Protein Kinase D Phosphorylation Research Articles

Proteinase-activated receptors 1 and 2 activate protein kinase D1 in human melanoma cells

Proteinase-activated receptors (PARs) are G-protein coupled receptors with seven transmembrane domains that are stimulated by a unique activation mechanism. The extracellular N-terminus is specifically cleaved by certain serine proteinases, which exposes a formerly latent peptide sequence. This is able to bind to the second extracellular loop of the receptor, leading to the transduction of signal events. Four PARs have been cloned so far. PAR1, PAR3 and PAR4 are stimulated by thrombin, whereas PAR2 can be activated by trypsin. In vitro, PARs can also be stimulated by synthetic peptides, which mimic the tethered ligand sequence. PAR1, but not PAR2, is expressed by primary melanocytes. It is well known that PAR1 is overexpressed in malignant melanomas, enhancing migration and metastasis. PAR2 also seems to play a role in melanoma progression and metastasis. The underlying mechanisms, however, still remain elusive. Stimulation of both receptors on the the human melanoma cell line WM9 led to rearrangement of integrin αvβ3 and phosphorylation of protein kinase D1 (PKD1). PKD1 is known to control persistent cell migration by regulating the intracellular integrin αvβ3 recycling pathway. Stable knockdown of PKD1 in WM9 cells led to an inhibition of cell proliferation, changes in cell shape and a downregulation of integrin αvβ3 on the cell surface. Moreover, cell migration was impaired. Taken together, we could show for the first time that both PAR1 and PAR2 are involved in melanoma cell migration and proliferation via activation of PKD1.

Protein Kinase D1–Mediated Phosphorylation and Subcellular Localization of β-Catenin

beta-Catenin is essential for E-cadherin-mediated cell adhesion in epithelial cells and also acts as a key cofactor for transcription activity. We previously showed that protein kinase D1 (PKD1), founding member of the PKD family of signal transduction proteins, is down-regulated in advanced prostate cancer and interacts with E-cadherin. This study provides evidence that PKD1 interacts with and phosphorylates beta-catenin at Thr(112) and Thr(120) residues in vitro and in vivo; mutation of Thr(112) and Thr(120) results in increased nuclear localization of beta-catenin and is associated with altered beta-catenin-mediated transcription activity. It is known that mutation of Thr(120) residue abolishes binding of beta-catenin to alpha-catenin, which links to cytoskeleton, suggesting that PKD1 phosphorylation of Thr(120) could be critical for cell-cell adhesion. Overexpression of PKD1 represses beta-catenin-mediated transcriptional activity and cell proliferation. Epistatic studies suggest that PKD1 and E-cadherin are within the same signaling pathway. Understanding the molecular basis of PKD1-beta-catenin interaction provides a novel strategy to target beta-catenin function in cells including prostate cancer.

PKD3 Is the Predominant Protein Kinase D Isoform in Mouse Exocrine Pancreas and Promotes Hormone-induced Amylase Secretion

The protein kinase D (PKD) family of serine/threonine kinases, which can be activated by gastrointestinal hormones, consists of three distinct isoforms that modulate a variety of cellular processes including intracellular protein transport as well as constitutive and regulated secretion. Although isoform-specific functions have been identified in a variety of cell lines, the expression and function of PKD isoforms in normal, differentiated secretory tissues is unknown. Here, we demonstrate that PKD isoforms are differentially expressed in the exocrine and endocrine cells of the pancreas. Specifically, PKD3 is the predominant isoform expressed in exocrine cells of the mouse and human pancreas, whereas PKD1 and PKD2 are more abundantly expressed in the pancreatic islets. Within isolated mouse pancreatic acinar cells, PKD3 undergoes rapid membrane translocation, trans-activating phosphorylation, and kinase activation after gastrointestinal hormone or cholinergic stimulation. PKD phosphorylation in pancreatic acinar cells occurs viaaCa2+-independent, diacylglycerol- and protein kinase C-dependent mechanism. PKD phosphorylation can also be induced by physiologic concentrations of secretagogues and by in vivo stimulation of the pancreas. Furthermore, activation of PKD3 potentiates MEK/ERK/RSK (RSK, ribosomal S6 kinase) signaling and significantly enhances cholecystokinin-mediated pancreatic amylase secretion. These findings reveal a novel distinction between the exocrine and endocrine cells of the pancreas and further identify PKD3 as a signaling molecule that promotes hormone-stimulated amylase secretion.

Protein kinase D1 mediates NF-kappaB activation induced by cholecystokinin and cholinergic signaling in pancreatic acinar cells.

The transcription factor NF-kappaB plays a critical role in inflammatory and cell death responses during acute pancreatitis. Previous studies in our laboratory demonstrated that protein kinase C (PKC) isoforms PKCdelta and epsilon are key regulators of NF-kappaB activation induced by cholecystokinin-8 (CCK-8), tumor necrosis factor-alpha, and ethanol. However, the downstream participants in regulating NF-kappaB activation in exocrine pancreas remain poorly understood. Here, we demonstrate that protein kinase D1 (PKD1) is a key downstream target of PKCdelta and PKCepsilon in pancreatic acinar cells stimulated by two major secretagogues, CCK-8 and the cholinergic agonist carbachol (CCh), and that PKD1 is necessary for NF-kappaB activation induced by CCK-8 and CCh. Both CCK-8 and CCh dose dependently induced a rapid and striking activation of PKD1 in rat pancreatic acinar cells, as measured by in vitro kinase assay and by phosphorylation at PKD1 activation loop (Ser744/748) or autophosphorylation site (Ser916). The phosphorylation and activation of PKD1 correlated with NF-kappaB activity stimulated by CCK-8 or CCh, as measured by NF-kappaB DNA binding. Either inhibition of PKCdelta or epsilon by isoform-specific inhibitory peptides, genetic deletion of PKCdelta and epsilon in pancreatic acinar cells, or knockdown of PKD1 by using small interfering RNAs in AR42J cells resulted in a marked decrease in PKD1 and NF-kappaB activation stimulated by CCK-8 or CCh. Conversely, overexpression of PKD1 resulted in augmentation of CCK-8- and CCh-stimulated NF-kappaB activation. Finally, the kinetics of PKD1 and NF-kappaB activation during cerulein-induced rat pancreatitis showed that both PKD1 and NF-kappaB activation were early events during acute pancreatitis and that their time courses of response were similar. Our results identify PKD1 as a novel early convergent point for PKCdelta and epsilon in the signaling pathways mediating NF-kappaB activation in pancreatitis.

Diacylglycerol kinase regulation of protein kinase D during oxidative stress-induced intestinal cell injury

We recently demonstrated that protein kinase D (PKD) exerts a protective function during oxidative stress-induced intestinal epithelial cell injury; however, the exact role of DAG kinase (DGK)ζ, an isoform expressed in intestine, during this process is unknown. We sought to determine the role of DGK during oxidative stress-induced intestinal cell injury and whether DGK acts as an upstream regulator of PKD. Inhibition of DGK with R59022 compound or DGKζ siRNA transfection decreased H 2O 2-induced RIE-1 cell apoptosis as measured by DNA fragmentation and increased PKD phosphorylation. Overexpression of kinase-dead DGKζ also significantly increased PKD phosphorylation. Additionally, endogenous nuclear DGKζ rapidly translocated to the cytoplasm following H 2O 2 treatment. Our findings demonstrate that DGK is involved in the regulation of oxidative stress-induced intestinal cell injury. PKD activation is induced by DGKζ, suggesting DGK is an upstream regulator of oxidative stress-induced activation of the PKD signaling pathway in intestinal epithelial cells.

Highlights From The Literature

Highlights From The Literature

Protein Kinase D Is a Key Regulator of Cardiomyocyte Lipoprotein Lipase Secretion After Diabetes

The diabetic heart switches to exclusively using fatty acid (FA) for energy supply and does so by multiple mechanisms including hydrolysis of lipoproteins by lipoprotein lipase (LPL) positioned at the vascular lumen. We determined the mechanism that leads to an increase in LPL after diabetes. Diazoxide (DZ), an agent that decreases insulin secretion and causes hyperglycemia, induced a substantial increase in LPL activity at the vascular lumen. This increase in LPL paralleled a robust phosphorylation of Hsp25, decreasing its association with PKCdelta, allowing this protein kinase to phosphorylate and activate protein kinase D (PKD), an important kinase that regulates fission of vesicles from the golgi membrane. Rottlerin, a PKCdelta inhibitor, prevented PKD phosphorylation and the subsequent increase in LPL. Incubating control myocytes with high glucose and palmitic acid (Glu+PA) also increased the phosphorylation of Hsp25, PKCdelta, and PKD in a pattern similar to that seen with diabetes, in addition to augmenting LPL activity. In myocytes in which PKD was silenced or a mutant form of PKCdelta was expressed, high Glu+PA were incapable of increasing LPL. Moreover, silencing of cardiomyocyte Hsp25 allowed phorbol 12-myristate 13-acetate to elicit a significant phosphorylation of PKCdelta, an appreciable association between PKCdelta and PKD, and a vigorous activation of PKD. As these cells also demonstrated an additional increase in LPL, our data imply that after diabetes, PKD control of LPL requires dissociation of Hsp25 from PKCdelta, association between PKCdelta and PKD, and vesicle fission. Results from this study could help in restricting cardiac LPL translocation, leading to strategies that overcome contractile dysfunction after diabetes.

Protein Kinase D-dependent Phosphorylation and Nuclear Export of Histone Deacetylase 5 Mediates Vascular Endothelial Growth Factor-induced Gene Expression and Angiogenesis

Vascular endothelial growth factor (VEGF) is essential for normal and pathological angiogenesis. However, the signaling pathways linked to gene regulation in VEGF-induced angiogenesis are not fully understood. Here we demonstrate a critical role of protein kinase D (PKD) and histone deacetylase 5 (HDAC5) in VEGF-induced gene expression and angiogenesis. We found that VEGF stimulated HDAC5 phosphorylation and nuclear export in endothelial cells through a VEGF receptor 2-phospholipase Cgamma-protein kinase C-PKD-dependent pathway. We further showed that the PKD-HDAC5 pathway mediated myocyte enhancer factor-2 transcriptional activation and a specific subset of gene expression in response to VEGF, including NR4A1, an orphan nuclear receptor involved in angiogenesis. Specifically, inhibition of PKD by overexpression of the PKD kinase-negative mutant prevents VEGF-induced HDAC5 phosphorylation and nuclear export as well as NR4A1 induction. Moreover, a mutant of HDAC5 specifically deficient in PKD-dependent phosphorylation inhibited VEGF-mediated NR4A1 expression, endothelial cell migration, and in vitro angiogenesis. These findings suggest that the PKD-HDAC5 pathway plays an important role in VEGF regulation of gene transcription and angiogenesis.

A Novel Tyrosine Phosphorylation Site in Protein Kinase D Contributes to Oxidative Stress-mediated Activation

Protein kinase D1 (PKD1) is a mediator of oxidative stress signaling where it regulates cellular detoxification and survival. Critical for the regulation of PKD1 activity in response to oxidative stress are Src- and Abl-mediated tyrosine phosphorylations that eventually lead to protein kinase Cdelta (PKCdelta)-mediated activation of PKD1. Here we identify Tyr95 in PKD1 as a previously undescribed phosphorylation site that is regulated by oxidative stress. Our data suggest that PKD1 phosphorylation at Tyr95 generates a binding motif for PKCdelta, and that oxidative stress-mediated PKCdelta/PKD interaction results in PKD1 activation loop phosphorylation and activation. We further analyzed all PKD isoforms for this mechanism and show that PKD enzymes PKD1 and PKD2 are targets for PKCdelta in response to oxidative stress, and that PKD3 is not a target because it lacks the relevant tyrosine residue that generates a PKCdelta interaction motif.

Regulation of protein kinase D activity in adult myocardium: Novel counter-regulatory roles for protein kinase Cε and protein kinase A

Protein kinase D (PKD) is activated downstream of protein kinase C (PKC) in many cell types, although little is known about the mechanisms that regulate PKD in adult myocardium. Exposure of cultured adult rat ventricular myocytes (ARVM) to phorbol 12-myristate 13-acetate (PMA; 100 nM for 5 min) activated PKD, as evidenced by significantly increased phosphorylation at Ser744/8 (PKC phosphorylation sites) and Ser916 (autophosphorylation site). PKD activation occurred concomitantly with translocation of the enzyme from the cytosolic to the particulate fraction. The role of PKC was confirmed by pretreatment (15 min) of ARVM with the PKC inhibitors GF109203X (1 μM) and Ro31-8220 (1 μM), both of which prevented PKD phosphorylation on subsequent exposure to PMA. Exposure of ARVM to endothelin-1 (ET1; 100 nM for 10 min) also activated PKD by a PKC-dependent mechanism. To determine the PKC isoform(s) involved in the ET1-induced PKD activation, ARVM were infected with adenoviral vectors encoding dominant-negative (DN) mutants of PKCα, PKCδ and PKCε. Expression of DN-PKCα and DN-PKCδ had little effect on ET1-induced PKD activation, whilst this was significantly attenuated by expression of DN-PKCε, indicating that PKCε plays a predominant role in the pertinent ET1 signaling pathway. Intriguingly, prior exposure to the adenylyl cyclase activator forskolin (1 μM for 5 min) or the β-adrenergic agonist isoprenaline (100 nM for 5 min) markedly attenuated ET1-induced PKD activation, but not PMA-induced PKD activation. The ET1-induced response was rescued when protein kinase A (PKA) was inhibited (H89, 10 μM) before exposure to isoprenaline. These results show that ET1-induced PKD activation in ARVM is mediated by PKC, primarily the PKCε isoform, and is suppressed by PKA activation.

Phospholipase C beta3 is a key component in the Gbetagamma/PKCeta/PKD-mediated regulation of trans-Golgi network to plasma membrane transport.

The requirement of DAG (diacylglycerol) to recruit PKD (protein kinase D) to the TGN (trans-Golgi network) for the targeting of transport carriers to the cell surface, has led us to a search for new components involved in this regulatory pathway. Previous findings reveal that the heterotrimeric Gbetagamma (GTP-binding protein betagamma subunits) act as PKD activators, leading to fission of transport vesicles at the TGN. We have recently shown that PKCeta (protein kinase Ceta) functions as an intermediate member in the vesicle generating pathway. DAG is capable of activating this kinase at the TGN, and at the same time is able to recruit PKD to this organelle in order to interact with PKCeta, allowing phosphorylation of PKD's activation loop. The most qualified candidates for the production of DAG at the TGN are PI-PLCs (phosphatidylinositol-specific phospholipases C), since some members of this family can be directly activated by Gbetagamma, utilizing PtdIns(4,5)P2 as a substrate, to produce the second messengers DAG and InsP3. In the present study we show that betagamma-dependent Golgi fragmentation, PKD1 activation and TGN to plasma membrane transport were affected by a specific PI-PLC inhibitor, U73122 [1-(6-{[17-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione]. In addition, a recently described PI-PLC activator, m-3M3FBS [2,4,6-trimethyl-N-(m-3-trifluoromethylphenyl)benzenesulfonamide], induced vesiculation of the Golgi apparatus as well as PKD1 phosphorylation at its activation loop. Finally, using siRNA (small interfering RNA) to block several PI-PLCs, we were able to identify PLCbeta3 as the sole member of this family involved in the regulation of the formation of transport carriers at the TGN. In conclusion, we demonstrate that fission of transport carriers at the TGN is dependent on PI-PLCs, specifically PLCbeta3, which is necessary to activate PKCeta and PKD in that Golgi compartment, via DAG production.

Protein Kinase D Selectively Targets Cardiac Troponin I and Regulates Myofilament Ca 2+ Sensitivity in Ventricular Myocytes

Protein kinase D (PKD) is a serine/threonine kinase with emerging myocardial functions; in skinned adult rat ventricular myocytes (ARVMs), recombinant PKD catalytic domain phosphorylates cardiac troponin I at Ser22/Ser23 and reduces myofilament Ca(2+) sensitivity. We used adenoviral gene transfer to determine the effects of full-length PKD on protein phosphorylation, sarcomere shortening and [Ca(2+)](i) transients in intact ARVMs. In myocytes transduced to express wild-type PKD, the heterologously expressed enzyme was activated by endothelin 1 (ET1) (5 nmol/L), as reflected by PKD phosphorylation at Ser744/Ser748 (PKC phosphorylation sites) and Ser916 (autophosphorylation site). The ET1-induced increase in cellular PKD activity was accompanied by increased cardiac troponin I phosphorylation at Ser22/Ser23; this measured approximately 60% of that induced by isoproterenol (10 nmol/L), which activates cAMP-dependent protein kinase (PKA) but not PKD. Phosphorylation of other PKA targets, such as phospholamban at Ser16, phospholemman at Ser68 and cardiac myosin-binding protein C at Ser282, was unaltered. Furthermore, heterologous PKD expression had no effect on isoproterenol-induced phosphorylation of these proteins, or on isoproterenol-induced increases in sarcomere shortening and relaxation rate and [Ca(2+)](i) transient amplitude. In contrast, heterologous PKD expression suppressed the positive inotropic effect of ET1 seen in control cells, without altering ET1-induced increases in relaxation rate and [Ca(2+)](i) transient amplitude. Complementary experiments in "skinned" myocytes confirmed reduced myofilament Ca(2+) sensitivity by ET1-induced activation of heterologously expressed PKD. We conclude that increased myocardial PKD activity induces cardiac troponin I phosphorylation at Ser22/Ser23 and reduces myofilament Ca(2+) sensitivity, suggesting that altered PKD activity in disease may impact on contractile function.

Lipopolysaccharide-induced protein kinase D activation mediated by interleukin-1β and protein kinase C

Protein kinase D (PKD), a newly described serine/threonine kinase, has been implicated in many signal transduction pathways. The present study was designed to determine whether and how PKD is activated in inflammation. The results demonstrated that lipopolysaccharide (LPS, 30 μg/ml) stimulated PKD and protein kinase C (PKC) phosphorylation in spinal neurons within 0.5 h, and the activation reached a maximum at 3 or 8 h and declined at 12 h. The phosphorylation could be inhibited by the selective inhibitors for PKC (100 nM), mainly for PKCα and PKCβ, suggesting the involvement of the PKC pathway. Particularly, PKCα might be critical for LPS-induced PKD activation since the PKCβ inhibitor (100 nM) observed no effect on the phosphorylation of PKD. Furthermore, the expression of interleukin-1β (IL-1β) was significantly induced by LPS within 0.5 h, and reached a maximum at 8 h. IL-1 receptor antagonist inhibited PKD and PKCs activation induced by LPS at a concentration of 50 nM and achieved maximum at 1000 nM. These results demonstrated for the first time that PKD could be activated by LPS in spinal neurons, might via the IL-1β/PKCα pathway. Additionally, immunostaining showed an increase in number of phosphorylated PKD-immunoreactive cells of adult spinal dorsal horn induced by intraplantar injected carrageenan (2 μg/100 μl), and antisense oligodeoxynucleotide to IL-1 receptor type I (50 μg/10 μl, intrathecal injected) inhibited the PKD activation, suggesting an involvement of IL-1β/PKD pathway in inflammation in adult spinal cord.

CCK causes PKD1 activation in pancreatic acini by signaling through PKC-δ and PKC-independent pathways

Protein kinase D1 (PKD1) is involved in cellular processes including protein secretion, proliferation and apoptosis. Studies suggest PKD1 is activated by various stimulants including gastrointestinal (GI) hormones/neurotransmitters and growth factors in a protein kinase C (PKC)-dependent pathway. However, little is known about the mechanisms of PKD1 activation in physiologic GI tissues. We explored PKD1 activation by GI hormones/neurotransmitters and growth factors and the mediators involved in rat pancreatic acini. Only hormones/neurotransmitters activating phospholipase C caused PKD1 phosphorylation (S916, S744/748). CCK activated PKD1 and caused a time- and dose-dependant increase in serine phosphorylation by activation of high- and low-affinity CCK A receptor states. Inhibition of CCK-stimulated increases in phospholipase C, PKC activity or intracellular calcium decreased PKD1 S916 phosphorylation by 56%, 62% and 96%, respectively. PKC inhibitors GF109203X/Go6976/Go6983/PKC-ζ pseudosubstrate caused a 62/43/49/0% inhibition of PKD1 S916 phosphorylation and an 87/13/82/0% inhibition of PKD1 S744/748 phosphorylation. Expression of dominant negative PKC-δ, but not PKC-ε, or treatment with PKC-δ translocation inhibitor caused marked inhibition of PKD phosphorylation. Inhibition of Src/PI3K/MAPK/tyrosine phosphorylation had no effect. In unstimulated cells, PKD1 was mostly located in the cytoplasm. CCK stimulated translocation of total and phosphorylated PKD1 to the membrane. These results demonstrate that CCK A receptor activation leads to PKD activation by signaling through PKC-dependent and PKC-independent pathways.

Requirement of Protein Kinase D Tyrosine Phosphorylation for VEGF-A165-induced Angiogenesis through Its Interaction and Regulation of Phospholipase Cγ Phosphorylation

Vascular endothelial cell growth factor-A(165) (VEGF-A(165)) is critical for angiogenesis. Although protein kinase C-mediated protein kinase D(PKD)activation was implicated in the response, the detailed mechanism remains unclear. In this study, we found that VEGF-A(165)-stimulated tyrosine phosphorylation of PKD and the dominant negative mutant of PKD, PKD(Y463F), inhibited VEGF-A(165)-induced human umbilical vein endothelial cell (HUVEC) proliferation. In addition, PKD(S738A/S742A) overexpression inhibited VEGF-induced HUVEC migration. Furthermore, knockdown of PKD by its specific small interfering RNA inhibited VEGF-induced HUVEC proliferation and migration. Moreover transfection of PKD(Y463F), PKD(S738A/S742A), or PKD-small interfering RNA blocked VEGF-induced angiogenesis in vivo. Our signaling experiments show that KDR not Flt-1 mediated PKD tyrosine phosphorylation and KDR tyrosine residues 951 and 1059 were required for VEGF-A(165)-stimulated PKD serine and tyrosine phosphorylation, respectively. Whereas G protein Gbetagamma subunits were required for both PKD serine phosphorylation and tyrosine phosphorylation, intracellular Ca(2+) mobilization was required for VEGF-A(165)-stimulated PKD tyrosine phosphorylation and phospholipase C (PLC) activity was required for PKD serine phosphorylation. Surprisingly, the PLC inhibitor did not inhibit PKD tyrosine phosphorylation. Instead, PKD tyrosine 463 was required for VEGF-A(165)-stimulated PLCgamma tyrosine phosphorylation. Moreover, PKD interacted with PLCgamma even in unstimulated cells, and PKD tyrosine 463 phosphorylation was not required for this interaction. Together, we demonstrate that PKD interacts with PLCgamma and becomes tyrosine phosphorylated upon VEGF stimulation, leading to PLCgamma activation and angiogenic response of VEGF-A(165).

Angiotensin II-Mediated Protein Kinase D Activation Stimulates Aldosterone and Cortisol Secretion in H295R Human Adrenocortical Cells

Protein kinases are important mediators in intracellular signaling. Angiotensin II is the most important modulator of adrenal zona glomerulosa cell physiology. Angiotensin II regulates steroidogenesis and proliferation among many other metabolic processes. H295R human adrenal cells are a widely used experimental model to study adrenal cell physiology and metabolism. We screened for protein kinase expression levels using the Kinetwork system in H295R cells after 3 h angiotensin II treatment. Protein kinase D (PKD) was the protein kinase that suffers the most dramatic changes. PKD is a member of a new class of serine/threonine protein kinases that is activated by phosphorylation. Our studies indicated that angiotensin II time- and dose-dependently increased PKD phosphorylation, which occurred within 2 min of angiotensin II treatment and at concentrations as low as 1 nm. PKD phosphorylation was also dose-dependently increased by the PKC activator phorbol 12-myristate 13-acetate. Angiotensin II-mediated PKD phosphorylation was blocked by several PKC inhibitors. Furthermore, PKCepsilon translocation inhibitor peptide decreased angiotensin II-mediated PKD phosphorylation, and PKCepsilon down-regulation by RNA interference also decreased PKD phosphorylation mediated by angiotensin II. Cotransfection of constitutively active PKD mutant constructs up-regulated aldosterone synthase and 11beta-hydroxylase expression in reporter assays. Constitutively active PKD mutants increased aldosterone and cortisol secretion under angiotensin II stimulatory conditions. This study reveals that PKD is an intracellular signaling mediator of angiotensin II regulation of steroidogenesis in human adrenal cells. These data provide new insights into the molecular mechanisms involved in angiotensin II-induced physiological and pathophysiological events in adrenal cells.

Regulation of type II PIP kinase by PKD phosphorylation

The type II PIP kinases phosphorylate the poorly understood inositol lipid PtdIns5 P, producing the multi-functional lipid product PtdIns(4,5) P 2. To investigate the regulation of these enzymes by phosphorylation, we partially purified a protein kinase from pig platelets that phosphorylated type IIα PIP kinase on an activation loop threonine residue, T376. Pharmacological studies suggested this protein kinase was protein kinase D (PKD), and in vitro experiments confirmed this identification. A phospho-specific antibody was developed and used to demonstrate phosphorylation of T376 in living cells, and its enhancement under conditions in which PKD was activated. Although we were unable to determine the effects of phosphorylation on PIP kinase activity directly, mutation of T376 to aspartate significantly inhibited enzyme activity. We conclude that the type II PIP kinases are physiological targets for PKD phosphorylation, and that this modification is likely to regulate inositol lipid turnover by inhibition of these lipid kinases.

Protein kinase D protects against oxidative stress-induced intestinal epithelial cell injury via Rho/ROK/PKC-δ pathway activation

Protein kinase D (PKD) is a novel protein serine kinase that has recently been implicated in diverse cellular functions, including apoptosis and cell proliferation. The purpose of our present study was 1) to define the activation of PKD in intestinal epithelial cells treated with H2O2, an agent that induces oxidative stress, and 2) to delineate the upstream signaling mechanisms mediating the activation of PKD. We found that the activation of PKD is induced by H2O2 in both a dose- and time-dependent fashion. PKD phosphorylation was attenuated by rottlerin, a selective PKC-delta inhibitor, and by small interfering RNA (siRNA) directed against PKC-delta, suggesting the regulation of PKD activity by upstream PKC-delta. Activation of PKD was also blocked by a Rho kinase (ROK)-specific inhibitor, Y-27632, as well as by C3, a Rho protein inhibitor, demonstrating that the Rho/ROK pathway also mediates PKD activity in intestinal cells. In addition, H2O2-induced PKC-delta phosphorylation was inhibited by C3 treatment, further suggesting that PKC-delta is downstream of Rho/ROK. Interestingly, H2O2-induced intestinal cell apoptosis was enhanced by PKD siRNA. Together, these results clearly demonstrate that oxidative stress induces PKD activation in intestinal epithelial cells and that this activation is regulated by upstream PKC-delta and Rho/ROK pathways. Importantly, our findings suggest that PKD activation protects intestinal epithelial cells from oxidative stress-induced apoptosis. These findings have potential clinical implications for intestinal injury associated with oxidative stress (e.g., necrotizing enterocolitis in infants).

Mitochondria Signal to Nucleus Through PKD

When cells are stressed by stimuli that lead to the production of reactive oxygen species (ROS) by the mitochondria, the abundance of mitochondrial manganese-dependent superoxide dismutase (MnSOD) is increased. However, a nuclear SOD2 gene encodes this mitochondrial enzyme; thus, the mitochondria must signal to the nucleus to trigger the expression of the SOD2 gene. Storz et al. show that protein kinase D (PKD), Src, Abl, and protein kinase Cγ (PKCγ) partially translocate to the mitochondria when cells are exposed to hydrogen peroxide or to agents that inhibit the mitochondrial respiratory chain [rotenone or diphenyleneiodonium chloride (DPI), which increase mitochondrial ROS production. Furthermore, PKD phosphorylation and activity was stimulated by these conditions that produce oxidative stress. RNA interference (RNAi) to decrease PKD abundance blocked the increase in expression of SOD2 (measured by an SOD2 reporter construct) in response to mitochondrial oxidative stress. The SOD2 gene is regulated by the Forkhead transcription factor FOXO3a and by the transcription factor NF-κB. Mutation of the FOXO3a regulatory site did not inhibit SOD2 induction in response to peroxide or mitochondrial oxidative stress. However, expression of a superdominant version of the inhibitor of κBα (IκBα) blocked the increase in MnSOD abundance in response to either mitochondrial oxidative stress or activated version of Src or PKD. When PKD was depleted with RNAi, the induction of an NF-κB reporter in response to peroxide or mitochondrial oxidative stress was blocked. This pathway from the mitochondria to the nucleus through PKD was important for cell survival during periods of ROS induction. Cells in which PKD or MnSOD was depleted with RNAi showed a higher frequency of cell death in response to doses of peroxide or DPI that did not cause cell death of control cells. P. Storz, H. Döppler, A. Toker, Protein kinase D mediates mitochondrion-to-nucleus signaling and detoxification from mitochondrial reactive oxygen species. Mol. Cell. Biol. 25 , 8520-8530 (2005). [Abstract] [Full Text]

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.