Signal transduction 1

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Signal transduction
Major definitions
 Signal transduction is a basic

Paracrine signaling occurs between local cells where the
signals elicit quick responses and last only a short amount of
time due to the degradation of the paracrine ligands.

Endocrine signaling occurs between distant cells and is
mediated by hormones released from specific endocrine
cells that travel to target cells, producing a slower, longlasting response.
process in molecular cell
biology involving the
conversion of a signal from
outside the cell to a
functional change within the
cell.
 ECB consider 4 general
pathways of signal
transduction: endocrine,
paracrine, synaptic and
contact dependent
 More generally, signaling can
be either via diffused
molecules or due to surfacesurface interactions
Intracellular signaling
Intracellular signaling
molecules can relay, amplify,
integrate and distribue the
incoming signal.
Participants of the intrecellular
signaling cascades:
 extracellular signals (soluble
ligands, other cell surface
receptors)
 Receptors
 Adaptor proteins
 Second messengers
(intracellular soluble
ligands)
 Effector proteins
Key proteins that control intracellular
signaling act as molecular switches
Alberts et al. ECB
Alberts et al. ECB
Kinases. Kinome
Major definitions
 Kinases are enzymes that catalyze the transfer of the γ-phosphate





of ATP (GTP) on a corresponding substrate
Particuarly, protein kinases catalyze the transfer on protein alcohol
(on Ser and Ther) and/or phenolic (on Tyr) groups
Kinases are classified by sequence comparison of their catalytic
domains, and in second turn by sequence similarity and domain
structure outside of the catalytic domains and biological functions
Human kinome is a set of all human kinases
It includes about 500 protein kinase(518 according to Manning et
al., 2002) and a lot of kinases specific to non-protenaceus
substrates
Most of kinase families are conserved in eukaryotes and
particularly in multicellular eukaryotes (144 of 189 for PK)
Classification
Group Family
Examples
AGC
17
70
AGCI. Cyclic-nucleotideregulated PK
PKA (cAMP-dependent PK)
PKG (Cyclic GMP-dependent PK)
AGCII. Diacylglycerolactivated/phsospholipiddependent PK
PKC (protein kinase C) including
Ca2+-dependent (cPKC) ,Ca2+indepdendent (nPKC), atypical
(aPKC)
AGCIII. Related to PKA and PKC
RAC
AGCIV. G-protein-coupledreceptor phosphorylating PK
βARK1 and βARK1 (βadrenergic receptor kinases) ,
GRK5 and GRK6
Classification
Group
Family
Examples
CAMK
33
84
CAMKI. Ca2+/calmoduline regulated PK
CAMKs
EG2K (Elongation factor 2 kinase)
Phosphorylase kinases (PhK-γM)
Myosin light chain kinases (MLCK)
MAP kinase-activated PK 2(MAPKAP2)
CAMKII. AMPK family
AMPK AMP-activated PK
CMGCI. Cyclin-dependent kinases (CDKs)
Cdc2, Cdk2 etc
CMGCII. MAPK (or Erk) family
Erk1, Erk2(extracellular signal regulated
kinases, known as p44 MAPK and p42 MAPK)
P38 (MPK2, HOG1-related protein)
Jnk1, Jnk2 (SAPKs, stress-activated protein
kinases)
CMGC
Classification
Group
Family
Examples
STE
MAPK cascade families, 3
51
Ste7/MAP2K/MEK
MEK1, MEK2 (MAP ERK
Kinases)
Ste11/MAP3K
MEKK
Ste20/MAP4K/Pak
p21(CDC42/Rac) activated
kinase
Group
Classification
Tyrosine kinase group
Family
Examples
30
95
PTK1. Src
Src (cellular homolog of Rous sarcoma virus
oncoprotein)
Yes ,Yrk, Fgr, Fyn, Lyn, Lck (Lymphoid T-cell
protein-tyrosine kinase)Blk
PTKII. Brk family
Breast expressed PK
PTKIII.Tec
Tec (Tyrosine kinase expressed in hepatocellular
carcinoma)
Emt (Itk, Tsk, expressed in mainly in T-cells)
PTKIV. Csk
C terminal Src kinase; negative regulator of Src
PTKVIII. Jak family
Janus kinases (Jaks), Tyk2 )Transducer of interferon
signals)
PTKIX. Ack
Ack CDC42Hs-associated kinase
PTKX. Fak
Fak Focal adhesion kinase
Group
Classification
Family
Tyrosine kinase 30
group
Examples
95
PTKXI. EGFR family
EGFR, HER2/c-neu (ErbB-2), Her3 (ErbB-3), Her4
(ErbB-4).
PTKXII. Eph/Elk/Eck receptor
family
Eph (erythropoeitin-producing hepatoma PK)
Eck (epithelial cell linase)
Elk (Epl like kinase detected in brain)
PTK. Tie
Tie
PTKXVI. Fibroblast growth factor
receptor family
FGFR1 (Flg, Cek1), FGFR2 (Bek, K-Sam)
PTKXVII. Insulin receptor family
InsR, IGF1R (insulin like growth factor receptor)
PTKXXII. Hepatocyte growth
factor receptor
HGFR (Met), Sea, Ron, Stk (stem cell derived
tyrosine kinase)
Classification
Group
Family
Examples
TKL
tyrosine kinase–like , 7
48
MLK
mixed-lineage kinase
LISK (LIMK/TESK)
RGC
IRAK
interleukin-1 (IL-1) receptor–
associated kinase],
Raf
Raf (cellular homolog of
retroviral oncogene product)
RIPK
receptor-interacting protein
kinase (RIP)],
STRK
activin and TGF-β receptors
Receptor guanylate cyclase, 1
5
Platelet-derived growth factor receptor
PDGFR, CSF1R (Colony
stimulating factor 1 receptor)
Classification
Group
Family
Examples
Others
37
106
Activin/TGFβ receptor family
ActR
13
40
Atypical PKs
Kinase domain structure
Hanks and Hunter, 1995
Functional activities of the kinase
 Binding and orientation of ATP
(GTP) as a complex with divalent
cation
 Binding of a target protein
 Enzymatic transfer of the γphosphate group to the acceptor
hydroxyl residue
Catalytic kinase domain consists of
300-350 aa and is organized in 12
dubdomain
The catalytic activity depends on
cation (Mg2+ or Mn2+ ) binding,
often on phosphorylaton within
the VIII subdomain and sometimes
on binding of a ligand or a
catalytic subunit
Figure 1. Two Views of the Structure of PKA [70]
Scheeff ED, Bourne PE (2005) Structural Evolution of the Protein Kinase–Like Superfamily. PLoS Comput Biol 1(5): e49. doi:10.1371/journal.pcbi.0010049
Figure 1 Dendrogram of 491 ePK domains from 478 genes.
G. Manning et al. Science 2002;298:1912-1934
Other kinases
Enzyme
Target
Aminoglycoside
3′ and/or 5′′ hydroxyl of
phosphotransferase APH(3′)- aminoglycoside antibiotics
IIIa
Functional role
Antibiotic inactivation
Choline kinase (CK)
Phosphatidylcholine
precursor
Phosphatidylcholine
synthesis
Phosphoinositide 3 kinases
(PI3Ks)
Phosphatidylinositol (PI) or
its forms
PtdIns(3)P, PI(3,4,5)P3
Type IIβ phosphatidylinositol phosphatidylinositol 5phosphate kinase (PIPKIIβ) phosphate (PI5P)
PI(4,5)P2
Figure 4. Proposed Phylogeny for the Kinase-Like Superfamily, Based on a Unified Bayesian Analysis
Both the Sequence Alignment in Figure 3 and the Structural Character Matrix in Table 2
Scheeff ED, Bourne PE (2005) Structural Evolution of the Protein Kinase–Like Superfamily. PLoS Comput Biol 1(5): e49. doi:10.1371/journal.pcbi.001004
Regulatory GTP-binding proteins
=regulatory GTPases=G-proteins
Major classes of regulatory GTPbinding proteins
 Trimeric G- proteins associated
with GTP—binding protein
coupled receptors
 Small (monomeric) G proteins
inactive
active
Heterotrimeric guanine-nucleotide
binding proteins (G-proteins)
 G-proteins are peripheraly proteins of the




 alpha- subunit is myristoylated and

can be palmytolated
 Gamma-subunitc is prenylated


plasma membrane
G-proteins provide signal coupling to seventransmembrane-surface-receptors known as
G-protein couplde receptors (GPCR).
G proteins are composed of monomers of
alpha, beta, and gamma subunits.
The alpha-subunit is a GTPase
The beta- and gamma-subunits are tightly
associated.
Receptor phosphorylation upon signal binding
mediate GDP/GTP exchange .
The GTP bound alpha-subunit dissociate from
beta- and gamma-subunits
Dissociated subunits initiate cellular response
by altering the activity of effectors
G-protein coupled receptors (GPCRs)
 GPCRs IS the largest family of integral




membrane proteins
About 800 GPCR genes are identified in the
human genome
GPCRs are comprised of seven transmembrane
a-spirals (TM), an extracellular N-terminus and
an intracellular C-terminal domain.
GPCR are activated by ligand binding that causes
conformation changes
Ligand-bound GPCR affects the G-protein
alpha-subunit decreasing its affinity to GDP
 GDP/GTP exchange take place due to a
Alberts et al., ESB
decreased affinity to GDP and higher intracellular
concentrations of GTP
 Approximately 30% of all current therapeutic
agents acting directly on GPCRs
GPCR ligands and effectors
Ligands
Effectors
Hormones:
epinephrine,
acetylcholine,
noradrenaline,
dopamine, histamine
Lipids:
prostaglandins,
leukotriens,
Lysophosphatidic
acid (LPA)
Chemokines
Regulatory peptides:
thrombin, bombesin,
bradykinin
Nucleotides
Adenylyl
cyclase, PKA,
PKC,
Phospholiases
(PLC), Rho
GTPase, PI3K,
ion channels
Biological functions
Cell proliferaion, differentiation,
migration, angiogenesis, cancer
G-protein functions
 The β-adrenergic receptor is




the GPCR for the hormone
epinephrine.
epinephrine and glucagon
binding activates β-adrenergic
receptor
β-adrenergic receptor
stimulate GTP/GDP transition
in a stimulatory G-protein
(Gs with alpha subunit Gsa ).
Gs activates adenylyl cyclases
to switch on cyclic-AMP
formation that results in PKA
activation etc.
GTP/GDP degradation stop the
cascade
GPCR regulatory proteins
 GPCR kinases (GRKs) and
British Journal of Pharmacology
Volume 165, Issue 6, pages 1717-1736, 22 FEB 2012 DOI:
10.1111/j.1476-5381.2011.01552.x
http://onlinelibrary.wiley.com/doi/10.1111/j.14765381.2011.01552.x/full#f1
arrestins causes receptor
desensitization (uncoupling)
from hetereotrimeric Gproteins (fast recycling) or
CME (slow recycling)
 Receptor activity-modifying
proteins (RAMPS) modify the
expression, and pharmacology
of calcitonin receptor and
calcitonin-like receptor
(CRLR)
 Regulators of G-protein
signalling (RGS) act as GTPase
activating proteins
Ion channels as integrators of G-protein mediated
signaling: Sympathetic stimulation in the heart
 Noradrenalin
intecrats with
βAR, activates
Gs
Atsushi Inanobe , Yoshihisa Kurachi
Biochimica et Biophysica Acta (BBA) - Biomembranes, Volume 1838, Issue 2, 2014, 521 - 531
increased heartbeat
Hoe to stop and what happens if it isn’t
gonna stop ?
Figure 2 Cholera pathogenesis and cholera toxin action
 A, B (cholera toxin subunits);
GM1 (GM1 ganglioside
receptor); Gsa (G protein alfa
subunit); AC (adenylate
cyclase); Gi (G protein);
cAMP (cyclic AMP); CFTR
(cystic fibrosis
transmembrane conductance
regulator).
 Cholera toxin blocks Gsa in
the GTP-bound state via a
reaction of ADP-ribosylation
Clemens, J. et al. (2011) New-generation vaccines against cholera
Nat. Rev. Gastroenterol. Hepatol. doi:10.1038/nrgastro.2011.174
Small regulatory GTPases = small G proteins = small GTP
binding proteins =Ras superfamily
 Small GTPAses are monomeric proteins
GDI
 GTP-bound form is active, GDP-bound




form is inactive
Three types of regulatory proteins control
small G-protein activity
GAP- GTPase activating proteins increase
its low intrinsic hydrolase activity to
trasfser G-protein into an INACTIVE
form
GDI- GTPase dissociation inhibitors
stabilize the GDP-bound, inactive state of
G proteins
GEF- guanine nucleotide exchange factors
accelerate nucleotide exchange in
response to cellular signals to transfer Gprotein into an ACTIVE form
Small G-protein families
Family
Control of
Ras
Gene expression
Rho
Rab
Gene expression; Cytosceleton
rearrangements
Vesicular transport
Sar1/Arf
Vesicular transport
Ran
Nuclear transport
Cell cycle
Second messengers
Second messengers
 Small intracellular molecules that amplify incoming signal
 cAMP
 Products of PtdIns2P degradation: Ins3P and DAG
 cGMP
Phospholipid signaling
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