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