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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.
Traditional models of PKCδ2 activation have focused on lipid cofactor binding to determinants in the N-terminal regulatory domain that anchor PKCδ to membranes and promote a conformational change that expels the autoinhibitory pseudosubstrate domain from the substrate-binding pocket. This effectively relieves autoinhibition and enables PKCδ-dependent phosphorylation of target substrates. However, we and others recently demonstrated that PKCδ also is dynamically regulated through phosphorylation at a conserved threonine residue in the activation loop (Thr505) (1, 2). Other PKCs require activation loop phosphorylation as a “priming” event to generate a catalytically competent enzyme. In contrast, PKCδ 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 PKCδ (1–5). The precise controls and consequences of the coordinate events that govern PKCδ phosphorylation and translocation in highly differentiated cells, such as cardiomyocytes, remain uncertain.
PKCδ 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 PKCδ in undifferentiated cell types (3). Our recent studies suggest that this model is not sufficient to describe the control of PKCδ-Thr505 phosphorylation in the heart, where the α1-adrenergic receptor agonist norepinephrine (NE) and PMA increase PKCδ-Thr505 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 PKCδ-Thr505 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 PKCδ-Thr505 kinases are examined in greater detail in this study.
All antibodies were from Cell Signaling Technology with the following exceptions: anti-PKCδ (Santa Cruz Bio-technology, Inc., Santa Cruz, CA); anti-Tyr(P), anti-PKCα, and anti-AKT (Upstate Biotechnology); anti-PKCε (BD Transduction); and anti-Src (Oncogene).
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 publications (6). 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 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).
PKCε−/− mouse embryonic fibroblast cell lines (MEFs) were described previously and generously provided by Dr. Peter Parker (7). PKCε−/− MEFs were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum and 100 μg/ml hygromycin at 37 °C in a 5% CO2 atmosphere.
Cell infections with adenoviral vectors that drive expression of wild-type (WT) or kinase-dead (KD) PKCδ, WT-PKCε, KD-PKCε, or β-galactosidase (as a control) were according to protocols published previously (8). Protein extracts were prepared 44–48 h following infections.
Immuno-blotting on lysates or immunoprecipitated PKCδ 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.
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 β-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), lysed by sonication, and centrifuged at 100,000 × g for 1 h. The supernatant was saved as the soluble fraction, and the particulate fraction was solubilized in SDS-PAGE sample buffer.
0.1 μg of recombinant human PKCδ was preincubated for 15 min at 30 °C in the absence or presence of Src kinase (0.66 units) in 160 μl of a reaction buffer containing 43 mM Tris-Cl, pH 7.5, 6.25 mM MgCl2, 10 mM MnCl2, 0.75 mM EDTA, 0.77 mM EGTA, 0.3 mM dithiothreitol, 125 mM NaCl, 5% glycerol, 0.006 Brij-35, 0.04 mM phenylmethylsulfonyl fluoride, 0.2 mM benzamidine, and γ-32P]ATP (13 μCi, 83 μM) in the absence or presence of phosphatidylserine/PMA. Samples were subjected to SDS-PAGE, autoradiography, and immunoblotting with the indicated antibodies.
Our initial experiments used a pharmacologic approach to identify the PKCδ-Thr505 phosphorylation mechanism in NE- and PMA-treated cardiomyocytes. Fig. 1A shows that PKCδ retains a low level of Thr505 phosphorylation in resting cardiomyocytes; PKCδ-Thr505 phosphorylation increases dynamically in response to treatment with either NE or PMA. These stimulus-induced increases in PKCδ-Thr505 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 3′-phosphoinositides that co-localize 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 PKCδ-Thr505 phosphorylation.
These pharmacologic studies implicate a nPKC activity in agonist-dependent PKCδ-Thr505 phosphorylation. Since they run counter to the prevailing notion that PKCδ-Thr505 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 PKCδ-Thr505 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 PKCδ (Thr505), AKT (Thr308), and PKD (PKD-Ser744/748) 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 activation loop phosphorylation is mediated by a nPKC (PKCδ or PKCε, depending upon the specific stimulus and cell type (11)); PKD phosphorylation via PDK-1-dependent mechanisms has not been reported.
Fig. 1B shows that H2O2 increases AKT-Thr308 phosphorylation via a PDK-1-dependent mechanism that is fully abrogated by a very low concentration of UCN-01 (<0.1 μM). PMA does not increase AKT-Thr308 phosphorylation, and H2O2-dependent AKT-Thr308 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-T308 kinase.
Fig. 1C shows that PMA and NE increase PKCδ-Thr505 and PKD-Ser744/748 phosphorylation via a mechanism that is blocked by 1–3 μM GF109203X. Although UCN-01 also inhibits the PMA- and NE-dependent increases in PKCδ-Thr505 and PKD-Ser744/748 phosphorylation, these inhibitory actions of UCN-01 are detected only at high concentrations (>10-fold higher that the UCN-01 concentrations required to abrogate H2O2-dependent AKT-Thr308 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-Thr308 phosphorylation (which is blocked by low UCN-01 concentrations but not by GF109203X) from PKC-dependent phosphorylation of PKD-Ser744/748 and PKCδ-Thr505 (which are blocked by GF109203X and only high UCN-01 concentrations), and 2) the dynamic cycling of PKCδ between a fully active (Thr505-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 PKCδ-Thr505 autophosphorylation reaction or PKCδ-T505 phosphorylation in trans by PKCε.
The evidence that PKCδ-Thr505 phosphorylation is dynamically controlled through an nPKC-dependent mechanism is at odds with the prevailing model that attributes activation loop phosphorylation (for PKCδ and other AGC kinases) to PDK-1. However, this discrepancy might be reconciled if PKCδ-Thr505 phosphorylation is controlled through dual mechanisms, with an nPKC activity contributing to the dynamic regulation of PKCδ-Thr505 phosphorylation in response to receptor activation and PDK-1 functioning to phosphorylate the activation loop site of newly synthesized PKCδ. Therefore, we used an adenovirus-mediated gene transfer strategy to examine activation loop phosphorylation on heterologously overexpressed WT- and KD-PKCδ enzymes. We previously showed that WT-PKCδ is expressed at levels ~7–8 times higher than endogenous PKCδ under these conditions. Fig. 2 shows that WT-PKCδ and KD-PKCδ are both constitutively Thr505-phosphorylated in resting cardiomyocytes, indicating that PKCδ activity is not absolutely required for activation loop phosphorylation. However, at similar MOIs, KD-PKCδ expression is consistently ~3–4 times lower than WT-PKCδ expression. Moreover, Thr505 phosphorylation of KD-PKCδ is reduced relative to WT-PKCδ, when protein loading is normalized for differences in protein expression. These results suggest that an autophosphorylation mechanism contributes to Thr505 phosphorylation on newly synthesized PKCδ in cells. This phosphorylation defect presumably limits KD-PKCδ 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-PKCδ-Thr505 phosphorylation; UCN-01 completely abrogates KD-PKCδ-Thr505 phosphorylation. Collectively, these results indicate that the activation loop site of newly synthesized PKCδ is phosphorylated via a dual mechanism involving both an autophosphorylation reaction and PDK-1-dependent phosphorylation in trans.
Our pharmacologic studies implicate an nPKC activity (either PKCδ-Thr505 autophosphorylation or PKCδ-Thr505 phosphorylation in trans by PKCε in PMA-dependent PKCδ-Thr505 phosphorylation. We used an adenovirus-mediated gene transfer strategy to overexpress PKCε and test the hypothesis that PKCε acts as a PKCδ -Thr505 kinase. Fig. 3A shows that WT-PKCε overexpression (MOI of 100 pfu/cell) increases PKCδ-Thr505 phosphorylation. This is not associated with a change in PKCδ protein abundance. It is specific to catalytically active PKCε; KD-PKCε and WT-PKCα do not increase PKCδ-Thr505 phosphorylation (Fig. 3 and data not shown).
Although these results could suggest that PKCε overexpression increases PKCδ phosphorylation by acting as a direct PKCδ-Thr505 kinase, other mechanisms are possible and were considered. Fig. 3 shows that Ad-WT-PKCε overexpression does not lead to any detectable changes in PDK-1 protein expression, PDK-1-Ser241 (activation loop) phosphorylation (panel A); Ad-wt-PKCε overexpression also does not increase PDK-1 activity, measured as basal or agonist-dependent AKT phosphorylation (panel B). These results effectively exclude an indirect effect of PKCε overexpression to regulate PKCδ via PDK-1.
Our previous studies showed that the PMA-dependent increase in PKCδ-Thr505 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 PKCε 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 PKCδ at membranes. To address this alternative mechanism for PKCε-dependent PKCδ-Thr505 phosphorylation, we compared the subcellular distributions of PKCα and PKCδ in resting and PMA-treated Ad-β-galactosidase and Ad-PKCε cultures. Fig. 4 shows that PKCα is recovered largely in the soluble fraction, whereas PKCδ partitions between the soluble and particulate fractions of resting Ad-β-galactosidase and Ad-PKCε cultures. These results argue that the effect of PKCε overexpression to increase PKCδ-Thr505 phosphorylation cannot readily be attributed to a gross change in PKCδ targeting to membranes. Rather, Fig. 4 shows that PKCε overexpression leads to dysregulated PKCδ-Thr505 phosphorylation. PKCδ-Thr505 immunoreactivity is confined to the pool of PKCδ that localizes to the particulate fraction following PMA treatment in Ad-β-galactosidase cultures. In contrast, PKCδ-Thr505 immunoreactivity is detected in both the soluble and particulate fractions of resting Ad-PKCε cultures. Fig. 4 also shows that PMA treatment for 24 h leads to the complete loss of PKC immuno-reactivity in Ad-β-galactosidase cultures, whereas the Thr505-phosphorylated form of PKCδ (and lesser amounts of PKCα) accumulates in the particulate fraction of Ad-PKCε cultures under these conditions. These results indicate that Ad-PKCε overexpression leads to a defect in PKC down-regulation.
The studies thus far identify PKCδ as a downstream target of the PKCε signaling pathway but neither implicate nor refute the role of PKCε as a direct PKCδ-Thr505 kinase. Therefore, additional mechanisms for nPKC cross-talk were considered. In particular, PKCδ is a well known target for regulated tyrosine phosphorylation. We previously demonstrated that H2O2 increases PKCδ phosphorylation at Tyr311 (8). Other studies identify PKCδ phosphorylation at Tyr332 in cells subjected to oxidative stress (15). Although PKCδ-Thr505 and tyro-sine phosphorylations are generally viewed as independently regulated events (and there was no a priori reason to anticipate that PKCε overexpression would lead to PKCδ tyrosine phosphorylation), Fig. 5A provides surprising evidence that WT-PKCε (but not KD-PKCε) markedly increases basal and H2O2-dependent PKCδ tyrosine phosphorylation. The PKCε-dependent increase in PKCδ tyrosine phosphorylation is detected with an anti-phospho-PKCδ-Tyr311 antibody (that can be used directly on cell extracts) as well as with anti-phospho-Tyr and anti-phospho-PKCδ-Tyr332 antibodies (that require immunoprecipitation; the anti-phospho-PKCδ-Tyr332 phosphorylation site-specific antibodies detects too many non-specific bands to be informative in studies on cell extracts).
We previously reported that H2O2 increases PKCδ tyrosine phosphorylation via an Src-dependent mechanism in cardio-myocytes (8). Fig. 5 shows that the Ad-PKCε-dependent increases in PKCδ-Tyr311 and -Tyr332 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-PKCε-dependent increment in PKCδ-Thr505 phosphorylation requires Src activity, since PKCδ-Thr505 phosphorylation is not increased in Ad-PKCε cultures treated with PP1 (which inhibits Src activity and PKCδ tyrosine phosphorylation). These studies provide novel evidence that PKCδ tyrosine and Thr505 phosphorylation are interdependent events. Our results indicate that PKCε overexpression leads to Src-dependent PKCδ tyrosine phosphorylation and that PKCδ tyrosine phosphorylation facilitates further PKCδ phosphorylation at Thr505 (although these results still do not discriminate a PKCδ autophosphorylation reaction from PKCδ phosphorylation in trans by PKCε).
Previous studies in genetically engineered mouse models have suggested that PKCε exerts an inhibitory control on PKCδ protein expression and/or phosphorylation, which is lost in the PKCε−/− mouse (i.e. PKCδ protein and/or phosphorylation is already increased in PKCε−/− cells) (16, 17). However, Fig. 6 shows that Ad-PKCε overexpression (at increasing MOIs) leads to a dose-dependent increase in PKCδ-Thr505 and -Tyr311 phosphorylation in PKCε−/−MEFs and primary cardiac fibroblast cultures. In these cells (which exhibit robust PKCε overexpression, even at relatively low MOIs), PKCδ-Thr505/Tyr311 phosphorylation increases without an associated change in PKCδ abundance at low MOI (20 pfu/cell), whereas PKCδ protein also accumulates as PKCδ overexpression levels increase (Fig. 6) (data not shown). These results emphasize that PKCδ is not necessarily constitutively activated in PKCε−/− cells and that PKCε overexpression leads to a general increase in PKCδ phosphorylation in many cell types, not just cardiomyocytes.
In vitro kinase assays with recombinant PKCδ and active Src provided novel evidence that PKCδ undergoes a Src-regulated Thr505 autophosphorylation reaction. Fig. 7 shows that PKCδ autophosphorylates at a very low rate in the absence of lipids; PKCδ autophosphorylation at Thr505 is increased by the addition of lipid micelles containing phosphatidylserine/PMA. Src induces only a trivial increase in PKCδ tyrosine phosphorylation without lipid cofactors. However, Src induces a prominent increase in PKCδ-Tyr311 and -Tyr332 phosphorylation when incubations are performed in the presence of PMA (which does not alter Src activity but rather induces a conformational change that renders PKCδ a better substrate for Src). Importantly, Fig. 7 provides unanticipated evidence that the Src-dependent increase in PKCδ-Tyr311/-Tyr332 phosphorylation is associated with an increase in in vitro PKCδ autophosphorylation at Thr505.
The in vitro studies suggest that Src (and PKCδ tyrosine phosphorylation) plays a critical role to link PKCε overexpression to increased PKCδ-Thr505 phosphorylation. Since PKCε overexpression leads to an increase in PKCδ-Thr505 phosphorylation in cardiomyocytes, cardiac fibroblasts, and PKCε−/− 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 PKCε-dependent mechanism leading to PKCδ-Thr505 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 PKCδ is recovered from SYF cells with a low level of Thr505 phosphorylation and no detectable Tyr311 phosphorylation. PKCε overexpression does alter PKCδ protein expression or PKCδ phosphorylation at Tyr311 or Thr505 in SYF cells. In contrast, PKCε overexpression induces a coordinate increase in PKCδ-Tyr311 and -Thr505 phosphorylation in Src+ cells, a SYF cell derivative engineered to overexpress Src. Collectively, these studies unambiguously implicate 1) Src as a physiologically relevant PKCδ-Tyr311 and -Tyr332 kinase and 2) Src-dependent PKCδ tyrosine phosphorylation as a mechanism that links PKCε overexpression to PKCδ-Thr505 phosphorylation.
PKCδ 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. PKCδ-Thr505 phosphorylation was originally attributed to PDK-1, based upon an early study showing that PDK-1 complexes with and phosphorylates PKCδ (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 PKCδ-Thr505 phosphorylation in cardiomyocytes. Results reported herein extend these findings by identifying PKCδ-Thr505 phosphorylation as an elaborately controlled mechanism that is regulated by PDK-1, PKCδ autophosphorylation, PKCε, and Src, depending upon cell context.
We previously demonstrated that endogenous PKCδ is recovered from resting cardiomyocytes with little to no activation loop phosphorylation. In contrast, PKCδ 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 PKCδ-Thr505 phosphorylation in cardiac cultures. Our studies show that WT-PKCδ and KD-PKCδ are both recovered with some level of activation loop phosphorylation, indicating that PKCδ activity is not absolutely required for PKCδ-Thr505 phosphorylation. However, KD-PKCδ exhibits a relatively low level of Thr505 phosphorylation, even when corrected for the reduced levels of KD-PKCδ protein expression. This residual KD-PKCδ-Thr505 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 PKCδ to generate the fully phosphorylated form of PKCδ during de novo enzyme synthesis. Although PKCδ-Thr505 phosphorylation is generally attributed to PDK-1 (and a role for PKCδ autophosphorylation is not generally considered), it is worth noting that current models implicating PDK-1 as a PKCδ-Thr505 kinase are based largely upon an early study that used a bacterially expressed PKCδ 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+/+and PDK1−/− ES cells (20, 21)). Of note, PKCδ protein is detected in PDK1−/− ES cells, although PKCδ 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, PKCε is completely dependent upon PDK-1 for activation loop phosphorylation; PKCε protein is not detectable in PDK1−/− ES cells (22).
Although PDK-1 cooperates with PKCδ to generate the fully phosphorylated form of PKCδ during de novo enzyme synthesis, our pharmacologic studies indicate that PDK-1 does not participate in the PMA- or α1-adrenergic receptor-dependent mechanism that dynamically increases PKCδ-Thr505 phosphorylation in cells. Here, PKCδ-Thr505 phosphorylation is attributable to an nPKC activity, either an autophosphorylation reaction or a trans phosphorylation by PKCε. We used an adenovirus-mediated overexpression strategy as an initial strategy to determine whether PKCε can act as a direct PKCδ-Thr505 kinase. Our studies show that PKCε overexpression leads to a robust increase in PKCδ-Thr505 phosphorylation. Had we stopped at this level of analysis, we might have concluded that PKCε acts as a direct in vivo PKCδ-Thr505 kinase (and in fact, we have no direct evidence to exclude a role for this mechanism under certain circumstances). However, further studies provided compelling evidence that PKCε increases PKCδ-Thr505 phosphorylation indirectly via a mechanism involving Src and Src-dependent PKCδ tyrosine phosphorylation. Specifically, we found that PKCε increases PKCδ-Thr505 phosphorylation in association with an increase in PKCδ phosphorylation at Tyr311 and Tyr332. We also found that the PKCε-dependent increase in PKCδ-Thr505 phosphorylation requires Src activity (and PKCδ tyrosine phosphorylation), since 1) PKCε does not increase PKCδ-Thr505 phosphorylation in cardiomyocytes treated with PP1, and 2) the PKCε-dependent increase in PKCδ-Thr505 phosphorylation is not detected in SYF cells (that lack Src activity), and it is restored by Src reexpression (in the Src+ cell line). In vitro studies exposed the underlying mechanism, showing that PKCδ undergoes a Thr505 autophosphorylation reaction that is facilitated when assays are performed in the presence of active Src (under conditions leading to PKCδ-Tyr311/Tyr332 phosphorylation). Collectively, these results add a new dimension to models of PKCδ signaling, showing that Src (and PKCδ tyrosine phosphorylation) controls PKCδ-Thr505 autophosphorylation in cells. These studies indicate that PKCδ is uniquely positioned to sense signaling inputs from both Src and PKCδ pathways in cells. These studies also indicate that tyrosine phosphorylation plays a fundamental role in the control of PKCδ activity. Although the full functional implications of PKCδ-Thr505 phosphorylation are not fully resolved, recent studies provide intriguing evidence that PKCδ-Thr505 phosphorylation “fine tunes” the enzymology of PKCδ by altering its substrate specificity (5). Hence, our studies suggest that activation loop phosphorylation might represent a final common mechanism to control the catalytic function of PKCδ in a variety of contexts, including in the context of oxidative stress and Src-dependent PKCδ tyrosine phosphorylation.
Many laboratories have come to rely on an adenovirus-mediated overexpression strategies to resolve PKCε and PKCδ actions in cells, at least in part to avoid using pharmacologic inhibitors, such as chelerythrine and rottlerin, that exert toxic/PKC-independent actions. PKCε overexpression protocols are generally validated by experiments showing that PKCδ overexpression does not lead to compensatory changes in the abundance of other PKC isoforms (23). Our studies emphasize that measurements of nPKC isoform protein expression are inadequate, since even relatively low levels of PKCε overexpression lead to coordinate increases in PKCδ phosphorylation at Thr505, Tyr311, and Tyr332. These results identify a serious limitation associated with the use of an adenovirus-mediated PKCε overexpression strategy to resolve the cellular actions of PKCε and PKCδ in cells (perhaps explaining some of the ambiguities identified in previous studies that have used this experimental strategy (24)).
Previous studies in genetically engineered mouse models have offered hints that PKCδ might be a downstream target of PKCε. Klein et al. (17) reported that PKCδ protein expression and Thr505 phosphorylation are increased in PKCε−/− (but not normal) hearts subjected to pressure overload. Although Klein et al. did not identify changes in base-line PKCδ protein or phosphorylation in the absence of a hypertrophic stimulus, Gray et al. (16) identified increased PKCδ expression and PKCδ localization to perinuclear structures (a sign of chronic PKCδ activation) in resting cardiomyocytes isolated from PKCε−/− mice. These previous studies in genetically engineered mouse models were interpreted as evidence that PKCε functions to inhibit PKCδ (and that this inhibitory effect of PKCε is lost in the PKCε−/− mouse). Based upon this literature, the effect of PKCε overexpression to increase PKCδ phosphorylation was surprising (and serves to emphasize that despite years of research, we still have attained only a rudimentary understanding of mechanisms that control nPKC isoform cross-talk in highly differentiated tissues). The effect of PKCε overexpression to induce a generalized increase PKCδ phosphorylation in many cell types (including cardiomyocytes, primary cardiac fibroblasts, Src+ cells, and PKCε−/− MEFs) suggests that the elevated levels of PKCδ protein and/or phosphorylation observed in PKCε−/− mice may reflect a compensatory response to total body PKCε knock-out from embryonic life onward and have little relevance to the physiologic control of PKCδ in cells. These results serve to underscore the potential pitfalls inherent in extrapolations based upon data obtained in genetic models in mice, where some aspects of the phenotype may not necessarily be physiologically relevant.
Our studies suggest that PKCδ is uniquely positioned within cellular signaling networks to integrate input from PKCε and Src signaling pathways. The precise molecular determinants that link PKCε overexpression to an increase in PKCδ-Tyr311/Tyr332 and Thr505 phosphorylation (that is not associated with any gross changes in Src family kinase or PDK-1 activity and may involve a PKCδ-targeted phosphatase) are the focus of ongoing studies. However, to the best of our knowledge, the Src-dependent mechanism that controls activation loop phosphorylation identified in this study is unique to PKCδ; similar Src-dependent mechanisms that control activation loop phosphorylation have not been described for other AGC kinases. Insofar as PKCδ-Thr505 phosphorylation has emerged as an important determinant of PKCδ specificity toward heterologous substrates (5), an intervention that prevents Src-dependent PKCδ phosphorylation might constitute a novel therapeutic strategy to selectively regulate only a subset of PKCδ actions in cells.
2The abbreviations used are: PKC, protein kinase C; PDK, phosphoinositide-dependent kinase; NE, norepinephrine; PMA, phorbol 12-myristate 13-acetate; MEF, mouse embryo fibroblast; WT, wild type; KD, kinase-dead; PKD, protein kinase D; nPKC, novel PKC; MOI, multiplicity of infection; pfu, plaque-forming units.
*This work was supported by United States Public Health Service-NHLBI, National Institutes of Health Grant HL77860.