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J. Biol. Chem., Vol. 277, Issue 17, 15021-15027, April 26, 2002

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Nitric Oxide (NO) Induces Nitration of Protein Kinase C (PKC), Facilitating PKC Translocation via Enhanced PKC-RACK2 Interactions

A NOVEL MECHANISM OF NO-TRIGGERED ACTIVATION OF PKC^*

Zarema Balafanova^§, Roberto Bolli^§, Jun Zhang^§, Yuting Zheng, Jason M. Pass^§, Aruni Bhatnagar^§, Xian-Liang Tang^§, Ouli Wang, Ernest Cardwell^§, and Peipei Ping^§¶

From the  Department of Physiology and Biophysics, University of Louisville, Louisville, Kentucky 40202 and the ^§ Department of Medicine, Division of Cardiology, Louisville, Kentucky 40202

Received for publication, December 28, 2001, and in revised form, February 4, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Activation of protein kinase C (PKC)  by nitric oxide (NO) has been implicated in the development of cardioprotection. However, the cellular mechanisms underlying the activation of PKC by NO remain largely unknown. Nitration of protein tyrosine residues has been shown to alter functions of a variety of proteins, and NO-derived peroxynitrite is known as a strong nitrating agent. In this investigation, we demonstrate that NO donors promote translocation and activation of PKC in an NO- and peroxynitrite-dependent fashion. NO induces peroxynitrite-mediated tyrosine nitration of PKC in rabbit cardiomyocytes in vitro, and nitrotyrosine residues were also detected on PKC in vivo in the rabbit myocardium preconditioned with NO donors. Furthermore, coimmunoprecipitation of PKC and its receptor for activated C kinase, RACK2, illustrated a peroxynitrite-dependent increase in PKC-RACK2 interactions in NO donor-treated cardiomyocytes. Moreover, using an enzyme-linked immunosorbent assay-based protein-protein interaction assay, PKC proteins treated with the peroxynitrite donor SIN-1 exhibited enhanced binding to RACK2 in an acellular environment. Our data demonstrate that post-translational modification of PKC by NO donors, namely nitration of PKC, facilitates its interaction with RACK2 and promotes translocation and activation of PKC. These findings offer a plausible novel mechanism by which NO activates the PKC signaling pathway.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Protein kinase C (PKC)¹ is a family of serine-threonine kinases that participate in numerous biological processes (1, 2). In the heart, activation of PKC reduces the myocardial ischemic injury, whereas inhibition of PKC abolishes ischemic preconditioning (3-5). Recently, it has been shown that this cardioprotective effect can be fully mimicked by modulating the activity of a single isozyme of this family, the  isoform of PKC (6-9). Multiple molecular events have been shown to have an activating effect on this enzyme, among which, of particular interest, is nitric oxide (NO). Although the effects of NO on PKC depend on its biological functions and on the cell types (10-14), NO-induced activation of PKC is well documented in the heart (15, 16). Several investigations have demonstrated that at doses that produce a cardioprotective effect, exogenous NO (released by NO donors) activates PKC in an isoform-specific manner (17-20). Furthermore, activation of this isozyme has been demonstrated to play an essential role in orchestrating the signal transduction events during NO-induced cardioprotection against ischemic injury (15, 21). However, the exact molecular mechanism(s) whereby NO activates PKC in the heart remain largely unknown.

As a relatively stable hydrophobic free radical gas, NO can readily diffuse through cell membranes (22). Within cells, NO itself and NO-derived reactive nitrogen species are capable of reacting with various molecular targets that include complex biological molecules, such as proteins, lipids, and DNA, as well as low molecular weight compounds (23). One of the important molecular targets of NO are protein tyrosine residues, which can be modified to fairly stable 3-nitrotyrosines upon reacting with nitrating species. Protein nitration is believed to be a selective process with respect to both the proteins and the specific protein tyrosine residues that can undergo this posttranslational modification (24). Nitration of protein tyrosine residues has been shown to alter the functions of a variety of proteins under physiological and pathophysiological conditions both in vitro and in vivo (23, 25). Posttranslational modification of tyrosine residues has been shown to play an important role in modulating the activity of several PKC isozymes (26-28), and analysis of the molecular sequence of PKC reveals that it harbors multiple tyrosine residues. Therefore, we hypothesized that nitration of PKC on tyrosine residues may contribute to NO-induced activation of PKC.

Peroxynitrite (ONOO) is a well characterized nitrating species that is produced under physiological conditions when NO reacts with superoxide anion (O) (29). Because the reaction of NO with O to form ONOO occurs at a near diffusion-limited rate, generation of ONOO will predominate in any setting where NO and O are released concomitantly. Considering the fact that that cardiac myocytes have several potential sources of O, which among others include mitochondria and NADPH oxidase (30), it stands to reason that ONOO can play a role as a mediator of NO donor-induced nitration of PKC.

Translocation and subcellular redistribution of PKC have been recognized as important molecular events for the activation of this isozyme (1, 2, 31). The particulate translocation of PKC is known to be facilitated by its interaction with a specific anchoring protein termed receptor for activated C kinase 2, or RACK2 (32). Importantly, the interaction of PKC with RACK2 is crucial for mediating PKC activation and function (6, 8, 33). Accordingly, we postulated that tyrosine nitration may serve to promote the interaction between PKC and its RACK2, thereby leading to enhanced expression and activity of PKC in the particulate fraction.

In the present study, we investigated whether NO induces PKC activation and translocation in adult cardiac myocytes and elucidated the cellular mechanisms of this event. Cardiomyocytes were treated with the NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP), which was previously shown to induce PKC activation and cardiac protection in vivo (15). The translocation and activation of PKC, as well as the posttranslational modification of this isozyme such as tyrosine nitration, were characterized both in cultured cardiac cells in vitro and in a rabbit model of NO preconditioning in vivo. We found that NO induces activation, translocation, and nitration of PKC. Furthermore, nitration of PKC enhances PKC-RACK2 interactions and facilitates PKC translocation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The present study was performed in accordance with 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 [NIH] 86-23).

Materials and Reagents-- M199 medium, fetal calf serum, penicillin, and streptomycin were obtained from Invitrogen. Type II collagenase was from Worthington. SNAP, SIN-1, and Ebselen were from Calbiochem. Mouse monoclonal antibody against PKC and horseradish peroxidase-conjugated goat anti-mouse secondary antibody were obtained from Transduction Laboratories (Lexington, KY). Mouse monoclonal anti-nitrotyrosine antibody and nitrotyrosine immunoblotting control were from Upstate Biotechnology (Lake Placid, NY). Rat monoclonal anti-TCP-1 (anti-RACK2) and horseradish peroxidase-conjugated rabbit anti-rat IgG were obtained from StressGen Biotechnologies Corp. (Victoria, British Columbia, Canada). Protein A/G PLUS-agarose beads were from Santa Cruz Biotechnology (Santa Cruz, CA). Enhanced chemiluminescence (ECL)-detecting reagents were obtained from Amersham Biosciences. Reagents for SDS-PAGE were from Bio-Rad Laboratories. All other reagents were from Sigma.

Isolation of Adult Rabbit Cardiac Myocytes-- Adult rabbit cardiac myocytes were isolated using a modification of the method of Haddad et al. (34) using collagenase type II digestion. This method yielded 80-85% rod-shaped cardiac myocytes, generating an average total of 4-6 × 10⁷ cells per rabbit heart. This method has been employed previously to study PKC-induced activation of mitogen-activated protein kinases in rabbit cardiomyocytes (35). In brief, isolated myocytes were plated onto laminin-coated 100-mm dishes at subconfluence (2 × 10⁶ cells/dish) and cultured overnight at 37 °C in M199 medium with 2% fetal bovine serum, penicillin, and streptomycin. The medium was replaced with serum-free M199 medium supplemented with taurine (5 mM), creatine (5 mM), and carnitine (5 mM), and the cells were cultured under the serum-starved conditions for 3 h prior to performing the treatments with a nitric oxide donor.

Experimental Protocol-- Cardiomyocytes obtained from the same rabbit were divided into the following groups: a control group, in which only the vehicle (Me₂SO) was added to the medium; three groups in which the cells were incubated for 40 min with different concentrations of the NO donor SNAP obtained by diluting the stock solution of SNAP in the culture medium (2, 20, or 100 µM final concentrations of SNAP); a group in which the cells were pretreated for 10 min with the NO scavenger oxyhemoglobin at a 50 µM concentration before the treatment with 20 µM SNAP; and a group in which the peroxynitrite scavenger Ebselen was added at a concentration of 2 µM 10 min prior to the treatment with 20 µM SNAP. The SNAP stock solution was prepared freshly by dissolving it in Me₂SO prior to each experiment. After the treatment, the medium was removed, the cells were rinsed twice with ice-cold PBS, scraped off the bottoms of the dishes, and frozen at 80 °C.

Cell Sample Preparation-- The cell samples were processed for the assessment of protein expression and phosphorylation activity of PKC. The myocytes were resuspended and homogenized by glass-glass homogenization in sample buffer containing 20 mM Tris·HCl (pH 7.5), 10 mM EGTA, 2 mM EDTA, 50 µg/ml phenylmethylsulfonyl fluoride, and a mixture of protease inhibitors (10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin A). The total cellular proteins in the homogenates were subjected to centrifugation at 45,000 × g for 40 min; the supernatants were removed (cytosolic fractions), and the pellets were resuspended in the above sample buffer (particulate fractions). The protein concentrations in the cytosolic and particulate fractions of the samples were determined using the method of Bradford.

Western Immunoblotting Analysis-- The subcellular distribution and translocation of PKC were assessed by standard Western immunoblotting techniques as previously described (15). Briefly, 100 µg of proteins from the cytosolic and the particulate fraction of each cell sample were subjected to SDS-PAGE on a 10% denaturing gel and were electroblotted onto nitrocellulose membrane. Gel transfer efficiency and equal loading of protein were monitored by Ponceau staining of the nitrocellulose membranes. Background blocking was performed by incubating the membranes with 5% nonfat milk in Tris-buffered saline. Monoclonal antibodies against PKC isoform  were used to assess its subcellular distribution and translocation. Monoclonal anti-nitrotyrosine antibodies were used to detect protein tyrosine nitration. Monoclonal anti-TCP-1 antibodies were used to determine RACK2 protein expression. The protein signal was visualized using standard ECL methods.

Immunoprecipitations-- To carry out immunoprecipitations, 5 µg of anti-PKC antibodies were incubated with 50 µl of protein A/G-agarose beads for 40 min at 4 °C as described previously (36). The anti-PKC antibodies were substituted with IgG in controls. The protein A/G-agarose-anti-PKC complex was washed three times with phosphate-buffered saline containing 0.1% Triton X-100 and incubated with 1000 µg of proteins from the particulate fractions of the cell samples overnight at 4 °C, after which the beads were washed three times with PBS containing 0.1% Triton X-100. The immunoprecipitates were subsequently subjected to Western immunoblotting using either anti-nitrotyrosine antibodies (to detect tyrosine nitration of PKC protein), anti-RACK2 antibodies (to assess its co-immunoprecipitation with PKC), or anti-PKC antibodies.

Measurement of PKC Isoform-selective Phosphorylation Activity-- The phosphorylation activity of PKC was determined using a previously employed method (15, 21). Briefly, 50 µg of proteins from the particulate fraction were immunoprecipitated overnight with PKC monoclonal antibodies. Subsequently, the immunoprecipitates were subjected to a phosphorylation assay using a PKC-selective peptide substrate (ERMRPRKRQGSVRRRV).

ELISA for the Assessment of PKC-RACK Interactions-- Recombinant PKC and RACK2 proteins were generated as described previously (36, 37), and the interactions of PKC and RACK2 were determined. For the PKC-RACK2 in vitro binding assay, 96-well flat-bottomed ELISA plates were coated with either 10, 100, 500, 5, 10, or 50 ng of recombinant purified RACK2 dissolved in PBS (50 µl/well) and incubated overnight at 4 °C. The wells were washed three times with PBS and blocked with 200 µl of 4% bovine serum albumin (w/v) blocking buffer for 2 h at 37 °C to minimize any nonspecific binding. After washing the ELISA plates three times with PBS, 40 ng of recombinant purified PKC was added to each well and incubated for 2 h at 37 °C (final volume, 50 µl per well). PKC protein had either been left untreated (control), pretreated with 2 µM concentration of the ONOO donor SIN-1 in 15 mM HEPES buffer (pH 7.4) for 30 min (SIN-1-treated), or pretreated with 60 µg/ml phosphatidylserine and 100 nM phorbol myristate acetate (PS plus PMA-treated) for 30 min at room temperature. After allowing binding of PKC to RACK2, the wells were washed three times with PBS, and 50 µl of anti-PKC primary antibody (1:400 in bovine serum albumin blocking buffer) were added to each well and incubated for 1 h at 37 °C. PBS containing 0.05% Tween 20 (PBST) was used to wash the plates three times to remove any weakly, nonspecifically bound proteins, after which 50 µl of horseradish peroxidase-conjugated anti-mouse antibodies (1:3500) were added to each well. The plates were again incubated for 1 h at 37 °C and then washed 3 times with PBST. The bound antibodies were detected by incubating the reaction wells with TMB detection reagent for 30 min at room temperature and, for a greater sensitivity, subsequent acidifying using 50 µl of 2 M H₂SO₄. The optical density was measured on an ELISA plate reader at a single wavelength of 450 nm.

In Vivo Rabbit Model of NO-induced Cardioprotection-- To explore the functional significance of PKC nitration, we used a well established conscious rabbit model of NO-induced cardioprotection as described previously (15, 38, 39). Briefly, male New Zealand White rabbits (Myrtle's Rabbitry Inc., Thompson Station, TN; 2.0-2.5 kg, age 3-4 months) were given the NO donor diethylenetriamine/NO (DETA/NO, 0.1 mg/kg intravenously every 25 min for 75 min; total dose of 0.4 mg/kg). This dose of DETA/NO has been shown to trigger rapid activation of PKC and to induce protection against myocardial infarction and stunning 24 h later (15, 38, 39). Cardiac tissue samples were taken at 30 min after the last dose of DETA/NO, a time point when marked PKC activation by DETA/NO was previously demonstrated (15, 21).

Statistical Analysis-- All data are reported as means ± S.E. Differences between groups were analyzed using Student's t test for unpaired data. A p value of <0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Effect of the NO Donor SNAP on the Subcellular Distribution of PKC-- We have previously reported that NO released by NO donors induces preconditioning in rabbit hearts by activating PKC in an isozyme-specific manner (15, 21). To assess the effects of NO donors on PKC in isolated adult rabbit cardiac myocytes, we treated the cells with SNAP, an NO donor that has been shown to afford cardioprotection in rabbits (15, 38, 39). Cardiac myocytes were incubated with one of the three increasing concentrations of SNAP (2, 20, and 100 µM) for 40 min, and the changes in the PKC expression in the particulate fraction were subsequently determined. SNAP treatment induced a significant, concentration-dependent, translocation of PKC from the cytosolic to the particulate fraction of the cells (Fig. 1). This effect of SNAP was similar to that induced by PMA (data not shown). Pretreatment of the cells with the NO scavenger oxyhemoglobin at a concentration of 50 µM prevented the SNAP-induced translocation of PKC, demonstrating that this effect of SNAP was due to NO release. Similarly, pretreatment of the cells with the peroxynitrite scavenger Ebselen at a concentration of 2 µM for 10 min prior to SNAP application attenuated the translocation of  protein, implicating a role of a secondary peroxynitrite formation in mediating the effects of SNAP on PKC.

View larger version (44K): [in this window] [in a new window]   Fig. 1.   Changes in the subcellular distribution of PKC in isolated adult rabbit myocytes, detected by Western immunoblotting. A, cells treated with the NO donor SNAP for 40 min exhibited a dose-dependent translocation of PKC protein from the cytosolic (lanes 2-4) to the particulate fraction (lanes 8-10) when compared with the vehicle-treated controls (lanes 1 and 7). This translocation was abrogated when the cells were pretreated with the NO scavenger OxyHb (lanes 5 and 11) or the ONOO scavenger Ebselen (lanes 6 and 12) prior to the application of SNAP. B, histogram representing the changes in the subcellular distribution of PKC as percent of total PKC protein expression.

Effects of SNAP on PKC Isoform-selective Phosphorylation Activity-- We next examined whether SNAP treatment of cardiomyocytes led to changes in the kinase activity of particulate PKC parallel to the changes in its subcellular distribution. The ability of PKC to phosphorylate its specific peptide substrate was measured in the particulate fractions of samples treated with different concentrations of SNAP. The results were compared with PKC activity in Me₂SO-treated control samples and samples that were pretreated with oxyhemoglobin or Ebselen. We found that SNAP induced a dose-dependent increase in the phosphorylation activity of PKC in the particulate fraction (Fig. 2). Pretreating cardiac myocytes with either oxyhemoglobin or Ebselen before treating them with 20 µM SNAP attenuated the SNAP-induced activation of PKC, suggesting that this effect was dependent on the release of NO and on the secondary formation of ONOO.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   PKC phosphorylation activity in the particulate fraction. SNAP treatment of cardiac myocytes caused a significant increase in the isoform-selective phosphorylation activity of the particulate PKC in a dose-dependent fashion when compared with vehicle-treated controls. Scavenging of either NO or ONOO with oxyhemoglobin or Ebselen, respectively, prevented the increase in PKC activity.

SNAP-induced Tyrosine Nitration of PKC in Cardiac Myocytes-- We then proceeded to test whether NO produced during SNAP treatment of myocytes induced a posttranslational modification of PKC on tyrosine residues. We postulated that the activation of PKC by SNAP, as manifested by both increased particulate PKC expression and increased PKC phosphorylation activity, might be associated with the secondary production of ONOO, which has been shown to induce nitration of tyrosine residues on proteins (23, 25). Therefore, formation of 3-nitrotyrosines on PKC in response to treatment with SNAP was evaluated by immunoprecipitating PKC from the particulate fractions and then immunoblotting the precipitates with anti-nitrotyrosine monoclonal antibodies. A 20 µM concentration of SNAP, which significantly activated PKC (Fig. 2), induced a greater than 3.3-fold increase in the amount of tyrosine nitration on this PKC isoform compared with controls (Fig. 3). Scavenging of NO by oxyhemoglobin or ONOO by Ebselen markedly attenuated the effect of SNAP. These data support the concept that SNAP-induced nitration of PKC on its tyrosine residues was mediated by release of NO and secondary formation of ONOO.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Nitration of PKC in cardiac myocytes treated with SNAP in vitro. A, upper panel shows Western blot analysis of PKC immunoprecipitated from the particulate fractions of cells that were treated with SNAP only (lane 3), pretreated with oxyhemoglobin (lane 4), or pretreated with Ebselen (lane 5) prior to incubation with SNAP. Anti-nitrotyrosine monoclonal antibodies were used to detect the amount of 3-nitrotyrosines formed on PKC protein. Lower panel shows the same blot but reprobed using anti-PKC antibody. B, histogram of the respective changes in the amounts of nitrotyrosine expressed as percent of vehicle-treated control.

SNAP-induced Tyrosine Nitration of PKC in the Rabbit Model of NO-induced Cardioprotection in Vivo-- To determine whether NO induces tyrosine nitration of PKC in vivo, we examined tissue samples from hearts treated with DETA/NO and control hearts treated with the vehicle. The dose of DETA/NO was previously documented to induce activation of PKC and protection against myocardial infarction (15, 39). 1000 µg of proteins from the particulate fractions were used to carry out immunoprecipitations utilizing anti-PKC antibodies. We observed a significant increase in the amount of tyrosine nitration on PKC in hearts treated with DETA/NO when compared with the controls (Fig. 4). These data demonstrate that tyrosine nitration of PKC occurs in vivo after administration of NO donors at doses that had been shown previously to induce preconditioning by activating PKC.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Nitration of PKC in the hearts of rabbits treated with DETA/NO in vivo. A, upper panel shows Western immunoblotting with anti-nitrotyrosine antibodies of PKC immunoprecipitated from cardiac samples of rabbits that underwent NO donor treatment. The amount of 3-nitrotyrosine formation on PKC increased significantly 30 min after NO donor administration (lanes 4 and 5) when compared with vehicle-treated controls (lanes 2 and 3). Lower panel shows the same blot but reprobed using anti-PKC antibody. B, histogram of the change in the amount of nitrotyrosines formed on PKC in vivo upon NO donor treatment expressed as percent of control.

SNAP Enhances PKC-RACK2 Interactions-- The goal of the next set of experiments was to assess whether SNAP modulated the ability of PKC to interact with its selective anchoring protein, '-COP (RACK2). When cardiac myocytes were incubated with 20 µM SNAP, the amount of RACK2 that co-immunoprecipitated with PKC-specific antibodies increased significantly (Fig. 5). This indicates that binding of PKC to its selective RACK increased upon treatment with exogenous NO. To investigate whether the increase in PKC-RACK2 interactions occurred via secondary ONOO formation, we pretreated the cells with 2 µM Ebselen prior to the application of SNAP. Scavenging this strong nitrating agent prevented the increase in the binding of PKC to RACK2 (Fig. 5).

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Co-immunoprecipitation of RACK2 and PKC. A, PKC-RACK2 interactions were assessed by immunoprecipitating PKC and subsequent probing of the precipitates with anti-RACK2 antibody, as described under "Experimental Procedures." Cardiac myocytes treated with SNAP exhibited enhanced PKC-RACK2 protein-protein interactions in the particulate fraction (lane 3) when compared with control (lane 2). Pretreatment with the ONOO scavenger Ebselen prevented this effect of SNAP (lane 4). No significant changes in the amount of PKC in the immunoprecipitates were detected. B, histogram depicting the amount of RACK2 bound to PKC as percent of control.

Lack of Effect of SNAP on RACK2-- When cardiomyocytes were incubated with increasing concentrations of the NO donor SNAP, no change was observed in the subcellular distribution of RACK2 when compared with untreated controls. In control samples, 79 ± 3% of total RACK2 was present in the particulate fraction. In cardiomyocytes treated with 2, 20, and 100 µM SNAP, the amounts of RACK2 in the particulate fractions were 81 ± 1, 77 ± 4, and 80 ± 3% of total RACK2 expression, respectively. Furthermore, we did not detect nitrotyrosine signals on RACK2 in NO donor-treated cells (data not shown).

Assessment of PKC-RACK2 Interactions by ELISA-- To investigate the physical interactions between PKC and RACK2, we generated recombinant PKC protein using a baculovirus expression system (37) and recombinant RACK2 protein using an expression vector (a gift from Dr. Daria Mochly-Rosen). The interaction between PKC and RACK2 was characterized using an ELISA assay. 96-well ELISA plates were precoated with increasing amounts of RACK2 protein (10, 100, 500, 5, 10, or 50 ng). PKC recombinant protein was either untreated (control), pretreated with 2 µM ONOO donor SIN-1 (SIN-1-treated) for 30 min, or preincubated with phosphatidylserine, at a concentration 60 µg/ml, and phorbol myristate acetate, at 100 nM concentration ((PS plus PMA)-treated) for 30 min. 40 ng of either untreated, SIN-1-, or (PS plus PMA)-treated PKC was added to each well (total volume, 50 µl). The plates were incubated for 2 h, allowing sufficient binding between PKC and RACK2 recombinant proteins.

As shown in Fig. 6, the amount of PKC bound to RACK2 increased with RACK2 in a dose-dependent fashion. Pretreating the recombinant PKC proteins with the ONOO donor SIN-1 significantly enhanced PKC interactions with RACK2, shifting the dose-response curve to the left at the higher concentrations (10 ng). SIN-1-treated PKC also exhibited increased maximal binding to RACK2 (Fig. 6). This latter effect of the ONOO donor was comparable with that of the known activators of PKC, PS, and PMA (Fig. 6). These data demonstrate that ONOO activates PKC directly, that is, in the absence of other molecules that are present in the cellular environment.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Effect of the ONOO donor SIN-1 on PKC affinity for RACK2, as assessed by ELISA. The wells of ELISA plates were precoated with increasing amounts of recombinant RACK2 protein (10-200 ng). Pretreatment of recombinant purified PKC with SIN-1 promoted PKC-RACK2 interactions in vitro in a manner similar to PS (60 µg/ml) and PMA (100 nM) pretreatment when compared with the untreated control PKC. The amount of PKC bound to RACK2 is expressed as optical density.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Although NO-induced activation of PKC has been well documented, the cellular and molecular mechanisms mediating this event remain virtually unknown. We hereby present evidence to demonstrate that NO may activate PKC via post-translational modification (nitration of tyrosine residues) of this isozyme, a phenomenon observed both in isolated cardiac cells in vitro and in a model of NO-induced cardiac protection in vivo. We also show that nitration of PKC enhances its interaction with the selective anchoring protein RACK2, an event that is critical for PKC translocation and activation. To the best of our knowledge, this is the first study delineating a direct molecular modification of PKC by NO in the heart. Given the ubiquitous role of NO and PKC in the regulation of cardiac function, these findings may have significant implications for a variety of NO- and PKC-mediated biological processes in the cardiovascular system.

Cardiac Signaling Mechanisms Underlying NO-induced Activation of PKC-- The effect of NO on the activity of PKC isozymes is controversial. The specific effects of NO on its biological molecular targets appear to be dependent upon the species, model, cell types, and the concentration of NO achieved (22). In the heart, NO donors given at doses that produce a cardiac protective effect have been shown to activate the  isozyme of PKC (15, 21). However, the mechanisms of this event have not been characterized. Covalent posttranslational modification, namely phosphorylation, of PKC along with binding of PKC to the lipid second messenger diacylglycerol are recognized as the two equally important mechanisms that regulate PKC activity (1, 40). The recently discovered 3-phosphoinositide-dependent kinase (PDK)-1 has been demonstrated to induce phosphorylation of the activation loop in conventional, novel, and atypical PKCs (41-43). Moreover, posttranslational modification of tyrosine residues by phosphorylation has emerged as an important mechanism modulating PKC activity (26-28).

Our study shows that NO released by the NO donor SNAP activates PKC in cultured myocytes in a dose-dependent fashion. PKC activation was manifested by both translocation of this isozyme from the cytosolic to the particulate fraction and by its increased phosphorylation activity in the particulate fraction. These in vitro results complement previous in vivo findings in which the NO donors SNAP and DETA/NO induced activation of PKC in conscious rabbits (15, 21) at doses that had been previously shown to mimic the protective effects of the late phase of ischemic preconditioning (39). The activating effect of SNAP on PKC was due to NO production because scavenging of NO with oxyhemoglobin prevented PKC activation. In addition to the release of NO by SNAP, secondary production of ONOO was shown to be important because Ebselen, an ONOO scavenger, attenuated the activation of  protein by SNAP.

Nitration as a Mechanism for NO-dependent Modulation of Protein Function-- Both exogenously and endogenously produced NO have been shown to nitrate tyrosine residues on a number of proteins (25). Such covalent posttranslational modification modulates protein functions. NO^· is a relatively stable free radical molecule that is characterized by high membrane diffusibility and ability to react with biological molecules, which confers to NO an important role in cell signaling. Various physiological and pathophysiological actions of NO or NO-generated species depend on the NO concentration and local redox state and include reactions with heme-containing proteins, iron-sulfur clusters, lipids, DNA bases, oxygen, superoxide, water, and nitrosation of thiols and amines (44). One of the characteristic and more irreversible reactions of NO is nitration of DNA nucleotides, lipids, and aromatic amino acids. The aromatic amino acid tyrosine seems to be especially susceptible to nitration. Consequently, formation of 3-nitrotyrosines as a result of the covalent modification of tyrosines by either endogenous or exogenous NO and NO-derived species, ONOO in particular, has received much attention.

Nitration of tyrosine residues is considered to be a stable (or even irreversible) posttranslational modification of proteins as opposed to another cellular mechanism of NO action, S-nitrosylation of protein cysteine residues. Tyrosine nitration of proteins is also a selective process with respect to both specific proteins and specific tyrosine residues that can undergo such modification (24). Importantly, tyrosine nitration has been demonstrated to be capable of altering protein functions both in vivo and in vitro, thus modulating protein functions. For example, nitration of manganese superoxide dismutase leads to its enzymatic inactivation in human renal allografts (45). ONOO-dependent formation of 3-nitrotyrosines on surfactant protein A decreases the ability of this protein to aggregate lipids (46). Likewise, the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3-kinase) in macrophages has been found to be nitrated on tyrosine residues by both ONOO and NO donors, and this nitration has been implicated in abrogating the interaction of different subunits of PI3-kinase (47). ONOO has been reported to induce tyrosine nitration and differential activation of the mitogen-activated protein kinases p38, JNK1/2, and ERK1/2 (48) and Src tyrosine kinases (49, 50).

In this study, we present evidence that nitration of PKC contributes to the activating effect of NO on PKC. Treatment of cardiac myocytes with NO donors resulted in the detection of nitrotyrosine residues on myocardial PKC concomitant with its activation. The nitration effect appeared to be selective, as the tyrosine-bearing protein RACK2 did not exhibit any detectable nitrotyrosine signal. Both formation of nitrotyrosines on PKC and the activation of the enzyme could be prevented by pretreating the cells with Ebselen, supporting the role of ONOO as a major nitrating agent. These data indicate that generation of ONOO secondary to SNAP treatment and subsequent nitration of tyrosine residues on PKC may play a pivotal role in mediating the activation of this novel PKC isoform by exogenous NO.

Activation and Translocation of PKC via Its Interaction with RACK-- Isozyme-specific subcellular localization of each individual PKC isoform is important for the specific activity and, therefore, functions of each member of the PKC family of enzymes. A plausible theory has been put forth stating that isozyme-selective anchoring proteins, or receptor proteins, target activated PKC isoforms to their specific subcellular locations in close proximity to their corresponding substrates, thus allowing substrate phosphorylation to occur (51). Specifically, RACK2 was identified as a selective anchoring protein for activated PKC (52). Disruption of RACK2 interactions with PKC inhibits norepinephrine- or PMA-induced negative chronotropic effects (33) and abrogates PKC-mediated protection against ischemic injury in several species (6, 8, 9). These findings stress the importance of PKC-RACK2 protein-protein interactions for mediating various cardiac functions that involve the  isoform of PKC.

We found that SNAP treatment resulted in increased binding of the activated PKC to its selective anchoring protein RACK2 in the particulate fraction of cardiomyocytes. The increase in PKC-RACK2 interaction was mediated by generation of ONOO because it was attenuated by Ebselen. Because SNAP treatment did not result in increased expression of RACK2 in the particulate fraction, the enhancement of PKC-RACK2 binding cannot be ascribed to increased availability of the anchoring protein for PKC. Importantly, SIN-1 promoted the interaction of isolated recombinant PKC and SIN-1, demonstrating that ONOO can activate PKC directly, in the absence of other cellular components. Taken together, these results support our hypothesis that exogenous NO induces nitration of PKC via the production of ONOO and that this event plays an important role in mediating the increase in active PKC-RACK2 binding.

Conclusions-- Regulation of kinase activity via posttranslational modification has been recently recognized as an important means for facilitating signal transduction in a variety of biological systems. Consistent with this concept, our results show that nitration of PKC protein by NO donors modulates its interaction with the receptor binding protein RACK2, thereby promoting particulate translocation of PKC and activating the PKC signaling pathway. The findings reported herein delineate a novel signaling mechanism by which NO activates PKC isozymes and illustrate an example of how a cascade of molecular reactions in signal transduction can be initiated by chemical modifications of an individual element.

	FOOTNOTES

^* This work was supported in part by NHLBI, National Institutes of Health Grants HL-63901 and HL-65431 (to P. P.) and HL-43151, HL-55757, and HL-68088 (to R. B.), American Heart Association National Center Grant-in-aid 9750721N (to P. P.), by the Commonwealth of Kentucky Research Challenge Trust Fund, and by the Jewish Hospital Research Foundation, Louisville, Kentucky.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.

^¶ To whom correspondence should be addressed: 570 South Preston St., Baxter Building, Suite 122, Cardiology Research, Louisville, KY 40202. Tel.: 502-852-8431; Fax: 502-852-8421; E-mail: ping@ntr.net.

Published, JBC Papers in Press, February 11, 2002, DOI 10.1074/jbc.M112451200

	ABBREVIATIONS

The abbreviations used are: PKC, protein kinase C; NO, nitric oxide; SNAP, S-nitroso-N-acetyl-DL-penicillamine; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; PS, phosphatidylserine; PMA, phorbol 12-myristate 13-acetate; DETA, diethylenetriamine; JNK, c-Jun NH₂-terminal kinase; ERK, extracellular signal-regulated kinase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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