Characterization%20of%20PKCe%20functional

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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
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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

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Embracing The Era of Proteomics

• Functional Proteomic Analysis of the PKCe
  Signaling System
• Strategies for Mapping A Functional Sub-proteome.

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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
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Investigations of Cardioprotective Signaling A
PKC centric view
Understanding cellular mechanisms
PKC
Searching for therapeutic targets
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Investigations of Cardioprotective Signaling A
PKC centric view
Understanding cellular mechanisms
The PKCe Subproteome
Searching for therapeutic targets
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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)
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BackgroundThe 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.

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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).

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Transgenic Activation of PKCe Reduces Myocardial
Infarct Size in Mice
Wild Type
PKCe TG (low levels)
Ping et al. J Clin Invest 2002
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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)
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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.
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Investigations of Cardioprotective Signaling A
PKC centric view
Understanding cellular mechanisms
The PKCe Subproteome
Searching for therapeutic targets
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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.
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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
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Characterization of Multi-protein Complexes
Subcellular Fractionation
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Liquid Chromatography
GST-PKCe pull down
Native Gel
Sucrose Gradient
Multiprotein Complexes
LC/MS/MS Protein Array Immunoblotting
SDS PAGE
EM Analysis
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Functional Proteomic Analysis of The PKCe
Signaling System Technology Platform

• Isolation of signaling complexes
• Chromatography analysis
• Co-immunoprecipitation assays
• Affinity pull-down assays
• Protein separation and identification
• 2DE or 1DE coupled with MALDI Mass
• 2DE or 1DE coupled with LC Mass-Mass
• Confirmation of functional roles in phenotypes
• WB, kinase activity, and protein interaction
  assays
• ELISA-based protein arrays
• Cell culture models
• Transgenic mouse models

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Protein Profiles for PKCe Signaling
Complexes Gel-Filtration Chromatography
1.3x103 kDa
Control Hearts
800
600
400
Absorbance _at_ 280 nm (mAU)
200
0
PKCe WB
Cardioprotected Hearts
1.3x103 kDa
551kDa
193kDa
Absorbance _at_ 280 nm (mAU)
400
0
50
100
150
200
250
Elution Volume (ml)
PKCe WB
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Immunoprecipitation Protocol
Anti-PKCe
IgG


Pre-Clear
Protein-G
OR
Beads
Tissue Lysate
Non-Specific Binding
PKCe
Members of PKCe Complexes
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GST-Based Affinity Pull-Down Protocol
GST
GST-PKCe


OR
Tissue Lysate
GST-PKCe
Non-Specific Binding
Members of PKCe Complexes
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Functional Proteomics 2D Electrophoresis
The sub-proteome of the PKCe signaling system in
the myocardium, isolated via PKCe
immunoprecipitation (IP)
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Functional Proteomics 1D Coupled with Mass
Spectrometric Analysis
Sypro Ruby-Stained Large Format SDS-PAGE Gel (10
Duracryl)
Urea / Thiourea Elution
Low pH Elution
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Functional Proteomics Mass Spectrometric
Analysis
Spot 85 2,4-dienoyl-CoA reductase (NADPH)
mitochondrial precursor (gi13385680) pI 9.10
MW 36 kDa
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
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Criteria For A Positive IdentificationMembers
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).

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RESULTSThe Sub-Proteome of the PKCe Signaling
Systemin the Murine Myocardium

• 93 total proteins identified
• 88 proteins of known function identified
• 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.
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Log 250k
pI 3
pI 12
PKC?
Cardiac PKC? Signaling Subproteome
Log 5k
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RESULT ONEPKCe forms signaling complexes of
various sizes, these complexes contain an array
of proteins that are classified into six
functionally distinct groups

• Structural and cytoskeletal proteins
• Stress-activated proteins
• Signaling elements
• Transcriptional/ translational factors
• Metabolism-related proteins
• 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)
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Log 250k
pI 3
pI 12
PKC?
Log 5k
Cardiac PKC? Signaling Subproteome
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RESULT TWORegulation 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)

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RESULT THREESubcellular 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)

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RESULT FOURCardioprotection 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 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)

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Strategies For Mapping The Cardiac
Proteome Characterization of multiple
functional subproteomes

1. Function
2. Protein Profiling
3. Spatial Profiling
4. Temporal Profiling