Functional Proteomic Analysis of Protein Kinase C ε Signaling Complexes in Preconditioning.
Studies from our laboratory and others have shown that transgenic (Tg) mice expressing low levels of active protein kinase C ε (PKCε) exhibit resistance to ischemic injury, a cardioprotected phenotype analogous to that observed during preconditioning. Although PKCε has been shown to activate multiple downstream targets in preconditioning, the molecular components that form PKCε signaling complexes and those that constitute the PKCε signaling architecture are unknown. We used a proteomic approach to characterize PKCε signaling complexes. PKCε monoclonal antibodies were used to immunoprecipitate cardiac tissues from PKCε Tg mice and wild type mice (n=10 each). Combining 2-D electrophoresis, MALDI mass spectrometry, and immunoblotting, so far we have identified 27 known and 12 unknown molecules in PKCε signaling complexes. These include signaling proteins (RACK2, Lck, Src, Pyk2, PI3 kinase (p170/p85), p38 MAPK, p54/p46 JNKs, ERKs, Hsp27/Hsp71, αB-crystallin, iNOS, eNOS), and structural proteins (cardiac α-actin, troponin T, α-tropomyosin, prohibitin, desmin, Lap2, caveolin-3). Many of these proteins were not previously suspected to be in PKCε immunocomplexes. In PKCε Tg mice, altered expression and post-translational modification were evident in at least 21 known and 10 uncharacterized molecules. These data show, for the first time, (i) that PKCe forms signaling complexes with multiple proteins in multiple subcellular compartments, suggesting heretofore-unrecognized functions of PKCε isozyme; and (ii) that cardioprotection is coupled with dynamic modulation of PKCε-associated proteins and recruitment of signaling molecules to PKCε complexes. Functional proteomic analysis of PKCε signaling complexes is a crucial step toward understanding PKCε-dependent signaling architecture and cardioprotection.
- Copyright © 2000 by American Heart Association