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Molecular Medicine |
and MAPK Form Signaling Modules in the Murine Heart
-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
| Abstract |
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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
| Introduction |
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(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.35 Furthermore, genetic cardiac expression of active PKC
6 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.1518 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.1922 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 oxideinduced 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.
| Materials and Methods |
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Antibodies
The sources of antibodies were as follows: anti-PKC
, ERK1, and p38 MAPK, Transduction Laboratories; anti-PKC
polyclonal, Sigma; antiprohibitin-1, Research Diagnostics; anti-JNK2, Santa Cruz Biotechnology; anti-JNK1, Pharmingen; and antiphospho-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.2325 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 Students t tests for unpaired data. A P value less than 0.05 was considered significant.
| Results |
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and MAPKs Co-Localize in Cardiac Mitochondria
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).
|
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 ActivityDependent and Independent Pathways
Activation of PKC
has been previously reported to stimulate ERKs and JNKs in cardiac cells.1517,26 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 Bads 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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, 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 kinases 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,5,1517,30 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.35 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 oxideinduced 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.
| Acknowledgments |
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| Footnotes |
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Received December 10, 2001; revision received January 24, 2002; accepted January 30, 2002.
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