Protein Kinase Cϵ (PKCϵ) and Src Control PKCδ Activation Loop Phosphorylation in Cardiomyocytes*

Protein kinase Cδ (PKCδ) is unusual among AGC kinases in that it does not require activation loop (Thr505) phosphorylation for catalytic competence. Nevertheless, Thr505 phosphorylation has been implicated as a mechanism that influences PKCδ activity. This study examines the controls of PKCδ-Thr505 phosphorylation in cardiomyocytes. We implicate phosphoinositide-dependent kinase-1 and PKCδ autophosphorylation in the “priming” maturational PKCδ-Thr505 phosphorylation that accompanies de novo enzyme synthesis. In contrast, we show that PKCδ-Thr505 phosphorylation dynamically increases in cardiomyocytes treated with phorbol 12-myristate 13-acetate or the α1-adrenergic receptor agonist norepinephrine via a mechanism that requires novel PKC isoform activity and not phosphoinositide-dependent kinase-1. We used a PKCϵ overexpression strategy as an initial approach to discriminate two possible novel PKC mechanisms, namely PKCδ-Thr505 autophosphorylation and PKCδ-Thr505 phosphorylation in trans by PKCϵ. Our studies show that adenovirus-mediated PKCϵ overexpression leads to an increase in PKCδ-Thr505 phosphorylation. However, this cannot be attributed to an effect of PKCϵ to function as a direct PKCδ-Thr505 kinase, since the PKCϵ-dependent increase in PKCδ-Thr505 phosphorylation is accompanied by (and dependent upon) increased PKCδ phosphorylation at Tyr311 and Tyr332. Further studies implicate Src in this mechanism, showing that 1) PKCϵ overexpression increases PKCδ-Thr505 phosphorylation in cardiomyocytes and Src+ cells but not in SYF cells (that lack Src, Yes, and Fyn and exhibit a defect in PKCδ-Tyr311/Tyr332 phosphorylation), and 2) in vitro PKCδ-Thr505 autophosphorylation is augmented in assays performed with Src (which promotes PKCδ-Tyr311/Tyr332 phosphorylation). Collectively, these results identify a novel PKCδ-Thr505 autophosphorylation mechanism that is triggered by PKCϵ overexpression and involves Src-dependent PKCδ-Tyr311/Tyr332 phosphorylation.

Protein kinase C␦ (PKC␦) is unusual among AGC kinases in that it does not require activation loop (Thr 505 ) phosphorylation for catalytic competence. Nevertheless, Thr 505 phosphorylation has been implicated as a mechanism that influences PKC␦ activity. This study examines the controls of PKC␦-Thr 505 phosphorylation in cardiomyocytes. We implicate phosphoinositide-dependent kinase-1 and PKC␦ autophosphorylation in the "priming" maturational PKC␦-Thr 505 phosphorylation that accompanies de novo enzyme synthesis. In contrast, we show that PKC␦-Thr 505 phosphorylation dynamically increases in cardiomyocytes treated with phorbol 12-myristate 13-acetate or the ␣ 1 -adrenergic receptor agonist norepinephrine via a mechanism that requires novel PKC isoform activity and not phosphoinositide-dependent kinase-1. We used a PKC⑀ overexpression strategy as an initial approach to discriminate two possible novel PKC mechanisms, namely PKC␦-Thr 505 autophosphorylation and PKC␦-Thr 505 phosphorylation in trans by PKC⑀. Our studies show that adenovirus-mediated PKC⑀ overexpression leads to an increase in PKC␦-Thr 505 phosphorylation. However, this cannot be attributed to an effect of PKC⑀ to function as a direct PKC␦-Thr 505 kinase, since the PKC⑀-dependent increase in PKC␦-Thr 505 phosphorylation is accompanied by (and dependent upon) increased PKC␦ phosphorylation at Tyr 311 and Tyr 332 . Further studies implicate Src in this mechanism, showing that 1) PKC⑀ overexpression increases PKC␦-Thr 505 phosphorylation in cardiomyocytes and Src ؉ cells but not in SYF cells (that lack Src, Yes, and Fyn and exhibit a defect in PKC␦-Tyr 311 /Tyr 332 phosphorylation), and 2) in vitro PKC␦-Thr 505 autophosphorylation is augmented in assays performed with Src (which promotes PKC␦-Tyr 311 /Tyr 332 phosphorylation). Collectively, these results identify a novel PKC␦-Thr 505 autophosphorylation mechanism that is triggered by PKC⑀ overexpression and involves Src-dependent PKC␦-Tyr 311 /Tyr 332 phosphorylation.
Traditional models of PKC␦ 2 activation have focused on lipid cofactor binding to determinants in the N-terminal regulatory domain that anchor PKC␦ to membranes and promote a conformational change that expels the autoinhibitory pseudosubstrate domain from the substrate-binding pocket. This effectively relieves autoinhibition and enables PKC␦-dependent phosphorylation of target substrates. However, we and others recently demonstrated that PKC␦ also is dynamically regulated through phosphorylation at a conserved threonine residue in the activation loop (Thr 505 ) (1,2). Other PKCs require activation loop phosphorylation as a "priming" event to generate a catalytically competent enzyme. In contrast, PKC␦ is catalytically active even without activation loop phosphorylation. Rather, activation loop phosphorylation plays a distinctive role to regulate the enzymology (activity, substrate specificity) of membrane-associated allosterically activated PKC␦ (1)(2)(3)(4)(5). The precise controls and consequences of the coordinate events that govern PKC␦ phosphorylation and translocation in highly differentiated cells, such as cardiomyocytes, remain uncertain.
PKC␦ activation loop phosphorylation has been attributed to phosphoinositide-dependent kinase-1 (PDK-1, a general AGC activation loop kinase) on the basis of studies examining in vitro phosphorylation events on heterologously overexpressed PKC␦ in undifferentiated cell types (3). Our recent studies suggest that this model is not sufficient to describe the control of PKC␦-Thr 505 phosphorylation in the heart, where the ␣ 1 -adrenergic receptor agonist norepinephrine (NE) and PMA increase PKC␦-Thr 505 phosphorylation via a mechanism that is blocked by GF109203X (a relatively nonselective inhibitor of most PKC isoforms), and not by Go6976 (an inhibitor that preferentially blocks calcium-sensitive PKC isoforms (1)). These results provided tentative evidence that the dynamic stimulus-dependent increase in PKC␦-Thr 505 phosphorylation is mediated by an nPKC isoform and not PDK-1 (which is a GF109203X-insensitive enzyme). Since this conclusion runs counter to the general consensus that PKC activation loop phosphorylations are via a PDK-1-dependent mechanism, the relative roles of PDK-1 and nPKC isoforms as PKC␦-Thr 505 kinases are examined in greater detail in this study.
Cell Culture-Cardiomyocytes were isolated from the hearts of 2-day-old Wistar rats by a trypsin dispersion procedure using a differential attachment procedure to enrich for cardiomyocytes followed by irradiation as detailed in previous publica-tions (6). The yield of cardiomyocytes typically is 2.5-3 ϫ 10 6 cells/neonatal ventricle. Cells were plated on protamine sulfate-coated culture dishes at a density of 5 ϫ 10 6 cells/100-mm dish. Experiments were performed on cultures grown for 5 days in minimal essential medium (Invitrogen) supplemented with 10% fetal calf serum and then serum-deprived for the subsequent 24 h. Primary cardiac fibroblast cultures were obtained from the cells adherent to the culture dishes during the preplating step, as described previously (6).
Immunoprecipitation and Immunoblot Analysis-Immunoblotting on lysates or immunoprecipitated PKC␦ was according to methods described previously or the manufacturer's instructions (6). In each figure, each panel represents the results from a single gel (exposed for a uniform duration); detection was with enhanced chemiluminescence. All results were replicated in at least four experiments on separate culture preparations.
Preparation of Soluble and Particulate Fractions-Cells were washed with phosphate-buffered saline and then immediately transferred to ice-cold homogenization buffer (20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 2 mM EGTA, 6 mM ␤-mercaptoethanol, 50 g/ml aprotinin, 48 g/ml leupeptin, 5 M pepstatin A, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM sodium vanadate, and 50 mM NaF), lysed by sonication, and centrifuged at 100,000 ϫ g for 1 h. The supernatant was saved as the soluble fraction, and the particulate fraction was solubilized in SDS-PAGE sample buffer.

RESULTS
PMA Promotes PKC␦-Thr 505 Phosphorylation via a Mechanism That Requires nPKC Activity and Not PDK-1-Our initial experiments used a pharmacologic approach to identify the PKC␦-Thr 505 phosphorylation mechanism in NE-and PMAtreated cardiomyocytes. Fig. 1A shows that PKC␦ retains a low level of Thr 505 phosphorylation in resting cardiomyocytes; PKC␦-Thr 505 phosphorylation increases dynamically in response to treatment with either NE or PMA. These stimulusinduced increases in PKC␦-Thr 505 phosphorylation are blocked by GF109203X (a general PKC isoform inhibitor) but not by Go6976 (which selectively blocks calcium-sensitive PKC isoforms, the PKC effector protein kinase D (PKD), and JAK2 (8,9)). Since some PDK-1-dependent phosphorylation mechanisms require the generation of 3Ј-phosphoinositides that colocalize PDK-1 with substrates at the plasma membrane, the effect of LY294002 (a phosphatidylinositol 3-kinase inhibitor) also was examined. Fig. 1A shows that LY294002 does not block agonist-dependent PKC␦-Thr 505 phosphorylation.
These pharmacologic studies implicate a nPKC activity in agonist-dependent PKC␦-Thr 505 phosphorylation. Since they run counter to the prevailing notion that PKC␦-Thr 505 phosphorylation is mediated by PDK-1 (a GF109203X-insensitive enzyme), we performed a more detailed analysis of the relative roles of PDK-1 and PKC isoforms as in vivo PKC␦-Thr 505 kinases. These studies took advantage of the distinct inhibitory profiles of GF109203X and UCN-01 (a 7-hydroxystaurosporine derivative that was first identified as a PKC inhibitor and subsequently characterized as an even better inhibitor of PDK-1 (10)) ( Fig. 1, B and C). Stimulus-dependent activation loop phosphorylation events on PKC␦ (Thr 505 ), AKT (Thr 308 ), and PKD (PKD-Ser 744/748 ) were examined in parallel. AKT activation loop phosphorylation was tracked as a control for PDK-1 inhibition by UCN-01, since AKT is a bona fide PDK-1 target. Similarly, PKD activation loop phosphorylation was included to control for PKC inhibition by GF109203X, since PKD activa- tion loop phosphorylation is mediated by a nPKC (PKC␦ or PKC⑀, depending upon the specific stimulus and cell type (11)); PKD phosphorylation via PDK-1-dependent mechanisms has not been reported. Fig. 1B shows that H 2 O 2 increases AKT-Thr 308 phosphorylation via a PDK-1-dependent mechanism that is fully abrogated by a very low concentration of UCN-01 (Ͻ0.1 M). PMA does not increase AKT-Thr 308 phosphorylation, and H 2 O 2 -dependent AKT-Thr 308 phosphorylation is not blocked by GF109203X. These results identify distinct inhibitory profiles for UCN-01 and GF109203X and implicate PDK-1 (and effectively exclude PKC isoforms) as the AKT-T 308 kinase. Fig. 1C shows that PMA and NE increase PKC␦-Thr 505 and PKD-Ser 744/748 phosphorylation via a mechanism that is blocked by 1-3 M GF109203X. Although UCN-01 also inhibits the PMA-and NE-dependent increases in PKC␦-Thr 505 and PKD-Ser 744/748 phosphorylation, these inhibitory actions of UCN-01 are detected only at high concentrations (Ͼ10-fold higher that the UCN-01 concentrations required to abrogate H 2 O 2 -dependent AKT-Thr 308 phosphorylation). This represents a promiscuous action of high UCN-01 concentrations to inhibit PKC isoforms. Collectively, these results indicate that 1) inhibitor studies with UCN-01 and GF109203X can be used to distinguish PDK-1-dependent AKT-Thr 308 phosphorylation (which is blocked by low UCN-01 concentrations but not by GF109203X) from PKC-dependent phosphorylation of PKD-Ser 744/748 and PKC␦-Thr 505 (which are blocked by GF109203X and only high UCN-01 concentrations), and 2) the dynamic cycling of PKC␦ between a fully active (Thr 505 -phosphorylated) and a less active (unphosphorylated) form in response to PMA or NE is mediated by a GF109203X-sensitive kinase with properties resembling a nPKC isoform. This could involve either a PKC␦-Thr 505 autophosphorylation reaction or PKC␦-T 505 phosphorylation in trans by PKC⑀.
PDK-1 Contributes to Activation Loop Phosphorylation during de Novo PKC␦ Synthesis-The evidence that PKC␦-Thr 505 phosphorylation is dynamically controlled through an nPKCdependent mechanism is at odds with the prevailing model that attributes activation loop phosphorylation (for PKC␦ and other AGC kinases) to PDK-1. However, this discrepancy might be reconciled if PKC␦-Thr 505 phosphorylation is controlled through dual mechanisms, with an nPKC activity contributing to the dynamic regulation of PKC␦-Thr 505 phosphorylation in response to receptor activation and PDK-1 functioning to phosphorylate the activation loop site of newly synthesized PKC␦. Therefore, we used an adenovirus-mediated gene transfer strategy to examine activation loop phosphorylation on heterologously overexpressed WT-and KD-PKC␦ enzymes. We previously showed that WT-PKC␦ is expressed at levels ϳ7-8 times higher than endogenous PKC␦ under these conditions. Fig. 2 shows that WT-PKC␦ and KD-PKC␦ are both constitutively Thr 505 -phosphorylated in resting cardiomyocytes, indicating that PKC␦ activity is not absolutely required for activation loop phosphorylation. However, at similar MOIs, KD-PKC␦ expression is consistently ϳ3-4 times lower than WT-PKC␦ expression. Moreover, Thr 505 phosphorylation of KD-PKC␦ is reduced relative to WT-PKC␦, when protein loading is normalized for differences in protein expression. These results suggest that an autophosphorylation mechanism contributes to Thr 505 phosphorylation on newly synthesized PKC␦ in cells. This phosphorylation defect presumably limits KD-PKC␦ expression, since priming phosphorylations play a role to stabilize the phosphatase-/protease-resistant conformation of the enzyme (12). Fig. 2 also shows that UCN-01 treatment (to inhibit PDK-1) results in a modest decrease in WT-PKC␦-Thr 505 phosphorylation; UCN-01 completely abrogates KD-PKC␦-Thr 505 phosphorylation. Collectively, these results indicate that the activation loop site of newly synthesized PKC␦ is phosphorylated via a dual mechanism involving both an autophosphorylation reaction and PDK-1-dependent phosphorylation in trans.
PKC⑀ Overexpression Increases PKC␦-Thr 505 Phosphorylation-Our pharmacologic studies implicate an nPKC activity (either PKC␦-Thr 505 autophosphorylation or PKC␦-Thr 505 phosphorylation in trans by PKC⑀) in PMA-dependent PKC␦-Thr 505 phosphorylation. We used an adenovirus-mediated gene transfer strategy to overexpress PKC⑀ and test the hypothesis that PKC⑀ acts as a PKC␦-Thr 505 kinase. Fig. 3A shows that WT-PKC⑀ overexpression (MOI of 100 pfu/cell) increases PKC␦-Thr 505 phosphorylation. This is not associated with a change in PKC␦ protein abundance. It is specific to catalytically active PKC⑀; KD-PKC⑀ and WT-PKC␣ do not increase PKC␦-Thr 505 phosphorylation ( Fig. 3 and data not shown).
Although these results could suggest that PKC⑀ overexpression increases PKC␦ phosphorylation by acting as a direct PKC␦-Thr 505 kinase, other mechanisms are possible and were considered. Fig. 3 shows that Ad-WT-PKC⑀ overexpression does not lead to any detectable changes in PDK-1 protein expression, PDK-1-Ser 241 (activation loop) phosphorylation (panel A); Ad-wt-PKC⑀ overexpression also does not increase PDK-1 activity, measured as basal or agonist-dependent AKT Cell extracts were then subjected to immunoblotting to compare PKC␦ protein abundance and PKC␦-Thr 505 (T 505 ) phosphorylation. Since WT-PKC␦ overexpression was ϳ5 times higher than KD-PKC␦ expression (at a similar MOI), protein loading was adjusted so that activation loop phosphorylation could be compared on similar amounts of heterologously overexpressed WT and KD enzymes. The figure shows that UCN-01 decreases WT-PKC␦-Thr 505 phosphorylation and completely abrogates KD-PKC␦-Thr 505 phosphorylation; KD-PKC␦-Thr(P) 505 immunoreactivity was below the limits of detection in UCN-01-treated cultures, even when protein loading was increased and gel exposure times were prolonged. AUGUST 10, 2007 • VOLUME 282 • NUMBER 32 phosphorylation (panel B). These results effectively exclude an indirect effect of PKC⑀ overexpression to regulate PKC␦ via PDK-1.

The Control of PKC␦-Thr 505 Phosphorylation in Cardiomyocytes
Our previous studies showed that the PMA-dependent increase in PKC␦-Thr 505 phosphorylation is confined to the pool of enzyme recovered in the particulate fraction. Therefore, we considered an alternative indirect mechanism for nPKC isoform cross-regulation involving an effect of PKC⑀ to regulate a lipid-modifying enzyme (such as a phospholipase C or diacylglycerol kinase (13,14)), leading to increased DAG levels and the stabilization of PKC␦ at membranes. To address this alternative mechanism for PKC⑀-dependent PKC␦-Thr 505 phosphorylation, we compared the subcellular distributions of PKC␣ and PKC␦ in resting and PMA-treated Ad-␤-galactosidase and Ad-PKC⑀ cultures. Fig. 4 shows that PKC␣ is recovered largely in the soluble fraction, whereas PKC␦ partitions between the soluble and particulate fractions of resting Ad-␤-galactosidase and Ad-PKC⑀ cultures. These results argue that the effect of PKC⑀ overexpression to increase PKC␦-Thr 505 phosphorylation cannot readily be attributed to a gross change in PKC␦ targeting to membranes. Rather, Fig. 4 shows that PKC⑀ overexpression leads to dysregulated PKC␦-Thr 505 phosphorylation. PKC␦-Thr 505 immunoreactivity is confined to the pool of PKC␦ that localizes to the particulate fraction following PMA treatment in Ad-␤-galactosidase cultures. In contrast, PKC␦-Thr 505 immunoreactivity is detected in both the soluble and particulate fractions of resting Ad-PKC⑀ cultures. Fig. 4 also shows that PMA treatment for 24 h leads to the complete loss of PKC immunoreactivity in Ad-␤-galactosidase cultures, whereas the Thr 505phosphorylated form of PKC␦ (and lesser amounts of PKC␣) accumulates in the particulate fraction of Ad-PKC⑀ cultures under these conditions. These results indicate that Ad-PKC⑀ overexpression leads to a defect in PKC down-regulation.
PKC⑀ Overexpression Increases PKC␦-Tyr 311 /Tyr 332 Phosphorylation-The studies thus far identify PKC␦ as a downstream target of the PKC⑀ signaling pathway but neither implicate nor refute the role of PKC⑀ as a direct PKC␦-Thr 505 kinase. Therefore, additional mechanisms for nPKC cross-talk were considered. In particular, PKC␦ is a well known target for regulated tyrosine phosphorylation. We previously demonstrated that H 2 O 2 increases PKC␦ phosphorylation at Tyr 311 (8). Other studies identify PKC␦ phosphorylation at Tyr 332 in cells subjected to oxidative stress (15). Although PKC␦-Thr 505 and tyrosine phosphorylations are generally viewed as independently regulated events (and there was no a priori reason to anticipate that PKC⑀ overexpression would lead to PKC␦ tyrosine phosphorylation), Fig. 5A provides surprising evidence that WT-PKC⑀ (but not KD-PKC⑀) markedly increases basal and H 2 O 2 -dependent PKC␦ tyrosine phosphorylation. The PKC⑀dependent increase in PKC␦ tyrosine phosphorylation is detected with an anti-phospho-PKC␦-Tyr 311 antibody (that can be used directly on cell extracts) as well as with anti-phospho-Tyr and anti-phospho-PKC␦-Tyr 332 antibodies (that require immunoprecipitation; the anti-phospho-PKC␦-Tyr 332   phosphorylation site-specific antibodies detects too many nonspecific bands to be informative in studies on cell extracts).
We previously reported that H 2 O 2 increases PKC␦ tyrosine phosphorylation via an Src-dependent mechanism in cardiomyocytes (8). Fig. 5 shows that the Ad-PKC⑀-dependent increases in PKC␦-Tyr 311 and -Tyr 332 phosphorylation are not accompanied by a detectable increase in Src protein or Src activity (tracked by an antibody that recognizes Src activation loop phosphorylation, a useful surrogate for Src activity). However, the Ad-PKC⑀-dependent increment in PKC␦-Thr 505 phosphorylation requires Src activity, since PKC␦-Thr 505 phosphorylation is not increased in Ad-PKC⑀ cultures treated with PP1 (which inhibits Src activity and PKC␦ tyrosine phosphorylation). These studies provide novel evidence that PKC␦ tyrosine and Thr 505 phosphorylation are interdependent events. Our results indicate that PKC⑀ overexpression leads to Src-dependent PKC␦ tyrosine phosphorylation and that PKC␦ tyrosine phosphorylation facilitates further PKC␦ phosphorylation at Thr 505 (although these results still do not discriminate a PKC␦ autophosphorylation reaction from PKC␦ phosphorylation in trans by PKC⑀).
Previous studies in genetically engineered mouse models have suggested that PKC⑀ exerts an inhibitory control on PKC␦ protein expression and/or phosphorylation, which is lost in the PKC⑀ Ϫ/Ϫ mouse (i.e. PKC␦ protein and/or phosphorylation is already increased in PKC⑀ Ϫ/Ϫ cells) (16,17). However, Fig. 6 shows that Ad-PKC⑀ overexpression (at increasing MOIs) leads to a dose-dependent increase in PKC␦-Thr 505 and -Tyr 311 phosphorylation in PKC⑀ Ϫ/Ϫ MEFs and primary cardiac fibroblast cultures. In these cells (which exhibit robust PKC⑀ overexpression, even at relatively low MOIs), PKC␦-Thr 505 /Tyr 311 phosphorylation increases without an associated change in PKC␦ abundance at low MOI (20 pfu/cell), whereas PKC␦ protein also accumulates as PKC⑀ overexpression levels increase (Fig. 6) (data not shown). These results emphasize that PKC␦ is not necessarily constitutively activated in PKC⑀ Ϫ/Ϫ cells and that PKC⑀ overexpression leads to a general increase in PKC␦ phosphorylation in many cell types, not just cardiomyocytes.

In vitro Kinase Assays Show that Src Phosphorylates PKC␦ at
Tyr 311 /Tyr 332 , Leading to Enhanced PKC␦ Autophosphorylation at Thr 505 -In vitro kinase assays with recombinant PKC␦ and active Src provided novel evidence that PKC␦ undergoes a Src-regulated Thr 505 autophosphorylation reaction. Fig. 7 shows that PKC␦ autophosphorylates at a very low rate in the absence of lipids; PKC␦ autophosphorylation at Thr 505 is increased by the addition of lipid micelles containing phosphatidylserine/PMA. Src induces only a trivial increase in PKC␦ tyrosine phosphorylation without lipid cofactors. However, Src induces a prominent increase in PKC␦-Tyr 311 and -Tyr 332 phosphorylation when incubations are performed in the presence of PMA (which does not alter Src activity but rather induces a conformational change that renders PKC␦ a better substrate for Src). Importantly, Fig. 7 provides unanticipated evidence that the Src-dependent increase in PKC␦-  Tyr 311 /-Tyr 332 phosphorylation is associated with an increase in in vitro PKC␦ autophosphorylation at Thr 505 .

PKC⑀ Does Not Promote PKC␦-Tyr 311 /Thr 505 Phosphorylation in SYF Cells; PKC⑀-dependent PKC␦-Tyr 311 /Thr 505 Phosphorylation Is Restored by Src Expression-The in vitro studies
suggest that Src (and PKC␦ tyrosine phosphorylation) plays a critical role to link PKC⑀ overexpression to increased PKC␦-Thr 505 phosphorylation. Since PKC⑀ overexpression leads to an increase in PKC␦-Thr 505 phosphorylation in cardiomyocytes, cardiac fibroblasts, and PKC⑀ Ϫ/Ϫ MEFs (i.e. this is a general mechanism that is not confined to cardiomyocytes), we reasoned that SYF cells might constitute an informative model to interrogate the role of Src in the in vivo PKC⑀-dependent mechanism leading to PKC␦-Thr 505 phosphorylation. SYF cells are a continuous fibroblast cell line generated from the embryos of mice lacking the three major Src family kinases, Src, Yes, and Fyn (18). Fig. 8 shows that PKC␦ is recovered from SYF cells with a low level of Thr 505 phosphorylation and no detectable Tyr 311 phosphorylation. PKC⑀ overexpression does alter PKC␦ protein expression or PKC␦ phosphorylation at Tyr 311 or Thr 505 in SYF cells. In contrast, PKC⑀ overexpression induces a coordinate increase in PKC␦-Tyr 311 and -Thr 505 phosphorylation in Src ϩ cells, a SYF cell derivative engineered to overexpress Src. Collectively, these studies unambiguously implicate 1) Src as a physiologically relevant PKC␦-Tyr 311 and -Tyr 332 kinase and 2) Src-dependent PKC␦ tyrosine phosphorylation as a mechanism that links PKC⑀ overexpression to PKC␦-Thr 505 phosphorylation.

DISCUSSION
PKC␦ was originally characterized as an allosterically activated enzyme that transduces signals from stimuli that trigger the hydrolysis of membrane phosphoinositides. However, recent studies identify additional dynamic regulatory controls through activation loop phosphorylation. PKC␦-Thr 505 phos-phorylation was originally attributed to PDK-1, based upon an early study showing that PDK-1 complexes with and phosphorylates PKC␦ (as well as the extensive literature implicating PDK-1 as a general activation loop kinase for a diverse array of AGC kinases (3,19)). However, we recently implicated a novel PKC activity in the dynamic agonist-dependent increase in PKC␦-Thr 505 phosphorylation in cardiomyocytes. Results reported herein extend these findings by identifying PKC␦-Thr 505 phosphorylation as an elaborately controlled mechanism that is regulated by PDK-1, PKC␦ autophosphorylation, PKC⑀, and Src, depending upon cell context.
We previously demonstrated that endogenous PKC␦ is recovered from resting cardiomyocytes with little to no activation loop phosphorylation. In contrast, PKC␦ retains high levels of activation loop phosphorylation when overexpressed (even at relatively modest levels) in cardiomyocyte cultures. We exploited this feature of the overexpressed enzyme to delineate the mechanisms that set basal PKC␦-Thr 505 phosphorylation in cardiac cultures. Our studies show that WT-PKC␦ and KD-PKC␦ are both recovered with some level of activation loop phosphorylation, indicating that PKC␦ activity is not absolutely required for PKC␦-Thr 505 phosphorylation. However, KD-PKC␦ exhibits a relatively low level of Thr 505 phosphorylation, even when corrected for the reduced levels of KD-PKC␦ protein expression. This residual KD-PKC␦-Thr 505 phosphorylation is completely abrogated by a low concentration of UCN-01 (that selectively inhibits PDK-1). Collectively, these results indicate that PDK-1 cooperates with PKC␦ to generate the fully phosphorylated form of PKC␦ during de novo enzyme synthesis. Although PKC␦-Thr 505 phosphorylation is generally attributed to PDK-1 (and a role for PKC␦ autophosphorylation is not generally considered), it is worth noting that current models implicating PDK-1 as a PKC␦-Thr 505 kinase are based largely upon an early study that used a bacterially expressed PKC␦ preparation that retained only very limited catalytic activity (3). In fact, there is ample evidence that related AGC kinases, such as PKA, can be processed to an active form via an autocatalytic mechanism in certain in vivo environments and that PDK1 is not necessarily rate-limiting for PKA activation loop phosphorylation (since PKA activation loop phosphorylation and enzyme activity are similar in PDK1 ϩ/ϩ and PDK1 Ϫ/Ϫ ES cells (20,21)). Of note, PKC␦ protein is detected in PDK1 Ϫ/Ϫ ES cells, although PKC␦ expression is reduced (presumably as a result of a relative activation loop phosphorylation defect and the associated C-terminal autophosphorylation defect that destabilizes the nascent enzyme (22)). In contrast, PKC⑀ is completely dependent upon PDK-1 for activation loop phosphorylation; PKC⑀ protein is not detectable in PDK1 Ϫ/Ϫ ES cells (22).
Although PDK-1 cooperates with PKC␦ to generate the fully phosphorylated form of PKC␦ during de novo enzyme synthesis, our pharmacologic studies indicate that PDK-1 does not participate in the PMA-or ␣ 1 -adrenergic receptor-dependent mechanism that dynamically increases PKC␦-Thr 505 phosphorylation in cells. Here, PKC␦-Thr 505 phosphorylation is attributable to an nPKC activity, either an autophosphorylation reaction or a trans phosphorylation by PKC⑀. We used an adenovirus-mediated overexpression strategy as an initial strat- FIGURE 8. PKC⑀ overexpression increases PKC␦-Tyr 311 (pY 311 ) and -Thr 505 (pT 505 ) phosphorylation in Src ؉ cells but not in SYF cells that lack Src, Yes, and Fyn expression. Shown is immunoblotting on cell extracts prepared from Src ϩ and SYF cells that were cultured and infected with Ad-PKC⑀ or Ad-␤-galactosidase and then treated with vehicle or PMA (300 nM for 20 min) as described under "Experimental Procedures."