Protein Kinase C ε–Src Modules Direct Signal Transduction in Nitric Oxide–Induced Cardioprotection
Complex Formation as a Means for Cardioprotective Signaling
Abstract—An essential role for protein kinase C ε (PKCε) has been shown in multiple forms of cardioprotection; however, there is a distinct paucity of information concerning the signaling architecture that is responsible for the manifestation of a protective phenotype. We and others have recently shown that signal transduction may proceed via the formation of signaling complexes (Circ Res. 2001;88:59–62). In order to understand if the assembly of multiprotein complexes is the manner by which signaling is conducted in cardioprotection, we designed a series of experiments to characterize the associations of Src tyrosine kinase with PKCε in a conscious rabbit model of nitric oxide (NO)-induced late preconditioning. Our data demonstrate that PKCε and Src can form functional signaling modules in vitro: PKCε interacts with Src; the association with PKCε activates Src; and adult cardiac cells receiving recombinant adenoviruses encoding PKCε exhibit increased Src activity. Furthermore, our results show that NO-induced late preconditioning involved PKCε-Src module formation and enhanced the enzymatic activity of PKCε-associated Src. Inhibition of PKC blocked cardioprotection, module formation, and PKCε-associated Src activity, providing direct evidence for a functional role of the PKCε-Src module in the orchestration of NO-induced cardioprotection in conscious rabbits.
The protein kinase C ε (PKCε) signaling system in cardioprotection has two conceptual components: (1) the molecules that are involved in PKCε signaling and (2) the manner in which these molecules interact with PKCε. The first of these components has been extensively studied,1 2 3 4 5 6 7 8 9 10 11 12 which has resulted in the identification of numerous signaling elements that may participate in PKCε-mediated preconditioning (PC). However, the latter component, that is, the specific infrastructure established by these signaling elements to engender cardioprotection, remains undefined. Evidence from recent studies in numerous cellular theaters suggests that signal transduction may proceed via the formation of multiprotein signaling complexes.13 14 15 16 17 Our laboratory has shown that PKCε forms signaling complexes with at least 36 proteins in the heart, and that PKCε-mediated cardioprotection is associated with dynamic modulation of these complexes.17 Despite this, the specific manner in which PKCε might interact with a member of its complex in order to perform functional signaling tasks is at present unknown.
Previous studies pertaining to the components of the signaling system in cardioprotection have implicated a role for tyrosine kinases.8 12 18 19 20 21 22 Our laboratory has reported that ischemic PC induces activation of Src in a PKC-dependent manner in conscious rabbits.8 Mounting evidence has demonstrated that the administration of a nitric oxide (NO) donor fully mimics the delayed cardioprotective effect afforded by ischemic PC.1 7 Furthermore, this pharmacological PC also utilizes a PKCε-dependent signaling mechanism.1 7 However, the ability of, and the mechanism used by, NO to activate Src and to modulate the involvement of Src in NO-induced late PC in the myocardium remains to be elucidated. It was recently found in murine fibroblasts that Src could be induced to autophosphorylate and become active by NO,23 which raises the possibility that NO may activate Src via separate, non-PKCε-dependent mechanisms in the heart. In rat cardiomyocytes, oxidative stress is effective in activating extracellular signal–regulated kinases (ERKs) via an Src-dependent pathway, a phenomenon which is not blocked by inhibitors of PKC and/or PKA.23 Therefore, it is unclear whether NO can activate Src in the myocardium, and whether this occurs via activation of PKC.
Analyses of PKCε signaling complexes in the murine myocardium revealed that Src, a member of the Src family of nonreceptor tyrosine kinases, resides in the PKCε complex.17 Although the individual roles of Src and PKCε have been extensively studied,2 24 25 26 27 28 29 it remains unknown if PKCε interacts with Src, and if so, whether their direct interaction promotes signal transduction, ie, whether association with PKCε activates Src. The fulfillment of these criteria would indicate that PKCε and Src constitute a signaling module.
Accordingly, we designed a study to comprehensively analyze the interactions of PKCε and Src in NO-induced late PC. We reasoned that if signaling in NO-induced PC proceeds by the assembly of a module containing PKCε and Src, then the association of Src with PKCε in this fashion might lead to increased enzymatic activity of Src. To test this, we analyzed the enzymatic activity and subcellular localization of Src and the association of Src with the PKCε signaling complex. The PKC inhibitor chelerythrine (CHE) was given, at a dose shown previously to block late NO-induced PC, to determine whether the formation of a PKCε-Src module is an essential part of the signaling architecture that protects the heart against injury. We found that NO-induced cardioprotection involves the formation of a module containing PKCε and Src in the particulate fraction. Association of Src with PKCε in this module dynamically enhances Src kinase activity during the late phase of protection.
Materials and Methods
Recombinant Proteins and Reagents
In vitro studies used cDNAs of wild-type PKCε (rabbit), wild-type Src (mouse), and a mutant of Src, which were cloned into the pAcGHLT vector, expressed in the baculovirus system, and purified to generate glutathione S-transferase (GST) fusion proteins (Pharmingen). Cold or [35S]-methionine–labeled recombinant proteins of wild-type PKCε, wild-type Src, and a mutant of Src (Y529F) were also made through in vitro transcription and translation using the TNT Quick-Coupled reticulocyte system (Promega). The GST-PKCε, GST-Src, and the in vitro–translated PKCε and Src were all verified to retain kinase activity (data not shown). Inhibitor sources were as follows: PKC inhibitors chelerythrine (Sigma) and GF109203X (Calbiochem); Src inhibitor PP2 (Calbiochem).
Assessing Protein-Protein Interactions Via GST Affinity Pull-Down Assays
Briefly, GST-recombinant proteins were immobilized on GST beads, mixed with either recombinant proteins of interest or rabbit myocardial homogenates, and incubated in binding buffer with various concentrations of NaCl, as previously reported.17 30 The GST-protein complexes were washed, resolved by SDS-PAGE, and analyzed via immunoblotting with antibodies against corresponding proteins. Parallel reactions were conducted using equal molar amounts of GST-null proteins in place of the tested GST-fusion proteins (negative control).
The Conscious Rabbit Model of DETA/NO-Induced Late PC
The present study was performed in accordance with the guidelines of the Animal Care and Use Committee of the University of Louisville School of Medicine and with the Guide for the Care and Use of Laboratory Animals (Department of Health and Human Services, publication No. [NIH] 86-23).
A well-established conscious rabbit model of NO-induced PC was used as previously described.7 31 32 Briefly, male New Zealand White rabbits (Myrtle’s Rabbitry Inc, Thompson Station, Tenn; 2.0 to 2.5 kg, age 3 to 4 months) were intravenously administered the NO donor diethylenetriamine/NO (DETA/NO, 0.1 mg/kg once every 25 minutes for 75 minutes; total dose of 0.4 mg/kg). This dose of DETA/NO has been shown to be sufficient to trigger activation of PKCε on day 1 and to protect against myocardial infarction and stunning on day 2.7 31 32 To determine whether inhibition of PKC, which has been demonstrated to block NO-induced PC, would affect PKCε-Src signaling module formation, the PKC inhibitor chelerythrine (CHE; dose 5 mg/kg) was given 5 minutes before the first DETA/NO injection.7 10 This dose of CHE has been shown to abolish NO-induced activation of PKCε and to abrogate NO-induced late cardioprotection.7 10 Rabbits in control groups received vehicle. To characterize PKCε-Src module formation during the temporal development of NO-induced PC, cardiac tissues were taken at two time points: (1) at 30 minutes after the last DETA/NO injection (day 1) and (2) at 24 hours after DETA/NO (day 2). Hearts were frozen and stored at −80°C. Before biochemical analysis, tissue samples were fractionated as previously reported.6
Recombinant Adenoviral-Mediated PKCε Transfection in Adult Rabbit Cardiomyocytes
Recombinant adenoviruses encoding active PKCε were used to produce activation of PKCε in cardiomyocytes. Generation of recombinant viruses and isolation of adult cardiomyocytes were performed as detailed previously.5 9
Immunoprecipitation and Western Blotting
PKCε and Src antibody-based immunoprecipitations were performed as previously documented.7 8 17 33 IgG (Sigma) was substituted to determine nonspecific binding. Immunoblotting was performed for PKCε and Src as previously described.6 17 33
PKCε and Src Activity Assays
The phosphorylation activity of PKCε and Src was determined as previously described.7 8 Briefly, cytosolic or particulate samples were immunoprecipitated overnight with either PKCε (Pharmingen) or Src (Santa Cruz) antibodies. The PKCε phosphorylation activity was determined by incubating immunoprecipitates with the PKCε-selective substrate (ERMRPRKRQGSVRRRV) in the PKCε phosphorylation cocktail.7 Src phosphorylation activity was assessed by incubating the immunoprecipitates with either the Src-selective peptide (KVEKIGEGTYGVVYK) (Upstate) or enolase (Sigma) as substrates in the Src phosphorylation cocktail.8
The PKCε-associated Src phosphorylation activity was determined by subjecting the PKCε-immunoprecipitated complex to the Src phosphorylation assay using the peptide as a substrate.
For ease of comparison, measurements of kinase activity or protein level in each individual experiment were expressed as a percentage of the average value for the respective control group. Differences among the experimental groups were analyzed using ANOVA. When necessary, post hoc contrasts and Student’s t tests for unpaired data using Bonferroni correction were performed.34 Data are reported as mean±SEM.
PKCε and Src Form a Functional Module
Recombinant PKCε and GST-Src proteins were used to determine whether PKCε and Src were binding partners. GST-Src was found to interact with PKCε in vitro (Figure 1⇓). The intensity of interaction between PKCε and Src varied with NaCl concentration, indicating that the binding between these two molecules is ionic in nature (Figure 1A⇓, lanes 1 through 3). Next, we determined whether Src colocalizes with PKCε in the rabbit myocardium. Western immunoblots for Src revealed that endogenous Src associated with the GST-PKCε complex (Figure 1B⇓). Interestingly, the association of Src with the GST-PKCε complexes was found to be subcellular location-specific, as only Src from the particulate fraction, and not that from the cytosolic, interacted with the GST-PKCε complex (Figure 1B⇓, lanes 8 and 9 and 3 and 4, respectively). To our knowledge, this is the first demonstration that PKCε can bind directly to Src.
To determine whether the conformation of Src affects its binding affinity for PKCε, we performed GST pull-down assays using GST-PKCε and in vitro–translated wild-type Src and the Y529F mutant Src, which exists in an open conformation (Figure 1C⇑). GST-PKCε exhibited stronger binding with the mutant Src than with wild-type Src, a phenomenon that appeared to be concentration-dependent (Figure 1C⇑). These data show that when Src is in its open configuration, it displays higher binding affinity for PKCε.
We then proceeded to assess whether the in vitro association of PKCε and Src results in the transduction of a signal, that is, whether activation of PKCε enhances Src activity. Active PKCε was incubated with recombinant Src and the Src-specific substrate protein enolase. Activity of Src, as assessed by its phosphorylation of enolase, was significantly higher in the presence of PKCε compared with that in the absence of PKCε (Figure 1D⇑), indicating that PKCε activates Src. To verify this result, we also performed Src phosphorylation activity assay using the Src-selective substrate peptide.8 These data confirmed the finding that PKCε induced activation of Src in vitro (Figure 1D⇑).
To further characterize the interaction of PKCε with Src in cardiomyocytes, we performed transfection experiments with adenoviruses encoding active PKCε.5 9 Adult rabbit cardiomyocytes receiving PKCε adenoviruses exhibited activation of PKCε5 9 and displayed a marked increase in Src kinase activity (235.8±8.8% of null vector; P<0.05) (Figure 1E⇑). Treatment with the PKC inhibitor GF109203X blocked activation of PKCε5 as well as PKCε-induced activation of Src (80.4±7% of null vector). As expected, Src activation was also blocked by the Src inhibitor PP2 (91.9±9.0% of null vector). These data indicate that, in adult cardiomyocytes, PKCε activation is sufficient to stimulate increased Src kinase activity.
Increased Association of Src With the PKCε Complex During the Development of NO-Induced Cardioprotection
We next examined whether the association of Src with the PKCε complex was concomitant with the development of cardioprotection in vivo. It has been well documented that NO-induced PC requires activation of PKC.1 7 Although tyrosine kinases have been implicated in PC,8 12 20 35 the manner in which Src participates in PKCε-dependent signaling during NO-induced PC is unknown. To address this issue, we determined whether NO-induced cardioprotective signaling involves the colocalization of Src and PKCε, and whether the interactions of PKCε and Src correspond with the temporal development of NO-induced PC. Rabbits were administered DETA/NO at a dose that has been previously demonstrated to generate a late phase of cardioprotection against infarction and stunning.1 7 31 32 We found that this dose of DETA/NO promoted recruitment of Src to the PKCε complex. The amount of Src protein associated with the PKCε complex was significantly increased at 30 minutes (187.0±1.1% of control; P<0.05) and at 24 hours (286.0±3.5% of control; P<0.05) after DETA/NO (Figure 2A⇓). These data suggest that PKCε-mediated signal transduction during the genesis (30 minutes) and the manifestation (24 hours) of NO-induced late PC involves an increased formation of PKCε-Src modules.
Assembly of PKCε-Src Modules Increases Src Enzymatic Activity In Vivo
In order to understand if, and the manner by which, complex formation might facilitate signal transduction in vivo, we determined the PKCε-associated Src kinase activity in hearts from conscious rabbits. Cardiac tissues were immunoprecipitated with PKCε antibodies and subjected to Src activity assay. In hearts obtained 30 minutes after DETA/NO administration, we observed a significant increase in PKCε-associated Src kinase activity (181.0±0.4% of control; P<0.05) (Figure 2B⇑). Of particular importance, in hearts obtained 24 hours after DETA/NO administration, we found a marked increase in the PKCε-associated Src kinase activity (1082±319.1% of control; P<0.05) (Figure 2B⇑), a magnitude of increase far exceeding that observed at 30 minutes. Importantly, when analysis of Src activity included both the non–PKCε-associated Src and the PKCε-associated Src (30 minutes: 141.5±2.6% of control; 24 hours: 277.0±5.4% of control; P<0.05) (Figure 2C⇑), the increase in Src activity induced by NO was significantly less compared with the activity of PKCε-associated Src alone. The basal Src activity measurements in control animals (data not shown) were comparable to those previously reported.8 Taken with the data shown in Figure 2B⇑, these findings indicate that the PKCε-associated Src accounted for the increased activity attributed to the particulate Src and exhibited higher activity than that of the non–PKCε-associated Src.
These observations indicate that within the PKCε complex, a module containing PKCε and Src is formed, and that assembly of this module facilitates the transduction of a signal by increasing the enzymatic activity of Src.
NO-Induced Late PC Involves Increased Particulate Expression of PKCε But Not Src
The subcellular alterations that could lead to increased module formation are undoubtedly extensive. However, four fundamental scenarios, designed to incorporate a range of conceivable biochemical changes that could result in increased module formation, are discussed in Figure 3⇓. In the present study, we measured the level of expression and activity of PKCε and Src during NO-induced PC to determine which, if any, of these mechanisms might contribute to increased module formation between PKCε and Src.
In agreement with our previous report,7 we found that DETA/NO induced PKCε translocation at 30 minutes after NO-induced PC (Figure 4A⇓). Moreover, we also observed significant translocation of PKCε at 24 hours after NO-induced PC (Figure 4A⇓). The increased particulate localization of PKCε at 24 hours appeared to be partially supported by an increase of total PKCε protein (196.3±20.0% of control; P<0.05), whereas total PKCε expression was not altered at 30 minutes after DETA/NO (data not shown). Furthermore, inhibition of PKC with CHE before DETA/NO treatment abolished NO-induced translocation of PKCε both at 30 minutes and at 24 hours (data not shown). CHE also abrogated the increased expression of PKCε protein that was seen 24 hours later (127.1±5.0% of control).
As shown in Figure 2B⇑, DETA/NO induced a significant increase in the PKCε-associated Src activity. We next examined the effect of NO on Src protein level at 30 minutes and 24 hours after DETA/NO. Our data show that Src expression remained unaltered at either 30 minutes or 24 hours after DETA/NO administration (Figure 4B⇑). Furthermore, DETA/NO did not cause a subcellular redistribution of Src kinase (Figure 4B⇑), in that the expression of Src was unchanged when either the total or the particulate fraction alone was considered at both 30 minutes (total Src: 8 3.6±6.0% of control; particulate Src: 105.0±0.9% of control) and at 24 hours (total Src: 121.4±21.7% of control; particulate Src: 102.4±0.4% of control). Treatment with CHE before DETA/NO treatment did not alter the expression of Src in either the total or particulate pool both at 30 minutes and 24 hours after DETA/NO administration (data not shown).
CHE Inhibits PKCε-Src Module Formation and Blocks Cardioprotection
Previous studies have demonstrated that CHE, given at a dose that inhibits PKCε activation, is sufficient to block the salubrious effects afforded by ischemic and NO-induced PC.7 10 In the present investigation, our data demonstrate that pretreatment with this same dose of CHE abolished DETA/NO-induced formation of PKCε-Src modules both at 30 minutes (123.4±3.9% of control) and at 24 hours (75.4±0.6% of control) (Figure 5⇓). In accordance with the disruption of module formation by CHE was the observation that CHE also attenuated the NO-induced increase in PKCε-associated Src activity at 30 minutes (132.3±18.9% of control) and at 24 hours (95.8±10.3% of control) (Figure 2B⇑). These data demonstrate that the activation of PKC by NO is a prerequisite for PKCε-Src module formation and function 24 hours later. Furthermore, the assembly of PKCε-Src modules was disrupted by the same mechanism that blocked cardioprotection, ie, inhibition of PKCε.7 10 Together, these findings suggest that the formation of PKCε-Src modules in response to NO is a mechanism by which the heart carries out cardioprotective signaling.
To our knowledge, this is the first study to demonstrate that the assembly of a signaling module is a means for cardiac cell signaling, and that the formation of signaling modules containing PKCε and Src is an essential step in NO-induced cardioprotection. There are a number of salient observations in this study. In the in vitro setting, our data demonstrate that PKCε interacts with Src, and that this interaction promotes the activation of Src. In cardiac cells and in the normal myocardium, our results show that formation of PKCε-Src modules serves to accomplish signal transduction. In addition, the formation of PKCε-Src modules is subcellular compartment-dependent, in that it only occurs in the particulate fraction. Finally, and of particular importance, we found that administration of an NO-donor promotes PKCε-Src module formation during the development of cardioprotection in conscious rabbits. In NO-induced PC, the configuration of this module facilitates signal transduction by increasing PKCε-associated Src kinase activity. Inhibition of PKC with CHE, at a dose that has previously been demonstrated to block NO-induced cardioprotection,7 10 was sufficient to abolish PKCε-Src module formation and PKCε-associated Src activity.
Module Formation as a Means for Signal Transduction
Recent advances in molecular technologies have allowed for the identification of molecules that participate in biochemical responses. However, the precise manner in which these molecules interact to transmit subcellular signals is unclear. Studies regarding signaling paradigms in noncardiac tissues have identified the assembly of signaling complexes as a means for signal transduction.13 14 15 16 Moreover, our laboratory has conducted an extensive characterization of the PKCε signaling complex in the murine myocardium and found that Src (along with ≈35 other proteins) is a member of this complex.17 In concert with these findings, we have proposed the “signaling module hypothesis” of PKCε,36 in which we suggest that the formation of stimulus- and subcellular location–specific signaling modules may be a mechanism whereby a multiprotein complex coordinates signal transduction.36
In the present study, we undertook the characterization of a module containing a serine/threonine kinase, PKCε, and a tyrosine kinase, Src. Our data show that the formation of this module enhanced PKCε-associated Src activity, indicating that the interaction of these two kinases promotes signal transduction. Molecular analyses of Src reveal that both the SH2 and the SH3 domains contain multiple PKC phosphorylation motifs (a.a. S/T-X-K/R). Several investigations have shown that the SH2 and SH3 domains are the preferred regions for interaction of Src tyrosine kinase with other proteins, including the epidermal growth factor receptors,37 the β3-adrenergic receptors,14 and the receptors for activated C kinases.27 To our knowledge, the present study represents the first demonstration that PKCε can directly interact with Src and that this interaction is sufficient to lead to the activation of Src.
NO-Induced Cardioprotection Requires PKCε-Src Module Formation
To determine whether the assembly of multiprotein complexes serves as a means for cardioprotective signal transduction, we characterized the formation of PKCε-Src modules during the genesis of NO-induced PC. We found that NO-induced late PC involves an increase in module formation between PKCε and Src. This increase in association was accompanied by a dramatic (10-fold) enhancement of PKCε-associated Src kinase activity only in the particulate fraction of the cell, indicating that the association of Src with the PKCε signaling complex is a subcellular location-specific occurrence. It was also found that the PKC inhibitor CHE, given at a dose shown previously to abrogate the infarct- and stunning-sparing effects of NO-induced PC, was sufficient to block the increased PKCε-Src module formation afforded by NO. In addition, this dose of CHE also attenuated the increase in PKCε-associated Src activity that was seen 30 minutes and 24 hours after NO treatment. To our knowledge, no previous study has identified the formation of signaling modules as a mechanism of accomplishing signal transduction in vivo. Furthermore, the findings suggest that the formation of signaling complexes is not a random association of proteins or an artifact of immunoprecipitation procedures. Rather, the fact that association with PKCε enhances Src enzymatic activity suggests that the formation of these modules is a highly organized event that facilitates the transmission of a subcellular signal. In view of the fact that both PKCε and Src have been shown to play necessary roles in NO-induced PC,7 34 the data herein indicate that PKCε-Src modules direct signal transduction in NO-induced cardioprotection.
While it has been shown that PKCε associates with a large number of other proteins within a signaling complex, it is rational to hypothesize that not all of the proteins within the PKCε complex participate in all of the physiological processes that involve PKCε. Accordingly, we propose that signaling complexes, like the PKCε complex characterized by our laboratory,17 which contain within them stimulus-responsive and subcellular location–specific modules, like the PKCε-Src module that is described herein, may represent a means by which the cell uses multifunctional signaling elements to perform distinct subcellular tasks.36 For example, the PKCε-Src signaling module is cardioprotective, whereas a different subset of proteins within the PKCε complex containing connexin43, that would constitute a distinct module, may interact to coordinate excitation-contraction coupling.38
Molecular Mechanisms Underlying Module Formation
To elucidate the molecular mechanisms responsible for increased module formation, we characterized the expression levels of PKCε and Src, as well as the nature of the affinity of these two molecules for each other. In these studies, we found that NO induced an increase in the particulate PKCε protein at 30 minutes and at 24 hours, with no change in particulate Src protein at either of these time points (Figure 2⇑). These data indicate that one of the mechanisms leading to increased complex formation between PKCε and Src is an increase in the ratio of PKCε protein to Src protein relative to basal conditions (Figure 3⇑). Furthermore, we found that PKCε and Src interactions only existed in the particulate fraction where Src has been reported to be in its open configuration.26 This evidence, combined with the in vitro finding that the open conformation of Src favors its interaction with PKCε (Figure 1C⇑), suggests that an altered affinity of Src for PKCε may also contribute to the formation of PKCε-Src modules (Figure 3⇑).
A challenge for the development of pharmacological strategies that target specific signaling events is the physiological ubiquity of many of the proteins involved in protective phenomena (ie, preconditioning). In addition to participating in cardioprotection, both PKCε and Src tyrosine kinase are also involved in numerous other signaling events across a variety of cell types. An essential step toward addressing this challenge is the characterization of the distinct manner in which proteins interact to define a module. This information would provide a level of specificity that could not be achieved solely from the biochemical properties of the individual molecules. Thus, rather than to activate a single molecule, which would impact multiple signaling events, future pharmacological and/or genetic interventions may be tailored to specifically recapitulate the endogenous interactions that constitute a protective module.
This study was supported by AHA EIG-40167N (P.P.), NIH HL-63901 (P.P.), NIH HL-65431 (P.P.), NIH HL-43151 (R.B.), NIH HL-55757 (R.B.), the University of Louisville Research Foundation, the Commonwealth of Kentucky Research Challenge Trust Fund, and Jewish Hospital Research Foundation.
Original received March 21, 2001; revision received May 9, 2001; accepted May 14, 2001.
↵1 These authors contributed equally to this work.
This manuscript was sent to Eugene Braunwald, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
- © 2001 American Heart Association, Inc.
Bolli R. The late phase of preconditioning. Circ Res. 2000;87:972–983.
Dorn GW II, Souroujon MC, Liron T, Chen CH, Gray MO, Zhou HZ, Csukai M, Wu G, Lorenz JN, Mochly-Rosen D. Sustained in vivo cardiac protection by a rationally designed peptide that causes ε protein kinase C translocation. Proc Natl Acad Sci U S A. 1999;96:12798–12803.
Liu GS, Cohen MV, Mochly-Rosen D, Downey JM. Protein kinase Cε is responsible for the protection of preconditioning in rabbit cardiomyocytes. J Mol Cell Cardiol. 1999;31:1937–1948.
Tong H, Chen W, Steenbergen C, Murphy E. Ischemic preconditioning activates phosphatidylinositol-3-kinase upstream of protein kinase C. Circ Res. 2000;87:309–315.
Li RC, Ping P, Zhang J, Wead WB, Cao X, Gao J, Zheng Y, Huang S, Han J, Bolli R. PKCε modulates NF-κB and AP-1 via mitogen-activated protein kinases in adult rabbit cardiomyocytes. Am J Physiol. 2000;279:H1679–H1689.
Ping P, Zhang J, Qiu Y, Tang XL, Manchikalapudi S, Cao X, Bolli R. Ischemic preconditioning induces selective translocation of protein kinase C isoforms ε and η in the heart of conscious rabbits without a subcellular redistribution of total protein kinase C activity. Circ Res. 1997;81:404–414.
Ping P, Takano H, Zhang J, Tang XL, Qiu Y, Li RC, Banerjee S, Dawn B, Balafanova Z, Bolli R. Isoform-selective activation of protein kinase C by nitric oxide in the heart of conscious rabbits: a signaling mechanism for both nitric oxide-induced and ischemia-induced preconditioning. Circ Res. 1999;84:587–604.
Ping P, Zhang J, Zheng YT, Li RC, Dawn B, Tang XL, Takano H, Balafanova Z, Bolli R. Demonstration of selective protein kinase C-dependent activation of Src and Lck tyrosine kinases during ischemic preconditioning in conscious rabbits. Circ Res. 1999;85:542–550.
Ping P, Zhang J, Cao X, Li RC, Kong D, Tang XL, Qiu Y, Manchikalapudi S, Auchampach JA, Black RG, Bolli R. PKC-dependent activation of p44/p42 MAPKs during myocardial ischemia-reperfusion in conscious rabbits. Am J Physiol. 1999;276:H1468–H1481.
Qiu Y, Ping P, Tang XL, Manchikalapudi S, Rizvi A, Zhang J, Takano H, Wu WJ, Teschner S, Bolli R. Direct evidence that protein kinase C plays an essential role in the development of late preconditioning against myocardial stunning in conscious rabbits and that ε is the isoform involved. J Clin Invest. 1998;101:2182–2198.
Xuan YT, Tang XL, Qiu Y, Banerjee S, Takano H, Han H, Bolli R. Biphasic response of cardiac NO synthase isoforms to ischemic preconditioning in conscious rabbits. Am J Physiol. 2000;279:H2360–H2371.
Nakano A, Cohen MV, Downey JM. Ischemic preconditioning: from basic mechanisms to clinical applications. Pharmacol Ther. 2000;86:263–275.
Husi H, Ward MA, Choudhary JS, Blackstock WP, Grant SG. Proteomic analysis of NMDA receptor-adhesion protein signaling complexes. Nat Neurosci. 2000;3:661–669.
Cao W, Luttrell LM, Medvedev AV, Pierce KL, Daniel KW, Dixon TM, Lefkowitz RJ, Collins S. Direct binding of activated c-Src to the β3-adrenergic receptor is required for MAP kinase activation. J Biol Chem. 2000;275:38131–38134.
Fraser ID, Cong M, Kim J, Rollins EN, Daaka Y, Lefkowitz RJ, Scott JD. Assembly of an A-kinase-anchoring protein-β2-adrenergic receptor complex facilitates receptor phosphorylation and signaling. Curr Biol. 2000;10:409–412.
Yasuda J, Whitmarsh AJ, Cavanagh J, Sharma M, Davis RJ. The JIP group of mitogen-activated protein kinase scaffold proteins. Mol Cell Biol. 1999;19:7245–7254.
Ping P, Zhang J, Pierce WM Jr, Bolli R. Functional proteomic analysis of protein kinase C ε signaling complexes in the normal heart and during cardioprotection. Circ Res. 2001;88:59–62.
Vahlhaus C, Schulz R, Post H, Rose J, Heusch G. Prevention of ischemic preconditioning only by combined inhibition of protein kinase C and protein tyrosine kinases in pigs. J Mol Cell Cardiol. 1998;30:197–209.
Baines CP, Wang L, Cohen MV, Downey JM. Protein tyrosine kinase is downstream of protein kinase C for ischemic preconditioning’s anti-infarct effect in the rabbit heart. J Mol Cell Cardiol. 1998;30:383–392.
Maulik N, Yoshida T, Zu YL, Sato M, Banerjee A, Das DK. Ischemic preconditioning triggers tyrosine kinase signaling: a potential role for MAPKAP kinase 2. Am J Physiol. 1998;275:H1857–H1864.
Fryer RM, Schultz JE, Hsu AK, Gross GJ. Importance of PKC and tyrosine kinase in single or multiple cycles of preconditioning in rat hearts. Am J Physiol. 1999;276:H1229–H1235.
Dana A, Skarli M, Papakrivopoulou J, Yellon DM. Adenosine A1 receptor induced delayed preconditioning in rabbits: induction of p38 MAPK activation and Hsp 27 phosphorylation via a tyrosine kinase- and protein kinase C-dependent mechanism. Circ Res. 2000;86:989–997.
Akhand AA, Pu M, Senga T, Kato M, Suzuki H, Miyata T, Hamaguchi M, Nakashima I. Nitric oxide controls Src kinase activity through a sulfhydryl group modification-mediated Tyr-527-independent and Tyr-416-linked mechanism. J Biol Chem. 1999;274:25821–25826.
Aikawa R, Komuro I, Yamazaki T, Zou Y, Kudoh S, Tanaka M, Shiojima I, Hiroi Y, Yazaki Y. Oxidative stress activates extracellular signal-regulated kinases through Src and Ras in cultured cardiac myocytes of neonatal rats. J Clin Invest. 1997;100:1813–1821.
Haendeler J, Berk BC. Angiotensin II mediated signal transduction: important role of tyrosine kinases. Regul Pept. 2000;95:1–7.
Sicheri F, Kuriyan J. Structures of Src-family tyrosine kinases. Curr Opin Struct Biol. 1997;7:777–785.
Chang BY, Conroy KB, Machleder EM, Cartwright CA. RACK1, a receptor for activated C kinase and a homolog of the β subunit of G proteins, inhibits activity of Src tyrosine kinases and growth of NIH 3T3 cells. Mol Cell Biol. 1998;18:3245–3256.
Dempsey EC, Newton AC, Mochly-Rosen D, Fields AP, Reyland ME, Insel PA, Messing RO. Protein kinase C isozymes and the regulation of diverse cell responses. Am J Physiol. 2000;279:L429—L438.
Takeishi Y, Ping P, Bolli R, Kirkpatrick DL, Hoit BD, Walsh RA. Transgenic overexpression of constitutively active protein kinase C ε causes concentric cardiac hypertrophy. Circ Res. 2000;86:1218–1223.
Phizicky EM, Fields S. Protein-protein interactions: methods for detection and analysis. Microbiol Rev. 1995;59:94–123.
Shinmura K, Tang XL, Takano H, Hill M, Bolli R. Nitric oxide donors attenuate myocardial stunning in conscious rabbits. Am J Physiol. 1999;277:H2495–H2503.
Takano H, Tang XL, Qiu Y, Guo Y, French BA, Bolli R. Nitric oxide donors induce late preconditioning against myocardial stunning and infarction in conscious rabbits via an antioxidant-sensitive mechanism. Circ Res. 1998;83:73–84.
Pass JM, Zheng YT, Wead WB, Zhang J, Li RC, Bolli R, Ping P. PKCε activation induces dichotomous cardiac phenotypes and modulates PKCε-RACK interactions and RACK expression. Am J Physiol. 2001;280:H946–H955.
Wallenstein S, Zucker CL, Fleiss JL. Some statistical methods useful in circulation research. Circ Res. 1980;47:1–9.
Tang XL, Kodani E, Takano H, Hill M, Ping P, Bolli R. Protein tyrosine kinase signaling is necessary for NO donor-induced late preconditioning against myocardial stunning in conscious rabbits. J Mol Cell Cardiol. Abstract. In press.
Vondriska TM, Klein JB, Ping P. Use of functional proteomics to investigate PKCε-mediated cardioprotection: the signaling module hypothesis. Am J Physiol. 2001;280:H1434–H1441.
Luttrell DK, Lee A, Lansing TJ, Crosby RM, Jung KD, Willard D, Luther M, Rodriquez M, Berman J, Gilmer TM. Involvement of pp60c-src with two major signaling pathways in human breast cancer. Proc Natl Acad Sci U S A. 1994;91:83–87.
Doble BW, Ping P, Kardami E. The ε subtype of protein kinase C is required for cardiomyocyte connexin-43 phosphorylation. Circ Res. 2000;86:293–301.