Functional Proteomic Analysis of Protein Kinase C ε Signaling Complexes in the Normal Heart and During Cardioprotection
Abstract—Using two-dimensional electrophoresis, mass spectrometry, immunoblotting, and affinity pull-down assays, we found that myocardial protein kinase C ε (PKCε) is physically associated with at least 36 known proteins that are organized into structural proteins, signaling molecules, and stress-responsive proteins. Furthermore, we found that the cardioprotection induced by activation of PKCε is coupled with dynamic modulation and recruitment of PKCε-associated proteins. The results suggest heretofore-unrecognized functions of PKCε and provide an integrated framework for the understanding of PKCε-dependent signaling architecture and cardioprotection.
The ε isoform of protein kinase C (PKCε) has recently become the focus of considerable interest because of its critical role in protecting the myocardium against ischemia/reperfusion injury.1 2 3 4 Cogent evidence indicates that the activation of PKCε is a pivotal event in the development of the cardioprotective effects of ischemic preconditioning.1 2 3 4 Moreover, transgenic overexpression of low levels of active PKCε2 3 or of a peptide that causes its activation4 results in a cardiac phenotype characterized by enhanced resistance to myocardial ischemia/reperfusion injury, ie, a phenotype analogous to that observed during preconditioning. Thus, recruitment of PKCε is both necessary and sufficient to confer cardioprotection, emphasizing the unique role of this isozyme in the pathophysiology of myocardial ischemia. However, the precise molecular signaling mechanisms underlying PKCε-dependent cardioprotection remain unknown.
In the complex molecular infrastructure that underlies preconditioning, PKCε appears to be an upstream element, orchestrating a series of signaling events that result in the recruitment of many of the downstream factors (kinases, transcription factors, and other proteins) involved in the acquisition of cardioprotection.1 2 3 4 Thus, identification of the molecules that participate in PKCε signaling may provide important insights into the molecular basis of both preconditioning and cardioprotection. In noncardiac cells, it is becoming apparent that signaling molecules operate in close proximity,5 forming complexes that aid the transmission of signals.5 However, virtually nothing is known regarding whether PKCε forms signaling complexes in the heart, and, if so, which specific proteins participate in PKCε-dependent signaling complexes. Elucidation of these issues is an indispensable first step toward unraveling the mechanism of PKCε-dependent cardioprotection.
Thus far, studies of signaling in preconditioning have adopted a conventional approach, in which the investigation is focused on one or few target kinase(s).1 2 3 4 Although this narrow approach has provided many important insights into the mechanism of preconditioning, it is inherently limited in its ability to offer a comprehensive and systematic analysis of the multiple signaling events that underlie this phenomenon. Although individual molecules may trigger important signaling responses, it is the coordinated interaction of multiple kinases within a signaling complex and the subsequent integrated action of multiple complexes that bring about the manifestation of a phenotype. In recent years, the development of proteomic technology has made it possible to examine multiple proteins and their interactions on a large scale.6 Two-dimensional (2D) gel electrophoresis coupled with advanced mass spectrometry allows high-throughput, systematic display of a complete protein profile and comprehensive assessment of multiple molecules in parallel.6 Rather than examining a single molecule in isolation, functional proteomic strategies are aimed at obtaining information regarding the expression profile of all proteins as well as the protein interactions within a signaling complex, thereby providing a holistic portrait of the entire signaling network.
Accordingly, in the present study, we adopted a novel functional proteomic approach to gain insights into the PKCε-dependent signaling events that lead to cardioprotection. We specifically sought to determine whether PKCε forms signaling complexes in the heart and to identify the components of these complexes. Using this approach, we have thus far identified 36 proteins physically associated with PKCε. Our data show that PKCε signaling complexes are associated with multiple subcellular compartments and that activation of PKCε induces dynamic modulation of the expression profile of these complexes.
Materials and Methods
Total cardiac tissue lysates from either 10 PKCε transgenic mice2 3 or 10 age-matched nontransgenic littermates were pooled as one sample. A total of 30 mice (3 samples) in each group were studied. PKCε signaling complexes were identified by coimmunoprecipitation with PKCε monoclonal antibodies (PharMingen), followed by 2D electrophoresis, matrix-assisted laser desorption ionization (MALDI) mass spectrometry, and Western immunoblotting. Formation of PKCε signaling complexes was then independently confirmed by a GST-PKCε–based affinity pull-down assay.6 Only those components whose localization in the PKCε complexes was confirmed with the latter assay are reported in the present study. Adult cardiomyocytes were isolated from 6 PKCε transgenic and 6 nontransgenic hearts to verify the expression of all molecules in this cell type. All experiments were performed using a protocol approved by the Institutional Animal Care and Use Committee. A detailed methodology is provided in the online data supplement (http://www.circresaha.org).
Results and Discussion
Expression of Structural Proteins in PKCε Signaling Complexes in Nontransgenic Mice
Proteomic analysis revealed the association of several structural proteins with PKCε signaling complexes (Table⇓, Figure⇓), suggesting that these complexes are widely distributed in multiple subcellular locations. Importantly, besides proteins residing in subcellular structures previously known to be associated with PKCε (Golgi apparatus, caveolae, and contractile filaments), expression profiling also identified nuclear apparatus proteins (Lap 2) and mitochondrial inner membrane proteins (prohibitin). Although previous investigations have indicated perinuclear expression of PKC, our results provide the first evidence that PKCε signaling complexes are associated with the inner mitochondrial and nuclear compartments. To exclude the possibility that the presence of these two structural proteins in PKCε complexes may have been an artifact associated with the immunoprecipitation procedure, we isolated cardiac cell nuclei, mitochondria, and caveolae. PKCε monoclonal antibodies revealed strong immunosignals from each of these preparations, confirming that PKCε signaling complexes do exist in these subcellular compartments. PKCε complexes were also found to be associated with the cytoskeletal proteins desmin, villin, and adaptin-β (Table⇓). Analysis of isolated cardiomyocytes confirmed the expression of all listed proteins in this cell type (Table⇓). The affiliation of PKCε with nuclear apparatus, inner mitochondrial membrane, and cytoskeletal proteins implies novel targets and previously unrecognized functions for this kinase. Our findings warrant further studies to determine whether PKCε plays a regulatory role in modulating the functions of these proteins and to establish the overall functional significance of the observed protein interactions.
Expression of Signaling and Stress Proteins in PKCε Signaling Complexes
As expected, a large number of signaling molecules claim residence in the PKCε complexes (Table⇑, Figure⇑). The potential downstream targets of PKCε, such as Src and Lck tyrosine kinases and mitogen-activated protein kinases (p38 MAPKs, JNKs, and ERKs), and upstream modulators of PKCε, such as PI3 kinases and their substrate, PKB/Akt, are included in this list. Interestingly, these kinases have also been implicated in preconditioning. Colocalization of these kinases in the PKCε signaling complexes indicates potentially important mechanisms for the transmission and integration of signals during preconditioning.
An intriguing finding is the presence of two isoforms of nitric oxide synthase (NOS) (inducible [iNOS] and constitutive [eNOS]) in the PKCε signaling complexes. In noncardiac tissues, it has been shown that tyrosine kinases are key regulators of iNOS and that Akt is a direct activator of eNOS. However, although both iNOS and eNOS contain multiple potential PKC phosphorylation sites (16 for mouse iNOS and 22 for mouse eNOS), virtually nothing is known regarding PKCε-dependent posttranslational modifications of NOS. Our observations provide the first evidence that iNOS and eNOS (both of which play a fundamental role in preconditioning) are physically associated with PKCε. Other stress-responsive proteins implicated in preconditioning, such as COX-2, Hif-1α, heme oxygenase-1, heat shock proteins (HSPs), and aldose reductase, were also found in the PKCε complexes (Table⇑). The coexistence of multiple signaling kinases with these stress proteins undoubtedly facilitates posttranslational regulatory events. Taken together, our data suggest a novel role of PKCε in the modulation of iNOS, eNOS, COX-2, Hif-1α, heme oxygenase-1, HSPs, and aldose reductase, and lay the groundwork for future in-depth and detailed investigations of the mechanisms responsible for the regulation of these proteins.
PKCε-Mediated Cardioprotection Is Associated With Posttranslational Modification and Recruitment of Proteins Into Its Signaling Complexes
To elucidate the molecular infrastructure that underlies PKCε-mediated cardioprotection, we examined transgenic mice expressing low levels of constitutively active PKCε (PKCε activity: 208±18% of negative littermates), which exhibit a cardioprotected phenotype (ie, enhanced resistance to myocardial ischemia/reperfusion injury) similar to that conferred by preconditioning. Proteomic analysis revealed that PKCε-mediated cardioprotection was associated with two striking changes in PKCε signaling complexes: (1) posttranslational modification of 24 of the 36 proteins identified and (2) altered protein expression of 35 of the 36 proteins identified (Table⇑, Figure⇑). Posttranslational modification was determined by a shift in pI in the 2D electrophoresis and/or by MALDI analysis.
Among the proteins that underwent posttranslational modification in PKCε transgenic mice, 23 of 24 contain potential PKC phosphorylation sites. Although PKC-dependent posttranslational modifications have been implicated in a number of biological functions, events specifically induced by activation of the ε isoform have never been defined. Our study represents the first investigation to identify PKCε-induced posttranslational modifications in the heart.
In addition to these posttranslational changes, the current proteomic analysis reveals that PKCε-mediated cardioprotection is associated with remarkable changes in both the quality and the quantity of the components constituting the PKCε signaling complexes (Table⇑). Specifically, we found that some proteins were recruited into the complexes whereas others were no longer part of them (Figure⇑). Among molecules that were recruited to join these complexes in the heart of PKCε transgenic mice, one group consists of new proteins that are not found in wild-type mice (eg, αB-crystallin [Figure⇑]), whereas the second group is composed of proteins that are found in wild-type mice but are expressed with much greater abundance in PKCε transgenic mice (eg, JNK2, eNOS, and adaptin-β [Table⇑]). These results suggest that PKCε-dependent cardioprotection requires not only an increase in physiological protein-protein interactions involving PKCε but also the development of new interactions that are absent in the naïve, unprotected state. Finally, our finding that molecules such as HSP 27, HSP 70, and MAPKAPK2, which have been implicated in preconditioning, are recruited to the PKCε complexes in transgenic mice (Table⇑, Figure⇑) provides additional evidence supporting the role of these proteins in cardioprotection.
This is the first investigation to use a functional proteomic strategy to systematically delineate PKCε signaling complexes in the heart. This approach has enabled us to demonstrate physical associations of PKCε with numerous structural, signaling, and stress-responsive proteins in the normal heart and to identify new interactions associated with cardioprotection in PKCε transgenic mice. Many of the proteins found to be physically associated with PKCε in this study were not previously known or suspected to interact with this kinase. Therefore, our results suggest novel, heretofore-unrecognized functions for PKCε in the regulation of a multitude of cellular proteins. Based on our findings, we propose that PKCε forms different complexes at different subcellular locations; further studies will be needed to define the composition of individual PKCε complexes in each specific subcellular compartments (eg, nuclei, mitochondria, etc). The recruitment of molecules into a signaling complex is known to be a mechanism to facilitate the interaction of these proteins and the integration of signal transduction. By deciphering the molecular infrastructure that underlies PKCε-dependent signaling in normal and protected myocardium, our observations provide the indispensable framework for future studies aimed at interrogating the functional significance of the observed protein-protein interactions. The information obtained with this proteomic analysis will expedite our understanding of PKCε-dependent cardioprotection and signaling. Given the multiplicity of PKCε functions, this study has broad implications for numerous biological processes in which PKCε has been implicated.
This study was supported in part by American Heart Association Grant EIG-40167N (P.P.), National Institutes of Health (NIH) Grants HL-63901 (P.P.), HL-65431 (P.P.), HL-43151 (R.B.), HL-55757 (R.B.), NIH 1S10RR11368-01A1 (W.P.), the Kentucky Physical Facilities Trust Fund, the University of Louisville Research Foundation, and the Jewish Hospital Research Foundation.
Original received September 12, 2000; revision received November 30, 2000; accepted November 30, 2000.
This manuscript was sent to Stephen F. Vatner, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
- © 2001 American Heart Association, Inc.
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