Mitochondrial PKCε and MAPK Form Signaling Modules in the Murine Heart
Enhanced Mitochondrial PKCε-MAPK Interactions and Differential MAPK Activation in PKCε-Induced Cardioprotection
Although activation of protein kinase C (PKC) ε and mitogen-activated protein kinases (MAPKs) are known to play crucial roles in the manifestation of cardioprotection, the spatial organization of PKCε signaling modules in naïve and protected myocardium remains unknown. Based on evidence that mitochondria are key mediators of the cardioprotective signal, we hypothesized that PKCε and MAPKs interact, and that they form functional signaling modules in mitochondria during cardioprotection. Both immunoblotting and immunofluorescent staining demonstrated that PKCε, ERKs, JNKs, and p38 MAPK co-localized with cardiac mitochondria. Moreover, transgenic activation of PKCε greatly increased mitochondrial PKCε expression and activity, which was concomitant with increased mitochondrial interaction of PKCε with ERKs, JNKs, and p38 as determined by co-immunoprecipitation. These complex formations appeared to be independent of PKCε activity, as the interactions were also observed in mice expressing inactive PKCε. However, although both active and inactive PKCε bound to all three MAPKs, increased phosphorylation of mitochondrial ERKs was only observed in mice expressing active PKCε but not in mice expressing inactive PKCε. Examination of potential downstream targets of mitochondrial PKCε-ERK signaling modules revealed that phosphorylation of the pro-apoptotic protein Bad was elevated in mitochondria. Together, these data show that PKCε forms subcellular-targeted signaling modules with ERKs, leading to the activation of mitochondrial ERKs. Furthermore, formation of mitochondrial PKCε-ERK modules appears to play a role in PKCε-mediated cardioprotection, in part by the phosphorylation and inactivation of Bad.
Protein kinase C ε (PKCε) is a pivotal signaling element in the genesis of ischemic preconditioning and myocardial protection against ischemic injury.1,2⇓ Biochemical analysis in rats and rabbits has demonstrated activation of PKCε in response to cardioprotective stimuli.3–5⇓⇓ Furthermore, genetic cardiac expression of active PKCε6 or a specific activator7 of PKCε confers a chronically cardioprotected phenotype in mice. However, the molecular signaling events that underlie PKCε-induced cardioprotection are still poorly understood.
An essential feature of the activation of PKCε during ischemic preconditioning is its subcellular redistribution.3,4⇓ This translocation to specific subcellular compartments is thought to be an important mechanism for PKCε to direct downstream signaling cascades and orchestrate protection.8 One compartment that has received considerable attention in the development of cardioprotection is the mitochondrion. Pharmacological studies have implicated a role for mitochondrial KATP channels in protection against ischemic injury9 and activation of PKC potentiates mitochondrial KATP channel opening.10 Moreover, preconditioning has been shown to preserve mitochondrial function11 and to reduce mitochondrial cytochrome c release and apoptosis12 in response to ischemia/reperfusion. Together, these data support an important role of mitochondria in cardioprotection and suggest a functional link between PKCε and this organelle. However, whether the signal initiated by PKCε is integrated and transmitted to the mitochondria remains unknown, and specific mitochondrial targets of PKCε have not been defined.
Recently, cardioprotection by the mitogen-activated protein kinase (MAPK) activator anisomycin was shown to be blocked by a mitochondrial KATP channel inhibitor.13 In contrast, opening of mitochondrial KATP channels has been shown to activate p38 MAPK.13 Regardless of the sequence, both studies would indicate participation of MAPKs at the mitochondrial level. However, although considerable attention has focused on MAPKs as potential transducers during preconditioning,1,2⇓ little is known regarding the precise identity of the MAPKs involved in PKCε-mediated cardioprotection. We and others have shown in rabbits and rats that activation of PKCε both in vivo and in vitro protects against ischemia via activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) members of the MAPK family.15–18⇓⇓⇓ Nevertheless, it remains unknown whether PKCε-mediated cardioprotection involves the activation of MAPKs at the mitochondrial level and, conversely, whether inhibition of PKCε affects the activation state of MAPKs.
A primary mechanism by which subcellular compartment-specific signal transduction is achieved is the formation of signaling complexes at the target subcellular location.19–22⇓⇓⇓ To this end, formation of a signaling module has been hypothesized to occur as the physical interaction of key molecules that leads to signal transduction.8,21,22⇓⇓ For example, a signaling module involving PKCε and Src tyrosine kinase has been shown to facilitate signal transduction during nitric oxide–induced cardioprotection in rabbits.23 Using proteomic analysis, we have recently found that ERKs, JNKs, and p38 MAPK are all constituents of the PKCε signaling complex in whole murine myocardial homogenates.24 Accordingly, we postulated that PKCε forms signaling modules with, and modulates the activity of, individual MAPKs at the level of the mitochondria, and that the formation of these mitochondrial PKCε-MAPK modules participates in PKCε-mediated cardioprotection.
The present study, therefore, was designed to comprehensively investigate the development of PKCε-MAPK signaling modules in mitochondria during cardioprotection. Specifically, we determined in the murine heart whether PKCε and individual MAPKs are expressed and co-localized in cardiac mitochondria and, if so, whether activation of PKCε modulates the activity of individual MAPKs (ie, whether they form signaling modules) in this organelle. To determine whether activation of PKCε plays a necessary role in the formation of PKCε-MAPK modules, these complexes were examined during both activation and inhibition of PKCε. We found that transgenic activation of PKCε enhances mitochondrial co-localization of PKCε with ERKs, JNKs, and p38, concomitantly with increased phosphorylation activity of both ERKs and p38 and that formation of PKCε-ERK modules was blocked by inhibition of PKCε. Furthermore, PKCε-ERK module formation was associated with enhanced phosphorylation of the pro-apoptotic protein Bad.
Materials and Methods
All procedures were performed in accordance with the Institutional Animal Care and Use Committee of the University of Louisville animal care guidelines, which conform with the Guide for the Care and Use of Laboratory Animals, published by the NIH.
The sources of antibodies were as follows: anti-PKCε, ERK1, and p38 MAPK, Transduction Laboratories; anti-PKCε polyclonal, Sigma; anti–prohibitin-1, Research Diagnostics; anti-JNK2, Santa Cruz Biotechnology; anti-JNK1, Pharmingen; and anti–phospho-p38, phospho-ERK, phospho-JNK, Bad, and phospho-Bad (Ser112), Cell Signaling.
Generation of PKCε Transgenic Mice
Cardiac-targeted PKCε transgenic mice were developed as previously described.24,25⇓ Briefly, the αMHC promoter was used to drive the cardiac-specific expression of either an active (A159E; AE) or a dominant-negative (K436R and A159E; DN) PKCε mutant in ICR strain mice. The AE line exhibits moderate increases in total PKCε activity whereas the DN line has reduced basal PKCε activity and both lines are free of hypertrophy and cardiac function is normal.25 Both nontransgenic and transgenic littermates were studied at 9 to 12 weeks.
Tissue Sample Preparation and Subcellular Fractionation
Mitochondria and cytosolic fractions were isolated by differential centrifugation. Frozen mouse hearts were homogenized in buffer containing 250 mmol/L sucrose, 10 mmol/L Tris-HCl (pH 7.4), 1 mmol/L EDTA, 1 mmol/L Na3VO4, 1 mmol/L NaF, and protease inhibitor cocktail (Roche). The homogenate was centrifuged at 1000g for 10 minutes to remove nuclei and debris. The supernatant was centrifuged at 10 000g for 30 minutes. The resultant supernatant was subsequently centrifuged at 100 000g for 1 hour to yield the cytosolic fraction. The 10 000g pellet, corresponding to the mitochondrial fraction, was resuspended and centrifuged again at 10 000g for 30 minutes. The washed mitochondria were then resuspended and homogenized. Protein concentrations were determined using the Bradford method. Cytosolic contamination of the mitochondrial fraction was less than 0.5% as measured by LDH activity. Our data indicated that freezing of the hearts did not compromise mitochondrial integrity, which was documented by measuring the release of mitochondrial cytochrome c into the cytosol.
Mitochondrial and cytosolic proteins (100 μg) were resolved on 10% to 15% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. Equal loading was confirmed by staining with Ponceau-S. After blocking with 5% nonfat milk, the membranes were immunoblotted using the ECL detection system (Amersham).
Mitochondrial or cytosolic proteins (500 μg) were incubated with 1 μg of ERK1, JNK2, or p38 antibody and 30 μL Protein A/G PLUS-Agarose (Santa Cruz) overnight at 4°C. The beads were then washed three times with buffer containing 150 mmol/L NaCl, 20 mmol/L Tris-Cl (pH 7.4), 10 mmol/L EDTA, 1% Nonidet-P40, 1 mmol/L Na3VO4, and protease inhibitors.23–25⇓⇓ The beads were suspended in sample buffer, boiled, centrifuged, and the supernatants subjected to immunoblotting.
PKCε Activity Assay
Phosphotransferase activity of PKCε was determined as described previously.4,23,25⇓⇓ Briefly, mitochondrial samples (150 μg) were immunoprecipitated overnight with anti-PKCε monoclonal antibody and the precipitates incubated with the PKCε-selective substrate (ERMRPRKRQGSVRRRV) in a phosphorylation cocktail for 15 minutes at 30°C. The amount of radioactivity incorporated was quantified by scintillation.
Immunofluorescent Confocal Microscopy
Isolated mitochondria were fixed in 4% paraformaldehyde and mounted onto gelatin-coated slides. The slides were boiled for 10 minutes in 10 mmol/L citrate buffer (pH 6.0), washed with PBS, and permeabilized/blocked with 7% goat serum, 5% nonfat milk, and 1% Triton X-100 for 10 minutes at 37°C. After washing, the mitochondria were blocked further with mouse Ig blocking reagent (Vector Laboratories) for 1 hour at RT. The slides were incubated with polyclonal PKCε antibody (1:100) overnight at 4°C, washed, and then incubated with monoclonal prohibitin-1 antibody (1:100) for 2 hours at 37°C. The slides were then washed and incubated with a mixed solution of secondary antibodies (TRITC-conjugated anti-rabbit and FITC-conjugated anti-mouse, 1:200) for 1 hour at 37°C. Confocal microscopy of the sections was done using a Zeiss LSM510 inverted confocal scanning laser microscope.
Data are expressed as mean±SEM. Groups were compared using Student’s t tests for unpaired data. A P value less than 0.05 was considered significant.
PKCε and MAPKs Co-Localize in Cardiac Mitochondria
We first determined whether PKCε and MAPK are present in cardiac mitochondria. Immunoblotting of cardiac mitochondrial extracts from nontransgenic (NTG) mice demonstrated the expression of PKCε, ERKs, JNKs, and p38 MAPK in this subcellular fraction (Figure 1). In all cases, the mitochondrial expression of the respective kinase was ≈10% to 20% of that observed in the cytosol. To confirm the mitochondrial expression of PKCε, we performed immunofluorescent confocal microscopy on murine cardiac mitochondria. Staining for PKCε, along with the mitochondrial marker prohibitin-1, demonstrated the association of PKCε with mitochondrial structures (Figure 2). Together, these data demonstrate that PKCε and the three major MAPKs co-localize with cardiac mitochondria.
Mitochondrial PKCε Expression and Activation Are Increased in PKCε Transgenic Mice
We next examined mitochondrial PKCε levels and phosphotransferase activity in the constitutively active (AE) and dominant-negative (DN) PKCε mouse lines. The AE line is chronically cardioprotected, exhibiting an ≈70% reduction in infarction when compared with wild-type mice,6 whereas in the DN line, infarction is no different from controls (P. Ping and R. Bolli, unpublished data, 1999). Figure 3A shows total myocardial expression of PKCε in NTG, AE, and DN mouse hearts. There was an ≈16-fold increase in total PKCε levels in the AE line and an ≈24-fold increase in the DN line when compared with controls. In the AE hearts, cytosolic PKCε expression was 13.0±0.4-fold greater than in NTG littermates (data not shown), but mitochondrial PKCε expression was elevated to a greater extent (26.5±1.6-fold; Figure 3B). This was confirmed by immunohistochemical staining of mitochondria for PKCε, which revealed a much more intense co-localization of PKCε with mitochondria in the AE mice when compared with NTG mice (Figure 2). The differential increase in cytosolic and mitochondrial PKCε expression observed in AE hearts was also present in DN hearts (21.3±0.7 versus 31.1±0.4-fold, respectively). Therefore, cardiac mitochondrial PKCε expression is preferentially increased over cytosolic expression in both the AE and DN lines, suggesting that the open configuration of PKCε is sufficient for its subcellular distribution in these mice. There were no significant changes in the expression levels of MAPKs in cardiac mitochondria of any mouse line, with the exception of ERK, which was increased ≈2-fold in the AE, but not the DN line (data not shown).
Mitochondrial PKCε activity was assessed by the phosphorylation of a PKCε-specific peptide following immunoprecipitation. The activity of PKCε was significantly increased in mitochondrial fractions of hearts from the AE mice (Figure 3C). In contrast, there was a significant decrease in basal PKCε activity in the mitochondria of DN hearts compared with NTG hearts.
Transgenic Activation of PKCε Enhances Mitochondrial PKCε-MAPK Module Formation
Formation of compartment-specific signaling modules is the primary mode by which signal transduction is implemented. Since all three MAPK families are part of the overall myocardial PKCε complex24 and since we observed mitochondrial co-localization of PKCε and MAPKs, we examined whether PKCε forms complexes with individual MAPKs at the mitochondrion. Mitochondrial lysates were immunoprecipitated with MAPK antibodies (Figure 4A) and blotted for PKCε (Figure 4B). Mitochondrial PKCε was found to interact with ERKs, JNKs, and p38 MAPK in both nontransgenic and transgenic hearts. Moreover, in AE hearts, the amount of PKCε co-precipitating with the mitochondrial MAPKs was increased strikingly. Interestingly, PKCε-MAPK interactions were also increased in the cytosolic fraction of AE mice but to a much lesser extent (Figure 4C). These data show that PKCε can form signaling complexes with individual MAPKs in cardiac mitochondria. Furthermore, the increased interaction appears to be due to increased PKCε protein rather than activity, as PKCε still bound MAPKs in DN mitochondria (Figure 4B).
PKCε Activates Mitochondrial ERKs and p38 MAPK via Kinase Activity–Dependent and –Independent Pathways
Activation of PKCε has been previously reported to stimulate ERKs and JNKs in cardiac cells.15–17,26⇓⇓⇓ Consequently, we assessed the effect of enhanced mitochondrial PKCε activity and complex formation on mitochondrial MAPK activity using phosphospecific antibodies. In AE mice, there was a marked increase in phosphorylation of mitochondrial ERKs, which was not observed in DN mice (Figure 5A). In contrast, there was no change in JNK phosphorylation in either AE or DN cardiac mitochondria when compared with NTG hearts (Figure 5C). Phosphorylation of mitochondrial p38 MAPK was also elevated in AE mouse hearts (Figure 5B). Surprisingly, however, this increase in p38 activity was still observed in DN mouse hearts. Thus, unlike ERK activation, PKCε-induced activation of mitochondrial p38 does not require PKCε activity. Importantly, these data suggest that ERKs, but not p38, contribute to the cardioprotective phenotype in these mice.
Phosphorylation of Bad Is Elevated in PKCε Transgenic Mice
We next explored mitochondrial targets of PKCε and ERKs that could play a role in cardioprotection. We focused on the pro-apoptotic protein Bad because both PKC and ERKs are known to prevent cell death via the induction of Ser112 phosphorylation of Bad.27,28⇓ This phosphorylation inhibits Bad’s ability to sequester protective Bcl proteins and induce cell death.29 Total mitochondrial levels of Bad remained unaltered in both AE and DN mice. However, examination of the phosphorylation status of mitochondrial Bad revealed a marked increase in phosphorylation of Ser112 in AE, but not DN, mice (Figure 6). Therefore, phosphorylation of Bad is correlated with the cardioprotective phenotype in the AE-PKCε transgenic mice.
Many individual signaling elements such as PKCε, and MAPKs, have been identified as playing essential roles in the signal transduction system utilized by cardioprotective stimuli.1,2⇓ However, the architectural arrangement of these elements both in relation to each other and in relation to specific subcellular environments remains unknown. The present study provides novel insight into the mitochondrial signaling infrastructure of the myocardium and its changes within the context of cardioprotection. We present the first piece of direct evidence demonstrating that activation of PKCε enhances mitochondrial co-localization of PKCε with MAPKs, increases phosphorylation of mitochondrial MAPKs, and promotes the formation of mitochondrial PKCε-MAPK signaling modules. This dynamic modulation of the PKCε-MAPK modules is associated with the inhibition of pro-apoptotic molecules and the genesis of a cardioprotective phenotype.
PKCε and MAPKs Are Associated With Cardiac Mitochondria
The genesis of cardioprotection requires the transduction of cellular signals to specific intracellular domains, including the mitochondria.1,2,9⇓⇓ Previous studies examining PKC and MAPK signaling in cardioprotection have generally separated cellular lysates into crude cytosolic and particulate fractions.3,4,7,23⇓⇓⇓ As the latter contains many organelles, including nuclei, Golgi, etc, mitochondrial-specific alterations in a kinase’s expression and/or activity during cardioprotection have not been comprehensively addressed.
While the expression and function of cardiac PKCε and MAPKs have been studied in other compartments,5,15–17,30⇓⇓⇓⇓ there is a paucity of data regarding the co-existence of PKCε and MAPKs isoforms in either cardiac or noncardiac mitochondria. Cellular fractionation has shown that PKCα, δ, γ, and ζ can associate with mitochondria in cultured cells31,32⇓ and immunohistochemical studies indicate that PKCε and PKCδ may affiliate with cardiac mitochondria in the rat.33,34⇓ Nevertheless, no previous studies have examined the formation of PKCε signaling modules in mitochondria. In the present study, using both biochemical and immunohistochemical analyses, we demonstrate that PKCε does indeed co-localize to mitochondria in the murine myocardium. In addition, our data show that all three major MAPK families, namely ERKs, JNKs, and p38, are expressed in cardiac mitochondria. This is in keeping with recent studies showing that ERKs and JNKs are associated with mitochondria in noncardiac cells,35,36⇓ although to our knowledge the present investigation is the first to show p38 expression in mitochondria. Therefore, the major components of the cardioprotective machinery are directly associated with this target subcellular domain.
Translocation of PKCε to particulate structures is viewed as one of the necessary early events in the development of preconditioning.3–5⇓⇓ We have recently shown that stimulation of rabbit hearts, either by ischemic preconditioning or phorbol ester, induces the translocation of PKCε to the mitochondria.37 Therefore, upregulation of mitochondrial PKCε may be a critical step in the modulation of mitochondrial targets and the genesis of cardioprotection. To address this question we utilized a genetic mouse model of cardioprotection whereby cardiac-specific expression of active PKCε confers resistance to ischemia/reperfusion.6 Mutation of Ala159 to Glu (AE) in the pseudosubstrate region of PKCε changes the enzyme to its open conformation, thus inducing its translocation to, and activation at, its subcellular target sites. Analysis of AE hearts showed that while both cytosolic and mitochondrial PKCε pools were increased when compared with controls, the increase in mitochondrial PKCε was ≈2-fold greater than that in the cytosol. This was confirmed by confocal microscopy, showing an increased intensity of PKCε staining in AE mitochondria. Interestingly, the confocal images revealed that in NTG hearts PKCε is concentrated in the periphery of the mitochondria, whereas in AE hearts PKCε also appears in the matrix. The reason for this is unknown. We speculate that placing PKCε in its open conformation may have facilitated its uptake into the matrix. The mitochondrial redistribution of PKCε does not appear to be dependent on kinase activity, as a similar relocalization of PKCε to the mitochondrial fraction was observed in DN mice. Since DN-PKCε possesses the same Ala159 mutation as the AE mutant but with an additional kinase-inactivating mutation, the open conformation of PKCε appears sufficient for its mitochondrial targeting. This is consistent with previous studies demonstrating that conformational modifications alter the ability of PKC isoforms to translocate to subcellular compartments.38,39⇓ Nevertheless, activity is required for cardioprotection as only the AE, but not the DN, mice are resistant to ischemia (P. Ping and B. Bolli, unpublished data, 1999). Hence, we found that mitochondrial PKCε activity was enhanced in the AE and attenuated in the DN hearts. Therefore, the cardioprotective phenotype is associated with a preferential increase in expression and activity of mitochondrial versus cytosolic PKCε.
Mitochondrial PKCε Forms Complexes With ERKs, JNKs, and p38 MAPK
The formation of subcellular compartment-specific multiprotein complexes has been shown as a mechanism by which cells integrate and transmit different extracellular signals.21,22⇓ In rat myocytes, PKCε interacts with and phosphorylates the gap junction protein connexin-43 in response to fibroblast growth factor,30 whereas in rabbits enhanced interaction of PKCε with Src in the particulate fraction is a prerequisite for nitric oxide–induced preconditioning.23 Thus, subcellular-specific module formation plays a critical role in myocardial signaling and cardioprotection. Importantly, ERKs, JNKs, and p38 MAPK are all constituents of the murine cardiac PKCε signaling complex.24 The present investigation presents strong evidence that the mitochondrion is one of the primary locations where PKCε-MAPK modules engage in signal transduction.
Immunoprecipitation of ERKs, JNKs, or p38 pulled down PKCε from cardiac mitochondrial lysates, demonstrating that PKCε interacts with all three MAPK subfamilies in the mitochondria. Interestingly, cytosolic associations were also observed for MAPKs, suggesting that in the basal state there is a weak, generalized interaction between PKCε and MAPKs throughout the cell. As expected, however, the level of PKCε association with ERKs, JNKs, and p38 was significantly upregulated in the AE-PKCε mice. In particular, the amount of PKCε co-precipitating was greater in the mitochondrial than the cytosolic fraction. This interaction does not require catalytic activity as the DN-PKCε was just as able to bind MAPKs. Therefore, while the physical interaction of PKCε with MAPKs may be necessary for cardioprotection, it is not sufficient. Placing PKCε in the open conformation enhances its ability to bind to Src40 implicating the configuration of PKCε as a critical determinant of its capacity to interact with proteins. This supports the conclusion that the enhanced interaction of PKCε with MAPKs seen in both the AE and DN-PKCε mice is due to the fact that both PKCε mutants are in the open conformation.
Mitochondrial PKCε-MAPK Complex Formation Induces Differential Activation of MAPKs
The formation of PKCε-MAPK signaling modules implies a functional coupling between mitochondrial PKCε and MAPKs. We found marked increases in mitochondrial MAPK phosphorylation with respect to ERKs and p38 MAPK in the AE-PKCε mice whereas JNK phosphorylation did not change, even though functional coupling between PKCε and JNKs has been observed in total cell lysates of rabbit cardiomyocytes.15,16⇓ As regulatory cross-talk between MAPKs can occur,41 the sustained activation of ERKs observed in the AE mice may lead to the activation/expression of proteins that negatively regulate the JNK signaling cascade. A potential limitation of transgenic overexpression is that the protein is expressed/activated in a sustained, rather than a transient, manner. Thus, in the present study, the phenotypic and signaling changes observed are in response to chronic PKCε activation and, therefore, may involve transcriptional as well as post-translation alterations.
To confirm that phosphorylation of mitochondrial ERKs and p38 in the AE-PKCε mice was PKCε activity-dependent, we also examined the MAPK phosphorylation status in the DN-PKCε mice. Dual phosphorylation of ERK was not different from controls in the DN-PKCε hearts, confirming that mitochondrial PKCε activity is required for the phosphorylation of mitochondrial ERKs. However, p38 phosphorylation remains increased in the DN-PKCε mice, indicating that phosphorylation of p38 was PKCε activity-independent. There is considerable controversy surrounding the identity of MAPKs in cardioprotection.1 Several studies have indicated that ERK may be critical15,17,18⇓⇓ whereas others have reported p38 as a mediator of protection.13,14,42⇓⇓ In the present study, the fact that p38 MAPK, but not ERK, is still phosphorylated in the DN line would indicate that mitochondrial ERK and not p38 is required for protection. However, as mentioned, sustained p38 MAPK activation is seen in this model. Thus, it is still plausible that in transient forms of protection, brief p38 activation may play a vital role. Moreover, as multiple p38 MAPKs are expressed in the heart,43 individual isoforms may exhibit opposing responses during activation and inhibition of PKCε activity, which was not detected using the commercially available phospho-p38 antibodies. Future studies are needed to discern the role of PKCε modules with respect to individual p38 isoforms.
Upregulation of Mitochondrial PKCε-ERK Modules Induces Phosphorylation of Bad
There are multiple mitochondrial proteins that could be targets of PKCε-ERK modules in the mitochondria, including components of the mitochondrial apoptotic machinery.27,28,36⇓⇓ In particular, activation of both PKCε and ERKs has been shown to induce phosphorylation of the pro-apoptotic protein Bad in noncardiac cells.27,28⇓ Bad acts by interacting with the anti-apoptotic protein Bcl-XL, thereby disabling the inhibition of apoptosis. However, phosphorylated Bad can no longer interact with Bcl-XL and, therefore, cannot induce cell death.29 Interestingly, mitochondria-anchored PKA has been shown to phosphorylate the pro-apoptotic protein Bad.44 Analysis of the phosphorylation status of mitochondrial Bad revealed that activation of PKCε-ERK was associated with phosphorylation of Bad in the AE-PKCε transgenic mice but not in the DN-PKCε animals. Therefore, a cascade consisting of PKCε-ERK-Bad exists at the level of the cardiac mitochondrion. This functional coupling between the PKCε-ERK signaling complex and Bad may contribute to cardioprotective actions of PKCε.
In summary, PKCε interacts with, as well as forms subcellular-specific signaling modules with, ERKs, JNKs, and p38 in cardiac mitochondria. The molecular interactions result in the activation of mitochondrial ERKs via a PKCε activity-dependent mechanism. Furthermore, formation of mitochondrial PKCε-ERK modules is coupled to the inactivation of Bad and may contribute to PKCε-mediated cardioprotection. Delineation of other protein-protein interactions within mitochondrial PKCε-MAPK signaling modules and downstream targets will hopefully provide greater insight into the molecular machinery underlying cardioprotection.
This study was supported by AHA Ohio Valley Affiliate Fellowship Award 0120412B (C.P.B.), AHA EIG-40167N (P.P.), NIH grants HL-63901 and HL-65431 (P.P.), NIH grants HL-43151, HL-55757, and HL-68088 (R.B.), University of Louisville Research Foundation, Jewish Hospital Research Foundation, and Commonwealth of Kentucky Research Challenge Trust Fund.
This manuscript was sent to Eugene Braunwald, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received December 10, 2001; revision received January 24, 2002; accepted January 30, 2002.
- ↵Baines CP, Pass JM, Ping P. Protein kinases and kinase-modulated effectors in the late phase of ischemic preconditioning. Basic Res Cardiol. 2001; 96: 207–218.
- ↵Ping P, Takano H, Zhang J, Tang XL, Qiu Y, Li RC, Banerjee S, Dawn B, Balafonova Z, Bolli R. Isoform-selective activation of protein kinase C by nitric oxide in the heart of conscious rabbits: a signaling mechanism for both nitric oxide-induced and ischemia-induced preconditioning. Circ Res. 1999; 84: 587–604.
- ↵Ping P, Zhang J, Qiu Y, Tang XL, Manchikalapudi S, Cao X, Bolli R. Ischemic preconditioning induces selective translocation of protein kinase C isoforms ε and η in the heart of conscious rabbits without subcellular redistribution of total protein kinase C activity. Circ Res. 1997; 81: 404–414.
- ↵Kawata H, Yoshida K, Kawamoto A, Kurioka H, Takase E, Sasaki Y, Hatanaka K, Kobayashi M, Ueyama T, Hashimoto T, Dohi K. Ischemic preconditioning upregulates vascular endothelial growth factor mRNA expression and neovascularization via nuclear translocation of protein kinase C ε in the rat ischemic myocardium. Circ Res. 2001; 88: 696–704.
- ↵Dorn GW2nd, Souroujon MC, Liron T, Chen CH, Gray MO, Zhou HZ, Csukai M, Wu G, Lorenz JN, Mochly-Rosen D. Sustained in vivo cardiac protection by a rationally designed peptide that causes ε protein kinase C translocation. Proc Natl Acad Sci U S A. 1999; 96: 12798–12803.
- ↵Vondriska TM, Klein JB, Ping P. Use of functional proteomics to investigate PKCε-mediated cardioprotection: the signaling module hypothesis. Am J Physiol. 2001; 280: H1434–H1441.
- ↵O’Rourke B. Myocardial KATP channels in preconditioning. Circ Res. 2000; 87: 845–855.
- ↵Sato T, O’Rourke B, Marban E. Modulation of mitochondrial ATP-dependent K+ channels by protein kinase C. Circ Res. 1998; 83: 110–114.
- ↵Fryer RM, Eells JT, Hsu AK, Henry MM, Gross GJ. Ischemic preconditioning in rats: role of mitochondrial KATP channel in preservation of mitochondrial function. Am J Physiol. 2000; 278: H305–H312.
- ↵Xu M, Wang Y, Hirai K, Ayub A, Ashraf M. Calcium preconditioning inhibits mitochondrial permeability transition and apoptosis. Am J Physiol. 2001; 280: H899–H908.
- ↵Pain T, Yang XM, Critz SD, Yue Y, Nakano A, Liu GS, Heusch G, Cohen MV, Downey JM. Opening of mitochondrial KATP channels triggers the preconditioned state by generating free radicals. Circ Res. 2000; 87: 460–466.
- ↵Li RC, Ping P, Zhang J, Wead WB, Cao X, Gao J, Zheng Y, Huang S, Han J, Bolli R. PKCε modulates NF-κB and AP-1 via mitogen-activated protein kinases in adult rabbit cardiomyocytes. Am J Physiol. 2000; 279: H1679–H1689.
- ↵Punn A, Mockridge JW, Farooqui S, Marber MS, Heads RJ. Sustained activation of p42/p44 mitogen-activated protein kinase during recovery from simulated ischaemia mediates adaptive cytoprotection in cardiomyocytes. Biochem J. 2000; 350: 891–899.
- ↵McDonald PH, Chow CW, Miller WE, Laporte SA, Field ME, Lin FT, Davis RJ, Lefkowitz RJ. β-Arrestin 2: a receptor-regulated MAPK scaffold for the activation of JNK3. Science. 2000; 290: 1574–1577.
- ↵Vondriska TM, Zhang J, Song C, Tang XL, Cao X, Baines CP, Pass JM, Wang S, Bolli R, Ping P. Protein kinase Cε-Src modules direct signal transduction in nitric oxide-induced cardioprotection: complex formation as a means for cardioprotective signaling. Circ Res. 2001; 88: 1306–1313.
- ↵Ping P, Zhang J, Pierce WM Jr, Bolli R. Functional proteomic analysis of protein kinase C ε signaling complexes in the normal heart and during cardioprotection. Circ Res. 2001; 88: 59–62.
- ↵Pass JM, Zheng Y, Wead WB, Zhang J, Li RC, Bolli R, Ping P. PKCε activation induces dichotomous cardiac phenotypes and modulates PKCε-RACK interactions and RACK expression. Am J Physiol Heart Circ Physiol. 2001; 280: H946–H955.
- ↵Heidkamp MC, Bayer AL, Martin JL, Samarel AM. Differential activation of mitogen-activated protein kinase cascades and apoptosis by protein kinase C ε and δ in neonatal rat ventricular myocytes. Circ Res. 2001; 89: 882–890; published online before print September 27, 2001, 10.1161/hh2201.099434.
- ↵Tan Y, Ruan H, Demeter MR, Comb MJ. p90RSK blocks bad-mediated cell death via a protein kinase C-dependent pathway. J Biol Chem. 1999; 274: 34859–34867.
- ↵Bonni A, Brunet A, West AE, Datta SR, Takasu MA, Greenberg ME. Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms. Science. 1999; 286: 1358–1362.
- ↵Zha J, Harada H, Osipov K, Jockel J, Waksman G, Korsmeyer SJ. BH3 domain of BAD is required for heterodimerization with BCL-XL and pro-apoptotic activity. J Biol Chem. 1997; 272: 24101–24104.
- ↵Doble BW, Ping P, Kardami E. The ε subtype of protein kinase C is required for cardiomyocyte connexin-43 phosphorylation. Circ Res. 2000; 86: 293–301.
- ↵Ruvolo PP, Deng X, Carr BK, May WS. A functional role for mitochondrial protein kinase Cα in Bcl2 phosphorylation and suppression of apoptosis. J Biol Chem. 1998; 273: 25436–25442.
- ↵Majumder PK, Pandey P, Sun X, Cheng K, Datta R, Saxena S, Kharbanda S, Kufe D. Mitochondrial translocation of protein kinase C δ in phorbol ester-induced cytochrome c release and apoptosis. J Biol Chem. 2000; 275: 21793–21796.
- ↵Fryer RM, Wang Y, Hsu AK, Gross GJ. Essential activation of PKC-δ in opioid-initiated cardioprotection. Am J Physiol. 2001; 280: H1346–H1353.
- ↵Wang Y, Ashraf M. Role of protein kinase C in mitochondrial KATP channel-mediated protection against Ca2+ overload injury in rat myocardium. Circ Res. 1999; 84: 1156–1165.
- ↵Kharbanda S, Saxena S, Yoshida K, Pandey P, Kaneki M, Wang Q, Cheng K, Chen YN, Campbell A, Sudha T, Yuan ZM, Narula J, Weichselbaum R, Nalin C, Kufe D. Translocation of SAPK/JNK to mitochondria and interaction with Bcl-xL in response to DNA damage. J Biol Chem. 2000; 275: 322–327.
- ↵Deng X, Ruvolo P, Carr B, May WS Jr. Survival function of ERK1/2 as IL-3-activated, staurosporine-resistant Bcl2 kinases. Proc Natl Acad Sci U S A. 2000; 97: 1578–1583.
- ↵Zhang J, Bolli R, Lalli J, Tang X-L, Li RCX, Zheng Y, Pass J, Ping P. Ischemic preconditioning and phorbol ester redistribute protein kinase C ε to the nucleus, sarcolemmal membranes, and mitochondria in rabbit myocardium. Circulation. 1999; 100: I-325.Abstract.
- ↵Vallentin A, Lo TC, Joubert D. A single point mutation in the V3 region affects protein kinase Cα targeting and accumulation at cell-cell contacts. Mol Cell Biol. 2001; 21: 3351–3363.
- ↵Song C, Vondriska TM, Wang GW, Klein JB, Cao X, Zhang J, Kang YJ, D’Souza S, Ping P. Molecular conformation dictates signaling module formation: example of PKCε and Src tyrosine kinase. Am J Physiol. 2002; 282: H1166–H1171.
- ↵Paumelle R, Tulasne D, Leroy C, Coll J, Vandenbunder B, Fafeur V. Sequential activation of ERK and repression of JNK by scatter factor/hepatocyte growth factor in madin-darby canine kidney epithelial cells. Mol Biol Cell. 2000; 11: 3751–3763.
- ↵Mocanu MM, Baxter GF, Yue Y, Critz SD, Yellon DM. The p38 MAPK inhibitor, SB203580, abrogates ischaemic preconditioning in rat heart but timing of administration is critical. Basic Res Cardiol. 2000; 95: 472–478.
- ↵Jiang Y, Chen C, Li Z, Guo W, Gegner JA, Lin S, Han J. Characterization of the structure and function of a new mitogen-activated protein kinase (p38β). J Biol Chem. 1996; 271: 17920–17926.