Characterization of PKCe functional
sub-proteome in the normal and protected
myocardium: strategies for mapping a functional
sub-proteome.
Peipei Ping
Departments of Physiology and Biophysics
Medicine, Division of Cardiology
University of California at Los Angeles
David Geffen School of Medicine at UCLA
Acknowledgments
Dr. Rick Edmondson
Dr. William Pierce
Dr. Joseph Loo
Dr. Julian Whitelegge
Dr. Sam Hanash
NHLBI
American Heart Association
Human Proteome Organization
Laubisch Foundation
Embracing The Era of Proteomics
•
Functional Proteomic Analysis of the PKCe
Signaling System;
•
Strategies for Mapping A Functional Subproteome.
Embracing The Era of Proteomics
Proteomics
Expression Proteomics
Functional Proteomics
…
Functional Proteomics
A functional sub-proteome is a biological entity
Investigation of protein function within a sub-proteome
Investigations of Cardioprotective Signaling
A PKC centric view
Understanding cellular mechanisms
targets
PKC
Phenotype
Searching for therapeutic targets
Investigations of Cardioprotective Signaling
A PKC centric view
Understanding cellular mechanisms
The PKCe
targets
Subproteome
Phenotype
Searching for therapeutic targets
Functional Proteomic Approach:
Linking Cellular Mechanisms to Phenotypes
One Cell Type:
Cardiac Cells
A sub-proteome:
The PKCe Signaling System
One Phenotype:
Protection Against Ischemic Injury
(Cardioprotection)
Background:
The PKC Hypothesis in Preconditioning
Preconditioning protects ischemic rabbit heart by
protein kinase C activation.
Ytrehus et al, Am J Physiol 1994
– Activation of PKC by PMA reduced myocardial infarct size, similar
to ischemic preconditioning.
– Inhibition of PKC by staurosporine or polymyxin B blocked ischemic
preconditioning-induced infarct-sparing effect.
Evidence Supporting an Essential Role of PKCe
in Cardiac Protection Against Ischemic Injury
• Ischemic preconditioning induces isoform-selective translocation and
activation of PKCe. Inhibition of PKCe abolishes protection against
myocardial infarction and stunning (Ping et al. 1997 Circ Res; Qiu
and Ping et al. 1998 JCI).
• Inhibition of PKCe translocation abrogates protection (Gray et al.
1997 JBC; Liu et al. 1999 JMCC).
• Translocation of PKCe facilitates sustained in vivo cardioprotection
(Dorn II et al. 1999 PNAS).
Transgenic Activation of PKCe Reduces
Myocardial Infarct Size in Mice
PKCe TG (low levels)
Wild Type
Ping et al. J Clin Invest 2002
Evidence Supporting The Existence Of
A Cardioprotective PKCe Signaling System
Candidate Molecules Proposed:
Receptors (ADO, AR, OP, B)
Channels (e.g., KATP, L-type calcium)
ROS
Lipo-oxygenase
PI3 Kinase
RACKs
PKB/Akt
PTKs
MAPKs
HSPs
Bcl2
NOS
COX-2
Transcriptional factors (AP-1, NF-kB)
Rather than examining a single molecule in
isolation, functional proteomic strategies enable
an unbiased investigation of multiple signaling
molecules and their protein-protein interactions
in parallel, and thereby, provide a holistic
portrait of the entire signaling system.
Investigations of Cardioprotective Signaling
A PKC centric view
Understanding cellular mechanisms
The PKCe
targets
Subproteome
Phenotype
Searching for therapeutic targets
Hypothesis
The PKCe signaling system is composed of
signaling complexes.
These complexes serve to bring molecules
into close vicinity and to facilitate signal
transduction during the genesis of a
cardioprotective phenotype.
Functional Proteomic Analysis of Signaling Systems:
Strategies and Approaches
1. Purification and isolation of a signaling system
(the sub-proteome)
2. Protein separation and identification
3. Confirmation of functional roles for the identified
proteins in the genesis of a phenotype
Characterization of Multi-protein Complexes
1
Subcellular Fractionation
2
GST-PKCe pull down
Liquid Chromatography
Sucrose Gradient
Native Gel
Multiprotein Complexes
EM Analysis
SDS PAGE
LC/MS/MS
Protein Array
Immunoblotting
Functional Proteomic Analysis of
The PKCe Signaling System: Technology Platform
1. Isolation of signaling complexes
•
•
•
Chromatography analysis
Co-immunoprecipitation assays
Affinity pull-down assays
2. Protein separation and identification
•
•
2DE or 1DE coupled with MALDI Mass
2DE or 1DE coupled with LC Mass-Mass
3. Confirmation of functional roles in phenotypes
•
•
WB, kinase activity, and protein interaction assays
ELISA-based protein arrays
•
•
Cell culture models
Transgenic mouse models
Protein Profiles for PKCe Signaling Complexes:
Gel-Filtration Chromatography
Absorbance @ 280 nm (mAU)
1.3x103 kDa
Control Hearts
800
600
95kDa
337kDa
400
200
0
Absorbance @ 280 nm (mAU)
PKCe WB
Cardioprotected
Hearts
800
1.3x103 kDa
551kDa
193kDa
600
400
200
0
0
50
PKCe WB
100
150
200
250
Elution Volume (ml)
Immunoprecipitation Protocol
IgG
Anti-PKCe
YYYYYY
Y Y
Y
Y YY
Pre-Clear
OR
PKCe
Members
of PKCe
Complexes
Y
Y
Tissue Lysate
Y
Y
Protein-G
Beads
+
+
Non-Specific
Binding
GST-Based Affinity Pull-Down Protocol
GST
Beads
GST-PKCe
GST
+
+
OR
Tissue Lysate
GST-PKCe
Members
of PKCe
Complexes
Non-Specific
Binding
Functional Proteomics:
2D Electrophoresis
The sub-proteome of the PKCe signaling system in the myocardium,
isolated via PKCe immunoprecipitation (IP)
Anti-PKCe Mouse IgG
Mouse IgG
IEF
IEF
M
W
M
W
pI3
pI10
pI3
pI10
Functional Proteomics:
1D Coupled with Mass Spectrometric Analysis
Sypro Ruby-Stained Large Format SDS-PAGE Gel (10 % Duracryl)
Low pH Elution
Spot 85
2,4-dienoyl-CoA reductase
(NADPH) precursor
Urea / Thiourea Elution
Functional Proteomics:
Mass Spectrometric Analysis
Spot 85: 2,4-dienoyl-CoA reductase (NADPH) mitochondrial precursor
(gi|13385680) pI: 9.10; MW: 36 kDa
y7
y5
110
b2 b3 b4 b5
Intensity × 104
100
90
F N I
80
70
60
b2
y6
b5
P I K
y8
b4
30
20
10
0
Q P G
y9 y8 y7 y6 y5
b3
50
40
I
y9
200
1
51
101
151
201
251
301
300
400
MALLGRAFFA
MLPPDAFQGK
EEISSKTGNK
SPSERLTPNG
ESGSGFVMPS
RLDPTGRFEK
GGEEVFLSGE
500
600
GVSRLPCDPG
VAFITGGGTG
VHAIRCDVRD
WKTITDIVLN
SSAKSGVEAM
EMIDRIPCGR
FNSLKKVTKE
m/z
700
PQRFFSFGTK
LGKAMTTFLS
PDMVHNTVLE
GTAYVTLEIG
NKSLAAEWGR
LGTMEELANL
EWDIIEGLIR
800
900
TLYQSKDAPQ
TLGAQCVIAS
LIKVAGHPDV
KQLIKAQKGA
YGMRFNIIQP
ATFLCSDYAS
KTKGS
1000
1100
SKFFQPVLKP
RNIDVLKATA
VINNAAGNFI
AFLAITTIYA
GPIKTKGAFS
WINGAVIRFD
Criteria For A Positive Identification:
Members of Signaling Complexes.
1. Identification is made in complexes purified via at least
two independent methods.
2. Mass spectrometry results are verified by either coimmunoprecipitation or protein arrays.
3. Functional assays ascertain the participation of the
molecules (Complex-bound protein exhibits biological functional
activity; or the association of a member with the complex modifies
its functional activity; altered activity, expression, or PTM of a
protein modulates the assembly of the complex).
RESULTS:
The Sub-Proteome of the PKCe Signaling System
in the Murine Myocardium
1. 93 total proteins identified
2. 88 proteins of known function identified
3. 5 unknown proteins identified
Edmondson et al. Mol Cell Proteomics 2002;
Ping et al. Circ Res 2001;
Vondriska & Zhang et al. Circ Res 2001;
Baines et al. Circ Res 2002;
Ping et al. J Clin Invest 2002.
Log 250k
pI 3
pI 12
PKCe
Log 5k
Cardiac PKCe Signaling Subproteome
RESULT ONE:
PKCe forms signaling complexes of various sizes,
these complexes contain an array of proteins that
are classified into six functionally distinct groups
1. Structural and cytoskeletal proteins
2. Stress-activated proteins
3. Signaling elements
4. Transcriptional/ translational factors
5. Metabolism-related proteins
6. PKC-interaction domain containing proteins
(e.g., PDZ)
(Ping et al. Circ Res 2001; Vondriska & Zhang et al.
Circ Res 2001; Baines et al. Circ Res 2002)
Log 250k
pI 3
pI 12
PKCe
Log 5k
Cardiac PKCe Signaling Subproteome
RESULT TWO:
Regulation of PKCe complex assembly
•
The assembly of PKCe complexes is dictated by the
molecular conformation of PKCe
(Song & Vondriska et al, Am J Physiol, 2002)
RESULT THREE:
Subcellular location dictates PKCe complex assembly
•
The composition of PKCe complexes is governed by
the subcellular location in which the complex resides
(Zhang et al and Baines et al, Circulation 2001; Baines et al, Circ Res
2002)
RESULT FOUR:
Cardioprotection is associated with dynamic
regulation of PKCe complexes
•
Multiple proteins were recruited to the PKCe
complexes, whereas others were discharged (Ping et al.
Circ Res 2001)
•
Multiple
proteins
underwent
modifications (Ping et al. Circ Res 2001)
•
Multiple
signaling
kinases
exhibited
phosphorylation activities (Vondriska & Zhang et
post-translational
altered
al. Circ Res
2001; Song & Vondriska et al. and Baines et al. Circulation 2001; Ping
et al. JCI 2002)
Strategies For Mapping The Cardiac Proteome:
Characterization of multiple functional subproteomes
I.
Function
II.
Protein Profiling
III. Spatial Profiling
IV. Temporal Profiling