Regulated nucleocytoplasmic transport of protein kinase D in response to G protein-coupled receptor activation

Protein kinase D (PKD)/protein kinase C mu is a serine/threonine protein kinase activated by growth factors, antigen-receptor engagement, and G protein-coupled receptor (GPCR) agonists via a phosphorylation-dependent mechanism that requires protein kinase C (PKC) activity. In order to investigate the dynamic mechanisms associated with GPCR signaling, the intracellular distribution of PKD was analyzed in live cells by imaging fluorescent protein-tagged PKD and in fixed cells by immunocytochemistry. We found that PKD shuttled between the cytoplasm and the nucleus in both fibroblasts and epithelial cells. Cell stimulation with mitogenic GPCR agonists that activate PKD induced a transient nuclear accumulation of PKD that was prevented by inhibiting PKC activity. The nuclear import of PKD requires its cys2 domain in conjunction with a nuclear import receptor, while its nuclear export requires its pleckstrin homology domain and a competent Crm1-dependent nuclear export pathway. This study thus characterizes the regulated nuclear transport of a signaling molecule in response to mitogenic GPCR agonists and positions PKD as a serine kinase whose kinase activity and intracellular localization is coordinated by PKC.     

Rapid protein kinase D translocation in response to G protein-coupled receptor activation. Dependence on protein kinase C.

Protein kinase D (PKD)/protein kinase C (PKC) mu is a serine/threonine protein kinase that can be activated by physiological stimuli like growth factors, antigen-receptor engagement and G protein-coupled receptor (GPCR) agonists via a phosphorylation-dependent mechanism that requires PKC activity. In order to investigate the dynamic mechanisms associated with GPCR signaling, the intracellular translocation of a green fluorescent protein-tagged PKD was analyzed by real-time visualization in fibroblasts and epithelial cells stimulated with bombesin, a GPCR agonist. We found that bombesin induced a rapidly reversible plasma membrane translocation of green fluorescent protein-tagged PKD, an event that can be divided into two distinct mechanistic steps. The first step, which is exclusively mediated by the cysteine-rich domain in the N terminus of PKD, involved its translocation from the cytosol to the plasma membrane. The second step, i.e. the rapid reverse translocation of PKD from the plasma membrane to the cytosol, required its catalytic domain and surprisingly PKC activity. These findings provide evidence for a novel mechanism by which PKC coordinates the translocation and activation of PKD in response to bombesin-induced GPCR activation.     

Intracellular redistribution of protein kinase D2 in response to G-protein-coupled receptor agonists

The protein kinase D (PKD) family consists of three serine/threonine protein kinases: PKC mu/PKD, PKD2, and PKC nu/PKD3. While PKD has been the focus of most studies to date, no information is available on the intracellular distribution of PKD2. Consequently, we examined the mechanism that regulates its intracellular distribution in human pancreatic carcinoma Panc-1 cells. Analysis of the intracellular steady-state distribution of fluorescent-tagged PKD2 in unstimulated cells indicated that this kinase is predominantly cytoplasmic. Cell stimulation with the G protein-coupled receptor agonist neurotensin induced a rapid and reversible plasma membrane translocation of PKD2 by a mechanism that requires PKC activity. In contrast to the other PKD isoenzymes, PKD2 activation did not induce its redistribution from the cytoplasm to the nucleus. Thus, this study demonstrates that the regulation of the distribution of PKD2 is distinct from other PKD isoenzymes, and suggests that the differential spatio-temporal localization of these signaling molecules regulates their specific signaling properties.     

G protein-coupled receptor-mediated phosphorylation of the activation loop of protein kinase D: dependence on plasma membrane translocation and protein kinase Cepsilon

Protein kinase D (PKD) is a serine/threonine protein kinase activated by G protein-coupled receptor (GPCR) agonists through an incompletely characterized mechanism that includes its reversible plasma membrane translocation and activation loop phosphorylation via a protein kinase C (PKC)-dependent pathway. To gain a better understanding of the mechanism regulating the activation of PKD in response to GPCR stimulation, we investigated the role of its rapid plasma membrane translocation on its activation loop phosphorylation and identified the endogenous PKC isozyme that mediates that event in vivo. We had found that the activation loop of a PKD mutant, with reduced affinity for diacylglycerol and phorbol esters, was only phosphorylated upon its plasma membrane association. We also found that the activation loop phosphorylation and rapid plasma membrane dissociation of PKD were inhibited either by preventing the plasma membrane translocation of PKCepsilon, through abolition of its interaction with receptor for activated C kinase, or by suppressing the expression of PKCepsilon via specific small interfering RNAs. Thus, this study demonstrates that the plasma membrane translocation of PKD, in response to GPCR stimulation, is necessary for the PKCepsilon-mediated phosphorylation of the activation loop of PKD and that this event requires the translocation of both kinases to the plasma membrane. Based on these and previous results, we propose a model of GPCR-mediated PKD regulation that integrates its changes in distribution, catalytic activity, and multisite phosphorylation.     

Vasopressin-induced intracellular redistribution of protein kinase D in intestinal epithelial cells

The spatio-temporal changes of signaling molecules in response to G protein-coupled receptors (GPCR) stimulation is a poorly understood process in intestinal epithelial cells. Here we investigate the dynamic mechanisms associated with GPCR signaling in living rat intestinal epithelial cells by characterizing the intracellular translocation of protein kinase D (PKD), a serine/threonine protein kinase involved in mitogenic signaling in intestinal epithelial cells. Analysis of the intracellular steady-state distribution of green fluorescent protein (GFP)-tagged PKD indicated that in non-stimulated IEC-18 cells, GFP-PKD is predominantly cytoplasmic. However, cell stimulation with the GPCR agonist vasopressin induces a rapid translocation of GFP-PKD from the cytosol to the plasma membrane that is accompanied by its activation via protein kinase C (PKC)-mediated process and posterior plasma membrane dissociation. Subsequently, active PKD is imported into the nuclei where it transiently accumulates before being exported into the cytosol by a mechanism that requires a competent Crm1 nuclear export pathway. These findings provide evidence for a mechanism by which PKC coordinates in intestinal epithelial cells the translocation and activation of PKD in response to vasopressin-induced GPCR activation.     

Protein kinase D potentiates DNA synthesis induced by Gq-coupled receptors by increasing the duration of ERK signaling in swiss 3T3 cells

Protein kinase D (PKD) potentiates cellular DNA synthesis in response to G protein-coupled receptor (GPCR) agonists but the mechanism(s) involved has not been elucidated. Here, we examined whether PKD overexpression in Swiss 3T3 cells regulates the activation/inactivation kinetics of the extracellular-regulated protein kinase (ERK) in response to the mitogenic GPCR agonists bombesin and vasopressin. Addition of bombesin or vasopressin to Swiss 3T3 cells overexpressing PKD induced a striking increase in the duration of MEK/ERK/RSK activation as compared with cultures of either control Swiss 3T3 cells or Swiss 3T3 cells expressing a kinase-inactive PKD mutant. In contrast, the duration of ERK activation in response to epidermal growth factor, which acts via protein kinase C/PKD-independent pathways, was not increased. Furthermore, bombesin or vasopressin promoted a striking increase in phosphorylation (at Ser-374) and accumulation of c-Fos (the c-fos proto-oncogene product) in Swiss 3T3 cells overexpressing wild-type (but not kinase-inactive) PKD. Inhibition of the sustained phase of ERK/RSK activation abrogated the increase in c-Fos accumulation and DNA synthesis induced by bombesin or vasopressin in PKD-overexpressing cells. Our results demonstrate that PKD selectively potentiates mitogenesis induced by bombesin or vasopressin in Swiss 3T3 cells by increasing the duration of MEK/ERK/RSK signaling.     

Protein kinase D2 potentiates MEK/ERK/RSK signaling, c-Fos accumulation and DNA synthesis induced by bombesin in Swiss 3T3 cells

Protein kinase D (PKD) plays an important role in mediating cellular DNA synthesis in response to G protein-coupled receptor (GPCR) agonists but the function of other isoforms of the PKD family has been much less explored. Here, we examined whether PKD2 overexpression in Swiss 3T3 cells facilitates DNA synthesis and the activation of the extracellular regulated protein kinase (ERK) pathway in response to the mitogenic GPCR agonist bombesin. We show that PKD2 overexpression markedly potentiated the ability of this agonist to induce DNA synthesis. Addition of bombesin to Swiss 3T3 cells overexpressing PKD2 also induced a striking increase in the duration of MEK/ERK/RSK activation as compared with cultures of control cells. In contrast, neither DNA synthesis nor the duration of ERK activation in response to epidermal growth factor, which acts via protein kinase C/PKD2-independent pathways, was increased. Furthermore, bombesin promoted a striking accumulation of c-Fos protein in cells overexpressing PKD2. Our study demonstrates that PKD2, like PKD, facilitates mitogenesis and supports the hypothesis that an increase in the duration of the ERK signaling leading to accumulation of immediate gene products is one of the mechanisms by which isoforms of the PKD family enhance re-initiation of DNA synthesis by Gq-coupled receptor activation.     

PKCepsilon-PKD1 signaling complex at Z-discs plays a pivotal role in the cardiac hypertrophy induced by G-protein coupling receptor agonists

Cardiac hypertrophy is triggered in response to mechanical stress and various neurohumoral factors, such as G-protein coupling receptor (GPCR) and gp130 cytokine receptor agonists. Recent studies have suggested cardiac Z-disc plays a pivotal role to regulate these cellular responses. Here, we demonstrate stimulations with GPCR agonists (norepinephrine, angiotensin II, and endothelin 1) and phorbol ester activated and translocated protein kinase D1 (PKD1) to the Z-discs in neonatal rat cardiomyocytes in a protein kinase C (PKC)-dependent manner, whereas gp130 agonist did not. Especially, upon the alpha-adrenergic receptor agonist stimulations, following the PKCepsilon-PKD1 complex formation, PKCepsilon-dependent activation of PKD1 was essential to induce hypertrophic responses. Constitutively active mutant of either PKD1 or PKCepsilon also induced cardiac hypertrophy ex vivo. Taken together, the PKCepsilon-PKD1 complex at Z-discs could play a pivotal role in the cardiac hypertrophy induced by GPCR agonists, at least alpha-adrenergic receptor agonist.     

Mitogenic signaling pathways induced by G protein-coupled receptors

G protein-coupled receptor (GPCR) agonists, including neurotransmitters, hormones, chemokines, and bioactive lipids, act as potent cellular growth factors and have been implicated in a variety of normal and abnormal processes, including development, inflammation, and malignant transformation. Typically, the binding of an agonistic ligand to its cognate GPCR triggers the activation of multiple signal transduction pathways that act in a synergistic and combinatorial fashion to relay the mitogenic signal to the nucleus and promote cell proliferation. A rapid increase in the activity of phospholipases C, D, and A2 leading to the synthesis of lipid-derived second messengers, Ca2+ fluxes and subsequent activation of protein phosphorylation cascades, including PKC/PKD, Raf/MEK/ERK, and Akt/mTOR/p70S6K is an important early response to mitogenic GPCR agonists. The EGF receptor (EGFR) tyrosine kinase has emerged as a transducer in the signaling by GPCRs, a process termed transactivation. GPCR signal transduction also induces striking morphological changes and rapid tyrosine phosphorylation of multiple cellular proteins, including the non-receptor tyrosine kinases Src, focal adhesion kinase (FAK), and the adaptor proteins CAS and paxillin. The pathways stimulated by GPCRs are extensively interconnected by synergistic and antagonistic crosstalks that play a critical role in signal transmission, integration, and dissemination. The purpose of this article is to review recent advances in defining the pathways that play a role in transducing mitogenic responses induced by GPCR agonists.     

Protein kinase C-independent activation of protein kinase D is involved in BMP-2-induced activation of stress mitogen-activated protein kinases JNK and p38 and osteoblastic cell differentiation

An important role for JNK* and p38 has recently been discovered in the differentiating effect of bone morphogenetic protein 2 (BMP-2) on osteoblastic cells. In this study, we investigated the molecular mechanism by which BMP-2 activates JNK and p38 in MC3T3-E1 osteoblastic cells. Activation of JNK and p38 induced by BMP-2 was blocked by the protein kinase C/protein kinase D (PKC/PKD) inhibitor Go6976 but not by the related compound, Go6983, a selective inhibitor of conventional PKCs. Associated with this inhibitory effect of Go6976, BMP-2 induced a selective and a dose-dependent Ser916 phosphorylation/activation of PKD, which was also blocked by Go6976. In contrast to the recently described PKC-dependent molecular mechanism involved in activation of PKD by G protein-coupled receptor agonists, BMP-2 did not induce a phosphorylation of PKD on Ser744/748. To further document an implication of PKD in activation of JNK and p38 induced by BMP-2, we constructed MC3T3-E1 cells stably expressing PKD antisense oligonucleotide (AS-PKD). In AS-PKD clones having low PKD levels, activation of JNK and p38 by BMP-2, but not of Smad1/5, was markedly impaired compared with empty vector transfected (V-PKD) cells. Analysis of osteoblastic cell differentiation in AS-PKD compared with V-PKD cells showed that mRNA and protein expressions of alkaline phosphatase and osteocalcin induced by BMP-2 were markedly reduced in AS-PKD. In conclusion, results presented in this study indicate that BMP-2 can induce activation of PKD in osteoblastic cells by a PKC-independent mechanism and that this kinase is involved in activation of JNK and p38 induced by BMP-2. Thus, this pathway, in addition to Smads, appears to be essential for the effect of BMP-2 on osteoblastic cell differentiation.     

Thrombin rapidly induces protein kinase D phosphorylation,and protein kinase C delta mediates the activation

Thrombin plays a critical role in hemostasis, thrombosis, and inflammation. However, the responsible intracellular signaling pathways triggered by thrombin are still not well defined. We report here that thrombin rapidly and transiently induces activation of protein kinase D (PKD) in aortic smooth muscle cells. Our data demonstrate that protein kinase C (PKC) inhibitors completely block thrombin-induced PKD activation, suggesting that thrombin induces PKD activation via a PKC-dependent pathway. Furthermore, our results show that thrombin rapidly induces PKC delta phosphorylation and that the PKC delta-specific inhibitor rottlerin blocks thrombin-induced PKD activation, suggesting that PKC delta mediates the thrombin-induced PKD activation. Using dominant negative approaches, we demonstrated that expression of a dominant negative PKC delta inhibits the phosphorylation and activation of PKD induced by thrombin, whereas neither PKC epsilon nor PKC zeta affects thrombin-induced PKD activation. In addition, our results of co-immunoprecipitation assays showed that PKD forms a complex with PKC delta in smooth muscle cells. Taken together, the findings of the present study demonstrate that thrombin induces activation of PKD and reveal a novel role of PKC delta in mediating thrombin-induced PKD activation in vascular smooth muscle cells.     

Mechanism of activation of protein kinase D2(PKD2) by the CCK(B)/gastrin receptor

Recently, we cloned a novel serine/threonine kinase termed protein kinase D2 (PKD2). PKD2 can be activated by phorbol esters both in vivo and in vitro but also by gastrin via the cholecystokinin/CCK(B) receptor in human gastric cancer cells stably transfected with the CCK(B)/gastrin receptor (AGS-B cells). Here we identify the mechanisms of gastrin-induced PKD2 activation in AGS-B cells. PKD2 phosphorylation in response to gastrin was rapid, reaching a maximum after 10 min of incubation. Our data demonstrate that gastrin-stimulated PKD2 activation involves a heterotrimeric G alpha(q) protein as well as the activation of phospholipase C. Furthermore, we show that PKD2 can be activated by classical and novel members of the protein kinase C (PKC) family such as PKC alpha, PKC epsilon, and PKC eta. These PKCs are activated by gastrin in AGS-B cells. Thus, PKD2 is likely to be a novel downstream target of specific PKCs upon the stimulation of AGS-B cells with gastrin. Our data suggest a two-step mechanism of activation of PKD2 via endogenously produced diacylglycerol and the activation of PKCs.     

Protein kinase C phosphorylates protein kinase D activation loop Ser744 and Ser748 and releases autoinhibition by the pleckstrin homology domain

Persistent activation of protein kinase D (PKD) via protein kinase C (PKC)-mediated signal transduction is accompanied by phosphorylation at Ser(744) and Ser(748) located in the catalytic domain activation loop, but whether PKC isoforms directly phosphorylate these residues, induce PKD autophosphorylation, or recruit intermediate upstream kinase(s) is unclear. Here, we explore the mechanism whereby PKC activates PKD in response to cellular stimuli. We first assessed in vitro PKC-PKD transphosphorylation and PKD activation. A PKD738-753 activation loop peptide was well phosphorylated by immunoprecipitated PKC isoforms, consistent with similarities between the loop and their known substrate specificities. A similar peptide with glutamic acid replacing Ser(748) was preferentially phosphorylated by PKCepsilon, suggesting that PKD containing phosphate at Ser(748) is rapidly targeted by this isoform at Ser(744). When incubated in the presence of phosphatidylserine, phorbol 12,13-dibutyrate and ATP, intact PKD slowly autophosphorylated in the activation loop but only at Ser(748). In contrast, addition of purified PKCepsilon to the incubation mixture induced rapid Ser(744) and Ser(748) phosphorylation, concomitant with persistent 2-3-fold increases in PKD activity, measured using reimmunoprecipitated PKD to phosphorylate an exogenous peptide, syntide-2. We also further examined pleckstrin homology domain-mediated PKD regulation to determine its relationship with activation loop phosphorylation. The high constitutive activity of the pleckstrin homology (PH) domain deletion mutant PKD-deltaPH was not abrogated by mutation of Ser(744) and Ser(748) to alanines, suggesting that one function of activation loop phosphorylation in the PKD activation mechanism is to relieve autoinhibition by the PH domain. These studies provide evidence of a direct PKCepsilon-PKD phosphorylation cascade and provide additional insight into the activation mechanism.     

Oxidative stress induces protein kinase C-mediated activation loop phosphorylation and nuclear redistribution of protein kinase D

Oxidative stress induced by cell treatments with H(2)O(2) activates protein kinase D (PKD) via a protein kinase C (PKC)-dependent signal transduction pathway (Waldron, R. T., and Rozengurt, E. (2000) J. Biol. Chem. 275, 17114-17121). Here we show that oxidative stress induces PKC-dependent activation loop Ser(744) and Ser(748) phosphorylation to mediate dose- and time-dependent activation of PKD, both endogenously expressed in Swiss 3T3 cells and stably overexpressed in Swiss 3T3-GFP.PKD cells. Although oxidative stress induced PKD activation loop phosphorylation and activation with identical kinetics, both were dose-dependently blocked by preincubation of cells with selective inhibitors of PKC (GF109203X and G?6983) or c-Src (PP2). Inhibition of Src tyrosine kinase activity eliminated oxidative stress-induced direct PKD tyrosine phosphorylation, but only partially attenuated activation loop phosphorylation and activation. Mutation of a putative tyrosine phosphorylation site on PKD, Tyr(469) to phenylalanine, had no effect on its activation by oxidative stress in transfected COS-7 cells. Similarly, a mutant with Tyr(469) replaced by aspartic acid had increased basal activity but was also further activated by oxidative stress. Thus, PKD tyrosine phosphorylation at this site neither produced full activation by itself nor was required for oxidative stress-induced activation mediated by activation loop phosphorylation. In addition to PKD activation, activation loop phosphorylation in response to oxidative stress also redistributed activated PKD to cell nuclei, as revealed by PKD indirect immunofluorescence, imaging of a PKD-green fluorescent protein fusion construct (GFP-PKD), and analysis of nuclear pellets. Cell preincubation with G?6983 strongly diminished H(2)O(2)-induced nuclear relocalization of GFP-PKD. Taken together, these results indicate that PKC-mediated PKD Ser(744) and Ser(748) phosphorylation induced by oxidative stress integrates PKD activation with redistribution to the nucleus.     

Stimulation of the neurokinin 3 receptor activates protein kinase C epsilon and protein kinase D in enteric neurons

Tachykinins, acting through NK(3) receptors (NK(3)R), contribute to excitatory transmission to intrinsic primary afferent neurons (IPANs) of the small intestine. Although this transmission is dependent on protein kinase C (PKC), its maintenance could depend on protein kinase D (PKD), a downstream target of PKC. Here we show that PKD1/2-immunoreactivity occurred exclusively in IPANs of the guinea pig ileum, demonstrated by double staining with the IPAN marker NeuN. PKCepsilon was also colocalized with PKD1/2 in IPANs. PKCepsilon and PKD1/2 trafficking was studied in enteric neurons within whole mounts of the ileal wall. In untreated preparations, PKCepsilon and PKD1/2 were cytosolic and no signal for activated (phosphorylated) PKD was detected. The NK(3)R agonist senktide evoked a transient translocation of PKCepsilon and PKD1/2 from the cytosol to the plasma membrane and induced PKD1/2 phosphorylation at the plasma membrane. PKCepsilon translocation was maximal at 10 s and returned to the cytosol within 2 min. Phosphorylated-PKD1/2 was detected at the plasma membrane within 15 s and translocated to the cytosol by 2 min, where it remained active up to 30 min after NK(3)R stimulation. PKD1/2 activation was reduced by a PKCepsilon inhibitor and prevented by NK(3)R inhibition. NK(3)R-mediated PKCepsilon and PKD activation was confirmed in HEK293 cells transiently expressing NK(3)R and green fluorescent protein-tagged PKCepsilon, PKD1, PKD2, or PKD3. Senktide caused membrane translocation and activation of kinases within 30 s. After 15 min, phosphorylated PKD had returned to the cytosol. PKD activation was confirmed through Western blotting. Thus stimulation of NK(3)R activates PKCepsilon and PKD in sequence, and sequential activation of these kinases may account for rapid and prolonged modulation of IPAN function.     

Protein kinase C-dependent protein kinase D activation modulates ERK signal pathway and endothelial cell proliferation by vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is essential for many angiogenic processes both in normal conditions and in pathological conditions. However, the signaling pathways involved in VEGF-induced angiogenesis are not well defined. Protein kinase D (PKD), a newly described serine/threonine protein kinase, has been implicated in many signal transduction pathways and in cell proliferation. We hypothesized that PKD would mediate VEGF signaling and function in endothelial cells. Here we found that VEGF rapidly and strongly stimulated PKD phosphorylation and activation in endothelial cells via VEGF receptor 2 (VEGFR2). The pharmacological inhibitors for phospholipase Cgamma (PLCgamma) and protein kinase C (PKC) significantly inhibited VEGF-induced PKD activation, suggesting the involvement of the PLCgamma/PKC pathway. In particular, PKCalpha was critical for VEGF-induced PKD activation since both overexpression of adenovirus PKCalpha dominant negative mutant and reduction of PKCalpha expression by small interfering RNA markedly inhibited VEGF-induced PKD activation. Importantly, we found that small interfering RNA knockdown of PKD and PKCalpha expression significantly attenuated ERK activation and DNA synthesis in endothelial cells by VEGF. Taken together, our results demonstrated for the first time that VEGF activates PKD via the VEGFR2/PLCgamma/PKCalpha pathway and revealed a critical role of PKD in VEGF-induced ERK signaling and endothelial cell proliferation.     

The nuclear import of protein kinase D3 requires its catalytic activity

The protein kinase D (PKD) family consists of three serine/threonine protein kinases termed PKD, PKD2, and PKD3, which are similar in overall structure and primary amino acid sequence. However, each isozyme displays a distinctive intracellular localization. Taking advantage of the structural homology and opposite nuclear localization of PKD2 and PKD3, we generated an extensive set of chimeric proteins between both isozymes to determine which PKD3 domain(s) mediates its nuclear localization. We found that the C-terminal region of PKD3, which contains its catalytic domain, is necessary but not sufficient for its nuclear localization. Real time imaging of a photoactivatable green fluorescent protein fused to PKD3 revealed that point mutations that render PKD3 catalytically inactive completely prevented its nuclear import despite its interaction with importin alpha and beta. We also found that activation loop phosphorylation of PKD3 did not require its nuclear localization, and it was not sufficient to promote the nuclear import of PKD3. These results identify a novel function for the kinase activity of PKD3 in promoting its nuclear entry and suggest that the catalytic activity of PKD3 may regulate its nuclear import through autophosphorylation and/or interaction with another protein(s).     

A novel protein kinase C target site in protein kinase D is phosphorylated in response to signals for cardiac hypertrophy

Protein kinase D (PKD) regulates cardiac myocyte growth and contractility through phosphorylation of proteins such as class IIa histone deacetylases (HDACs) and troponin I (TnI). In response to agonists that activate G-protein-coupled receptors (GPCRs), PKD is phosphorylated by protein kinase C (PKC) on two serine residues (Ser-738 and Ser-742 in human PKD1) within an activation loop of the catalytic domain, resulting in stimulation of PKD activity. Here, we identify a novel PKC target site located adjacent to the auto-inhibitory pleckstrin homology (PH) domain in PKD. This site (Ser-412 in human PKD1) is conserved in each of the three PKD family members and is efficiently phosphorylated by multiple PKC isozymes in vitro. Employing a novel anti-phospho-Ser-412-specific antibody, we demonstrate that this site in PKD is rapidly phosphorylated in primary cardiac myocytes exposed to hypertrophic agonists, including norepinephrine (NE) and endothelin-1 (ET-1). Differential sensitivity of this event to pharmacological inhibitors of PKC, and data from in vitro enzymatic assays, suggest a predominant role for PKCδ in the control of PKD Ser-412 phosphorylation. Together, these data suggest a novel, signal-dependent mechanism for controlling PKD function in cardiac myocytes.     

Lysophosphatidic acid rapidly induces protein kinase D activation through a pertussis toxin-sensitive pathway

Protein kinase D(PKD) is a serine-threonine protein kinase with distinct structuralfeatures and enzymological properties. Herein we demonstrate thatlysophosphatidic acid (LPA) induces rapid PKD activation in mouse Swiss3T3 and Rat-1 cells. LPA induced PKD activation in aconcentration-dependent fashion with maximal stimulation (7.6-fold)achieved at 5 µM. Treatment of Swiss 3T3 cells with the proteinkinase C (PKC) inhibitors GF-I, Ro-31-8220, and Gö-7874completely abrogated PKD activation induced by LPA at concentrationsthat did not inhibit PKD activity when added directly to the in vitrokinase assays. PKD activation induced by LPA was attenuated markedlyand selectively by prior exposure of either Swiss 3T3 or Rat-1 cells topertussis toxin (PTx) in a concentration-dependent manner. In contrast,treatment with the protein tyrosine kinase inhibitor genistein, the MEKinhibitor PD-098059, or the phosphoinositide 3-kinase inhibitorwortmannin did not affect PKD activation in response to LPA. Theseresults provide the first example of PTx-sensitive and PKC-dependentPKD activation and identify a novelG_i-dependent event in the action of LPA.