Published online before print February 7, 2002, doi: 10.1161/01.RES.0000012702.90501.8D
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© 2002 American Heart Association, Inc.
Molecular Medicine |
Mitochondrial PKC
and MAPK Form Signaling Modules in the Murine Heart
Enhanced Mitochondrial PKC-MAPK Interactions and Differential MAPK Activation in PKC-Induced Cardioprotection
From the Department of Physiology and Biophysics and the Department of Medicine/Division of Cardiology, University of Louisville, and the Jewish Hospital Heart and Lung Institute, Louisville, Ky.
Correspondence to Peipei Ping, PhD, Department of Physiology and Biophysics and Division of Cardiology, Suite 122, Baxter Building, 570 S Preston St, Louisville, KY 40202. E-mail ping{at}ntr.net or peipeiping@hotmail.com
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Abstract |
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Although activation of protein kinase C (PKC)
and mitogen-activated protein kinases (MAPKs) are known to play crucial roles in the manifestation of cardioprotection, the spatial organization of PKC signaling modules in naïve and protected myocardium remains unknown. Based on evidence that mitochondria are key mediators of the cardioprotective signal, we hypothesized that PKC and MAPKs interact, and that they form functional signaling modules in mitochondria during cardioprotection. Both immunoblotting and immunofluorescent staining demonstrated that PKC, ERKs, JNKs, and p38 MAPK co-localized with cardiac mitochondria. Moreover, transgenic activation of PKC greatly increased mitochondrial PKC expression and activity, which was concomitant with increased mitochondrial interaction of PKC with ERKs, JNKs, and p38 as determined by co-immunoprecipitation. These complex formations appeared to be independent of PKC activity, as the interactions were also observed in mice expressing inactive PKC. However, although both active and inactive PKC bound to all three MAPKs, increased phosphorylation of mitochondrial ERKs was only observed in mice expressing active PKC but not in mice expressing inactive PKC. Examination of potential downstream targets of mitochondrial PKC-ERK signaling modules revealed that phosphorylation of the pro-apoptotic protein Bad was elevated in mitochondria. Together, these data show that PKC forms subcellular-targeted signaling modules with ERKs, leading to the activation of mitochondrial ERKs. Furthermore, formation of mitochondrial PKC-ERK modules appears to play a role in PKC-mediated cardioprotection, in part by the phosphorylation and inactivation of Bad.
Key Words: mitochondria • protein-protein interactions • functional proteomics • signaling modules • cardioprotection
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Introduction |
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Protein kinase C
(PKC) 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 PKC in response to cardioprotective stimuli.3–5 Furthermore, genetic cardiac expression of active PKC6 or a specific activator7 of PKC confers a chronically cardioprotected phenotype in mice. However, the molecular signaling events that underlie PKC-induced cardioprotection are still poorly understood.
An essential feature of the activation of PKC during ischemic preconditioning is its subcellular redistribution.3,4 This translocation to specific subcellular compartments is thought to be an important mechanism for PKC 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 PKC and this organelle. However, whether the signal initiated by PKC is integrated and transmitted to the mitochondria remains unknown, and specific mitochondrial targets of PKC 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 PKC-mediated cardioprotection. We and others have shown in rabbits and rats that activation of PKC 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 PKC-mediated cardioprotection involves the activation of MAPKs at the mitochondrial level and, conversely, whether inhibition of PKC 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 PKC 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 PKC signaling complex in whole murine myocardial homogenates.24 Accordingly, we postulated that PKC forms signaling modules with, and modulates the activity of, individual MAPKs at the level of the mitochondria, and that the formation of these mitochondrial PKC-MAPK modules participates in PKC-mediated cardioprotection.
The present study, therefore, was designed to comprehensively investigate the development of PKC-MAPK signaling modules in mitochondria during cardioprotection. Specifically, we determined in the murine heart whether PKC and individual MAPKs are expressed and co-localized in cardiac mitochondria and, if so, whether activation of PKC modulates the activity of individual MAPKs (ie, whether they form signaling modules) in this organelle. To determine whether activation of PKC plays a necessary role in the formation of PKC-MAPK modules, these complexes were examined during both activation and inhibition of PKC. We found that transgenic activation of PKC enhances mitochondrial co-localization of PKC with ERKs, JNKs, and p38, concomitantly with increased phosphorylation activity of both ERKs and p38 and that formation of PKC-ERK modules was blocked by inhibition of PKC. Furthermore, PKC-ERK module formation was associated with enhanced phosphorylation of the pro-apoptotic protein Bad.
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Materials and Methods |
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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.
Antibodies
The sources of antibodies were as follows: anti-PKC, ERK1, and p38 MAPK, Transduction Laboratories; anti-PKC 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 PKC Transgenic Mice
Cardiac-targeted PKC transgenic mice were developed as previously described.24,25 Briefly, the 
MHC promoter was used to drive the cardiac-specific expression of either an active (A159E; AE) or a dominant-negative (K436R and A159E; DN) PKC mutant in ICR strain mice. The AE line exhibits moderate increases in total PKC activity whereas the DN line has reduced basal PKC 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.
Western Blotting
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).
Co-Immunoprecipitation
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.
PKC Activity Assay
Phosphotransferase activity of PKC was determined as described previously.4,23,25 Briefly, mitochondrial samples (150 µg) were immunoprecipitated overnight with anti-PKC monoclonal antibody and the precipitates incubated with the PKC-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 PKC 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.
Statistical Analysis
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.
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Results |
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PKC
and MAPKs Co-Localize in Cardiac MitochondriaWe first determined whether PKC
and MAPK are present in cardiac mitochondria. Immunoblotting of cardiac mitochondrial extracts from nontransgenic (NTG) mice demonstrated the expression of PKC, 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 PKC, we performed immunofluorescent confocal microscopy on murine cardiac mitochondria. Staining for PKC, along with the mitochondrial marker prohibitin-1, demonstrated the association of PKC with mitochondrial structures (Figure 2). Together, these data demonstrate that PKC and the three major MAPKs co-localize with cardiac mitochondria.
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Mitochondrial PKC Expression and Activation Are Increased in PKC Transgenic Mice
We next examined mitochondrial PKC levels and phosphotransferase activity in the constitutively active (AE) and dominant-negative (DN) PKC 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 PKC in NTG, AE, and DN mouse hearts. There was an 16-fold increase in total PKC levels in the AE line and an 24-fold increase in the DN line when compared with controls. In the AE hearts, cytosolic PKC expression was 13.0±0.4-fold greater than in NTG littermates (data not shown), but mitochondrial PKC expression was elevated to a greater extent (26.5±1.6-fold; Figure 3B). This was confirmed by immunohistochemical staining of mitochondria for PKC, which revealed a much more intense co-localization of PKC with mitochondria in the AE mice when compared with NTG mice (Figure 2). The differential increase in cytosolic and mitochondrial PKC 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 PKC expression is preferentially increased over cytosolic expression in both the AE and DN lines, suggesting that the open configuration of PKC 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).
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Mitochondrial PKC activity was assessed by the phosphorylation of a PKC-specific peptide following immunoprecipitation. The activity of PKC was significantly increased in mitochondrial fractions of hearts from the AE mice (Figure 3C). In contrast, there was a significant decrease in basal PKC activity in the mitochondria of DN hearts compared with NTG hearts.
Transgenic Activation of PKC Enhances Mitochondrial PKC-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 PKC complex24 and since we observed mitochondrial co-localization of PKC and MAPKs, we examined whether PKC forms complexes with individual MAPKs at the mitochondrion. Mitochondrial lysates were immunoprecipitated with MAPK antibodies (Figure 4A) and blotted for PKC (Figure 4B). Mitochondrial PKC was found to interact with ERKs, JNKs, and p38 MAPK in both nontransgenic and transgenic hearts. Moreover, in AE hearts, the amount of PKC co-precipitating with the mitochondrial MAPKs was increased strikingly. Interestingly, PKC-MAPK interactions were also increased in the cytosolic fraction of AE mice but to a much lesser extent (Figure 4C). These data show that PKC can form signaling complexes with individual MAPKs in cardiac mitochondria. Furthermore, the increased interaction appears to be due to increased PKC protein rather than activity, as PKC still bound MAPKs in DN mitochondria (Figure 4B).
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PKC Activates Mitochondrial ERKs and p38 MAPK via Kinase Activity–Dependent and –Independent Pathways
Activation of PKC has been previously reported to stimulate ERKs and JNKs in cardiac cells.15–17,26 Consequently, we assessed the effect of enhanced mitochondrial PKC 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, PKC-induced activation of mitochondrial p38 does not require PKC activity. Importantly, these data suggest that ERKs, but not p38, contribute to the cardioprotective phenotype in these mice.
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Phosphorylation of Bad Is Elevated in PKC Transgenic Mice
We next explored mitochondrial targets of PKC 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-PKC transgenic mice.
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Discussion |
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Many individual signaling elements such as PKC
, 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 PKC enhances mitochondrial co-localization of PKC with MAPKs, increases phosphorylation of mitochondrial MAPKs, and promotes the formation of mitochondrial PKC-MAPK signaling modules. This dynamic modulation of the PKC-MAPK modules is associated with the inhibition of pro-apoptotic molecules and the genesis of a cardioprotective phenotype.
PKC 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 PKC and MAPKs have been studied in other compartments,5,15–17,30 there is a paucity of data regarding the co-existence of PKC and MAPKs isoforms in either cardiac or noncardiac mitochondria. Cellular fractionation has shown that PKC, 
, 
, and 
can associate with mitochondria in cultured cells31,32 and immunohistochemical studies indicate that PKC and PKC may affiliate with cardiac mitochondria in the rat.33,34 Nevertheless, no previous studies have examined the formation of PKC signaling modules in mitochondria. In the present study, using both biochemical and immunohistochemical analyses, we demonstrate that PKC 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 PKC 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 PKC to the mitochondria.37 Therefore, upregulation of mitochondrial PKC 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 PKC confers resistance to ischemia/reperfusion.6 Mutation of Ala159 to Glu (AE) in the pseudosubstrate region of PKC 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 PKC pools were increased when compared with controls, the increase in mitochondrial PKC was 2-fold greater than that in the cytosol. This was confirmed by confocal microscopy, showing an increased intensity of PKC staining in AE mitochondria. Interestingly, the confocal images revealed that in NTG hearts PKC is concentrated in the periphery of the mitochondria, whereas in AE hearts PKC also appears in the matrix. The reason for this is unknown. We speculate that placing PKC in its open conformation may have facilitated its uptake into the matrix. The mitochondrial redistribution of PKC does not appear to be dependent on kinase activity, as a similar relocalization of PKC to the mitochondrial fraction was observed in DN mice. Since DN-PKC possesses the same Ala159 mutation as the AE mutant but with an additional kinase-inactivating mutation, the open conformation of PKC 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 PKC 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 PKC.
Mitochondrial PKC 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, PKC interacts with and phosphorylates the gap junction protein connexin-43 in response to fibroblast growth factor,30 whereas in rabbits enhanced interaction of PKC 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 PKC signaling complex.24 The present investigation presents strong evidence that the mitochondrion is one of the primary locations where PKC-MAPK modules engage in signal transduction.
Immunoprecipitation of ERKs, JNKs, or p38 pulled down PKC from cardiac mitochondrial lysates, demonstrating that PKC 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 PKC and MAPKs throughout the cell. As expected, however, the level of PKC association with ERKs, JNKs, and p38 was significantly upregulated in the AE-PKC mice. In particular, the amount of PKC co-precipitating was greater in the mitochondrial than the cytosolic fraction. This interaction does not require catalytic activity as the DN-PKC was just as able to bind MAPKs. Therefore, while the physical interaction of PKC with MAPKs may be necessary for cardioprotection, it is not sufficient. Placing PKC in the open conformation enhances its ability to bind to Src40 implicating the configuration of PKC as a critical determinant of its capacity to interact with proteins. This supports the conclusion that the enhanced interaction of PKC with MAPKs seen in both the AE and DN-PKC mice is due to the fact that both PKC mutants are in the open conformation.
Mitochondrial PKC-MAPK Complex Formation Induces Differential Activation of MAPKs
The formation of PKC-MAPK signaling modules implies a functional coupling between mitochondrial PKC and MAPKs. We found marked increases in mitochondrial MAPK phosphorylation with respect to ERKs and p38 MAPK in the AE-PKC mice whereas JNK phosphorylation did not change, even though functional coupling between PKC 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 PKC activation and, therefore, may involve transcriptional as well as post-translation alterations.
To confirm that phosphorylation of mitochondrial ERKs and p38 in the AE-PKC mice was PKC activity-dependent, we also examined the MAPK phosphorylation status in the DN-PKC mice. Dual phosphorylation of ERK was not different from controls in the DN-PKC hearts, confirming that mitochondrial PKC activity is required for the phosphorylation of mitochondrial ERKs. However, p38 phosphorylation remains increased in the DN-PKC mice, indicating that phosphorylation of p38 was PKC 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 PKC activity, which was not detected using the commercially available phospho-p38 antibodies. Future studies are needed to discern the role of PKC modules with respect to individual p38 isoforms.
Upregulation of Mitochondrial PKC-ERK Modules Induces Phosphorylation of Bad
There are multiple mitochondrial proteins that could be targets of PKC-ERK modules in the mitochondria, including components of the mitochondrial apoptotic machinery.27,28,36 In particular, activation of both PKC 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 PKC-ERK was associated with phosphorylation of Bad in the AE-PKC transgenic mice but not in the DN-PKC animals. Therefore, a cascade consisting of PKC-ERK-Bad exists at the level of the cardiac mitochondrion. This functional coupling between the PKC-ERK signaling complex and Bad may contribute to cardioprotective actions of PKC.
In summary, PKC 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 PKC activity-dependent mechanism. Furthermore, formation of mitochondrial PKC-ERK modules is coupled to the inactivation of Bad and may contribute to PKC-mediated cardioprotection. Delineation of other protein-protein interactions within mitochondrial PKC-MAPK signaling modules and downstream targets will hopefully provide greater insight into the molecular machinery underlying cardioprotection.
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Acknowledgments |
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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.
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Footnotes |
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This manuscript was sent to Eugene Braunwald, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Received December 10, 2001; revision received January 24, 2002; accepted January 30, 2002.
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C. Ballard-Croft, G. Kristo, Y. Yoshimura, E. Reid, B. J. Keith, R. M. Mentzer Jr., and R. D. Lasley Acute adenosine preconditioning is mediated by p38 MAPK activation in discrete subcellular compartments Am J Physiol Heart Circ Physiol, March 1, 2005; 288(3): H1359 - H1366. [Abstract] [Full Text] [PDF]
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J. Zhang, C. P. Baines, C. Zong, E. M. Cardwell, G. Wang, T. M. Vondriska, and P. Ping Functional proteomic analysis of a three-tier PKC{varepsilon}-Akt-eNOS signaling module in cardiac protection Am J Physiol Heart Circ Physiol, February 1, 2005; 288(2): H954 - H961. [Abstract] [Full Text] [PDF]
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J. J. Martindale, J. A. Wall, D. M. Martinez-Longoria, P. Aryal, H. A. Rockman, Y. Guo, R. Bolli, and C. C. Glembotski Overexpression of Mitogen-activated Protein Kinase Kinase 6 in the Heart Improves Functional Recovery from Ischemia in Vitro and Protects against Myocardial Infarction in Vivo J. Biol. Chem., January 7, 2005; 280(1): 669 - 676. [Abstract] [Full Text] [PDF]
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 J. M. V. Mammen, J. C. Song, J. Yoo, P. S. Kim, H. W. Davis, M. I. Calvo, R. T. Worrell, K. S. Matlin, and J. B. Matthews Differential subcellular targeting of PKC-{epsilon} in response to pharmacological or ischemic stimuli in intestinal epithelia Am J Physiol Gastrointest Liver Physiol, January 1, 2005; 288(1): G135 - G142. [Abstract] [Full Text] [PDF]
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 D. H. Korzick, J. C. Hunter, M. K. McDowell, M. D. Delp, M. M. Tickerhoof, and L. D. Carson Chronic Exercise Improves Myocardial Inotropic Reserve Capacity Through {alpha}1-Adrenergic and Protein Kinase C-Dependent Effects in Senescent Rats J. Gerontol. A Biol. Sci. Med. Sci., November 1, 2004; 59(11): 1089 - 1098. [Abstract] [Full Text] [PDF]
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X. Wang, C. Yin, L. Xi, and R. C. Kukreja Opening of Ca2+-activated K+ channels triggers early and delayed preconditioning against I/R injury independent of NOS in mice Am J Physiol Heart Circ Physiol, November 1, 2004; 287(5): H2070 - H2077. [Abstract] [Full Text] [PDF]
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M. Zaugg and M. C. Schaub Cellular mechanisms in sympatho-modulation of the heart Br. J. Anaesth., July 1, 2004; 93(1): 34 - 52. [Abstract] [Full Text] [PDF]
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 G. G. Corbucci, C. Perrino, G. Donato, A. Ricchi, B. Lettieri, G. Troncone, C. Indolfi, M. Chiariello, and E. V. Avvedimento Transient and reversible deoxyribonucleic acid damage in human left ventricle under controlled ischemia and reperfusion J. Am. Coll. Cardiol., June 2, 2004; 43(11): 1992 - 1999. [Abstract] [Full Text] [PDF]
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V. U. Rao, H. Shiraishi, and P. J. McDermott PKC-{epsilon} regulation of extracellular signal-regulated kinase: a potential role in phenylephrine-induced cardiocyte growth Am J Physiol Heart Circ Physiol, June 1, 2004; 286(6): H2195 - H2203. [Abstract] [Full Text] [PDF]
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H. Tong, H. A. Rockman, W. J. Koch, C. Steenbergen, and E. Murphy G Protein-Coupled Receptor Internalization Signaling Is Required for Cardioprotection in Ischemic Preconditioning Circ. Res., April 30, 2004; 94(8): 1133 - 1141. [Abstract] [Full Text] [PDF]
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D. J. Lips, O. F. Bueno, B. J. Wilkins, N. H. Purcell, R. A. Kaiser, J. N. Lorenz, L. Voisin, M. K. Saba-El-Leil, S. Meloche, J. Pouyssegur, et al. MEK1-ERK2 Signaling Pathway Protects Myocardium From Ischemic Injury In Vivo Circulation, April 27, 2004; 109(16): 1938 - 1941. [Abstract] [Full Text] [PDF]
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R. A. Kaiser, O. F. Bueno, D. J. Lips, P. A. Doevendans, F. Jones, T. F. Kimball, and J. D. Molkentin Targeted Inhibition of p38 Mitogen-activated Protein Kinase Antagonizes Cardiac Injury and Cell Death Following Ischemia-Reperfusion in Vivo J. Biol. Chem., April 9, 2004; 279(15): 15524 - 15530. [Abstract] [Full Text] [PDF]
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P. Rosini, G. De Chiara, P. Bonini, M. Lucibello, M. E. Marcocci, E. Garaci, F. Cozzolino, and M. Torcia Nerve Growth Factor-dependent Survival of CESS B Cell Line Is Mediated by Increased Expression and Decreased Degradation of MAPK Phosphatase 1 J. Biol. Chem., April 2, 2004; 279(14): 14016 - 14023. [Abstract] [Full Text] [PDF]
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 J. Feng, C. Bianchi, J. Li, and F. W. Sellke Improved profile of bad phosphorylation and caspase 3 activation after blood versus crystalloid cardioplegia Ann. Thorac. Surg., April 1, 2004; 77(4): 1384 - 1389. [Abstract] [Full Text] [PDF]
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 Y. Takagi, J. Du, X.-Y. Ma, I. Nakashima, and F. Nagase Phorbol 12-Myristate 13-Acetate Protects Jurkat Cells from Methylglyoxal-Induced Apoptosis by Preventing c-Jun N-Terminal Kinase-Mediated Leakage of Cytochrome c in an Extracellular Signal-Regulated Kinase-Dependent Manner Mol. Pharmacol., March 1, 2004; 65(3): 778 - 787. [Abstract] [Full Text] [PDF]
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A. P Halestrap, S. J Clarke, and S. A Javadov Mitochondrial permeability transition pore opening during myocardial reperfusion--a target for cardioprotection Cardiovasc Res, February 15, 2004; 61(3): 372 - 385. [Abstract] [Full Text] [PDF]
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F. Eefting, B. Rensing, J. Wigman, W. J. Pannekoek, W. M. Liu, M. J. Cramer, D. J Lips, and P. A Doevendans Role of apoptosis in reperfusion injury Cardiovasc Res, February 15, 2004; 61(3): 414 - 426. [Abstract] [Full Text] [PDF]
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G. Nowak, D. Bakajsova, and G. L. Clifton Protein kinase C-{epsilon} modulates mitochondrial function and active Na+ transport after oxidant injury in renal cells Am J Physiol Renal Physiol, February 1, 2004; 286(2): F307 - F316. [Abstract] [Full Text] [PDF]
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E. Murphy Primary and Secondary Signaling Pathways in Early Preconditioning That Converge on the Mitochondria to Produce Cardioprotection Circ. Res., January 9, 2004; 94(1): 7 - 16. [Abstract] [Full Text] [PDF]
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M. Joyeux-Faure, C. Arnaud, D. Godin-Ribuot, and C. Ribuot Heat stress preconditioning and delayed myocardial protection: what is new? Cardiovasc Res, December 1, 2003; 60(3): 469 - 477. [Abstract] [Full Text] [PDF]
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B. P. S. Kang, A. Urbonas, A. Baddoo, S. Baskin, A. Malhotra, and L. G. Meggs IGF-1 inhibits the mitochondrial apoptosis program in mesangial cells exposed to high glucose Am J Physiol Renal Physiol, November 1, 2003; 285(5): F1013 - F1024. [Abstract] [Full Text] [PDF]
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P. Ping Identification of Novel Signaling Complexes by Functional Proteomics Circ. Res., October 3, 2003; 93(7): 595 - 603. [Abstract] [Full Text] [PDF]
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D. M. YELLON and J. M. DOWNEY Preconditioning the Myocardium: From Cellular Physiology to Clinical Cardiology Physiol Rev, October 1, 2003; 83(4): 1113 - 1151. [Abstract] [Full Text] [PDF]
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J. N. Weiss, P. Korge, H. M. Honda, and P. Ping Role of the Mitochondrial Permeability Transition in Myocardial Disease Circ. Res., August 22, 2003; 93(4): 292 - 301. [Abstract] [Full Text] [PDF]
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C. P. Baines, C.-X. Song, Y.-T. Zheng, G.-W. Wang, J. Zhang, O.-L. Wang, Y. Guo, R. Bolli, E. M. Cardwell, and P. Ping Protein Kinase C{epsilon} Interacts With and Inhibits the Permeability Transition Pore in Cardiac Mitochondria Circ. Res., May 2, 2003; 92(8): 873 - 880. [Abstract] [Full Text] [PDF]
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L. G. Kevin, A. K. S. Camara, M. L. Riess, E. Novalija, and D. F. Stowe Ischemic preconditioning alters real-time measure of O2 radicals in intact hearts with ischemia and reperfusion Am J Physiol Heart Circ Physiol, February 1, 2003; 284(2): H566 - H574. [Abstract] [Full Text] [PDF]
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C. Steenbergen, C. A. Afshari, J. G. Petranka, J. Collins, K. Martin, L. Bennett, A. Haugen, P. Bushel, and E. Murphy Alterations in apoptotic signaling in human idiopathic cardiomyopathic hearts in failure Am J Physiol Heart Circ Physiol, January 1, 2003; 284(1): H268 - H276. [Abstract] [Full Text] [PDF]
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K. A. Detillieux, F. Sheikh, E. Kardami, and P. A. Cattini Biological activities of fibroblast growth factor-2 in the adult myocardium Cardiovasc Res, January 1, 2003; 57(1): 8 - 19. [Abstract] [Full Text] [PDF]
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O. F. Bueno and J. D. Molkentin Involvement of Extracellular Signal-Regulated Kinases 1/2 in Cardiac Hypertrophy and Cell Death Circ. Res., November 1, 2002; 91(9): 776 - 781. [Abstract] [Full Text] [PDF]
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U. Schwanke, I. Konietzka, A. Duschin, X. Li, R. Schulz, and G. Heusch No ischemic preconditioning in heterozygous connexin43-deficient mice Am J Physiol Heart Circ Physiol, October 1, 2002; 283 (4): H1740 - H1742. [Abstract] [Full Text] [PDF]
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 R. D. Edmondson, T. M. Vondriska, K. J. Biederman, J. Zhang, R. C. Jones, Y. Zheng, D. L. Allen, J. X. Xiu, E. M. Cardwell, M. R. Pisano, et al. Protein Kinase C {epsilon} Signaling Complexes Include Metabolism- and Transcription/Translation-related Proteins: Complimentary Separation Techniques With LC/MS/MS Mol. Cell. Proteomics, June 1, 2002; 1(6): 421 - 433. [Abstract] [Full Text] [PDF]
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