine significantly inhibited both this recruitment and
mTORC1 activation but did not affect mTOR levels in the endosome
preparations. Perhaps mTOR dissociates from endosomes/lysosomes only under
more extreme amino acid deprivation. Interestingly we observed an EGFdependent decrease in RagA/C coincident with the recruitment of Rheb to our
endosome preparations (Figure 3.13B). A decrease in lysosome-bound Rag,
consequent to mTORC1 activation, was previously observed by others [274]. We
suggest that this reflects the displacement of the Rag heterodimer consequent to
binding to and activation of mTOR by Rheb (Figure 4.1).
Our observations suggest that EGF induces an increase of lysosomal
proteolysis which releases essential amino acids from lysosomes thus sustaining
cytosolic amino acid levels and contributing to mTORC1 activation (Figure 4.1).
During preparation of this manuscript, Zoncu et al proposed that the vATPase
complex senses amino acids accumulating in the lysosomal lumen leading to
mTORC1 translocation and signaling [314]. This model, which places the active
vATPase downstream of amino acids, could explain some of our observations;
however other findings argue extra-lysosomal amino acids play an important role.
Thus we observed no or minimal effect of the vATPase inhibitor bafilomycin on
cycloheximide induced mTOR activation suggesting that this activation was
evoked by extra-lysosomal amino acids which accumulate in the presence of
cycloheximide. Furthermore the acidotropic agent chloroquine, which has no
inhibitory effect on vATPase activity [125], mimicked the effect on mTORC1 of
141
inhibiting vATPase by bafilomycin. This strongly suggests that vacuolar
acidification promoted by vATPase recruitment but not vATPase activity itself
facilitates mTORC1 activation by EGF.
In our study we found that Rheb not mTORC1 was recruited to late
endosomes and lysosomes by EGF. Because inhibiting acidification by
chloroquine blunted Rheb recruitment then it is possible that the recruitment of
Rheb is mediated by EGF-induced acidification in concert with Rheb activation
(i.e. conversion to RhebGTP) via TSC2 inhibition. Since EGF induced vacuolar
acidification would appear to assure intracellular amino acid sufficiency, and
growth factor-dependent mTORC1 activation is strongly dependent on the
presence of essential amino acids [257, 301], it is possible that the amino acid
availability is sensed by Rheb and promotes Rheb translocation to late
ENs/lysosomes to activate mTORC1 signaling. One should also consider the
possibility of cell/tissue specific mechanisms involved in mTORC1 activation as
exemplified perhaps by our studies in rat liver versus those in cultured HEK 293
cells [314].
Summary and Conclusion
In summary this work is the first to show that EGF induces the recruitment of
V1 subunits to the V0 domain to generate increased vATPase holoenzyme leading
to increased vacuolar acidification. We propose that this leads to increased
lysosomal proteolysis which assures an adequate supply of intracellular amino
acids which would otherwise be depleted by augmented protein synthesis under
the anabolic response to growth stimulation [315]. At the same time, the EGF142
induced vacuolar acidification is accompanied by Rheb recruitment. The
abrogation of vacuolar acidification (viz. by bafilomycin) resulted in a decline of
intracellular essential amino acid levels leading to an inhibition of EGF induced
mTORC1 and mitogenesis. Thus, in addition to activating TORC1 via both
increased Akt and Erk activation, EGF- induced vacuolar acidification may reflect
a mechanism for maintaining adequate intracellular amino acid levels and Rheb
targeting essential for the continuing activation of mTORC1 [270] (Figure 4.1).
143
Figure 4.1
Figure 4.1 Model of EGF induced mTORC1 activation.
EGF activation of mTORC1 involves the inhibition of TSC2 activity consequent
to Akt and Erk activation, leading to RhebGTP formation. In addition EGF induces
the rapid recruitment of V1 extrinsic to V0 intrinsic subunits to generate increased
vATPase holoenzyme in late endosomes/lysosomes (ENs/Lys). This leads to
increased vacuolar acidification and proteolysis which releases amino acids (AA)
from Lys to cytosol. Sustained levels of cytosolic essential amino acid enable
RhebGTP translocation to late ENs/Lys where it displaces Rag heterodimers (viz.
RagA/C) to associate with and activate mTORC1. Bars indicate inhibition and
arrows indicate activation. Broken lines indicate possible roles for intra-lysosomal
AA in mTORC1 activation and for Akt/Erk in vATPase holoenzyme
formation/activation.
144
Chapter 5
Contribution to Original Knowledge
145
In this thesis, I have presented the following original results:
 EGF promotes recruitment of V1 subunits of vATPase to late endosomeslysosomes and increases vacuolar acidification in primary rat hepatocytes.
 Inhibiting vATPase-mediated acidification ...

inhibits EGF-stimulated mitogenesis in primary rat hepatocytes

has no effects on EGF-induced Akt and Erk signaling, but inhibits
EGF-induced mTORC1 activation in primary rat hepatocytes,
HepG2 and FAO cells

inhibits PRAS40 phosphorylation in primary rat hepatocytes and
HepG2 cells

decreases the intracellular concentrations of essential amino acids
in EGF-treated primary rat hepatocytes
 Cycloheximide (a translation elongation inhibitor) blunts the decrease of
intracellular essential amino acid levels in EGF-treated hepatocytes
following vATPase inhibition, resulting in the complete reversal of
mTORC1 inhibition.
 MG132, a specific proteasome inhibitor, mimics the effect of vATPase
inhibition on mTORC1 activation.
 mTORC1 is highly concentrated in rat liver endosomes and lysosomes.
 EGF induces the recruitment of Rheb but not mTOR to rat liver
endosomes/lysosomes.
 In vivo administration of chloroquine inhibits EGF-induced recruitment of
Rheb and mTORC1 activation.
146
Chapter 6
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147
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