Characterization of PKC e functional sub-proteome in the normal and protected myocardium: strategies for mapping a f

1. 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

2. 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

3. Embracing The Era of Proteomics • Functional Proteomic Analysis of the PKCe Signaling System; • Strategies for Mapping A Functional Sub-proteome.

4. 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

5. targets Phenotype Investigations of Cardioprotective Signaling A PKC centric view Understanding cellular mechanisms PKC Searching for therapeutic targets

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

7. 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)

8. 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.

9. Evidence Supporting an Essential Role of PKCein 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).

10. Transgenic Activation of PKCe Reduces Myocardial Infarct Size in Mice PKCe TG (low levels) Wild Type Ping et al. J Clin Invest 2002

11. 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)

12. 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.

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

14. 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.

15. 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

16. 1 Characterization of Multi-protein Complexes Subcellular Fractionation 2 GST-PKCe pull down Liquid Chromatography Native Gel Sucrose Gradient Multiprotein Complexes LC/MS/MS Protein Array Immunoblotting EM Analysis SDS PAGE

17. 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

18. 95kDa 337kDa 800 600 200 0 Protein Profiles for PKCe Signaling Complexes: Gel-Filtration Chromatography 1.3x103 kDa Control Hearts 800 600 400 Absorbance @ 280 nm (mAU) 200 0 PKCe WB Cardioprotected Hearts 1.3x103 kDa 551kDa 193kDa Absorbance @ 280 nm (mAU) 400 0 50 100 150 200 250 ElutionVolume (ml) PKCe WB

19. Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Immunoprecipitation Protocol Anti-PKCe IgG + + Pre-Clear Protein-G OR Beads Tissue Lysate Non-Specific Binding PKCe Members of PKCe Complexes

20. GST Beads GST-Based Affinity Pull-Down Protocol GST GST-PKCe + + OR Tissue Lysate GST-PKCe Non-Specific Binding Members of PKCe Complexes

21. Anti-PKCe Mouse IgG Mouse IgG IEF IEF MW MW pI3 pI3 pI10 pI10 Functional Proteomics: 2D Electrophoresis The sub-proteome of the PKCe signaling system in the myocardium, isolated via PKCe immunoprecipitation (IP)

22. Spot 85 2,4-dienoyl-CoA reductase (NADPH) precursor Functional Proteomics: 1D Coupled with Mass Spectrometric Analysis Sypro Ruby-Stained Large Format SDS-PAGE Gel (10 % Duracryl) Urea / Thiourea Elution Low pH Elution

23. y7 b2 b3 b4 b5 F N I I Q P G P I K y9 y8 y7 y6 y5 y5 110 100 90 b2 80 70 b3 60 Intensity × 104 y6 50 b5 y8 40 b4 30 20 y9 10 0 200 300 400 500 600 700 800 900 1000 1100 m/z Functional Proteomics: Mass Spectrometric Analysis Spot 85: 2,4-dienoyl-CoA reductase (NADPH) mitochondrial precursor (gi|13385680) pI:9.10; MW: 36 kDa 1 MALLGRAFFA GVSRLPCDPG PQRFFSFGTK TLYQSKDAPQ SKFFQPVLKP 51 MLPPDAFQGK VAFITGGGTGLGKAMTTFLS TLGAQCVIAS RNIDVLKATA 101 EEISSKTGNK VHAIRCDVRD PDMVHNTVLE LIKVAGHPDV VINNAAGNFI 151 SPSERLTPNG WKTITDIVLN GTAYVTLEIG KQLIKAQKGA AFLAITTIYA 201 ESGSGFVMPS SSAKSGVEAM NKSLAAEWGR YGMRFNIIQPGPIKTKGAFS 251 RLDPTGRFEK EMIDRIPCGR LGTMEELANL ATFLCSDYAS WINGAVIRFD 301 GGEEVFLSGE FNSLKKVTKE EWDIIEGLIR KTKGS

24. Criteria For A Positive Identification:Members of Signaling Complexes. • Identification is made in complexes purified via at least two independent methods. • Mass spectrometry results are verified by either co-immunoprecipitation or protein arrays. • 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).

25. RESULTS:The 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.

26. Log 250k pI 3 pI 12 PKC Cardiac PKC Signaling Subproteome Log 5k

27. RESULT ONE:PKCe 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)

28. Log 250k pI 3 pI 12 PKC Log 5k Cardiac PKC Signaling Subproteome

29. 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)

30. 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)

31. 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 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)

32. Strategies For Mapping The Cardiac Proteome: Characterization of multiple functional subproteomes • Function • Protein Profiling • Spatial Profiling • Temporal Profiling