Cross-regulation of Novel Protein Kinase C (PKC) Isoform Function in Cardiomyocytes

ROLE OF PKCepsilon IN ACTIVATION LOOP PHOSPHORYLATIONS AND PKCdelta IN HYDROPHOBIC MOTIF PHOSPHORYLATIONS*

Vitalyi O. RybinDagger , Abdelkarim SabriDagger §, Jacob ShortDagger , Julian C. Braz, Jeffery D. Molkentin, and Susan F. SteinbergDagger ||**

From the Departments of Dagger  Pharmacology and || Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032 and the  Department of Pediatrics, University of Cincinnati, Children's Hospital Medical Center, Cincinnati, Ohio 45229

Received for publication, December 11, 2002, and in revised form, January 30, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Recent studies identify conventional protein kinase C (PKC) isoform phosphorylations at conserved residues in the activation loop and C terminus as maturational events that influence enzyme activity and targeting but are not dynamically regulated by second messengers. In contrast, this study identifies phorbol 12-myristoyl 13-acetate (PMA)- and norepinephrine-induced phosphorylations of PKCepsilon (at the C-terminal hydrophobic motif) and PKCdelta (at the activation loop) as events that accompany endogenous novel PKC (nPKC) isoform activation in neonatal rat cardiomyocytes. Agonist-induced nPKC phosphorylations are prevented (and the kinetics of PMA-dependent PKC down-regulation are slowed) by pharmacologic inhibitors of nPKC kinase activity. PKCdelta is recovered from PMA-treated cultures with increased in vitro lipid-independent kinase activity (and altered substrate specificity); the PMA-dependent increase in PKCdelta kinase activity is attenuated when PKCdelta activation loop phosphorylation is prevented. To distinguish roles of individual nPKC isoforms in nPKC phosphorylations, wild-type (WT) and dominant negative (DN) PKCdelta and PKCepsilon mutants were introduced into cardiomyocyte cultures using adenovirus-mediated gene transfer. WT-PKCdelta and WT-PKCepsilon are highly phosphorylated at activation loop and hydrophobic motif sites, even in the absence of allosteric activators. DN-PKCdelta is phosphorylated at the activation loop but not the hydrophobic motif; DN-PKCepsilon is phosphorylated at the hydrophobic motif but not the activation loop. Collectively, these results identify a role for PKCepsilon in nPKC activation loop phosphorylations and PKCdelta in nPKC hydrophobic motif phosphorylations. Agonist-induced nPKC isoform phosphorylations that accompany activation/translocation of the enzyme contribute to the regulation of PKCdelta kinase activity, may influence nPKC isoform trafficking/down-regulation, and introduce functionally important cross-talk for nPKC signaling pathways in cardiomyocytes.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein kinase C (PKC)1 constitutes a family of serine/threonine kinases that play key roles in signal transduction by receptors coupled to phosphoinositide hydrolysis (1). The basic primary structure of all PKC isozymes consists of a single polypeptide chain with N-terminal regulatory and C-terminal catalytic domains. PKC isoforms are subdivided into three distinct subfamilies based upon structural differences in their N-terminal regulatory domains. Conventional PKC isoforms (cPKCs) contain an autoinhibitory pseudosubstrate domain followed by two membrane-targeting modules, the C1 domain that binds diacylglycerol and phosphatidylserine (PS) and the C2 domain that binds anionic lipids in a calcium-dependent manner. Novel PKC (nPKC) isoforms lack the C2 domain and are maximally activated by diacylglycerol/phorbol ester in the absence of calcium; atypical PKCs are regulated by lipids but are not activated by calcium and diacylglycerol. Current models of PKC isoform activation focus largely on the allosteric changes that are induced by cofactor binding to N-terminal membrane-targeting modules; these conformational changes expel the autoinhibitory pseudosubstrate domain from the substrate binding pocket, relieve autoinhibition, and allow PKC to phosphorylate target substrates.

In addition to allosteric activation mechanisms, recent studies identify a series of ordered "priming" phosphorylations that convert nascent PKC isoforms into the mature forms that reside in the cytosol and can be activated by diacylglycerol and calcium (2). The focus of most studies has been on cPKC isoform phosphorylations in heterologous expression systems. Here, the first of three sequential phosphorylations is at a conserved threonine in the activation loop and is under the control of phosphoinositide-dependent kinase (PDK-1) (3, 4). According to current models, activation loop phosphorylation introduces a negative charge that critically aligns residues in the catalytic site and is required for catalytic competence of the enzyme. This enables two subsequent phosphorylations at conserved residues in the C-terminal V5 domain (a predicted autophosphorylation in a proline-flanked "turn motif" and an additional phosphorylation in a hydrophobic FXXFS/TF/Y motif 19 residues C-terminal to the turn motif). Phosphorylations at these two C-terminal sites are believed to lock the enzyme in a closed, catalytically competent, stabilized, and protease/phosphatase-resistant conformation (2, 5). Although nPKC isoforms undergo a series of equivalent priming phosphorylations, there has been only limited progress defining the order of these phosphorylation events, the identity of the regulatory kinase(s) that operate on the activation loop and C-terminal sites, and the consequences of multisite phosphorylations on nPKC isoforms. Current concepts regarding nPKC isoform phosphorylation mechanisms are based largely on studies of serum-deprived cells maintained in suspension culture because nPKC isoforms remain highly phosphorylated in most adherent cell types grown in the presence of serum (6). However, in the course of studies on adherent neonatal cardiomyocyte cultures, we identified dynamically regulated serine/threonine phosphorylations of PKCdelta and PKCepsilon which accompany ligand-dependent PKC activation. Given the paucity of information regarding the regulation of nPKC phosphorylation in differentiated cell types (including cardiomyocytes) and the importance of nPKC isoforms as components of the signaling pathways leading to hypertrophy, apoptosis, and ischemic preconditioning in the heart, this study examines the regulation and consequences of nPKC isoform phosphorylations in cardiomyocytes.

    EXPERIMENTAL PROCEDURES
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Materials-- Antibodies against total and phosphorylated forms of PKC were from the following sources. Phospho-PKC-Pan, PKCdelta -pS643, and PKCdelta -pT505 were from Cell Signaling Technology. PKCalpha and PKCepsilon were from Invitrogen. PKCdelta was from Santa Cruz Biotechnology. The PKCdelta substrate peptide (delta -peptide) and the PKCepsilon substrate peptide (epsilon -peptide) were purchased from Calbiochem and Upstate Biotechnology, respectively. PMA was from Sigma. All other chemicals were reagent grade.

Cardiomyocyte Culture and Transfection-- Cardiomyocytes were isolated from the hearts of 2-day-old Wistar rats by a trypsin dispersion procedure according to a protocol that incorporates a differential attachment procedure to enrich for cardiomyocytes followed by irradiation as described previously (7). The yield of cardiomyocytes typically is 2.5-3 × 106 cells/neonatal ventricle. Cells were plated on protamine sulfate-coated culture dishes at a density of 5 × 106 cells/100-mm dish. Experiments were performed on cultures grown for 5 days in minimum Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum and then serum-deprived for the subsequent 24 h. PKC overexpression was accomplished as described previously (8). Cardiomyocyte infections were for 2 h with adenoviral vectors that contain nearly the full-length Ad5 genome (lacking regions E1 and E3) and a cytomegalovirus promoter driving expression of wild-type or dominant negative mouse PKCdelta (K436R substitution at the ATP binding site of the catalytic domain), wild-type or dominant negative rabbit PKCepsilon (analogous K376R substitution at the ATP binding), or beta -galactosidase (as a control). Protein extracts were prepared 24 h later, in some experiments after a 15-min treatment with 200 nM PMA or vehicle.

Immunoblot Analysis-- Immunoblot analysis was performed on whole cell extracts or soluble and particulate fractions prepared according to methods described previously (9, 10). Briefly, cells were washed with phosphate-buffered saline and then transferred immediately to ice-cold homogenization buffer (20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 2 mM EGTA, 6 mM beta -mercaptoethanol, 50 µg/ml aprotinin, 48 µg/ml leupeptin, 5 µM pepstatin A, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM sodium vanadate, and 50 mM NaF); cells were lysed by sonication and centrifuged at 100,000 × g for 1 h. The supernatant was saved as the soluble fraction. PKC isoforms were extracted from the pellets by incubation on ice for 10 min in homogenization buffer containing 1% Triton X-100 followed by centrifugation at 100,000 × g for 30 min at 4 °C. Electrophoresis was on 8% SDS-polyacrylamide gels followed by transfer to nitrocellulose for immunoblotting with a panel of antibodies that recognize total PKCalpha , PKCdelta , or PKCepsilon protein expression (identified previously as the phorbol ester-sensitive PKC isoforms expressed by cardiomyocyte cultures (9, 11)), PKCdelta phosphorylated at the activation loop (anti-PKCdelta -pT505), PKCdelta phosphorylated at the turn motif (anti-PKCdelta -pS643), or an antibody that detects all phorbol ester-sensitive PKC isoforms phosphorylated at the conserved hydrophobic motif serine (anti-phospho-PKC-Pan; Fig. 1). The specificity of the anti-PKC antibodies has been established previously (10, 12); the specificity of all anti-phospho-PKC antibodies was validated in control experiments showing that anti-phospho-PKC immunoreactivity is stripped by treatment of samples with alkaline phosphatase (data not shown) and is down-regulated along with PMA-dependent down-regulation of the cognate protein (see Fig. 3). Bands were detected by enhanced chemiluminescence, with each panel in each figure from a single gel exposed for a uniform duration.


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Fig. 1.   The conserved sequences surrounding the priming phosphorylation sites in PKCalpha , PKCdelta , and PKCepsilon . The bold type denotes the phosphorylation sites identified by the anti-PKCdelta -pT505, anti-PKCdelta -pS643, and anti-phospho-PKC-Pan antibodies used in this study.

Immunoprecipitation and Immunocomplex PKCdelta Assays-- After incubation with the compounds indicated in the figures, cells were lysed in homogenization buffer (20 mM Tris-Cl, pH 7.5, 2 mM EDTA, 2 mM EGTA, 0.5 mM dithiothreitol, 1 µg/ml aprotinin, 5.5 µg/ml leupeptin, 0.25 mM phenylmethylsulfonyl fluoride, 1 µM pepstatin A, 0.1 mM sodium orthovanadate, 0.2% Triton X-100). Lysates were centrifuged at 4 °C for 15 min at 1,500 × g. For immunoprecipitation, supernatant from five 100-mm dishes was incubated with 5 µg of anti-PKCdelta antibody for 1 h at 4 °C followed by the addition of 200 µl of protein A/G PLUS-agarose beads (Santa Cruz Biotechnology) and incubation for 3 h at 4 °C. The immunoprecipitates were washed twice with washing buffer (10 mM Tris-Cl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.5 mM dithiothreitol, 150 mM NaCl) and suspended in 1.15 ml of storage buffer (18 mM Tris-Cl, pH 7.5, 1.8 mM EDTA, 1.8 mM EGTA, 0.45 mM dithiothreitol, 225 mM NaCl, 10% glycerol).

Immunoprecipitates (from 0.3 mg of protein assay) were used in immunocomplex kinase assays carried out in 200 µl of a reaction mixture containing buffer (26 mM Tris, pH 7.5, 5 mM MgCl2, 0.6 mM EGTA, 0.6 mM EDTA, 0.5 µM protein kinase inhibitor, 10 µM PP1, and 0.25 mM dithiothreitol) in the absence and presence of 80 µg/ml PS plus 160 ng/ml PMA with 0.5 mg/ml histone III-S, 50 µM epsilon -peptide, or 50 µM delta -peptide as substrate. epsilon -Peptide is a synthetic peptide that corresponds to the pseudosubstrate domain of PKCepsilon , with a phosphorylatable serine for alanine substitution (ERMRPRKRQGSVRRRV). delta -Peptide is a highly specific PKCdelta substrate that corresponds to amino acids 422-443 of murine eEF-1alpha (RFAVRDMRQTVAVGVIKAVDKK). Reactions were initiated by the addition 13 µCi of 66 µM [gamma -32P]ATP and were performed in quadruplicate at 30 °C for 16 min. Assays were terminated by placing samples on ice followed by centrifugation at 1,500 × g for 10 min at 4 °C. 40 µl of supernatant was spotted onto phosphocellulose filter papers (P-81), which were immediately dropped into water, washed five times for 5 min, and counted for radioactivity. Pellets were subjected to SDS-PAGE and immunoblotting for PKCdelta to normalize for any minor differences in immunoprecipitated enzyme.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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PMA Stimulates PKCdelta and PKCepsilon Phosphorylation in Cardiomyocytes-- PKC phosphorylation is increasingly recognized as a mechanism to regulate cPKC targeting and catalytic activity. However, there is still only limited information on the inputs that promote nPKC phosphorylation or its importance as a mechanism to regulate nPKC targeting and activity; PKC isoform phosphorylation in cardiomyocytes has never been considered. Therefore, PKC isoform phosphorylation was examined in cardiomyocyte cultures at rest and after stimulation with PMA. Fig. 2A shows that anti-phospho-PKC-Pan (an antibody that recognizes phorbol ester-sensitive PKC isoforms phosphorylated at the conserved hydrophobic motif) identifies three major bands that comigrate with PKCdelta , PKCalpha , and PKCepsilon (~78, 82, and 96 kDa, respectively), corresponding to PKCdelta phosphorylated at Ser662, PKCalpha phosphorylated at Ser657, and PKCepsilon phosphorylated at Ser729. Atypical PKC isoforms contain an acidic Glu in place of the Ser phosphoacceptor site in the hydrophobic motif and are not detected by the anti-phospho-PKC-Pan antibody (2).


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Fig. 2.   PMA-dependent phosphorylation and translocation of phorbol ester-sensitive PKC isoforms in cardiomyocytes. Day 5 cardiomyocyte cultures were serum starved for 24 h followed by stimulation for 20 min with vehicle or 100 nM PMA. Whole lysates (A) or soluble and particulate fractions (C and D) were subjected to SDS-PAGE and immunoblot (IB) analysis with antibodies that recognize phosphorylated (A and D) or total (C) PKC protein. In C, PKC isoforms are presented according to their molecular masses (with the slowest migrating PKCepsilon at the top and the most rapidly migrating PKCdelta at the bottom), to facilitate comparisons of total and phosphorylated proteins (C and D). Results are representative of data obtained on four or five separate culture preparations. B, extracts from cardiomyocyte cultures stimulated for 20 min with 100 nM PMA were subjected to immunoprecipitation (IP) with antibodies that recognize RSK2 or PKCepsilon as indicated. The pelleted proteins were probed for RSK, PKCepsilon , and anti-phospho-PKC-Pan immunoreactivity as indicated. Similar results were obtained in two experiments on separate culture preparations.

The most prominent immunoreactive species detected with anti-phospho-PKC-Pan in resting cardiomyocyte cultures corresponds to PKCalpha ; PKCalpha -Ser657 phosphorylation is not detectably altered by PMA. PKCdelta phosphorylation at the hydrophobic domain (Ser662) is also detected in quiescent cardiomyocyte cultures; treatment with PMA slows the migration of PKCdelta in SDS-PAGE (characteristic of a phosphorylation-induced change in electrophoretic mobility) but induces little to no increase in hydrophobic motif phosphorylation. This suggests that PMA promotes PKCdelta phosphorylation at a site that is distinct from Ser662. Therefore, immunoblot analysis also was performed with antibodies that recognize PKCdelta phosphorylated at Ser643 in the turn motif (anti-PKCdelta -pS643) or Thr505 in the activation loop (anti-PKCdelta -pT505). Fig. 2A shows that PKCdelta -pS643 immunoreactivity is similar in quiescent and PMA-stimulated cultures (i.e. PKCdelta phosphorylations at the C-terminal turn and hydrophobic motifs are relatively stable modifications that are not influenced by PMA-dependent activation). In contrast, there is little to no PKCdelta -pT505 immunoreactivity in quiescent cultures; PKCdelta phosphorylation at the Thr505 activation loop site is increased markedly in association with PMA-dependent PKCdelta activation.

Anti-phospho-PKC-Pan detects low levels of immunoreactivity that comigrates with PKCepsilon in quiescent cardiomyocyte cultures; immunoreactivity with PKCepsilon -like mobility is considerably more abundant and (in some experiments) resolved as a doublet (similar to the appearance of PKCepsilon protein; compare Fig. 2, A and C) in cultures stimulated with PMA. Because PKCepsilon is recovered from many cell types as a mixture of species (with phosphorylation-induced differences in electrophoretic mobility (13)), the doublet recognized by anti-phospho-PKC-Pan could represent a mixture of Ser729-phosphorylated PKCepsilon species with distinct phosphorylation patterns at other residues. Alternatively, the doublet could represent a phosphorylated form of PKCepsilon and a phosphorylated form of a distinct gene product that comigrates with PKCepsilon and also contains a phosphorylatable hydrophobic motif sequence. The most likely candidate is 90-kDa ribosomal S6 kinase (RSK), which is reported to undergo PMA-dependent phosphorylation at a consensus FRGFS386F hydrophobic motif sequence (14, 15). To determine whether RSK contributes to anti-phospho-PKC-Pan immunoreactivity, RSK and PKCepsilon were immunoprecipitated individually from PMA-treated cultures and probed for anti-phospho-PKC-Pan immunoreactivity. Fig. 2B shows that anti-phospho-PKC-Pan immunoreactivity is recovered from PMA-treated cardiomyocyte cultures along with PKCepsilon but not with RSK, effectively excluding RSK as the source of anti-phospho-PKC-Pan immunoreactivity in cardiomyocyte cultures.

To examine PKC isoform phosphorylation in the context of activation-dependent redistribution of the enzyme to membranes, immunoblot analysis also was performed on cell extracts partitioned into soluble and particulate fractions. Fig. 2C shows that PKCalpha protein is recovered largely from the soluble fraction of quiescent cardiomyocyte cultures and the particulate fraction of cultures stimulated with PMA. Immunodetection of PKCalpha with the anti-phospho-PKC-Pan antibody essentially tracks total PKCalpha protein (Fig. 2D; i.e. Ser657-phosphorylated PKCalpha is recovered largely from the soluble fraction of quiescent cultures and the particulate fraction of cultures stimulated with PMA). These results are consistent with the experiments on whole cell lysates (Fig. 2A), which show no PMA-dependent change in PKCalpha -Ser657 phosphorylation.

PKCdelta protein is recovered from both soluble and particulate fractions of resting cardiomyocytes, but the phosphorylated forms of PKCdelta are confined to the particulate fraction (Fig. 2, C and D); phosphorylated forms of PKCdelta are not detected in the soluble fraction, even with increased protein loading and prolonged exposures of the gel. PKCdelta phosphorylation at the C terminus (Ser662 and Ser643 in the hydrophobic and turn motifs, respectively) is similar in resting and PMA-stimulated cultures. In contrast, PKCdelta phosphorylation at Thr505 in the activation loop increases during stimulation with PMA, in association with PMA-dependent translocation of PKCdelta protein to membranes (and the PMA-induced decrease in its mobility in SDS-PAGE).

PKCepsilon also distributes between soluble and particulate fractions of quiescent cardiomyocytes. PKCepsilon -Ser729 phosphorylation is detected at trace levels in quiescent cardiomyocyte cultures and at markedly increased levels in both the soluble and particulate fractions of cultures treated with PMA (Fig. 2D). A phosphorylated form of PKCepsilon in the soluble fraction of PMA-treated cultures was not anticipated because the bulk of PKCepsilon protein is recovered from the particulate fraction. However, C-terminal phosphorylations have been described as modifications that promote the release of PKC from membranes and facilitate its down-regulation (16); the highly phosphorylated form of PKCepsilon recovered in the soluble fraction of PMA-treated cardiomyocytes could represent a pool of PKCepsilon newly released from membranes.

Fig. 3 (left panel) examines the kinetics of PMA-induced phosphorylations and translocation of PKC isoforms in greater detail. PMA-dependent translocation of PKC isoforms, phosphorylation of PKCepsilon at Ser729, and phosphorylation of PKCdelta at Thr505 are rapid events that are readily detected at 5 min (the first time point measured). Phosphorylations of PKCepsilon at Ser729 and PKCdelta at Thr505 are stable modifications; both decline in parallel with the agonist-induced down-regulation of these enzymes.


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Fig. 3.   The kinetics of PMA-dependent translocation and phosphorylation of phorbol ester-sensitive PKC isoforms. Cardiomyocytes were pretreated for 60 min with vehicle or 5 µM GF109203X followed by stimulation with vehicle or 100 nM PMA for the indicated intervals. Immunoblot analysis was on soluble and particulate fractions with antibodies that recognize total PKCalpha , PKCdelta , and PKCepsilon as well as anti-phospho-PKC-Pan and anti-PKCdelta -pT505 as described in Fig. 2. Similar results were obtained in three experiments performed on separate culture preparations.

Kinases That Mediate nPKC Isoform Phosphorylation-- There is mounting evidence that PKC phosphorylations are mediated by at least two distinguishable signaling pathways (2, 6). Largely based upon in vitro studies of isolated PKC isoforms (or studies of PKC isoforms heterologously expressed in undifferentiated cell types), activation loop phosphorylation has been attributed to the actions of PDK-1 (at least for PKCalpha , beta , and delta ), and the C-terminal phosphorylations have been characterized as intramolecular autophosphorylations (2, 4, 17, 18). These results predict that certain PKC phosphorylations would require intact intrinsic PKC activity. Therefore, initial studies examined the effects of GF109203X (an inhibitor of cPKCs and nPKCs (19)). Figs. 3 (right panel) and 4 show that GF109203X prevents PMA-dependent phosphorylation of PKCepsilon at Ser729. Somewhat unexpectedly, GF109203X also prevents PMA-dependent phosphorylation of PKCdelta at Thr505 (as well as the PMA-dependent shift in PKCdelta mobility). The inhibitory effects of GF109203X (on PKCepsilon phosphorylation at Ser729, PKCdelta phosphorylation at Thr505, and the PKCdelta mobility shift) are mimicked by Go6983 (another bisindolylmaleimide that inhibits cPKCs and nPKCs) but not by Go6976 (an indolocarbazole which selectively inhibits cPKCalpha but not nPKCs, Fig. 4). PMA-dependent phosphorylations of PKCepsilon at Ser729 and PKCdelta at Thr505 are not inhibited by the phosphoinositide 3-kinase inhibitor LY294002 or the Src family kinase inhibitor PP1 (Fig. 5, under conditions where the efficacy of LY294002 and PP1 was validated, data not shown). These results identify a role for nPKC catalytic activity in the PMA-dependent phosphorylations of native PKCepsilon (at Ser729) and PKCdelta (at Thr505) in cardiomyocytes.


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Fig. 4.   The role of PKC isoforms in PMA-dependent phosphorylations of PKCepsilon and PKCdelta . Cardiomyocytes were pretreated with 5 µM GF109203X, Go6983, or Go6976 (30 min for each) and then incubated for 30 min with 100 nM PMA. Particulate fractions (containing the bulk of the PKC isoform immunoreactivity) were subjected to SDS-PAGE and immunoblot analysis with anti-phospho-PKC-Pan, anti-PKCdelta -pT505, and anti-PKCdelta (to normalized for total protein). Results are representative of data obtained in three separate experiments on separate cultures.


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Fig. 5.   PMA-dependent phosphorylation of PKCdelta and PKCepsilon requires intact nPKC kinase activity. Cardiomyocytes were pretreated for 30 min with vehicle, 10 µM LY294002, 5 µM GF109203X, or 10 µM PP1. Stimulations were with vehicle or 100 nM PMA for 10 min. Immunoblot analysis on total cell extracts was according to methods described in Fig. 4, with similar results obtained in three separate experiments on separate culture preparations.

The acute PMA-dependent translocation of PKCepsilon to the particulate fraction is similar in cultures treated with PMA alone or PMA plus GF109203X (Fig. 3). These results indicate that phosphorylation of PKCepsilon at Ser729 is not required for PKCepsilon association with membranes. However, Fig. 3 shows that GF109203X slows the kinetics of PMA-induced PKC isoform down-regulation (compare levels of PKCalpha , PKCdelta , and PKCepsilon immunoreactivity in the particulate fractions of cultures treated for 24 h with PMA alone versus PMA plus GF109203X). These results suggest that PKC kinase activity and/or the resultant PKC isoform phosphorylations influence the kinetics of down-regulation. To examine more carefully the effect of PKC inhibitors on the kinetics of PKC isoform down-regulation, PKC isoform abundance was compared in resting cultures (as a reference for total PKC isoform immunoreactivity), cultures treated for 31 h with PMA alone, and cultures treated for 31 h with PMA plus PKC inhibitors. Fig. 6 shows that PMA effectively down-regulates phorbol ester-sensitive PKCalpha , PKCdelta , and PKCepsilon and that PMA-dependent down-regulation of PKCalpha , PKCdelta , and (to a lesser extent) PKCepsilon is impaired by GF109203X and Go6983 (general inhibitors of cPKC and nPKC kinase activities) but not by Go6976 (a selective cPKC isoform inhibitor). This identifies a requirement for intact nPKC isoform kinase activity and suggests two possible mechanisms. Because some studies identify C-terminal PKC isoform phosphorylations as prerequisites for the release of PKC from membranes and its subsequent down-regulation (16), nPKC kinase inhibitors might slow PKCepsilon down-regulation by preventing its C-terminal phosphorylation. However, the effect of nPKC isoform inhibitors to slow the kinetics of PKCalpha and PKCdelta down-regulation (i.e. to regulate PKCalpha in trans) suggests a more general role for nPKC kinase activity in the events that govern PKC trafficking/down-regulation (20, 21).


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Fig. 6.   PMA-dependent down-regulation of nPKC isoforms is attenuated by GF109203X and Go6983 but not by Go6976. Cardiomyocytes were pretreated with vehicle or 5 µM GF109203X, Go6983, or Go6976 (for 30 min each) followed by stimulation with PMA for 30 min (first lane) or 31 h (second through fifth lanes) in the absence or continued presence of the PKC inhibitors. Immunoblot analysis of whole extracts was with anti-PKCalpha , anti-PKCdelta , and anti-PKCepsilon . Similar results were obtained in three separate experiments on separate culture preparations.

NE Promotes the Phosphorylation of PKCdelta and PKCepsilon in Cardiomyocytes-- PMA promotes high affinity irreversible interactions of PKC isoforms with membranes. Because PMA does not necessarily mimic the effects of physiologic G protein-coupled receptor agonists, the studies next examined the effects of alpha 1-adrenergic receptor activation with norepinephrine (NE). Fig. 7 shows that NE promotes the phosphorylation of PKCepsilon at Ser729 and PKCdelta at Thr505. PKCepsilon phosphorylation at Ser729 is detected in both soluble and particulate fractions of NE-stimulated cultures but only after a lag of ~10 min; the intensity of PKCepsilon phosphorylation at Ser729 is similar in cultures treated for 10 min with either NE or PMA. In contrast, PKCdelta phosphorylation at Thr505 is confined to the particulate fraction, is detected at a much earlier time point (0.5 min), and is sustained for at least 20 min. PKCdelta phosphorylation at Thr505 is more prominent in cultures treated with PMA than cultures treated with NE. The NE-dependent increases in PKCepsilon phosphorylation at Ser729 and PKCdelta phosphorylation at Thr505 are inhibited by the alpha 1-adrenergic receptor antagonist prazosin or GF109203X (and not by the beta -adrenergic receptor antagonist propranolol or Go6976; data not shown).


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Fig. 7.   NE promotes nPKC isoform phosphorylation. Cardiomyocytes were stimulated with vehicle, 10-5 M NE for the indicated intervals, or 100 nM PMA for 10 min, as a positive control. Immunoblot analysis was of soluble and particulate fractions (for anti-phospho-PKC-Pan) or whole cell lysates (for anti-PKCdelta -pT505). Similar results were obtained in four separate experiments on separate culture preparations.

PKC Activity Measurements-- Because activation loop phosphorylation is viewed as a mechanism to regulate PKC kinase activity, the catalytic activity of PKCdelta recovered from quiescent cultures (with little to no PKCdelta -Thr505 phosphorylation) and cultures treated for 10 min with PMA (with demonstrable PKCdelta -Thr505 phosphorylation) was compared; assays were performed under basal conditions and with lipid (PS plus PMA) added to the in vitro incubations. To determine whether PMA-dependent activation/phosphorylation leads to a change in PKCdelta substrate specificity, we measured the incorporation of 32Pi from [gamma -32P]ATP into a panel of substrates, including delta -peptide and epsilon -peptide (model substrates for PKCdelta and PKCepsilon , respectively) as well as histone (generally considered to be a better substrate for cPKC isoforms than for nPKC isoforms).

Fig. 8A shows that PKCdelta immunopurified from quiescent cardiomyocyte cultures exhibits little to no lipid-independent kinase activity toward any substrate; histone, delta -peptide, and epsilon -peptide kinase activities are increased markedly by the addition of lipid (PS/PMA) to the in vitro kinase assays (although surprisingly, 32Pi incorporation into delta -peptide is consistently relatively modest compared with the activity measured with epsilon -peptide or histone as substrates). PKCdelta is recovered from PMA-treated cultures with markedly different cofactor requirements, substrate specificity, and activity. Here, lipid-independent kinase activity toward all substrates is increased significantly. However, the increment in lipid-independent histone kinase activity is relatively modest in magnitude (~40%); the addition of PS/PMA to the in vitro kinase assay results in ~3-fold further increase in histone phosphorylation, whereas the level of histone phosphorylation by PKCdelta recovered from PMA-stimulated cultures is relatively low level compared with the robust histone phosphorylation induced by PKCdelta recovered from quiescent cultures (i.e. histone is a particularly poor substrate for PKCdelta recovered from PMA-stimulated cultures). In contrast, PKCdelta is recovered from PMA-activated cultures as a markedly activated (largely lipid-independent) epsilon -peptide kinase. The pattern for PKCdelta phosphorylation of delta -peptide is intermediate. Lipid-independent delta -peptide kinase activity approximately doubles when cultures are treated with PMA; the addition of PS/PMA to the in vitro kinase assay results in ~2-fold further increase in delta -peptide phosphorylation. Importantly, control experiments established that the phosphorylations of delta - and epsilon -peptide (which do not contain tyrosines) and histone are mediated by PKC rather than another kinase that coimmunoprecipitates with PKCdelta ; all phosphorylations are inhibited completely by the addition of 5 µM GF109203X to the in vitro kinase assay buffer.


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Fig. 8.   PKCdelta is recovered from PMA-activated cardiomyocytes with altered substrate-specificity, cofactor requirements, and activity. A, cultures were treated with vehicle or 300 nM PMA and then subjected to extraction, immunoprecipitation, and immunocomplex PKCdelta kinase assays with histone, delta -peptide, or epsilon -peptide as substrate, assayed in the absence or presence of PS/PMA, as described under "Experimental Procedures." B, cultures were treated with vehicle, 300 nM PMA, or PMA + GF109203X (each for 20 min). Kinase activity was assayed with epsilon -peptide as substrate in the absence or presence of PS/PMA. The levels of lipid-independent PKCdelta kinase activity recovered from unstimulated cultures, cultures stimulated with PMA alone, and cultures stimulated with PMA + GF109203X differed significantly from each other (p < 0.05). In each case, results are the mean ± S.E. of quadruplicate measurements, with similar results obtained in three separate experiments on separate culture preparations.

Because PMA-dependent changes in PKCdelta kinase activity could be caused by phosphorylation at Thr505 in the activation loop or other PMA-induced regulatory modifications of the enzyme (such as tyrosine phosphorylation or the release of chelated zinc ions from the cysteine-rich zinc finger domains (22, 23)), we compared PKCdelta kinase activity in cultures treated with PMA alone and cultures treated with PMA plus GF109203X (to prevent Thr505 activation loop phosphorylation). The effect of PMA to reduced basal and PS/PMA-stimulated histone phosphorylation was not prevented when PMA stimulation was in the presence of GF109203X (data not shown). However, Fig. 8B shows that the effect of PMA to render PKCdelta highly lipid-independent toward epsilon -peptide as substrate is significantly attenuated when cultures are treated with PMA plus GF109203X. Nevertheless, lipid-independent epsilon -peptide phosphorylation by PKCdelta recovered from PMA/GF109203X-treated cultures remains significantly higher than the base line values recovered from quiescent cultures. Control experiments excluded the trivial explanation that GF109203X inhibition results from carryover of drug from the culture medium into the in vitro kinase reaction; PKCdelta kinase activity is not reduced when cultures are treated with GF109203X alone (data not shown). Collectively, these results indicate that PMA profoundly alters the cofactor requirements and substrate specificity of PKCdelta and that the effects of PMA result from Thr505 phosphorylation as well as other mechanisms.

PKC Phosphorylation in Cardiomyocytes That Overexpress Wild-type and Kinase-inactive Dominant Negative nPKC Isoforms-- The studies with pharmacologic inhibitors identify a nPKC catalytic activity requirement for PMA-dependent phosphorylations of PKCdelta at Thr505 in the activation loop and PKCepsilon at Ser729 in the hydrophobic motif. Adenovirus-mediated gene transfer, with constructs that drive expression of wild-type and dominant negative mutants of PKCdelta and PKCepsilon (full-length proteins with single Lys right-arrow Arg substitutions in their ATP binding sites), were used to resolve the relative roles of individual nPKC isoforms in this process. The integrity of these PKC-encoding adenoviruses was established in a previous study (8). Fig. 9A shows that protocols were designed to achieve comparable levels of WT and DN nPKC isoform overexpression, with exogenous proteins overexpressed at levels that are 5-7 times higher than the cognate endogenous PKC isoforms (as reported previously (8)). Overexpression of WT- or DN-PKCepsilon has no obvious effect on expression of the endogenous PKCdelta protein; WT-PKCdelta overexpression also does not alter endogenous PKCepsilon expression. However, DN-PKCdelta overexpression promotes the accumulation of endogenous PKCepsilon .


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Fig. 9.   Phosphorylation of wild-type and kinase-inactive nPKC isoforms overexpressed in cardiomyocytes. Adenovirus-mediated gene transfer was used to overexpress WT-PKCdelta , DN-PKCdelta , WT-PKCepsilon , DN-PKCepsilon , and beta -galactosidase (beta -gal) as a control. A, immunoblot analysis of total cell lysates was used to compare total PKCdelta and PKCepsilon expression, PKCdelta activation loop phosphorylation (with anti-PKCdelta -pT505), and hydrophobic domain phosphorylation (anti-phospho-PKC-Pan). B-D, cultures were serum starved for 24 h, treated with vehicle or 100 nM PMA, and then partitioned into soluble and particulate fractions. Immunoblot analysis was used to detect PKCdelta activation loop phosphorylation (with anti-PKCdelta -pT505; B), hydrophobic domain phosphorylation (anti-phospho-PKC-Pan; C), and turn motif phosphorylation (anti-PKCdelta -pS643; D). Similar results were obtained in two separate experiments on separate culture preparations.

The anti-PKCdelta -pT505 antibody was used to explore the role of nPKC kinase activity in activation loop phosphorylation. Fig. 9A shows that the anti-PKCdelta -pT505 antibody readily detects WT and DN-PKCdelta . Although native PKCdelta is phosphorylated at Thr505 only in the particulate fraction of PMA-stimulated cardiomyocytes (Fig. 2), overexpressed WT-PKCdelta is highly Thr505-phosphorylated in both the soluble and particulate fractions (and in both resting and PMA-stimulated cultures, Fig. 9B). These results indicate that the tight regulation of PKCdelta phosphorylation at Thr505 in the activation loop is lost during PKCdelta overexpression. Because DN-PKCdelta is phosphorylated at the activation loop, PKCdelta cannot be required for Thr505 phosphorylation. Of note, the anti-PKCdelta -pT505 antibody detects a prominent band that comigrates with PKCepsilon in cultures that overexpress WT-PKCepsilon (Fig. 9A). Given the high degree of primary sequence homology surrounding the activation loop phosphorylation site in PKC isoforms (see Fig. 1), this band is presumed to represent cross-reactivity with PKCepsilon overexpressed at high levels and phosphorylated at the activation loop. Precedent for this type of cross-reactivity (with an antibody raised to Thr497 in the activation loop of PKCalpha recognizing phosphorylated forms of PKCbeta I and PKCbeta II) has been published. In this context, it is noteworthy that DN-PKCepsilon , which is overexpressed at similarly high levels, is not detected by the antibody that recognizes activation loop phosphorylation. This suggests that the activation loop site of DN-PKCepsilon is not phosphorylated (and that the nPKC kinase activity requirement for PMA-dependent PKCdelta activation loop phosphorylation reflects a requirement for PKCepsilon catalytic activity).

A potential role for PKCdelta or PKCepsilon kinase activity in nPKC isoform C-terminal phosphorylations was explored in a similar fashion. Fig. 9, A and C, show that WT-PKCdelta and WT-PKCepsilon are highly phosphorylated at their hydrophobic motifs; WT-PKCdelta also is highly phosphorylated at Ser643 in the turn motif (Fig. 9D). Although C-terminal phosphorylations of endogenous PKCdelta are confined to the particulate fraction, overexpressed WT-PKCdelta is phosphorylated at the C-terminal regulatory sites in both the soluble and particulate fractions (identifying deregulated C-terminal phosphorylation of overexpressed PKCdelta ). Fig. 9, A and C, shows that DN-PKCepsilon is highly phosphorylated at the hydrophobic motif, effectively excluding an intramolecular autophosphorylation mechanism. In contrast, DN-PKCdelta is not phosphorylated at the hydrophobic motif. In the context of the pharmacologic data that identifies a requirement for nPKC kinase activity for hydrophobic motif phosphorylation, these results are most consistent with a model that attributes PKCdelta hydrophobic motif to an intramolecular autophosphorylation and characterizes PKCepsilon hydrophobic motif phosphorylation as a reaction that requires intact PKCdelta kinase activity. Collectively, these results are most consistent with a model that incorporates a high level of nPKC isoform cross-regulation, with PKCepsilon catalytic activity required for nPKC activation loop phosphorylations and PKCdelta catalytic activity required for nPKC hydrophobic motif phosphorylations, as schematized in Fig. 10.


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Fig. 10.   Model for the cross-regulation of nPKC isoform phosphorylation in cardiomyocytes.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It is almost a decade since PKC isoforms first emerged as important regulatory elements in fundamental physiological processes in cardiomyocytes, including hypertrophic signaling, ion channel regulation, and ischemic preconditioning. The recognition that cardiomyocytes coexpress multiple PKC isoforms with unique modes of activation, localization, and substrate specificity has provided the impetus to consider PKC isoforms as promising targets for the therapy of cardiovascular disorders. Strategies to modulate PKC function have focused on mechanisms that alter the abundance, activity, and/or subcellular localization of each isoform. Although ordered phosphorylations of key serine/threonine residues at the activation loop and C terminus have been identified as important regulatory events required for full maturation of catalytically competent cPKCs, knowledge of the controls and consequences of regulatory phosphorylations on nPKCs is more limited, and PKC phosphorylation in cardiomyocytes has never been considered. This study provides novel evidence that activation loop and hydrophobic domain phosphorylations constitute highly regulated events that impact on the catalytic activity, substrate specificity, and perhaps even trafficking/down-regulation of nPKCs in cardiomyocytes.

Consistent with previous literature that identifies PKCalpha phosphorylation as a priming event completed during maturation of the enzyme and not dynamically regulated by second messengers (2), PKCalpha hydrophobic motif phosphorylation was detected in resting cardiomyocytes and was not increased by PMA. In contrast, certain PKCdelta and PKCepsilon phosphorylations were dynamically regulated by PMA and NE. The rapid kinetics of ligand-induced nPKC isoform phosphorylations in cardiomyocytes (measured in minutes) contrasts with the much slower kinetics for phosphorylation of heterologously expressed nPKC isoforms in suspension and/or serum-deprived HEK293 cell cultures (6, 24). The most obvious potential explanation for these discrepant results is that phosphorylation events on native and overexpressed nPKC isoforms (and in differentiated cardiomyocytes versus undifferentiated cell lines) are regulated differently. The excessive/dysregulated phosphorylation patterns identified for nPKC isoforms overexpressed in cardiomyocytes (which differ strikingly from the phosphorylation pattern of the native cardiomyocyte enzymes) lend support to this formulation. It is tempting to speculate that dynamically regulated nPKC phosphorylations are widespread components of the nPKC isoform activation mechanism (and that these events are best appreciated in studies of native enzymes in highly differentiated cell types; they are obscured in studies of overexpressed enzymes).

This study demonstrates that PKCdelta is phosphorylated at hydrophobic and turn motifs in resting cardiomyocytes. C-terminal phosphorylations on PKCdelta appear to be stable modifications that are not regulated during PMA-dependent activation or down-regulation. In contrast, PKCepsilon retains little hydrophobic motif phosphorylation in resting cardiomyocytes; the hydrophobic motif of PKCepsilon becomes phosphorylated during allosteric activation by agonist. cPKC C-terminal phosphorylations are described as intramolecular autophosphorylations (25); the mechanism(s) for PKCepsilon hydrophobic motif phosphorylation has been disputed. Cenni et al. (24) characterized this event as an autophosphorylation reaction based upon evidence that WT-PKCepsilon overexpressed in HEK293 cells is phosphorylated at its hydrophobic motif (Ser729) but kinase-inactive PKCepsilon is not, PKCepsilon -Ser729 phosphorylation is reduced in cells incubated with GF109203X, and PKCepsilon autophosphorylates at Ser729 in vitro. However, others attribute PKCepsilon hydrophobic motif phosphorylation to a heterologous kinase complex (not inhibited by GF109203X (6)). This study identifies PMA-dependent phosphorylation of native PKCepsilon at the hydrophobic motif as an event that requires nPKC kinase activity in cardiomyocytes. An autophosphorylation reaction is highly unlikely because kinase-inactive PKCepsilon is phosphorylated at its hydrophobic motif when overexpressed in cardiomyocytes (a result that differs from the previous findings in HEK293 cells (24) and could suggest differences related to the cell types harboring the kinase-inactive PKC mutant). Rather, the observation that DN-PKCdelta is not phosphorylated at its hydrophobic motif when overexpressed in cardiomyocytes suggests that PKCdelta hydrophobic motif phosphorylation results from an autophosphorylation reaction and that PKCdelta kinase activity contributes to PKCepsilon hydrophobic motif phosphorylation in trans. However, it is worth noting that this model assumes a similar pharmacology for the phosphorylation of native and overexpressed nPKC isoforms. Although this assumption seems reasonable (and underlies much of the literature that has used a molecular strategy to characterize nPKC isoform phosphorylation mechanisms), it might deserve more careful scrutiny given the distinct phosphorylation patterns for native and overexpressed nPKCs in cardiomyocytes identified in this study.

There is mounting evidence that hydrophobic motif phosphorylations promote the release of cPKCs from membranes, allowing for reversible stimulation by physiologic agonists and/or trafficking to intracellular membranes for PMA-dependent down-regulation (26). Studies reported herein are consistent with a similar regulatory role for PKCepsilon hydrophobic motif phosphorylation in cardiomyocytes. We demonstrate that PMA induces a stable increase in PKCepsilon hydrophobic motif phosphorylation (which declines in parallel with PMA-dependent down-regulation of the protein) and that the kinetics of PMA-dependent PKCepsilon down-regulation is markedly slowed when PKCepsilon hydrophobic motif phosphorylation is prevented by nPKC isoform inhibitors. In the context of the pharmacologic and molecular evidence implicating PKCdelta kinase activity in the pathway leading to PKCepsilon hydrophobic motif phosphorylation, these results suggest a plausible explanation for the accumulation of PKCepsilon in cardiomyocytes that overexpress DN-PKCdelta ; any intervention that impairs PKCdelta kinase activity would reduce PKCepsilon hydrophobic motif phosphorylation and lead to defective PKCepsilon processing. However, the observation that PKCdelta and PKCalpha accumulate in cells treated with nPKC kinase inhibitors argues that nPKC kinases play an additional (more general) role in the processing/down-regulation of all PKC isoforms. A general requirement for nPKC kinase activity in PKC processing events is noteworthy, given the growing interest in nPKC isoform inhibitors for the treatment of cardiac disorders. An effect of nPKC kinase inhibitors to promote PKC isoform accumulation (even in kinase-inhibited forms) could lead to important physiologic consequences, given recent evidence that kinase-inactive PKCdelta induces apoptosis (27).

These studies indicate that PKCdelta is not phosphorylated detectably at Thr505 (the activation loop) in quiescent cardiomyocyte cultures; PKCdelta -Thr505 phosphorylation is increased dynamically in the particulate fraction of ligand-stimulated cultures. Concepts regarding the requirements for PKCdelta -Thr505 phosphorylation have recently evolved. Initial studies identified a requirement for allosteric activators (diacylglycerol/PMA) and membranes that contain PDK-1 and phosphatidylinositol (3,4,5)P3 (17). More recent studies indicate that complex formation between PDK-1 and the C terminus of membrane-localized unphosphorylated PKC (in an open conformation to expose the activation loop residue) is sufficient (28, 29). The lack of any requirement for phosphoinositide 3-kinase or 3'-phosphoinositide binding to the pleckstrin homology domain of PDK-1 is consistent with the pharmacology of PKCdelta -Thr505 phosphorylation in cardiomyocytes, which is not blocked by a phosphoinositide 3-kinase inhibitor. However, the observation that PKCdelta -Thr505 phosphorylation is inhibited by GF109203X and that kinase-inactive PKCepsilon displays defective activation loop phosphorylation (whereas kinase-inactive PKCdelta does not) suggests that PKCepsilon kinase activity is required for nPKC activation loop phosphorylations in cardiomyocytes. This result was unanticipated, based upon previous studies of in vitro phosphorylation events or phosphorylation of heterologously expressed enzymes in undifferentiated cell types. However, the result is consistent with recent evidence that PKCepsilon phosphorylates the activation loop of PKCµ (and that in vitro and in vivo requirements for activation loop phosphorylation can differ (30)).

The functional importance of PKCdelta -Thr505 phosphorylation remains disputed. Although activation loop phosphorylation is required for the maturation of catalytically competent cPKCs, PKCdelta expressed in bacteria is not phosphorylated at Thr505 and is enzymatically active (presumably because an acidic glutamic acid at position 500 assumes the role of the phosphorylated threonine in cPKC isoforms (31)). Although wild-type and the PKCdelta -T505A mutant exhibit similar Km values for substrates and inhibition by staurosporine-related blockers (and both undergo autophosphorylation and Src-dependent tyrosine phosphorylation (32)), Thr505 phosphorylation has been identified as a regulated event that enhances PKCdelta activity (17). Our studies used a pharmacologic strategy to assess the functional significance of PKCdelta activation loop phosphorylation and reached a similar conclusion. PKCdelta is recovered from PMA-stimulated cardiomyocytes as a lipid-independent enzyme with altered substrate specificity; the PMA-dependent modulation of PKCdelta kinase activity is attenuated when activation loop phosphorylation is prevented. However, some regulatory effects of PMA persist in this context. This is not entirely surprising because PMA also modulates PKCdelta kinase activity via other mechanisms (including by releasing tetrahedrically coordinated zinc ions from regulatory cysteine-rich motifs and promoting Src-dependent tyrosine phosphorylation of PKCdelta (22, 23)).

The observation that PKCdelta activation loop phosphorylation accompanies (and is required for optimal) PKCdelta activation deserves emphasis for two major reasons. First, PKC isoform translocation generally is considered a hallmark of PKC activation; this study demonstrates that PKCdelta activation may be incomplete unless accompanied by phosphorylation (i.e. translocation may be an imperfect surrogate marker for PKC activation). The converse scenario, PKCdelta activation without translocation, also may pertain to certain clinical conditions; our recent studies identify H2O2-dependent activation of PKCdelta in association with its release from membranes in cardiomyocytes.2 The imperfect relationship between PKC translocation and activation raises questions as to whether the pharmaceuticals that have been designed to block PKC phosphorylations by inhibiting PKC translocation are complete inhibitors of PKC cardiac actions. Second, our results suggest that nPKC isoform phosphorylations are functionally interdependent. PKCdelta activation loop phosphorylation (which is required for optimal PKCdelta activation) requires intact PKCepsilon kinase activity, whereas PKCepsilon hydrophobic motif phosphorylation (which influences its down-regulation) requires intact PKCdelta kinase activity. These results identify a serious theoretical obstacle to the design of in vivo nPKC isoform selective inhibitors. Pharmaceuticals with even high levels of in vitro PKC isoform specificity may not retain in vivo selectively for a single isoform. This considerably more elaborate paradigm for nPKC function in cardiomyocytes, which incorporates phosphorylations as a component of nPKC activation, suggests new challenges and opportunities for the development of PKC targeted therapeutic agents.

    ACKNOWLEDGEMENT

We thank Ema Stasko for preparing the myocyte cultures.

    FOOTNOTES

* This work was supported by United States Public Health Service Grant HL-64639 through the NHLBI, National Institutes of Health.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.

§ Present address: University of Alabama at Birmingham.

** To whom correspondence should be addressed: Dept. of Pharmacology, College of Physicians and Surgeons, Columbia University, 630 West 168 St., New York, NY 10032. Tel.: 212-305-4297; Fax: 212-305-8780; E-mail: sfs1@columbia.edu.

Published, JBC Papers in Press, January 31, 2003, DOI 10.1074/jbc.M212644200

2 V. O. Rybin and S. F. Steinberg, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: PKC, protein kinase C; cPKC, conventional protein kinase C; DN, dominant negative; HEK, human embryonic kidney; NE, norepinephrine; nPKC, novel protein kinase C; PDK, phosphoinositide-dependent kinase; PMA, phorbol 12-myristate 13- acetate; pS, phosphoserine; PS, phosphatidylserine; pT, phosphothreonine; RSK, ribosomal S6 kinase; WT, wild type. PP1, 4- amino-5-(4-methylphenyl)-7-(t-butyl)-pyrazolo[3,4-d]pyramidine.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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