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.     

Tyrosine phosphorylation of protein kinase D in the pleckstrin homology domain leads to activation

Protein kinase D (PKD) is a member of the AGC family of Ser/Thr kinases and is distantly related to protein kinase C (PKC). Formerly known as PKCmu, PKD contains protein domains not found in conventional PKC isoforms. A functional pleckstrin homology (PH) domain is critical for the regulation of PKD activity. Here we report that PKD is tyrosine-phosphorylated within the PH domain, leading to activation. This phosphorylation is mediated by a pathway that consists of the Src and Abl tyrosine kinases and occurs in response to stimulation with pervanadate and oxidative stress. Mutational analysis revealed three tyrosine phosphorylation sites (Tyr(432), Tyr(463), and Tyr(502)), which are regulated by the Src-Abl pathway, and phosphorylation of only one of these (Tyr(463)) leads to PKD activation. By using a phospho-specific antibody, we show that Abl directly phosphorylates PKD at Tyr(463) in vitro, and in cells phosphorylation of this site is sufficient to mediate full activation of PKD. Mutation of the other two sites, Tyr(432) and Tyr(502), had no significant influence on PKD activity. These data reveal a tyrosine phosphorylation-dependent activation mechanism for PKD and suggest that this event contributes to the release of the autoinhibitory PKD PH domain leading to kinase activation and downstream responses.     

Protein kinase D1: a new component in TLR9 signaling

Protein kinase D1 (PKD1) is expressed ubiquitously and regulates diverse cellular processes such as oxidative stress, gene expression, cell survival, and vesicle trafficking. However, the presence and function of PKD1 in monocytic cells are currently unknown. In this study, we provide evidence that PKD1 is involved in TLR9 signaling in macrophages. Class B-type CpG DNA (CpG-B DNA) induced activation of PKD1 via a pathway that is dependent on endosomal pH, TLR9, MyD88, and IL-1R-associated kinase 1 in macrophages. Upon CpG-B DNA stimulation, PKD1 interacted with the TLR9/MyD88/IL-1R-associated kinase/TNFR-associated factor 6 complex. Knockdown of PKD1 revealed that PKD1 is required for activation of NF-kappaB and MAPKs, and subsequent expression of cytokines in response to CpG-B DNA. Our findings identify PKD1 as a key signaling modulator in TLR9-mediated macrophage activation.     

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.     

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.     

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.     

DAP kinase regulates JNK signaling by binding and activating protein kinase D under oxidative stress

The stress-activated kinase JNK mediates key cellular responses to oxidative stress. Here we show that DAP kinase (DAPk), a cell death promoting Ser/Thr protein kinase, plays a main role in oxidative stress-induced JNK signaling. We identify protein kinase D (PKD) as a novel substrate of DAPk and demonstrate that DAPk physically interacts with PKD in response to oxidative stress. We further show that DAPk activates PKD in cells and that induction of JNK phosphorylation by ectopically expressed DAPk can be attenuated by knocking down PKD expression or by inhibiting its catalytic activity. Moreover, knockdown of DAPk expression caused a marked reduction in JNK activation under oxidative stress, indicating that DAPk is indispensable for the activation of JNK signaling under these conditions. Finally, DAPk is shown to be required for cell death under oxidative stress in a process that displays the characteristics of caspase-independent necrotic cell death. Taken together, these findings establish a major role for DAPk and its specific interaction with PKD in regulating the JNK signaling network under oxidative stress.     

Hepatitis C virus NS5A and subgenomic replicon activate NF-kappaB via tyrosine phosphorylation of IkappaBalpha and its degradation by calpain protease

Hepatitis C virus nonstructural protein 5A (NS5A) has been implicated in the HCV antiviral resistance, replication, and transactivation of cellular gene expression. We have recently shown that HCV NS5A activates NF-kappaB via oxidative stress (22). In this study, we investigate the molecular mechanism(s) of NF-kappaB activation in response to oxidative stress induced by NS5A protein. In contrast to the classic Ser32,36 phosphorylation of IkappaBalpha, we report here that tyrosine phosphorylation of IkappaBalpha at Tyr42 and Tyr305 residues is induced by the HCV NS5A and the subgenomic replicons in the NF-kappaB activation process. Use of IkappaBalpha-Tyr42,305 double mutant provided the evidence for their key role in the activation of NF-kappaB. Activation of NF-kappaB was blocked by a series of tyrosine kinase inhibitors but not by IkappaB kinase inhibitor BAY 11-7085. More specifically, a ZAP-70 knock-out cell line expressing NS5A and other nonstructural proteins respectively prevented the NF-kappaB activation, indicating the involvement of ZAP-70 as a probable tyrosine kinase in the activation process. Evidence is also presented for the possible role of calpain proteases in the NS5A-induced IkappaBalpha degradation. These studies collectively define an alternate pathway of NF-kappaB activation by NS5A alone or in the context of the HCV subgenomic replicon. Constitutive activation of NF-kappaB by HCV has implications in the chronic liver disease including hepatocellular carcinoma associated with HCV infection.     

Protein Kinase D1 (PKD1) Phosphorylation Promotes Dopaminergic Neuronal Survival during 6-OHDA-Induced Oxidative Stress

Oxidative stress is a major pathophysiological mediator of degenerative processes in many neurodegenerative diseases including Parkinson’s disease (PD). Aberrant cell signaling governed by protein phosphorylation has been linked to oxidative damage of dopaminergic neurons in PD. Although several studies have associated activation of certain protein kinases with apoptotic cell death in PD, very little is known about protein kinase regulation of cell survival and protection against oxidative damage and degeneration in dopaminergic neurons. Here, we characterized the PKD1-mediated protective pathway against oxidative damage in cell culture models of PD. Dopaminergic neurotoxicant 6-hydroxy dopamine (6-OHDA) was used to induce oxidative stress in the N27 dopaminergic cell model and in primary mesencephalic neurons. Our results indicated that 6-OHDA induced the PKD1 activation loop (PKD1S744/S748) phosphorylation during early stages of oxidative stress and that PKD1 activation preceded cell death. We also found that 6-OHDA rapidly increased phosphorylation of the C-terminal S916 in PKD1, which is required for PKD1 activation loop (PKD1S744/748) phosphorylation. Interestingly, negative modulation of PKD1 activation by RNAi knockdown or by the pharmacological inhibition of PKD1 by kbNB-14270 augmented 6-OHDA-induced apoptosis, while positive modulation of PKD1 by the overexpression of full length PKD1 (PKD1^WT) or constitutively active PKD1 (PKD1^S744E/S748E) attenuated 6-OHDA-induced apoptosis, suggesting an anti-apoptotic role for PKD1 during oxidative neuronal injury. Collectively, our results demonstrate that PKD1 signaling plays a cell survival role during early stages of oxidative stress in dopaminergic neurons and therefore, positive modulation of the PKD1-mediated signal transduction pathway can provide a novel neuroprotective strategy against PD.     

Activation of hematopoietic progenitor kinase 1 involves relocation, autophosphorylation, and transphosphorylation by protein kinase D1

Adaptive immune signaling can be coupled to stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) and NF-kappaB activation by the hematopoietic progenitor kinase 1 (HPK1), a mammalian hematopoiesis-specific Ste20 kinase. To gain insight into the regulation of leukocyte signal transduction, we investigated the molecular details of HPK1 activation. Here we demonstrate the capacity of the Src family kinase Lck and the SLP-76 family adaptor protein Clnk (cytokine-dependent hematopoietic cell linker) to induce HPK1 tyrosine phosphorylation and relocation to the plasma membrane, which in lymphocytes results in recruitment of HPK1 to the contact site of antigen-presenting cell (APC)-T-cell conjugates. Relocation and clustering of HPK1 cause its enzymatic activation, which is accompanied by phosphorylation of regulatory sites in the HPK1 kinase activation loop. We show that full activation of HPK1 is dependent on autophosphorylation of threonine 165 and phosphorylation of serine 171, which is a target site for protein kinase D (PKD) in vitro. Upon T-cell receptor stimulation, PKD robustly augments HPK1 kinase activity in Jurkat T cells and enhances HPK1-driven SAPK/JNK and NF-kappaB activation; conversely, antisense down-regulation of PKD results in reduced HPK1 activity. Thus, activation of major lymphocyte signaling pathways via HPK1 involves (i) relocation, (ii) autophosphorylation, and (iii) transphosphorylation of HPK1 by PKD.     

Requirement of protein kinase D tyrosine phosphorylation for VEGF-A165-induced angiogenesis through its interaction and regulation of phospholipase Cgamma 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).