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Characterization of PKC functional
sub-proteome in the normal and protected
myocardium: strategies for mapping a functional
sub-proteome.
Peipei Ping
Departments of Physiology and BiophysicsMedicine, Division of Cardiology
University of California at Los AngelesDavid Geffen School of Medicine at UCLA
Dr. Rick EdmondsonDr. William Pierce
Dr. Joseph LooDr. Julian Whitelegge
Dr. Sam Hanash
NHLBIAmerican Heart Association
Human Proteome OrganizationLaubisch Foundation
Acknowledgments
Embracing The Era of Proteomics
• Functional Proteomic Analysis of the PKC Signaling System;
• Strategies for Mapping A Functional Sub-proteome.
Embracing The Era of Proteomics
Proteomics Expression ProteomicsFunctional Proteomics
…
Functional ProteomicsA functional sub-proteome is a biological entity
Investigation of protein function within a sub-proteome
Searching for therapeutic targets
Understanding cellular mechanisms
Investigations of Cardioprotective Signaling
A PKC centric view
targets PhenotypePKC
Searching for therapeutic targets
Understanding cellular mechanisms
Investigations of Cardioprotective Signaling
A PKC centric view
targets PhenotypeThe PKC
Subproteome
Functional Proteomic Approach:Linking Cellular Mechanisms to Phenotypes
One Cell Type: Cardiac Cells
A sub-proteome: The PKC 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 PKC in Cardiac Protection Against Ischemic Injury
• Ischemic preconditioning induces isoform-selective translocation and activation of PKC Inhibition of PKC abolishes protection against myocardial infarction and stunning (Ping et al. 1997 Circ Res; Qiu and Ping et al. 1998 JCI).
• Inhibition of PKC translocation abrogates protection (Gray et al. 1997 JBC; Liu et al. 1999 JMCC).
• Translocation of PKC facilitates sustained in vivo cardioprotection (Dorn II et al. 1999 PNAS).
Transgenic Activation of PKC Reduces Myocardial Infarct Size in Mice
Wild TypePKC TG (low levels)
Ping et al. J Clin Invest 2002
Evidence Supporting The Existence Of A Cardioprotective PKC Signaling System
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-B)
Candidate Molecules Proposed:
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.
Searching for therapeutic targets
Understanding cellular mechanisms
Investigations of Cardioprotective Signaling
A PKC centric view
targets PhenotypeThe PKC
Subproteome
The PKC 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.
Hypothesis
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
Subcellular Fractionation
Liquid ChromatographyGST-PKC pull down
SDS PAGE
Native Gel
EM Analysis
Sucrose Gradient
Multiprotein Complexes
LC/MS/MSProtein Array
Immunoblotting
1 2
Functional Proteomic Analysis of The PKC 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
Abs
orba
nce
@ 2
80 n
m (
mA
U)
Elution Volume (ml)
Protein Profiles for PKC Signaling Complexes:Gel-Filtration Chromatography
Cardioprotected Hearts
800
600
400
200
0
Control Hearts
Abs
orba
nce
@ 2
80 n
m (
mA
U)
PKC WB
PKC WB
400
200
800
600
0
0 50 100 150 200 250
1.3x103 kDa
337kDa95kDa
1.3x103 kDa551kDa
193kDa
Pre-Clear
YYYYYY+YY YYY Y +
IgGAnti-PKC
PKC
YY
Y
Y
Y
Y
YY
Y
Y
Y
Y
Non-SpecificBinding
ORProtein-G
Beads
Tissue Lysate
Membersof PKC
Complexes
Immunoprecipitation Protocol
++GSTGST-PKC
GST-PKC
Membersof PKC
Complexes
Non-SpecificBinding
OR
GSTBeads
Tissue Lysate
GST-Based Affinity Pull-Down Protocol
Functional Proteomics:
2D Electrophoresis
The sub-proteome of the PKC signaling system in the myocardium,isolated via PKC immunoprecipitation (IP)
Anti-PKC Mouse IgGMouse IgG
pI3 pI10
IEF
MW
IEF
MW
pI3 pI10
Functional Proteomics:
1D Coupled with Mass Spectrometric Analysis
Low pH Elution Urea / Thiourea Elution
Sypro Ruby-Stained Large Format SDS-PAGE Gel (10 % Duracryl)
Spot 852,4-dienoyl-CoA reductase (NADPH) precursor
1 MALLGRAFFA GVSRLPCDPG PQRFFSFGTK TLYQSKDAPQ SKFFQPVLKP 51 MLPPDAFQGK VAFITGGGTG LGKAMTTFLS TLGAQCVIAS RNIDVLKATA 101 EEISSKTGNK VHAIRCDVRD PDMVHNTVLE LIKVAGHPDV VINNAAGNFI 151 SPSERLTPNG WKTITDIVLN GTAYVTLEIG KQLIKAQKGA AFLAITTIYA 201 ESGSGFVMPS SSAKSGVEAM NKSLAAEWGR YGMRFNIIQP GPIKTKGAFS 251 RLDPTGRFEK EMIDRIPCGR LGTMEELANL ATFLCSDYAS WINGAVIRFD 301 GGEEVFLSGE FNSLKKVTKE EWDIIEGLIR KTKGS
200 300 400 500 600 700 800 900 1000 1100m/z0
10
2030
4050
607080
90100
110
Inte
nsi
ty ×
104
b2 b3 b4 b5 F N I I Q P G P I K
y9 y8 y7 y6 y5 b2
b3
y5
b5
y7
y8
y9
b4
y6
Spot 85: 2,4-dienoyl-CoA reductase (NADPH) mitochondrial precursor (gi|13385680) pI: 9.10; MW: 36 kDa
Functional Proteomics:
Mass Spectrometric Analysis
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 co-
immunoprecipitation 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 PKC Signaling Systemin 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 5k
Log 250k
Cardiac PKCCardiac PKC Signaling Subproteome Signaling Subproteome
PKCPKC
pI 3 pI 12
RESULT ONE:
PKC 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)
PKCPKC
Log 5k
pI 3 pI 12
Cardiac PKCCardiac PKC Signaling Subproteome Signaling Subproteome
Log 250k
RESULT TWO:
Regulation of PKC complex assembly
• The assembly of PKC complexes is dictated by the
molecular conformation of PKC (Song & Vondriska et al, Am J Physiol, 2002)
RESULT THREE:
Subcellular location dictates PKC complex assembly
• The composition of PKC 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 PKC complexes
• Multiple proteins were recruited to the PKC complexes, whereas others were discharged (Ping et al.
Circ Res 2001)
• Multiple proteins underwent post-translational
modifications (Ping et al. Circ Res 2001)
• Multiple signaling kinases exhibited altered
phosphorylation activities (Vondriska & Zhang et 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