Eukaryotic elongation factor 1A2 cooperates with phosphatidylinositol-4 kinase III beta to stimulate production of filopodia through increased phosphatidylinositol-4,5 bisphosphate generation

Eukaryotic elongation factor 1 alpha 2 (eEF1A2) is a transforming gene product that is highly expressed in human tumors of the ovary, lung, and breast. eEF1A2 also stimulates actin remodeling, and the expression of this factor is sufficient to induce the formation of filopodia, long cellular processes composed of bundles of parallel actin filaments. Here, we find that eEF1A2 stimulates formation of filopodia by increasing the cellular abundance of cytosolic and plasma membrane-bound phosphatidylinositol-4,5 bisphosphate [PI(4,5)P(2)]. We have previously reported that the eEF1A2 protein binds and activates phosphatidylinositol-4 kinase III beta (PI4KIIIbeta), and we find that production of eEF1A2-dependent PI(4,5)P(2) and generation of filopodia require PI4KIIIbeta. Furthermore, PI4KIIIbeta is itself capable of activating both the production of PI(4,5)P(2) and the creation of filopodia. We propose a model for extrusion of filopodia in which eEF1A2 activates PI4KIIIbeta, and activated PI4KIIIbeta stimulates production of PI(4,5)P(2) and filopodia by increasing PI4P abundance. Our work suggests an important role for both eEF1A2 and PI4KIIIbeta in the control of PI(4,5)P(2) signaling and actin remodeling.     

Binding of elongation factor eEF1A2 to phosphatidylinositol 4-kinase beta stimulates lipid kinase activity and phosphatidylinositol 4-phosphate generation

Eukaryotic protein translation elongation factor 1 alpha 2 (eEF1A2) is an oncogene that transforms mammalian cell lines and increases their tumorigenicity in nude mice. Increased expression of eEF1A2 occurs during the development of breast, ovarian, and lung cancer. Here, we report that eEF1A2 directly binds to and activates phosphatidylinositol 4-kinase III beta (PI4KIIIbeta), an enzyme that converts phosphatidylinositol to phosphatidylinositol 4-phosphate. Purified recombinant eEF1A2 increases PI4KIIIbeta lipid kinase activity in vitro, and expression of eEF1A2 in rat and human cells is sufficient to increase overall cellular phosphatidylinositol 4-kinase activity and intracellular phosphatidylinositol 4-phosphate abundance. siRNA-mediated reduction in eEF1A2 expression concomitantly reduces phosphatidylinositol 4-kinase activity. This identifies a physical and functional relationship between eEF1A2 and PI4KIIIbeta.     

Distinct Golgi populations of phosphatidylinositol 4-phosphate regulated by phosphatidylinositol 4-kinases

Phosphatidylinositol 4-phosphate (PI4P) regulates biosynthetic membrane traffic at multiple steps and differentially affects the surface delivery of apically and basolaterally destined proteins in polarized cells. Two phosphatidylinositol 4-kinases (PI4Ks) have been localized to the Golgi complex in mammalian cells, type III PI4Kbeta (PI4KIIIbeta) and type II PI4Kalpha (PI4KIIalpha). Here we report that PI4KIIIbeta and PI4KIIalpha localize to discrete subcompartments of the Golgi complex in Madin-Darby canine kidney (MDCK) cells. PI4KIIIbeta was enriched in early Golgi compartments, whereas PI4KIIalpha colocalized with markers of the trans-Golgi network (TGN). To understand the temporal and spatial control of PI4P generation across the Golgi complex, we quantitated the steady state distribution of a fluorescent PI4P-binding domain relative to cis/medial Golgi and TGN markers in transiently transfected MDCK cells. The density of the signal from this PI4P reporter was roughly 2-fold greater in the early Golgi compartments compared with that of the TGN. Furthermore, this ratio could be modulated in vivo by overexpression of catalytically inactive PI4KIIIbeta and PI4KIIalpha or in vitro by the PI4KIIIbeta inhibitor wortmannin. Our data suggest that both PI4KIIIbeta and PI4KIIalpha contribute to the compartmental regulation of PI4P synthesis within the Golgi complex. We discuss our results with respect to the kinetic effects of modulating PI4K activity on polarized biosynthetic traffic in MDCK cells.     

Phosphatidylinositol 3-kinase and protein kinase D1 specifically cooperate to negatively regulate the insulin-like growth factor signaling pathway

Insulin receptor substrate-1 (IRS-1) is a key protein in the insulin-like growth factor (IGF) signaling whose tyrosine phosphorylation by the type 1 IGF receptor is necessary for the recruitment and activation of the downstream effectors. Through the analysis of cross-talks occurring between different tyrosine kinase receptor-dependent signaling pathways, we investigated how two growth factors [epidermal growth factor (EGF) and fibroblast growth factor (FGF)] could modulate the IGF-I-induced IRS-1 tyrosine phosphorylation and its downstream signaling. EGF and FGF inhibited IGF-I-stimulated tyrosine phosphorylation of IRS-1 and the subsequent IGF-I-induced phosphatidylinositol 3-kinase (PI 3-kinase) activity. These EGF- and FGF-inhibitory effects were dependent on both PI 3-kinase and protein kinase D1 (PKD1) signaling pathways but independent on the extracellular signal-regulated kinase (ERK) pathway. PKD1, which was activated independently of the PI 3-kinase pathway, associated with IRS-1 in response to EGF or FGF. Unlike PI 3-kinase, PKD1 did not mediate the EGF- or FGF-induced-IRS-1 serine 307 phosphorylation which was described to inhibit IRS-1. Interestingly, specific inhibition of either PI 3-kinase or PKD1 totally impaired EGF- or FGF-induced inhibition of IGF-I-stimulated IRS-1 tyrosine phosphorylation. This indicated that serine 307 phosphorylation of IRS-1 is not sufficient per se to inhibit the IGF signaling pathway and demonstrated for the first time that the negative regulation of IRS-1 requires the coordinated action of PI 3-kinase and PKD1. This further suggests that PKD1 may be an attractive target for innovative strategies that target the IGF signaling pathway.     

Protein kinase D is a downstream target of protein kinase Ctheta

Protein kinase D (PKD/PKCmu immunoprecipitated from either COS-7 cells or Jurkat T lymphocytes transiently transfected with a constitutively active mutant of PKCtheta AE (PKCthetaAE) exhibited a marked increase in basal activity. In contrast, coexpression of constitutively active mutant of PKCzeta does not induce PKD activation in both types of cells. PKCthetaAE does not induce kinase activity in immunocomplexes of PKD kinase-deficient mutants PKDK618N or PKDD733A. PKD activation in response to PKCthetaAE signaling was completely prevented by treatment with the protein kinase C (PKC) inhibitors, GF I or Ro 31-8220, or by mutation of Ser-744 and Ser-748 to Ala in the kinase activation loop of PKD. Our results show that PKD is a downstream target of the theta isoform of PKC in both COS-7 cells and lymphocytes. The regulation of PKD by PKCtheta reveals a new pathway in the signaling network existing between multiple members of the PKC superfamily and PKD.     

Protein kinase D regulates cofilin activity through p21-activated kinase 4

Dynamic reorganization of the actin cytoskeleton at the leading edge is required for directed cell migration. Cofilin, a small actin-binding protein with F-actin severing activities, is a key enzyme initiating such actin remodeling processes. Cofilin activity is tightly regulated by phosphorylation and dephosphorylation events that are mediated by LIM kinase (LIMK) and the phosphatase slingshot (SSH), respectively. Protein kinase D (PKD) is a serine/threonine kinase that inhibits actin-driven directed cell migration by phosphorylation and inactivation of SSH. Here, we show that PKD can also regulate LIMK through direct phosphorylation and activation of its upstream kinase p21-activated kinase 4 (PAK4). Therefore, active PKD increases the net amount of phosphorylated inactive cofilin in cells through both pathways. The regulation of cofilin activity at multiple levels may explain the inhibitory effects of PKD on barbed end formation as well as on directed cell migration.     

PKD is a kinase of Vps34 that mediates ROS-induced autophagy downstream of DAPk

Autophagy, a process in which cellular components are engulfed and degraded within double-membrane vesicles termed autophagosomes, has an important role in the response to oxidative damage. Here we identify a novel cascade of phosphorylation events, involving a network of protein and lipid kinases, as crucial components of the signaling pathways that regulate the induction of autophagy under oxidative stress. Our findings show that both the tumor-suppressor death-associated protein kinase (DAPk) and protein kinase D (PKD), which we previously showed to be phosphorylated and consequently activated by DAPk, mediate the induction of autophagy in response to oxidative damage. Furthermore, we map the position of PKD within the autophagic network to Vps34, a lipid kinase whose function is indispensable for autophagy, and demonstrate that PKD is found in the same molecular complex with Vps34. PKD phosphorylates Vps34, leading to activation of Vps34, phosphatydilinositol-3-phosphate (PI(3)P) formation, and autophagosome formation. Consistent with its identification as a novel inducer of the autophagic machinery, we show that PKD is recruited to LC3-positive autophagosomes, where it localizes specifically to the autophagosomal membranes. Taken together, our results describe PKD as a novel Vps34 kinase that functions as an effecter of autophagy under oxidative stress.     

Protein kinase D specifically mediates apoptosis signal-regulating kinase 1-JNK signaling induced by H2O2 but not tumor necrosis factor

Although both tumor necrosis factor (TNF) and H2O2 induce activation of c-Jun N-terminal kinase (JNK) kinase cascades, it is not known whether they utilize distinct intracellular signaling pathways. In this study, we first examined a variety of pharmacological inhibitors on TNF and H2O2-induced JNK activation. Go6983 or staurosporine, which inhibits protein kinase C isoforms had no effects on TNF or H2O2-induced JNK activation. However, Go6976 and calphostin, which can inhibit protein kinase C as well as protein kinase D (PKD), blocked H2O2- but not TNF-induced JNK activation, suggesting that PKD may be specifically involved in H2O2-induced JNK activation. Consistently, H2O2, but not TNF, induced phosphorylation of PKD and translocation of PKD from endothelial cell membrane to cytoplasm where it associates with the JNK upstream activator, apoptosis signal-regulating kinase 1 (ASK1). The association is mediated through the pleckstrin homology domain of PKD and the C-terminal domain of ASK1. Inhibition of PKD by Go6976 or by small interfering RNA of PKD blocked H2O2-induced ASK1-JNK activation and endothelial cell apoptosis. Interestingly, H2O2 induced 14-3-3 binding to PKD via the phospho-Ser-205/208 and phospho-Ser-219/223 and H2O2-induced 14-3-3 binding of PKD was specifically blocked by Go6976 but not by Go6983. More significantly, the 14-3-3-binding defective forms of PKD failed to associate with ASK1 and to activate JNK signaling, highlighting the importance of 14-3-3 binding of PKD in H2O2-induced activation of ASK1-JNK cascade. Thus, our data have identified PKD as a critical mediator in H2O2- but not TNF-induced ASK1-JNK signaling.     

Identification of protein kinase D as a novel contraction-activated kinase linked to GLUT4-mediated glucose uptake, independent of AMPK

Contraction-induced glucose uptake is only partly mediated by AMPK activation. We examined whether the diacylglycerol-sensitive protein kinase D (PKD; also known as novel PKC isoform mu) is also involved in the regulation of glucose uptake in the contracting heart. As an experimental model, we used suspensions of cardiac myocytes, which were electrically stimulated to contract or treated with the contraction-mimicking agent oligomycin. Induction of contraction at 4 Hz in cardiac myocytes or treatment with 1 microM oligomycin enhanced (i) autophosphorylation of PKD at Ser916 by 5.1- and 3.8-fold, respectively, (ii) phosphorylation of PKD's downstream target cardiac-troponin-I (cTnI) by 2.9- and 2.1-fold, respectively, and (iii) enzymatic activity of immunoprecipitated PKD towards the substrate peptide syntide-2 each by 1.5-fold. Although AMPK was also activated under these same conditions, in vitro phosphorylation assays and studies with cardiac myocytes from AMPKalpha2(-/-) mice indicated that activation of PKD occurs independent of AMPK activation. CaMKKbeta, and the cardiac-specific PKC isoforms alpha, delta, and epsilon were excluded as upstream kinases for PKD in contraction signaling because none of these kinases were activated by oligomycin. Stimulation of glucose uptake and induction of GLUT4 translocation in cardiac myocytes by contraction and oligomycin each were sensitive to inhibition by the PKC/PKD inhibitors staurosporin and calphostin-C. Together, these data elude to a role of PKD in contraction-induced GLUT4 translocation. Finally, the combined actions of PKD on cTnI phosphorylation and on GLUT4 translocation would efficiently link accelerated contraction mechanics to increased energy production when the heart is forced to increase its contractile activity.     

PKD Regulates Membrane Fission to Generate TGN to Cell Surface Transport Carriers

The serine/threonine protein kinase D (PKD) is recruited to the trans-Golgi network (TGN) by binding diacylglycerol (DAG) and the ARF1 GTPase. PKD, at the TGN, promotes the production of phosphatidylinositol-4 phosphate (PI4P) by activating the lipid kinase phophatidylinositol 4-kinase IIIß (PI4KIIIß). PI4P recruits proteins such as oxysterol-binding protein 1 (OSBP) and ceramide transport protein (CERT) that control sphingolipid and sterol levels at the TGN. CERT mediated transport of ceramide to the TGN, we suggest, is used for increasing the local production and concentration of DAG. Once the crucial concentration of DAG is achieved, OSBP and CERT dissociate from the TGN on phosphorylation by PKD and DAG is sequentially converted into phosphatidic acid (PA) and lyso-PA (LPA). Therefore, the net effect of the activated PKD at the TGN is the sequential production of the modified lipids DAG, PA, and LPA that are necessary for membrane fission to generate cell surface specific transport carriers.     

The role of protein kinase D in neurotensin secretion mediated by protein kinase C-alpha/-delta and Rho/Rho kinase

Neurotensin (NT) is a gut peptide that plays an important role in gastrointestinal (GI) secretion, motility, and growth as well as the proliferation of NT receptor positive cancers. Secretion of NT is regulated by phorbol ester-sensitive protein kinase C (PKC) isoforms-alpha and -delta and may involve protein kinase D (PKD). The purpose of our present study was: (i) to define the role of PKD in NT release from BON endocrine cells and (ii) to delineate the upstream signaling mechanisms mediating this effect. Here, we demonstrate that small interfering RNA (siRNA) targeted against PKD dramatically inhibited both basal and PMA-stimulated NT secretion; NT release is significantly increased by overexpression of PKD. PKC-alpha and -delta siRNA attenuated PKD activity, whereas overexpression of PKC-alpha and -delta enhanced PKD activity. Rho kinase (ROK) siRNA significantly inhibited NT secretion, whereas overexpression of ROKalpha effectively increased NT release. Rho protein inhibitor C3 dramatically inhibited both NT secretion and PKD activity. In conclusion, our results demonstrate that PKD activation plays a central role in NT peptide secretion; upstream regulators of PKD include PKC-alpha and -delta and Rho/ROK. Importantly, our results identify novel signaling pathways, which culminate in gut peptide release.     

Oxidative stress induces protein kinase D activation in intact cells. Involvement of Src and dependence on protein kinase C

Protein kinase D (PKD) is a protein serine kinase that is directly stimulated in vitro by phorbol esters and diacylglycerol in the presence of phospholipids, and activated by phorbol esters, neuropeptides, and platelet-derived growth factor via protein kinase C (PKC) in intact cells. Recently, oxidative stress was shown to activate transfected PKC isoforms via tyrosine phosphorylation, but PKD activation was not demonstrated. Here, we report that oxidative stress initiated by addition of H(2)O(2) (0.15-10 mm) to quiescent Swiss 3T3 fibroblasts activates PKD in a dose- and time- dependent manner, as measured by autophosphorylation and phosphorylation of an exogenous substrate, syntide-2. Oxidative stress also activated transfected PKD in COS-7 cells but not a kinase-deficient mutant PKD form or a PKD mutant with critical activating serine residues 744 and 748 mutated to alanines. Genistein, or the specific Src inhibitors PP-1 and PP-2 (1-10 micrometer) inhibited H(2)O(2)-mediated PKD activation by 45%, indicating that Src contributes to this signaling pathway. PKD activation by H(2)O(2) was also selectively potentiated by cotransfection of PKD together with an active form of Src (v-Src) in COS-7 cells, as compared with PDB-mediated activation. The specific phospholipase C inhibitor, partly blocked H(2)O(2)-mediated but not PDB-mediated PKD activation. In contrast, PKC inhibitors blocked H(2)O(2) or PDB-mediated PKD activation essentially completely, suggesting that whereas Src mediates part of its effects via phospholipase C activation, PKC acts more proximally as an upstream activator of PKD. Together, these studies reveal that oxidative stress activates PKD by initiating distinct Src-dependent and -independent pathways involving PKC.     

Protein kinase CK2 and protein kinase D are associated with the COP9 signalosome

The COP9 signalosome (CSN) purified from human erythrocytes possesses kinase activity that phosphoryl ates proteins such as c-Jun and p53 with consequence for their ubiquitin (Ub)-dependent degradation. Here we show that protein kinase CK2 (CK2) and protein kinase D (PKD) co-purify with CSN. Immunoprecipitation and far-western blots reveal that CK2 and PKD are in fact associated with CSN. As indicated by electron microscopy with gold-labeled ATP, at least 10% of CSN particles are associated with kinases. Kinase activity, most likely due to CK2 and PKD, co-immuno precipitates with CSN from HeLa cells. CK2 binds to DeltaCSN3(111-403) and CSN7, whereas PKD interacts with full-length CSN3. CK2 phosphorylates CSN2 and CSN7, and PKD modifies CSN7. Both CK2 and PKD phosphorylate c-Jun as well as p53. CK2 phosphoryl ates Thr155, which targets p53 to degradation by the Ub system. Curcumin, emodin, DRB and resveratrol block CSN-associated kinases and induce degradation of c-Jun in HeLa cells. Curcumin treatment results in elevated amounts of c-Jun-Ub conjugates. We conclude that CK2 and PKD are recruited by CSN in order to regulate Ub conjugate formation.     

A retroviral-derived peptide phosphorylates protein kinase D/protein kinase Cmu involving phospholipase C and protein kinase C

CKS-17, a synthetic peptide representing a unique amino acid motif which is highly conserved in retroviral transmembrane proteins and other immunoregulatory proteins, induces selective immunomodulatory functions, both in vitro and in vivo, and activates intracellular signaling molecules such as cAMP and extracellular signal-regulated kinases. In the present study, using Jurkat T-cells, we report that CKS-17 phosphorylates protein kinase D (PKD)/protein kinase C (PKC) mu. Total cell extracts from CKS-17-stimulated Jurkat cells were immunoblotted with an anti-phospho-PKCmu antibody. The results show that CKS-17 significantly phosphorylates PKD/PKCmu in a dose- and time-dependent manner. Treatment of cells with the PKC inhibitors GF 109203X and Ro 31-8220, which do not act directly on PKD/PKCmu, attenuates CKS-17-induced phosphorylation of PKD/PKCmu. In contrast, the selective protein kinase A inhibitor H-89 does not reverse the action of CKS-17. Furthermore, a phospholipase C (PLC) selective inhibitor, U-73122, completely blocks the phosphorylation of PKD/PKCmu by CKS-17 while a negative control U-73343 does not. In addition, substitution of lysine for arginine residues in the CKS-17 sequence completely abrogates the ability of CKS-17 to phosphorylate PKD/PKCmu. These results clearly indicate that CKS-17 phosphorylates PKD/PKCmu through a PLC- and PKC-dependent mechanism and that arginine residues play an essential role in this activity of CKS-17, presenting a novel modality of the retroviral peptide CKS-17 and molecular interaction of this compound with target cells.     

Protein kinase D interacts with Golgi via its cysteine-rich domain

Protein kinase D (PKD)/protein kinase Cmu is a serine/threonine protein kinase that has been localized in the cytosol and in several intracellular compartments including Golgi, mitochondria and plasma membrane. Using real time imaging of fluorescent protein (GFP)-tagged PKD, we have found that the accumulation of PKD in the Golgi compartment, following a temperature shift from 37 to 20 degrees C, was mediated by the cysteine-rich domain (CRD) of PKD. The CRD of PKD also mediates its interaction with the plasma membrane, further supporting the conclusion that the CRD of PKD may act as a subcellular localization signal.     

Regulation of Oxysterol-binding Protein Golgi Localization through Protein Kinase D–mediated Phosphorylation

Protein kinase D (PKD) plays a critical role at the trans-Golgi network by regulating the fission of transport carriers destined for the plasma membrane. Two known Golgi-localized PKD substrates, PI4-kinase IIIβ and the ceramide transfer protein CERT, mediate PKD signaling to influence vesicle trafficking to the plasma membrane and sphingomyelin synthesis, respectively. PKD is recruited and activated at the Golgi through interaction with diacylglycerol, a pool of which is generated as a by-product of sphingomyelin synthesis from ceramide. Here we identify a novel substrate of PKD at the Golgi, the oxysterol-binding protein OSBP. Using a substrate-directed phospho-specific antibody that recognizes the optimal PKD consensus motif, we show that PKD phosphorylates OSBP at Ser240 in vitro and in cells. We further show that OSBP phosphorylation occurs at the Golgi. Phosphorylation of OSBP by PKD does not modulate dimerization, sterol binding, or affinity for PI(4)P. Instead, phosphorylation attenuates OSBP Golgi localization in response to 25-hydroxycholesterol and cholesterol depletion, impairs CERT Golgi localization, and promotes Golgi fragmentation.     

Protein kinase D (PKD) activation in intact cells through a protein kinase C-dependent signal transduction pathway.

Protein kinase D (PKD) is a serine/threonine protein kinase that is directly stimulated in vitro by phorbol esters and diacylglycerol in the presence of phospholipids. Here, we examine the regulation of PKD in living cells. Our results demonstrate that tumour-promoting phorbol esters, membrane-permeant diacylglycerol and serum growth factors rapidly induced PKD activation in immortalized cell lines (e.g. Swiss 3T3 and Rat-1 cells), in secondary cultures of mouse embryo fibroblasts and in COS-7 cells transiently transfected with a PKD expression construct. PKD activation was maintained during cell disruption and immunopurification and was associated with an electrophoretic mobility shift and enhanced 32P incorporation into the enzyme, but was reversed by treatment with alkaline phosphatase. PKD was activated, deactivated and reactivated in response to consecutive cycles of addition and removal of PDB. PKD activation was completely abrogated by exposure of the cells to the protein kinase C inhibitors GF I and Ro 31-8220. In contrast, these compounds did not inhibit PKD activity when added directly in vitro. Co-transfection of PKD with constitutively activated mutants of PKCs showed that PKCepsilon and eta but not PKCzeta strongly induced PKD activation in COS-7 cells. Thus, our results indicate that PKD is activated in living cells through a PKC-dependent signal transduction pathway.     

A novel kinase inhibitor establishes a predominant role for protein kinase D as a cardiac class IIa histone deacetylase kinase

Class IIa histone deacetylases (HDACs) repress genes involved in pathological cardiac hypertrophy. The anti-hypertrophic action of class IIa HDACs is overcome by signals that promote their phosphorylation-dependent nuclear export. Several kinases have been shown to phosphorylate class IIa HDACs, including calcium/calmodulin-dependent protein kinase (CaMK), protein kinase D (PKD) and G protein-coupled receptor kinase (GRK). However, the identity of the kinase(s) responsible for phosphorylating class IIa HDACs during cardiac hypertrophy has remained controversial. We describe a novel and selective small molecule inhibitor of PKD, bipyridyl PKD inhibitor (BPKDi). BPKDi blocks signal-dependent phosphorylation and nuclear export of class IIa HDACs in cardiomyocytes and concomitantly suppresses hypertrophy of these cells. These studies define PKD as a principal cardiac class IIa HDAC kinase.     

A plasma membrane pool of phosphatidylinositol 4-phosphate is generated by phosphatidylinositol 4-kinase type-III alpha: studies with the PH domains of the oxysterol binding protein and FAPP1

The PH domains of OSBP and FAPP1 fused to GFP were used to monitor PI(4)P distribution in COS-7 cells during manipulations of PI 4-kinase (PI4K) activities. Both domains were associated with the Golgi and small cytoplasmic vesicles, and a small fraction of OSBP-PH was found at the plasma membrane (PM). Inhibition of type-III PI4Ks with 10 microM wortmannin (Wm) significantly reduced but did not abolish Golgi localization of either PH domains. Downregulation of PI4KIIalpha or PI4KIIIbeta by siRNA reduced the localization of the PH domains to the Golgi and in the former case any remaining Golgi localization was eliminated by Wm treatment. PLC activation by Ca2+ ionophores dissociated the domains from all membranes, but after Ca2+ chelation, they rapidly reassociated with the Golgi, the intracellular vesicles and with the PM. PM association of the domains was significantly higher after the Ca2+ transient and was abolished by Wm pretreatment. PM relocalization was not affected by down-regulation of PI4KIIIbeta or -IIalpha, but was inhibited by down-regulation of PI4KIIIalpha, or by 10 microM PAO, which also inhibits PI4KIIIalpha. Our data suggest that these PH domains detect PI(4)P formation in extra-Golgi compartments under dynamic conditions and that various PI4Ks regulate PI(4)P synthesis in distinct cellular compartments.     

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.