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Am J Physiol Heart Circ Physiol 288: H954-H961, 2005. First published November 4, 2004; doi:10.1152/ajpheart.00756.2004
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Functional proteomic analysis of a three-tier PKC{varepsilon}-Akt-eNOS signaling module in cardiac protection

Jun Zhang,1 Christopher P. Baines,2 Chenggong Zong,1 Ernest M. Cardwell,1 Guangwu Wang,1 Thomas M. Vondriska,1 and Peipei Ping1

1Division of Cardiology, Departments of Physiology and Medicine, University of California at Los Angeles, Los Angeles, California; and 2Division of Molecular Cardiovascular Biology, Children’s Hospital Medical Center, Cincinnati, Ohio

Submitted 27 July 2004 ; accepted in final form 28 September 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cardiac protective signaling networks have been shown to involve PKC{varepsilon}. However, the molecular mechanisms by which PKC{varepsilon} interacts with other members of these networks to form task-specific modules remain unknown. Among 93 different PKC{varepsilon}-associated proteins that have been identified, Akt and endothelial nitric oxide (NO) synthase (eNOS) are of importance because of their independent abilities to promote cell survival and prevent cell death. The simultaneous association of PKC{varepsilon}, Akt, and eNOS has not been examined, and, in particular, the formation of a module containing these three proteins and the role of such a module in the regulation of NO production and cardiac protection are unknown. The present study was undertaken to determine whether these molecules form a signaling module and, thereby, play a collective role in cardiac signaling. Using recombinant proteins in vitro and PKC{varepsilon} transgenic mouse hearts, we demonstrate the following: 1) PKC{varepsilon}, Akt, and eNOS interact and form signaling modules in vitro and in the mouse heart. Activation of either PKC{varepsilon} or Akt enhances the formation of PKC{varepsilon}-Akt-eNOS signaling modules. 2) PKC{varepsilon} directly phosphorylates and enhances activation of Akt in vitro, and PKC{varepsilon} activation increases phosphorylation and activation of Akt in PKC{varepsilon} transgenic mouse hearts. 3) PKC{varepsilon} directly phosphorylates eNOS in vitro, and this phosphorylation enhances eNOS activity. Activation of PKC{varepsilon} in vivo increased phosphorylation of eNOS at Ser1177, indicating eNOS activation. This study characterizes, for the first time, the physical, as well as functional, coupling of PKC{varepsilon}, Akt, and eNOS in the heart and implicates these PKC{varepsilon}-Akt-eNOS signaling modules as critical signaling elements during PKC{varepsilon}-induced cardiac protection.

signaling network; protein-protein interactions; phosphorylation; cardioprotection; preconditioning; proteomics

INTRACELLULAR SIGNALING NETWORKS are composed of protein modules specified to execute distinct tasks (5, 17, 35). In the heart, complex cellular phenotypes, such as resistance to ischemic cell death, involve activation of various signaling molecules. Despite this information, the manner in which the actions of these individual proteins are integrated into functional modules has only begun to be understood. Numerous studies have demonstrated modules composed of two molecules; however, the next level of hierarchical structure, i.e., a three-tier module, remains scarcely defined. Although "three-tier" modules have been described in certain canonical signaling responses (e.g., MAPKs), there is a paucity of investigations demonstrating simultaneous association of three molecules as a generalized organizational pattern to recruit proteins with disparate functions together for shared signaling actions. The present study was designed to address this limitation of our understanding.

Protein kinase C{varepsilon} (PKC{varepsilon}) has been well documented to play an important role in the genesis of cardioprotection (11, 22). In particular, previous studies from our laboratory have shown that activation of PKC{varepsilon} in the heart is sufficient to significantly reduce myocardial infarction due to coronary artery occlusion (26, 27). Moreover, Akt/protein kinase B (PKB) (14, 15, 32) and endothelial nitric oxide (NO) synthase (eNOS) (31) have been independently implicated as protective molecules in the setting of oxidative stress and ischemic injury to the myocardium. As described above, however, the information gained about these molecules through previous investigations was acquired in isolation [e.g., transgenic activation of PKC{varepsilon} is sufficient to reduce infarct size (26)] or in a binary sense [e.g., regulation of NO production through eNOS by Akt (3, 15, 34)]. To our knowledge, no previous investigations examined the possibility that these three molecules, PKC{varepsilon}, Akt, and eNOS, together constitute a module, the assembly of which is a critical mechanism of protective signaling in the heart. In the present study, we took a combined proteomic and biochemical approach to characterize native protein complexes containing PKC{varepsilon}, Akt, and eNOS. We examined PKC{varepsilon}-Akt-eNOS signaling modules in vitro and in the mouse heart in terms of molecular architecture (i.e., protein-protein interactions) and signal transduction (i.e., posttranslational modification and alteration of enzymatic activity). Our findings indicate formation of a three-tier module in the heart and suggest that such signaling units, similar to that formed by PKC{varepsilon}, Akt, and eNOS, may represent a mechanism utilized to manipulate multipurpose signaling proteins to carry out distinct tasks in the myocyte.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
All procedures were performed in accordance with the Animal Research Committee guidelines at the University of California, Los Angeles, and the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health.

Materials. Recombinant active Akt-1, nonactive Akt-1, and pleckstrin homology (PH) domain of Akt-1, and PH domain-deleted Akt-1 fusion proteins corresponding to human Akt-1/PKB-{alpha} were purchased from Upstate Biotechnology (Lake Placid, NY). Recombinant human PKC{varepsilon} and bovine eNOS were purchased from Biomol (Plymouth Meeting, PA) and Calbiochem (San Diego, CA), respectively. Anti-Akt-1 polyclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PKC{varepsilon} and anti-eNOS monoclonal antibodies were purchased from BD Pharmingen (San Diego, CA). Anti-phospho-Akt (Thr308 and Ser473), anti-phospho-eNOS (Ser1177), and anti-phosphoglycogen synthase kinase (GSK)-3{beta} (Ser21/9) monoclonal antibodies and Akt kinase assay kit were obtained from Cell Signaling Technology (Beverly, MA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

PKC{varepsilon} transgenic mice. The transgenic mice with cardiac-specific activation of PKC{varepsilon} used in this study exhibit ~6.2-fold overexpression of PKC{varepsilon} and have been previously described (26). Transgenic mice and their nontransgenic littermates were used at 9–12 wk of age.

Immunoprecipitation. Immunoprecipitation was performed as previously described (2, 12, 26–28, 33, 36). Briefly, covalently cross-linked PKC{varepsilon} monoclonal antibodies were incubated with protein samples overnight at 4°C. Immunocomplexes were then washed three times with buffer containing 150 mM NaCl, 20 mM Tris·HCl (pH 7.4), 10 mM EDTA, 1% (vol/vol) Nonidet P-40 (NP-40), 1 mM Na3VO4, and a protease inhibitor cocktail (Roche, Indianapolis, IN). After the final wash, the protein sample was eluted from the beads by resuspension in Laemmli buffer, boiled, and then subjected to SDS-PAGE.

Immunoblotting. Standard protocols were applied for immunoblotting (2, 26–28, 36). Briefly, after SDS-PAGE separation, proteins were transferred to nitrocellulose membranes and blotted in 5% milk or 5% bovine serum albumin (BSA) for phosphospecific antibodies in Tris-buffered saline supplemented with 0.5% Tween 20 (TBS-T: 10 mM Tris·HCl, pH 7.5, 100 mM NaCl, and 0.5% Tween 20). The membranes were then immunoblotted using the enhanced chemiluminescence detection system (Amersham, Piscataway, NJ).

Gel filtration chromatography. Gel filtration chromatography was carried out as described elsewhere (8) with a few modifications. Two mouse hearts were homogenized in buffer A containing 20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 150 mM NaCl, 0.2 mM EDTA, 0.5% (vol/vol) NP-40, and a cocktail of protease inhibitors. The samples were dialyzed overnight at 4°C in buffer A without NP-40 and clarified before chromatography. The samples were then loaded onto a precalibrated Sephacryl S-400 column (26 mm diameter, 70 cm length; XK 26/70, Amersham) using buffer A without NP-40 as running buffer, and 10-ml fractions were collected. The proteins were subjected to PKC{varepsilon} immunoprecipitation followed by Akt and eNOS immunoblottings. Thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), and aldolase (158 kDa; Amersham) were used as molecular standards.

Affinity pull-down assay. Glutathione S-transferase (GST) affinity pull-down assays were performed as previously reported (26, 28, 36). Briefly, functionally viable GST-PKC{varepsilon} recombinant protein was generated using the baculovirus system (BD Pharmingen) and incubated with recombinant Akt or eNOS proteins in binding buffer containing 0.5% (vol/vol) Triton X-100, 20 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, and a cocktail of protease inhibitors overnight at 4°C. The beads were washed three times, and the proteins were eluted with glutathione elution buffer (BD Pharmingen).

Phosphorylation of Akt or eNOS by PKC{varepsilon}. Recombinant Akt or eNOS was incubated with recombinant PKC{varepsilon} in reaction buffer containing 0.03 mg/ml L-{alpha}-phosphatidyl-L-serine (PS), 2.5 µg/ml phorbol 12-myristate 13-acetate (PMA), 3.5 mM dithiothreitol, 100 µM ATP, 6.5 mM MgCl2, 50 mM Tris·HCl, pH 7.5, and 0.2 µCi of [{gamma}-32P]ATP (for in vitro phosphorylation assay) at 30°C for 30 min (33). The reaction was terminated by addition of Laemmli buffer and boiling for 5 min. The proteins were then separated by SDS-PAGE, and the phosphorylation signal was detected by autoradiography.

Akt activity assay. Akt kinase activity was measured using a kit from Cell Signaling Technology according to the manufacturer’s instructions. Briefly, Akt immunocomplexes were incubated with 1 µg of GSK-3{beta} fusion protein in the presence of ATP in buffer containing 25 mM Tris·HCl (pH 7.5), 5 mM {beta}-glycerolphosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, and 10 mM MgCl2 at 30°C for 30 min. Phosphorylation of GSK-3{beta} was measured by Western blotting using a phospho-GSK-3{beta} (Ser21/9) antibody.

eNOS activity assay. eNOS activity was measured as NO2/NO3 production using a colorimetric assay kit from Calbiochem. Briefly, recombinant eNOS, in the presence and absence of PKC{varepsilon}, was incubated in buffer containing 1 mM NADPH, 10 mM arginine, 1 mM CaCl2, 3 µM tetrahydrobiopterin, 1 µM flavin adenine dinucleotide, 1 µM flavin adenine mononucleotide, and 25 mM Tris·HCl (pH 7.4) for 30 min at 30°C. The resulting nitrate was converted to nitrite with nitrate reductase treatment. Total nitrite was measured by the Griess method and quantified by a Wallace 1420 multilabel counter (16).

Statistical analysis. Values are means ± SE. Differences among the experimental groups were analyzed using one-way ANOVA. If the ANOVA showed an overall significance, post hoc contrasts were performed with Student’s t-test (27, 37). P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Formation of PKC{varepsilon}-Akt-eNOS modules in vitro. Formation of this three-tier module was initially examined using recombinant proteins in vitro.

We first determined the ability of these three proteins to directly interact in a binary fashion. GST-PKC{varepsilon} fusion proteins were incubated with recombinant Akt or eNOS, GST pull-down was performed, and the products were separated by SDS-PAGE and subjected to Western blotting for Akt or eNOS. Figure 1 demonstrates direct interaction between PKC{varepsilon} and Akt in vitro. Two further observations regarding this interaction warrant remark. 1) GST-PKC{varepsilon} preferentially interacted with recombinant active Akt (130.0 ± 2.3% of nonactive Akt) vs. nonactive Akt with no treatment (Fig. 1A). 2). Recombinant PH-deleted Akt and Akt PH domains were used to demonstrate that the interaction between PKC{varepsilon} and Akt occurred via the PH domain of Akt (Fig. 1B). Interaction of Akt and eNOS is well established and was also observed in this study (data not shown).



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  		Fig. 1. PKC{varepsilon} directly interacts with Akt and endothelial nitric oxide (NO) synthase (eNOS) in vitro. Glutathione S-transferase (GST)-PKC{varepsilon} was incubated with recombinant Akt or eNOS, subjected to SDS-PAGE, and immunoblotted for Akt or eNOS. A: GST-PKC{varepsilon} favors interaction with active (lanes 1 and 2), compared with nonactive (lanes 3 and 4), Akt. GST without insert proteins (GST-null) exhibited minimal interactions with active or nonactive Akt (lanes 6 and 7, respectively). B: GST-PKC{varepsilon} favors interaction with the Akt pleckstrin homology (PH) domain (lanes 3 and 4) compared with PH domain-deleted Akt (Akt{Delta}PH; lanes 1 and 2). Lanes 6 and 7, positive controls (direct loading of recombinant proteins) for Akt{Delta}PH and Akt PH domain, respectively. C: GST-PKC{varepsilon} directly interacts with eNOS (lanes 1 and 2). GST without insert proteins exhibited minimal interactions with eNOS (lane 4). Results are representative of ≥3 independent experiments. +, Presence of a protein; –, absence of protein.

 
Next, we investigated the formation of this module when all three components were present. Akt and eNOS were incubated in the presence or absence of PKC{varepsilon}, and the sample was subjected to immunoprecipitation for eNOS and immunoblotting for Akt. The data indicate that addition of PKC{varepsilon} increases the interaction between eNOS and Akt (Fig. 2A). To determine the role of PKC{varepsilon} activation in its ability to interact with Akt and eNOS, these respective proteins were incubated with PKC{varepsilon} in the presence or absence of the potent PKC activators PMA and PS, and the samples were subjected to immunoprecipitation for PKC{varepsilon} and immunoblotting for Akt or eNOS. Figure 2B shows that activation of PKC{varepsilon} with PMA and PS enhances its interaction with Akt and eNOS. Similarly, to examine the effect of Akt’s activation on its interaction with PKC{varepsilon} and eNOS, these respective proteins were incubated with active or nonactive Akt recombinant protein and then subjected to immunoprecipitation and immunoblotting. Interestingly, Akt activation not only enhanced its interaction with PKC{varepsilon} (Fig. 2C, bottom), but, also, activation of Akt enhanced the interaction between PKC{varepsilon} and eNOS (Fig. 2C, top). These data suggest that activation of Akt is a key event to facilitate assembly of PKC{varepsilon}-Akt-eNOS modules.



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  		Fig. 2. Activation of PKC{varepsilon} or Akt enhances PKC{varepsilon}-Akt-eNOS module formation in vitro. A: recombinant Akt and eNOS were incubated in the absence (lane 1) or presence (lane 2) of PKC{varepsilon} and then subjected to immunoprecipitation (IP) for eNOS and immunoblotting (IB) for PKC{varepsilon} or Akt. PKC{varepsilon} is sufficient to increase interactions between eNOS and Akt. B: recombinant PKC{varepsilon} was incubated with Akt and eNOS in the absence (lane 1) or presence (lane 2) of the PKC activators phorbol 12-myristate 13-acetate (PMA) and phosphatidyl-L-serine (PS) and then subjected to immunoprecipitation for PKC{varepsilon} and immunoblotting for eNOS or Akt. Activation of PKC{varepsilon} with PMA and PS increases its affinity for Akt and eNOS. C: active (lane 2) or nonactive (lane 3) Akt recombinant proteins were incubated with PKC{varepsilon} and eNOS and then subjected to immunoprecipitation for PKC{varepsilon} and immunoblotting for Akt or eNOS. Activation of Akt increases its interaction with PKC{varepsilon} and increases interaction of PKC{varepsilon} with eNOS. Results are representative of ≥3 independent experiments, and IgG and beads-alone controls demonstrated negligible signal (data not shown). +, Presence of a protein or lipids; –, absence of protein or lipids.

 
Formation of PKC{varepsilon}-Akt-eNOS modules in the mouse heart. Having established formation of functional PKC{varepsilon}-Akt-eNOS modules in vitro, we next wanted to explore the assembly of, and signal transduction by, these modules in the heart. Previous studies showed that Akt and eNOS are present in cardiac PKC{varepsilon} signaling complexes (28). Despite this information, nothing was known regarding the nature of interactions between PKC{varepsilon} and Akt or eNOS (i.e., whether they modulate each other’s activity or posttranslational modification state), the architecture of the signaling complexes formed by these molecules, and whether the three localize in the same signaling unit in the heart.

To assess native, i.e., intact, complexes that have not been disrupted by harsh detergents or heating and, thus, maintain their endogenous interactions, mouse hearts were homogenized and separated via gel filtration chromatography. With this method, intact myocardial protein complexes are separated on the basis of their physical size. Individual gel filtration fractions were immunoprecipitated with PKC{varepsilon} antibodies to isolate the native complexes containing PKC{varepsilon} from the given elution fraction (representative of a given molecular size). This last step is essential, because although some native complexes of a given size may contain PKC{varepsilon}, other complexes of identical or similar sizes most certainly exist that do not contain PKC{varepsilon} but may coelute from the column nonetheless. The isolated PKC{varepsilon} immunocomplexes were then separated by denaturing SDS-PAGE and immunoblotted for PKC{varepsilon}, Akt, and eNOS. Figure 3A shows the Western immunoblot analysis of the chromatographic elution fractions after immunoprecipitation for PKC{varepsilon} in the wild-type nontransgenic mouse heart. The data indicate that Akt and eNOS associate with PKC{varepsilon} in multiple fractions, suggesting that these three molecules interact with each other in a variety of different-sized multiprotein complexes. Next, the identical analysis was performed using hearts from PKC{varepsilon} transgenic mice (Fig. 3B), which are inherently resistant to ischemic injury (26). We observed a shift toward a higher molecular weight (i.e., a lower elution fraction) in expression patterns of PKC{varepsilon}-Akt-eNOS signaling modules from PKC{varepsilon} transgenic mice compared with those from nontransgenic mouse hearts, indicating that, during protection, PKC{varepsilon}-Akt-eNOS signaling modules are assembled within native complexes of greater molecular size.



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  		Fig. 3. Formation of PKC{varepsilon}-Akt-eNOS signaling modules in vivo. Nontransgenic (A) or PKC{varepsilon} transgenic (B) mouse hearts were homogenized and subjected to gel filtration chromatography using a Sephacryl S400 column and then subjected to immunoprecipitation for PKC{varepsilon} and immunoblotting for PKC{varepsilon}, Akt, or eNOS. Fraction numbers are indicated above blots, with larger numbers indicative of later elution fractions and, therefore, smaller molecular masses (MW). Dashed lines, areas of salient correlation in protein content of PKC{varepsilon}, Akt, and eNOS. Cardiac protection is associated with formation of larger complexes containing PKC{varepsilon}-Akt-eNOS modules, as demonstrated by leftward shift of immunoblotting signal for the 3 proteins toward lower elution fractions. Results are representative of ≥3 independent experiments.

 
Signal transduction through a three-tier PKC{varepsilon}-Akt-eNOS module. To investigate signal transduction by PKC{varepsilon}-Akt-eNOS modules, the effect of these proteins to posttranslationally modify each other and to influence each other’s enzymatic activity was determined.

Recombinant Akt or eNOS was incubated with recombinant PKC{varepsilon} in the presence of the PKC activators PMA and PS and [{gamma}-32P]ATP. PKC{varepsilon} was found to directly phosphorylate Akt and eNOS (Fig. 4A). Next, the effect of these PKC{varepsilon}-induced modifications on the activation status of Akt and eNOS was examined. Akt phosphorylation activity directed at the well-known substrate GSK-3{beta} was significantly enhanced in the presence of PKC{varepsilon} (Fig. 4B), suggesting that the PKC{varepsilon}-dependent phosphorylation of Akt (Fig. 4A) leads to increased Akt kinase activity. Similarly, addition of PKC{varepsilon} significantly enhanced eNOS activity, as measured by NO2/NO3 production (178.8 ± 11%, P < 0.05 vs. eNOS alone) compared with eNOS alone (Fig. 4B). These data suggest that the PKC{varepsilon}-dependent phosphorylation of eNOS (Fig. 4A) triggers the increase in eNOS activity.



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  		Fig. 4. PKC{varepsilon} phosphorylates and enhances activity of Akt and eNOS. A: recombinant Akt (A) or eNOS (B) was incubated with recombinant PKC{varepsilon} (lane 2) in the presence of PKC activators PMA and PS and [{gamma}-32P]ATP. After SDS-PAGE separation, phosphorylation was visualized by autoradiography. Lanes 1 and 3 demonstrate minimal background phosphorylation signal. B: after incubation with PKC{varepsilon}, Akt (top; lanes 2 and 3) and eNOS (bottom) activities were assessed by the ability of Akt to phosphorylate glycogen synthase kinase (GSK)-3{beta} or eNOS to produce NO2/NO3. In the absence of PKC{varepsilon}, nonactive Akt does not phosphorylate GSK-3{beta} (lane 1). Results are representative of ≥3 independent experiments. +, Presence of a protein; –, absence of a protein.

 
Akt is known to be activated by phosphorylation of Thr308 within its activation loop, a modification that stabilizes the active conformation of the molecule. Subsequently, phosphorylation of Ser473 at the carboxyl terminal is essential for full activation of Akt. Although it is well known that phosphoinositide-dependent kinase (PDK)-1 is responsible for Thr308 phosphorylation, the upstream kinase(s) that catalyzes the phosphorylation of Ser473 has not been defined. In this study, we tested whether PKC{varepsilon} could be a putative "candidate PDK-2" that targets Ser473 for phosphorylation and completes the activation process of Akt. Indeed, addition of PKC{varepsilon} in vitro resulted in a 58.3 ± 2.8% increase in Akt phosphorylation at Ser473 above that seen in the absence of PKC{varepsilon} (using a site-specific antibody to phospho-Ser473; Fig. 5), suggesting that PKC{varepsilon} may be a kinase responsible for PDK-2 activity directed at Akt. This possibility was further supported by the data described below from PKC{varepsilon} transgenic mice.



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  		Fig. 5. PKC{varepsilon} phosphorylates Akt on Ser473 in vitro. PKC{varepsilon} was incubated with nonactive Akt (lanes 3 and 4), and Ser473 phosphorylation was detected by site-specific antibody-based immunoblotting (lanes 1 and 2, nonactive Akt negative control). In the absence of PKC{varepsilon} (lanes 5 and 6), Akt is not phosphorylated at Ser473. Results are representative of ≥3 independent experiments. +, Presence of a protein; –, absence of a protein.

 
Role of PKC{varepsilon}-Akt-eNOS modules in PKC{varepsilon}-induced cardiac protection. To confirm the functional importance of these posttranslational modifications observed in vitro, Akt and eNOS phosphorylation were also examined in the mouse heart. First, PKC{varepsilon} cardiac-protected transgenic mice hearts were used to test the effect of activation of PKC{varepsilon} on Akt by examining the two conserved phosphorylation sites of Akt. Using the site-specific phosphor-antibodies of Akt, we found that phosphorylation of Akt on both activation sites, Thr308 (336.1 ± 15.1% vs. nontransgenic) and Ser473 (181.3 ± 18.2% vs. nontransgenic), was significantly increased in PKC{varepsilon} transgenic mice compared with nontransgenic animals (Fig. 6A). These alterations occurred in the absence of any change in Akt protein level (Fig. 6A). Importantly, in agreement with the foregoing in vitro data, PKC{varepsilon} activation was concomitant not only with posttranslational modification of Akt, but also with enhanced total Akt kinase activity (Fig. 6B, top). Moreover, when only PKC{varepsilon}-associated Akt activity was examined (i.e., by immunoprecipitation of PKC{varepsilon} and an Akt kinase activity assay; Fig. 6B, bottom), the increase was even greater than the increase in total Akt activity (i.e., PKC{varepsilon}-associated and non-PKC{varepsilon}-associated Akt activity) in the protected mice. These data strongly support the theory that association of Akt with this module in the mouse heart significantly potentiates its activity.



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  		Fig. 6. Activation of PKC{varepsilon} leads to phosphorylation and activation of Akt in vivo. A: nontransgenic (NTG) and PKC{varepsilon} transgenic (TG) mouse heart lysates were immunoblotted for Akt, PKC{varepsilon}, and site-specific phospho-Akt (Ser473 and Thr308). Activation of PKC{varepsilon} in vivo leads to increased total Akt protein and increased phosphorylation at both residues critical for its activation. No change in total Akt protein level was observed in PKC{varepsilon} transgenic mice (bottom blot). B: total Akt activity (top blot) and PKC{varepsilon}-associated Akt activity (determined after PKC{varepsilon} immunoprecipitation; bottom blot) were measured by the ability of Akt to phosphorylate its well-characterized substrate GSK-3{beta}. PKC{varepsilon} activation in vivo (PKC{varepsilon} TG) leads to increased enzymatic activity of Akt compared with nontransgenic control. Results are representative of ≥3 independent experiments.

 
It is well documented that active Akt can phosphorylate eNOS at Ser1177, resulting in activation of eNOS and production of NO. Therefore, phosphorylation of eNOS in PKC{varepsilon} transgenic hearts was examined to determine whether functional coupling of PKC{varepsilon} to Akt resulted in downstream activation of eNOS, as suggested by the in vitro findings. Phosphorylation of eNOS on Ser1177 was drastically enhanced in the PKC{varepsilon} transgenic mice (407.8 ± 43.7%, P < 0.05 vs. nontransgenic; Fig. 7 ), indicating that activation of PKC{varepsilon} increased eNOS activity in vivo. Meanwhile, total eNOS protein expression was also significantly increased in PKC{varepsilon} transgenic mice (140.9 ± 7.2%, P < 0.05 vs. nontransgenic).



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  		Fig. 7. PKC{varepsilon} activates eNOS in vivo. Nontransgenic and PKC{varepsilon} transgenic mouse heart lysates were immunoblotted for Ser1177-phosphorylated eNOS and for total eNOS. PKC{varepsilon} activation promotes a modest increase in total eNOS protein and a dramatic increase in Ser1177 phosphorylation of eNOS. Results are representative of ≥3 independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Many subcellular functions, such as signal transduction, are accomplished by multiprotein complexes. These complexes are differentially assembled within cells in response to given stimuli, and they coordinate modules of proteins (≥2 interacting molecules) that are targeted to carry out specific tasks. The functions of these modules, therefore, are engendered by the properties of the molecules within them and the interactions among these individual components. Several recent studies have emphasized the role of modules containing two proteins as effectors of signaling tasks in the setting of cardiac protection. However, the next level of hierarchical structure, i.e., integration of a third molecule into these modules, is poorly understood. On the basis of findings from other laboratories suggesting the potential for a three-tier module containing PKC{varepsilon}, Akt, and eNOS, we designed the present study to functionally characterize this signaling unit containing two kinases and an NO-generating enzyme.

The molecular architecture of, and signal transduction by, PKC{varepsilon}-Akt-eNOS signaling modules was first examined in the in vitro setting. The role of Akt to activate eNOS has been established (3, 15, 25, 38), and several investigations have suggested that PKC signaling can directly influence Akt activity (12, 20, 21, 23). However, the role of the {varepsilon}-isoform of PKC in modulation of Akt is completely unknown, and formation of a module containing PKC{varepsilon}, Akt, and eNOS has never been studied. In the present study, all three molecules were found to form protein-protein interactions with each other in a pairwise fashion, but, importantly, the interaction was significantly potentiated when all three molecules were present (Fig. 2). We found that PKC{varepsilon} preferentially interacts with the PH domain of Akt, congruent with previous studies in noncardiac cells, indicating that this domain of Akt is critical for its activation (4). Moreover, we found that the PH domain of Akt appears not to be necessary for binding of eNOS to Akt in vitro (data not shown), suggesting that the interactions of PKC{varepsilon} and eNOS with Akt are noncompeting; i.e., they occur through different domains. These data suggest that the module concept, whereby two or more molecules are recruited into close apposition for signal transduction, is tenable, because a linear, or "pathway," model may not involve simultaneous interaction of more than two molecules.

To determine whether these interactions lead to signal transduction, we examined the ability of PKC{varepsilon} to posttranslationally modify Akt and eNOS and to regulate their activity. The findings demonstrate that PKC{varepsilon} phosphorylates Akt and leads to increased Akt activity (as evidenced by GSK-3{beta} phosphorylation; Fig. 4). Similarly, PKC{varepsilon} phosphorylates and activates eNOS, leading to increased NO2/NO3 production. Thus PKC{varepsilon} is physically and functionally coupled to Akt and eNOS in vitro, providing the impetus to examine this module in vivo.

Accordingly, we next examined PKC{varepsilon}-Akt-eNOS modules in a line of PKC{varepsilon} transgenic mice with cardiac-specific overexpression of an active mutant of PKC{varepsilon} that display a powerful inherent resistance to ischemia-reperfusion injury (26). We wanted to examine the presence and characteristics of PKC{varepsilon}-Akt-eNOS modules in these PKC{varepsilon} mice, with the goal to understand the role of these modules in a cardiac-protective phenotype.

Indeed, our in vitro findings were substantiated by the studies in murine hearts: activation of PKC{varepsilon} (PKC{varepsilon} transgenic mice) was associated with increased formation of functional PKC{varepsilon}-Akt-eNOS modules. PKC{varepsilon} mice demonstrated enhanced posttranslational modification of Akt and eNOS, as well as enhanced activation of these molecules (analogous to the in vitro experiments in which PKC{varepsilon} was activated with PMA and PS). The phosphorylation and inactivation of GSK-3{beta} observed in this study is in agreement with that documented during cardiac protection by other investigators (34). Recombinant PKC{varepsilon} alone could increase the phosphorylation of GSK-3{beta} (Fig. 4B). However, this was considerably less than when Akt was also present, suggesting that the preferred mechanism of activation of GSK-3{beta} by PKC{varepsilon} in this module is indirect, i.e., through Akt. Therefore, although we cannot rule out that the increase in phosphorylation of GSK-3{beta} in the PKC{varepsilon} transgenic mice is at least partly due to a direct action by PKC{varepsilon}, we believe that this is more likely achieved through Akt activation. Additional investigations are necessary to elucidate the functional importance of the direct effects of PKC{varepsilon} on GSK-3{beta} phosphorylation during ischemic injury and protection.

Using nondenaturing gel filtration liquid chromatography (to separate protein complexes) followed by immunoprecipitation for PKC{varepsilon} (to isolate PKC{varepsilon}-containing native complexes), we were able to examine intact protein complexes containing PKC{varepsilon}. To determine which of these complexes contained Akt and eNOS, we separated the complexes immunoprecipitated from the gel filtration fractions by SDS-PAGE and subjected them to Western blotting for Akt and eNOS (Fig. 3). The data indicate that PKC{varepsilon}, Akt, and eNOS reside together in multiple native complexes in the mouse heart and that these complexes display a large molecular weight range. Furthermore, PKC{varepsilon}-induced cardiac protection (i.e., PKC{varepsilon} transgenic mice) was associated with increased localization of these PKC{varepsilon}-Akt-eNOS modules to higher-molecular-weight complexes. To our knowledge, these findings are the first to demonstrate formation of native complexes in the myocardium that are modulated with regard to their molecular components (here, PKC{varepsilon}-Akt-eNOS modules) concomitant with changes in the phenotype of the organ. These findings are of salient interest, because they suggest that native protein complexes, such as PKC{varepsilon} complexes containing modules such as the PKC{varepsilon}-Akt-eNOS module described here, are mechanisms of signal transduction and are not solely artifacts of biochemical analyses. Taken with the striking data described above regarding posttranslational modifications and enhanced enzymatic activity engendered by assembly of this signaling unit, these findings support PKC{varepsilon}-Akt-eNOS modules as critical components of PKC{varepsilon} protective signal transduction. Because each component within the complex contributes to the complex formation, it is very likely that expression changes in Akt and/or eNOS may also influence assembly of this module. Consequently, future studies are required to investigate Akt- and eNOS-induced changes in PKC{varepsilon}-Akt-eNOS module formation, potentially with use of transgenic mouse models with cardiac-specific overexpression of Akt or eNOS (6, 9).

In contrast to our findings, other investigators reported that in A549 and HEK293 cells the phosphatidylinositol 3-kinase-Akt signaling pathway was regulated by PKC in a negative manner (10, 39). PMA-induced apoptosis was accompanied by an inhibition of Akt activity (29). Similarly, PKC activation inhibited eNOS activity by attenuating eNOS phosphorylation on Ser1177 and increasing phosphorylation of Thr495 in cardiovascular endothelial cells (24, 25). The present findings suggest that cardiac PKC{varepsilon} serves as a positive regulator of the formation and regulation of PKC{varepsilon}-Akt-eNOS signaling modules in mouse hearts. Possible reasons for these differences include isoform-specific functions of PKC and/or distinct cell types. These findings highlight the versatility of the enzymes to participate in a host of distinct cellular processes and reemphasize the importance of studying modules of proteins (and correlating these findings with a phenotype) that are regulated in a cell type- and stimulus-specific manner.

One caveat with the present studies is the use of the PKC{varepsilon} transgenic mouse model. This is a chronic genetic model in which the protein has been overexpressed for most of the animal’s postnatal life. This activation can in turn induce long-standing genetic and proteomic changes in the heart. Moreover, overexpression of a protein can lead to its mislocalization to different subcellular compartments. Consequently, caution needs to be exercised in extrapolation of the present data to more acute models of cardioprotection such as ischemic preconditioning or adenosine administration or physiological alterations in PKC{varepsilon} signaling. Future studies will examine changes in module formation under these conditions. A second caveat is that PKC{varepsilon} may not be the predominant "protective" isoform in other animal models. Studies from our laboratory have indicated that PKC{varepsilon} is the major mediator of protection in mice and rabbits (27, 28). However, PKC-{alpha} appears to be more important in dogs and pigs (19, 30), as does PKC-{delta} in the setting of opioid-induced protection (13). Whether other isoforms of PKC also form a similar signaling module with Akt and eNOS in these species remains to be determined.

Akt has two conserved phosphorylation sites, Thr308 and Ser473, which must be modified to induce full activation. The upstream kinase that phosphorylates Thr308 is PDK-1. However, the protein kinase responsible for Ser473 phosphorylation is unknown and has been tentatively named "PDK-2" (7). Several candidates have been proposed, including atypical PKC (40), lipid raft-associated activity (18), or simply the autophosphorylation processes that are triggered by PDK-1 activity (1). Because of the data in the present study indicating a link between PKC{varepsilon} and Akt, we examined the ability of PKC{varepsilon} to phosphorylate Akt at Ser473, thereby behaving as the aforementioned PDK-2. Interestingly, we found that PKC{varepsilon} can, in fact, phosphorylate Akt at Ser473 in vitro (Fig. 5). Taken with the data demonstrating increased Ser473 phosphorylation of Akt in PKC{varepsilon} transgenic mice (Fig. 6), these findings strongly suggest that PKC{varepsilon} is a candidate for the in vivo "PDK-2 activity" associated with the cardiac-protected phenotype displayed by these PKC{varepsilon} mice. Moreover, these findings have important implications for regulation of the PKC{varepsilon}-Akt-eNOS module. Specifically, once Akt has been primed by PDK-1, integration into the PKC{varepsilon}-Akt-eNOS module would theoretically allow for full activation of Akt on the basis of the PDK-2 activity ascribed here to PKC{varepsilon}. Further experimentation is required to fully establish or refute this concept.

In summary, we have demonstrated assembly of PKC{varepsilon}-Akt-eNOS signaling modules in vitro and in the mouse heart. These modules appear to be involved in cardiac protection and provide a mechanistic link between these three proteins previously associated with such protection. The concept of modules of proteins is not new; indeed, the field of MAPK signaling, for instance, has long recognized the importance of conserved mechanisms of activation. A departure from previous investigations that is offered by these studies is the principle that multipurpose signaling proteins, themselves not constrained to mandatory activation pathways, can be manipulated by the cell to form distinct signaling modules. In other words, module formation may be an organizational tool of the intracellular signaling network to elicit specialized responses.


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 MATERIALS AND METHODS
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This study was supported in part by National Heart, Lung, and Blood Institute Grants HL-69301 and HL-65431 (P. Ping), American Heart Association Grants 0120412B (C. P. Baines) and 0110053B (T. M. Vondriska), and the Laubisch Endowment at UCLA.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. Ping, Cardiovascular Research Laboratories, Depts. of Physiology and Medicine, Div. of Cardiology, David Geffen School of Medicine at UCLA, Rm. 1619 MRL Bldg., Los Angeles, CA 90095 (E-mail: peipeiping{at}earthlink.net)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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