Serine/threonine-protein kinase that converts transient diacylglycerol (DAG) signals into prolonged physiological effects downstream of PKC, and is involved in the regulation of MAPK8/JNK1 and Ras signaling, Golgi membrane integrity and trafficking, cell survival through NF-kappa-B activation, cell migration, cell differentiation by mediating HDAC7 nuclear export, cell proliferation via MAPK1/3 (ERK1/2) signaling, and plays a role in cardiac hypertrophy, VEGFA-induced angiogenesis, genotoxic-induced apoptosis and flagellin-stimulated inflammatory response. Phosphorylates the epidermal growth factor receptor (EGFR) on dual threonine residues, which leads to the suppression of epidermal growth factor (EGF)-induced MAPK8/JNK1 activation and subsequent JUN phosphorylation. Phosphorylates RIN1, inducing RIN1 binding to 14-3-3 proteins YWHAB, YWHAE and YWHAZ and increased competition with RAF1 for binding to GTP-bound form of Ras proteins (NRAS, HRAS and KRAS). Acts downstream of the heterotrimeric G-protein beta/gamma-subunit complex to maintain the structural integrity of the Golgi membranes, and is required for protein transport along the secretory pathway. In the trans-Golgi network (TGN), regulates the fission of transport vesicles that are on their way to the plasma membrane. May act by activating the lipid kinase phosphatidylinositol 4-kinase beta (PI4KB) at the TGN for the local synthesis of phosphorylated inositol lipids, which induces a sequential production of DAG, phosphatidic acid (PA) and lyso-PA (LPA) that are necessary for membrane fission and generation of specific transport carriers to the cell surface. Under oxidative stress, is phosphorylated at Tyr-463 via SRC-ABL1 and contributes to cell survival by activating IKK complex and subsequent nuclear translocation and activation of NFKB1. Involved in cell migration by regulating integrin alpha-5/beta-3 recycling and promoting its recruitment in newly forming focal adhesion. In osteoblast differentiation, mediates the bone morphogenic protein 2 (BMP2)-induced nuclear export of HDAC7, which results in the inhibition of HDAC7 transcriptional repression of RUNX2. In neurons, plays an important role in neuronal polarity by regulating the biogenesis of TGN-derived dendritic vesicles, and is involved in the maintenance of dendritic arborization and Golgi structure in hippocampal cells. May potentiate mitogenesis induced by the neuropeptide bombesin or vasopressin by mediating an increase in the duration of MAPK1/3 (ERK1/2) signaling, which leads to accumulation of immediate-early gene products including FOS that stimulate cell cycle progression. Plays an important role in the proliferative response induced by low calcium in keratinocytes, through sustained activation of MAPK1/3 (ERK1/2) pathway. Downstream of novel PKC signaling, plays a role in cardiac hypertrophy by phosphorylating HDAC5, which in turn triggers XPO1/CRM1-dependent nuclear export of HDAC5, MEF2A transcriptional activation and induction of downstream target genes that promote myocyte hypertrophy and pathological cardiac remodeling. Mediates cardiac troponin I (TNNI3) phosphorylation at the PKA sites, which results in reduced myofilament calcium sensitivity, and accelerated crossbridge cycling kinetics. The PRKD1-HDAC5 pathway is also involved in angiogenesis by mediating VEGFA-induced specific subset of gene expression, cell migration, and tube formation. In response to VEGFA, is necessary and required for HDAC7 phosphorylation which induces HDAC7 nuclear export and endothelial cell proliferation and migration. During apoptosis induced by cytarabine and other genotoxic agents, PRKD1 is cleaved by caspase-3 at Asp-378, resulting in activation of its kinase function and increased sensitivity of cells to the cytotoxic effects of genotoxic agents. In epithelial cells, is required for transducing flagellin-stimulated inflammatory responses by binding and phosphorylating TLR5, which contributes to MAPK14/p38 activation and production of inflammatory cytokines. May play a role in inflammatory response by mediating activation of NF-kappa-B. May be involved in pain transmission by directly modulating TRPV1 receptor.
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.
Protein kinase D (PKD) is known to participate in various cellular functions, including secretory vesicle fission from the Golgi and plasma membrane-directed transport. Here, we report on expression and function of PKD in hippocampal neurons. Expression of an enhanced green fluorescent protein (EGFP)-tagged PKD activity reporter in mouse embryonal hippocampal neurons revealed high endogenous PKD activity at the Golgi complex and in the dendrites, whereas PKD activity was excluded from the axon in parallel with axonal maturation. Expression of fluorescently tagged wild-type PKD1 and constitutively active PKD1(S738/742E) (caPKD1) in neurons revealed that both proteins were slightly enriched at the trans-Golgi network (TGN) and did not interfere with its thread-like morphology. By contrast, expression of dominant-negative kinase inactive PKD1(K612W) (kdPKD1) led to the disruption of the neuronal Golgi complex, with kdPKD1 strongly localized to the TGN fragments. Similar findings were obtained from transgenic mice with inducible, neuron-specific expression of kdPKD1-EGFP. As a prominent consequence of kdPKD1 expression, the dendritic tree of transfected neurons was reduced, whereas caPKD1 increased dendritic arborization. Our results thus provide direct evidence that PKD activity is selectively involved in the maintenance of dendritic arborization and Golgi structure of hippocampal neurons.
J. Immunol. 178, 5735-5743 (2007)[PubMed:17442957]
Protein kinase D (PKD), also called protein kinase C (PKC)mu, is a serine-threonine kinase that is involved in diverse areas of cellular function such as lymphocyte signaling, oxidative stress, and protein secretion. After identifying a putative PKD phosphorylation site in the Toll/IL-1R domain of TLR5, we explored the role of this kinase in the interaction between human TLR5 and enteroaggregative Escherichia coli flagellin in human epithelial cell lines. We report several lines of evidence that implicate PKD in TLR5 signaling. First, PKD phosphorylated the TLR5-derived target peptide in vitro, and phosphorylation of the putative target serine 805 in HEK 293T cell-derived TLR5 was identified by mass spectrometry. Furthermore, mutation of serine 805 to alanine abrogated responses of transfected HEK 293T cells to flagellin. Second, TLR5 interacted with PKD in coimmunoprecipitation experiments, and this association was rapidly enhanced by flagellin treatment. Third, pharmacologic inhibition of PKC or PKD with Gö6976 resulted in reduced expression and secretion of IL-8 and prevented the flagellin-induced activation of p38 MAPK, but treatment with the PKC inhibitor Gö6983 had no significant effects on these phenotypes. Finally, involvement of PKD in the p38-mediated IL-8 response to flagellin was confirmed by small hairpin RNA-mediated gene silencing. Together, these results suggest that phosphorylation of TLR5 by PKD may be one of the proximal elements in the cellular response to flagellin, and that this event contributes to p38 MAPK activation and production of inflammatory cytokines in epithelial cells.
Dysfunction and loss of insulin-producing pancreatic beta cells represent hallmarks of diabetes mellitus. Here, we show that mice lacking the mitogen-activated protein kinase (MAPK) p38delta display improved glucose tolerance due to enhanced insulin secretion from pancreatic beta cells. Deletion of p38delta results in pronounced activation of protein kinase D (PKD), the latter of which we have identified as a pivotal regulator of stimulated insulin exocytosis. p38delta catalyzes an inhibitory phosphorylation of PKD1, thereby attenuating stimulated insulin secretion. In addition, p38delta null mice are protected against high-fat-feeding-induced insulin resistance and oxidative stress-mediated beta cell failure. Inhibition of PKD1 reverses enhanced insulin secretion from p38delta-deficient islets and glucose tolerance in p38delta null mice as well as their susceptibility to oxidative stress. In conclusion, the p38delta-PKD pathway integrates regulation of the insulin secretory capacity and survival of pancreatic beta cells, pointing to a pivotal role for this pathway in the development of overt diabetes mellitus.
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.
The capsaicin receptor VR1 is a polymodal nociceptor activated by multiple stimuli. It has been reported that protein kinase C plays a role in the sensitization of VR1. Protein kinase D/PKCmu is a member of the protein kinase D serine/threonine kinase family that exhibits structural, enzymological, and regulatory features distinct from those of the PKCs, with which they are related. As part of our effort to optimize conditions for evaluating VR1 pharmacology, we found that treatment of Chinese hamster ovary (CHO) cells heterologously expressing rat VR1 (CHO/rVR1) with butyrate enhanced rVR1 expression and activity. The expression of PKCmu and PKCbeta1, but not of other PKC isoforms, was also enhanced by butyrate treatment, suggesting the possibility that these two isoforms might contribute to the enhanced activity of rVR1. In support of this hypothesis, we found the following. 1) Overexpression of PKCmu enhanced the response of rVR1 to capsaicin and low pH, and expression of a dominant negative variant of PKCmu reduced the response of rVR1. 2) Reduction of endogenous PKCmu using antisense oligonucleotides decreased the response of exogenous rVR1 expressed in CHO cells as well as of endogenous rVR1 in dorsal root ganglion neurons. 3) PKCmu localized to the plasma membrane when overexpressed in CHO/rVR1 cells. 4) PKCmu directly bound to rVR1 expressed in CHO cells as well as to endogenous rVR1 in dorsal root ganglia or to an N-terminal fragment of rVR1, indicating a direct interaction between PKCmu and rVR1. 5) PKCmu directly phosphorylated rVR1 or a longer N-terminal fragment (amino acids 1-118) of rVR1 but not a shorter one (amino acids 1-99). 6) Mutation of S116A in rVR1 blocked both the phosphorylation of rVR1 by PKCmu and the enhancement by PKCmu of the rVR1 response to capsaicin. We conclude that PKCmu functions as a direct modulator of rVR1.
Protein kinase C (PKC) mu is a novel member of the PKC family that differs from the other isozymes in structural and biochemical properties. The precise function of PKCmu is not known. The present studies demonstrate that PKCmu is cleaved during apoptosis induced by 1-beta-d-arabinofuranosylcytosine (ara-C) and other genotoxic agents. PKCmu cleavage is blocked in cells that overexpress the anti-apoptotic Bcl-x(L) protein or the baculovirus p35 protein. Our results demonstrate that PKCmu is cleaved by caspase-3 at the CQND(378)S site. Cleavage of PKCmu is associated with release of the catalytic domain and activation of its kinase function. We also show that, unlike the cleaved fragments of PKCdelta and theta, overexpression of the PKCmu catalytic domain is not lethal. Cells stably expressing the catalytic fragment of PKCmu, however, are more sensitive to apoptosis induced by genotoxic stress. In addition, expression of the caspase-resistant PKCmu mutant partially inhibits DNA damage-induced apoptosis. These findings demonstrate that PKCmu is cleaved by caspase-3 and that expression of the catalytic domain sensitizes cells to the cytotoxic effects of ara-C and other anticancer agents.
VEGF has been shown to regulate endothelial cell (EC) proliferation and migration. However, the nuclear mediators of the actions of VEGF in ECs have not been fully defined. We show that VEGF induces the phosphorylation of three conserved serine residues in histone deacetylase 7 (HDAC7) via protein kinase D, which promotes nuclear export of HDAC7 and activation of VEGF-responsive genes in ECs. Expression of a signal-resistant HDAC7 mutant protein in ECs inhibits proliferation and migration in response to VEGF. These results demonstrate that phosphorylation of HDAC7 serves as a molecular switch to mediate VEGF signaling and endothelial function.
In Rat-1 fibroblasts epidermal growth factor (EGF), but not platelet-derived growth factor (PDGF) stimulates the activity of the c-Jun N-terminal kinase (JNK). Moreover, PDGF induced suppression of EGF-mediated JNK activation, apparently through protein kinase C (PKC) activation. Further analysis revealed that PKD was specifically activated by PDGF but not EGF in Rat-1 cells. In SF126 glioblastoma cells, however, EGF and PDGF synergistically activated JNK, while neither PDGF nor EGF stimulated PKD activity. In this cell line, overexpression of PKD blocked EGF- and PDGF-induced JNK activation. Mutational analysis further revealed that the EGFR mutant (T654/669E) was incapable of activating JNK and provided evidence that PKD-mediated dual phosphorylation of these critical threonine residues leads to suppression of EGF-induced JNK activation. Our results establish a novel crosstalk mechanism which allows signal integration and definition in cells with many different RTKs.
The activation of the transcription factor NF-kappaB is critical for a number of physiological responses. Here, we provide evidence for a signaling pathway that mediates NF-kappaB activation in response to oxidative stress. We show that tyrosine phosphorylation of protein kinase D (PKD) at Y463 in the Pleckstrin Homology (PH) domain is mediated by the Src and Abl tyrosine kinase signaling pathway, and that this is both necessary and sufficient to activate NF-kappaB in response to oxidative stress. PKD activates NF-kappaB through the IKK complex and more specifically, IKKbeta, leading to I(kappa)B(alpha) degradation. We also present evidence that this pathway is required for increased cellular survival in response to oxidative stress. We propose a model in which protection from oxidative stress-induced cell death requires the tyrosine phosphorylation of PKD leading to the activation of the transcription factor NF-kappaB.
Dysfunction and loss of insulin-producing pancreatic beta cells represent hallmarks of diabetes mellitus. Here, we show that mice lacking the mitogen-activated protein kinase (MAPK) p38delta display improved glucose tolerance due to enhanced insulin secretion from pancreatic beta cells. Deletion of p38delta results in pronounced activation of protein kinase D (PKD), the latter of which we have identified as a pivotal regulator of stimulated insulin exocytosis. p38delta catalyzes an inhibitory phosphorylation of PKD1, thereby attenuating stimulated insulin secretion. In addition, p38delta null mice are protected against high-fat-feeding-induced insulin resistance and oxidative stress-mediated beta cell failure. Inhibition of PKD1 reverses enhanced insulin secretion from p38delta-deficient islets and glucose tolerance in p38delta null mice as well as their susceptibility to oxidative stress. In conclusion, the p38delta-PKD pathway integrates regulation of the insulin secretory capacity and survival of pancreatic beta cells, pointing to a pivotal role for this pathway in the development of overt diabetes mellitus.
Evidence
2:
Inferred from Physical InteractionIntAct
We identified the multifunctional chaperon protein p32 as a protein kinase C (PKC)-binding protein interacting with PKCalpha, PKCzeta, PKCdelta, and PKC mu. We have analyzed the interaction of PKC mu with p32 in detail, and we show here in vivo association of PKC mu, as revealed from yeast two-hybrid analysis, precipitation assays using glutathione S-transferase fusion proteins, and reciprocal coimmunoprecipitation. In SKW 6.4 cells, PKC mu is constitutively associated with p32 at mitochondrial membranes, evident from colocalization with cytochrome c. p32 interacts with PKC mu in a compartment-specific manner, as it can be coimmunoprecipitated mainly from the particulate and not from the soluble fraction, despite the presence of p32 in both fractions. Although p32 binds to the kinase domain of PKC mu, it does not serve as a substrate. Interestingly, PKC mu-p32 immunocomplexes precipitated from the particulate fraction of two distinct cell lines, SKW 6.4 and 293T, show no detectable substrate phosphorylation. In support of a kinase regulatory function of p32, addition of p32 to in vitro kinase assays blocked, in a dose-dependent manner, aldolase but not autophosphorylation of PKC mu, suggesting a steric hindrance of substrate within the kinase domain. Together, these findings identify p32 as a novel, compartment-specific regulator of PKC mu kinase activity.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionIntAct
We identified the multifunctional chaperon protein p32 as a protein kinase C (PKC)-binding protein interacting with PKCalpha, PKCzeta, PKCdelta, and PKC mu. We have analyzed the interaction of PKC mu with p32 in detail, and we show here in vivo association of PKC mu, as revealed from yeast two-hybrid analysis, precipitation assays using glutathione S-transferase fusion proteins, and reciprocal coimmunoprecipitation. In SKW 6.4 cells, PKC mu is constitutively associated with p32 at mitochondrial membranes, evident from colocalization with cytochrome c. p32 interacts with PKC mu in a compartment-specific manner, as it can be coimmunoprecipitated mainly from the particulate and not from the soluble fraction, despite the presence of p32 in both fractions. Although p32 binds to the kinase domain of PKC mu, it does not serve as a substrate. Interestingly, PKC mu-p32 immunocomplexes precipitated from the particulate fraction of two distinct cell lines, SKW 6.4 and 293T, show no detectable substrate phosphorylation. In support of a kinase regulatory function of p32, addition of p32 to in vitro kinase assays blocked, in a dose-dependent manner, aldolase but not autophosphorylation of PKC mu, suggesting a steric hindrance of substrate within the kinase domain. Together, these findings identify p32 as a novel, compartment-specific regulator of PKC mu kinase activity.
Evidence
2:
Inferred from Physical InteractionIntAct
Prostate cancer (PC) patients die from progression to androgen independence (AI) and chemoresistance (CR). Protein kinase Cmu (PKCmu) a novel member of the PKC family of signal transduction proteins is downregulated in AI PC. Studying PKCmu interactors in the yeast two-hybrid system identified metallothionein 2A (MT 2A) as an interactor of PKCmu kinase domain (KD) in PC, which was quantified by beta-gal assay, confirmed in PC cells by immunoprecipitation, and PKCmu-MT 2A co-localization in vivo by immunofluorescence studies. PKCmu domain interaction studies revealed that MT 2A interacted strongly with KD, relatively weakly with C1, and failed to interact with C2, PH or kinase mutant domains. Peptide library and in silico analysis strongly suggest that MT 2A is a novel substrate of PKCmu and our data indicate that the PKCmu-MT 2A interaction depends on PKCmu kinase activity. Because metallothioneins are associated with cell proliferation and CR, the PKCmu-MT 2A interaction may contribute to CR and/or AI in PC.
Evidence
3:
Inferred from Physical InteractionUniProtKB
Centaurin-alpha(1) is a member of the family of ADP-ribosylation factors (ARF) GTPase activating proteins (GAPs), although ARF GAP activity has not yet been demonstrated. The human homologue, centaurin-alpha(1) functionally complements the ARF GAP activity of Gcs1 in yeast. Although Gcs1 is involved in the formation of actin filaments in vivo, the function of centaurin remains elusive. We have identified a number of novel centaurin-alpha(1) binding partners; including CKIalpha and nucleolin. In this report, we have focused on the interaction of centaurin-alpha(1) with PKC. All groups of PKC associate directly through their cysteine rich domains. Centaurin-alpha(1) is also a substrate for all PKC classes and we have identified the two sites of phosphorylation. This is the first report of a kinase that phosphorylates centaurin-alpha(1).
Evidence
4:
Inferred from Physical InteractionIntAct
HSP90 is a molecular chaperone that associates with numerous substrate proteins called clients. It plays many important roles in human biology and medicine, but determinants of client recognition by HSP90 have remained frustratingly elusive. We systematically and quantitatively surveyed most human kinases, transcription factors, and E3 ligases for interaction with HSP90 and its cochaperone CDC37. Unexpectedly, many more kinases than transcription factors bound HSP90. CDC37 interacted with kinases, but not with transcription factors or E3 ligases. HSP90::kinase interactions varied continuously over a 100-fold range and provided a platform to study client protein recognition. In wild-type clients, HSP90 did not bind particular sequence motifs, but rather associated with intrinsically unstable kinases. Stabilization of the kinase in either its active or inactive conformation with diverse small molecules decreased HSP90 association. Our results establish HSP90 client recognition as a combinatorial process: CDC37 provides recognition of the kinase family, whereas thermodynamic parameters determine client binding within the family.
Evidence
5:
Inferred from Physical InteractionIntAct
The cadherin family of transmembrane glycoproteins plays a critical role in cell-to-cell adhesion and cadherin dysregulation is strongly associated with cancer metastasis and progression. In this study, we report a novel interaction between protein kinase D1 [PKD1; formerly known as protein kinase C mu (PKCmu)] and E-cadherin. PKD1 is a serine/threonine-specific kinase known to play a role in multiple cellular processes including apoptosis, cytoskeleton remodeling, and invasion. Our study shows that PKD1 colocalizes with E-cadherin at cell junctions in LNCaP prostate cancer cells and coimmunoprecipitates with E-cadherin from lysates of LNCaP cells. In vitro kinase assays have shown that PKD1 phosphorylates E-cadherin. Inhibition of PKD1 activity by the selective inhibitor Gö6976 in LNCaP cells resulted in decreased cellular aggregation and overexpression of PKD1 in C4-2 prostate cancer cells increased cellular aggregation and decreased cellular motility. We also validated the PKD1 and E-cadherin colocalization in human prostate cancer tissue by confocal laser scanning microscopy. Our study has identified E-cadherin as a novel substrate of PKD1, and phosphorylation of E-cadherin by PKD1 is associated with increased cellular aggregation and decreased cellular motility in prostate cancer. Because both E-cadherin and PKD1 are known to be dysregulated in prostate cancer, our study identified an important protein-protein interaction influencing the signal transduction system associated with cell adhesion in prostate cancer.
Evidence
6:
Inferred from Physical InteractionIntAct
Dysfunction and loss of insulin-producing pancreatic beta cells represent hallmarks of diabetes mellitus. Here, we show that mice lacking the mitogen-activated protein kinase (MAPK) p38delta display improved glucose tolerance due to enhanced insulin secretion from pancreatic beta cells. Deletion of p38delta results in pronounced activation of protein kinase D (PKD), the latter of which we have identified as a pivotal regulator of stimulated insulin exocytosis. p38delta catalyzes an inhibitory phosphorylation of PKD1, thereby attenuating stimulated insulin secretion. In addition, p38delta null mice are protected against high-fat-feeding-induced insulin resistance and oxidative stress-mediated beta cell failure. Inhibition of PKD1 reverses enhanced insulin secretion from p38delta-deficient islets and glucose tolerance in p38delta null mice as well as their susceptibility to oxidative stress. In conclusion, the p38delta-PKD pathway integrates regulation of the insulin secretory capacity and survival of pancreatic beta cells, pointing to a pivotal role for this pathway in the development of overt diabetes mellitus.
Proteomic analysis identified HSP27 phosphorylation as a major change in protein phosphorylation stimulated by Vascular Endothelial Growth Factor (VEGF) in Human Umbilical Vein Endothelial Cells (HUVEC). VEGF-induced HSP27 phosphorylation at serines 15, 78 and 82, but whereas HSP27 phosphorylation induced by H2O2 and TNFalpha was completely blocked by the p38 kinase inhibitor, SB203580, VEGF-stimulated serine 82 phosphorylation was resistant to SB203580 and small interfering(si)RNA-mediated knockdown of p38 kinase and MAPKAPK2. The PKC inhibitor, GF109203X, partially reduced VEGF-induced HSP27 serine 82 phosphorylation, and SB203580 plus GF109203X abolished phosphorylation. VEGF activated Protein Kinase D (PKD) via PKC, and siRNAs targeted to PKD1 and PKD2 inhibited VEGF-induced HSP27 serine 82 phosphorylation. Furthermore recombinant PKD selectively phosphorylated HSP27 at serine 82 in vitro, and PKD2 activated by VEGF in HUVECs also phosphorylated HSP27 selectively at this site. Knockdown of HSP27 and PKDs markedly inhibited VEGF-induced HUVEC migration and tubulogenesis, whereas inhibition of the p38 kinase pathway using either SB203580 or siRNAs against p38alpha or MAPKAPK2, had no significant effect on the chemotactic response to VEGF. These findings identify a novel pathway for VEGF-induced HSP27 serine 82 phosphorylation via PKC-mediated PKD activation and direct phosphorylation of HSP27 by PKD, and show that PKDs and HSP27 play major roles in the angiogenic response to VEGF.
J. Biol. Chem. 269, 6140-6148 (1994)[PubMed:8119958]
We have isolated the full-length cDNA of a novel human serine/threonine protein kinase gene. The deduced protein sequence shows strong homology to conserved domains of members of the protein kinase C (PKC) subfamily. Homologies reside in the duplex zinc-finger-like cysteine-rich motif and in the protein kinase domain. The lack of the C2 domain of the Ca(2+)-dependent PKCs and the presence of a unique NH2-terminal sequence with a potential signal peptide and a transmembrane domain suggest that PKC mu is a novel member of the subgroup of atypical PKCs. An open reading frame coding for 912 amino acids directs an in vitro translation product with an apparent M(r) of 115,000. In vitro phorbol ester binding studies and kinase assays with lysates of cells overexpressing PKC mu showed phorbol ester-independent kinase activity, autophosphorylation, and, in normal rat kidney (NRK) cells, predominant phosphorylation of a 30-kDa protein at serine residues. Southern analysis revealed that PKC mu is a single copy gene located on human chromosome 21. There is constitutive low level expression of the human PKC mu gene in normal tissues with a single transcript of 3.8 kilobases and elevated expression levels in selected tumor cell lines. These data suggest a role of PKC mu in signal transduction pathways related to growth control.
A programmed cell death process which begins when a cell receives an internal (e.g. DNA damage) or external signal (e.g. an extracellular death ligand), and proceeds through a series of biochemical events (signaling pathways) which typically lead to rounding-up of the cell, retraction of pseudopodes, reduction of cellular volume (pyknosis), chromatin condensation, nuclear fragmentation (karyorrhexis), plasma membrane blebbing and fragmentation of the cell into apoptotic bodies. The process ends when the cell has died. The process is divided into a signaling pathway phase, and an execution phase, which is triggered by the former.
J. Biol. Chem. 269, 6140-6148 (1994)[PubMed:8119958]
We have isolated the full-length cDNA of a novel human serine/threonine protein kinase gene. The deduced protein sequence shows strong homology to conserved domains of members of the protein kinase C (PKC) subfamily. Homologies reside in the duplex zinc-finger-like cysteine-rich motif and in the protein kinase domain. The lack of the C2 domain of the Ca(2+)-dependent PKCs and the presence of a unique NH2-terminal sequence with a potential signal peptide and a transmembrane domain suggest that PKC mu is a novel member of the subgroup of atypical PKCs. An open reading frame coding for 912 amino acids directs an in vitro translation product with an apparent M(r) of 115,000. In vitro phorbol ester binding studies and kinase assays with lysates of cells overexpressing PKC mu showed phorbol ester-independent kinase activity, autophosphorylation, and, in normal rat kidney (NRK) cells, predominant phosphorylation of a 30-kDa protein at serine residues. Southern analysis revealed that PKC mu is a single copy gene located on human chromosome 21. There is constitutive low level expression of the human PKC mu gene in normal tissues with a single transcript of 3.8 kilobases and elevated expression levels in selected tumor cell lines. These data suggest a role of PKC mu in signal transduction pathways related to growth control.
Any process that results in a change in state or activity of a cell (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of oxidative stress, a state often resulting from exposure to high levels of reactive oxygen species, e.g. superoxide anions, hydrogen peroxide (H2O2), and hydroxyl radicals.
The activation of the transcription factor NF-kappaB is critical for a number of physiological responses. Here, we provide evidence for a signaling pathway that mediates NF-kappaB activation in response to oxidative stress. We show that tyrosine phosphorylation of protein kinase D (PKD) at Y463 in the Pleckstrin Homology (PH) domain is mediated by the Src and Abl tyrosine kinase signaling pathway, and that this is both necessary and sufficient to activate NF-kappaB in response to oxidative stress. PKD activates NF-kappaB through the IKK complex and more specifically, IKKbeta, leading to I(kappa)B(alpha) degradation. We also present evidence that this pathway is required for increased cellular survival in response to oxidative stress. We propose a model in which protection from oxidative stress-induced cell death requires the tyrosine phosphorylation of PKD leading to the activation of the transcription factor NF-kappaB.
Any process that results in a change in state or activity of a cell (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a vascular endothelial growth factor stimulus.
Early growth response 3 (Egr3) is a member of a zinc-finger transcription factor subfamily, which we previously found to be strongly upregulated by vascular endothelial growth factor (VEGF)-A in an oligonucleotide microarray screen of endothelial cells. Here, we show that Egr3 is the predominant Egr family member upregulated by VEGF in endothelial cells at 45 min, and that VEGF induced a rapid increase in Egr-dependent transcriptional activation mediated via its major signalling receptor, VEGFR2/KDR, and the protein kinase C (PKC) pathway. VEGF-induced Egr3 gene expression was also mediated in part via a PKC-dependent activation of protein kinase D. Inhibition of Egr3 gene expression by RNA interference was effective in inhibiting basal and VEGF-induced Egr3 gene expression, and it also inhibited VEGF-mediated endothelial cell proliferation, migration and tubulogenesis. These findings indicate that Egr3 has an essential downstream role in VEGF-mediated endothelial functions leading to angiogenesis and may have particular relevance for adult angiogenic processes involved in vascular repair and neovascular disease.
Evidence
2:
Inferred from Genetic InteractionBHF-UCL
VEGF (vascular endothelial growth factor) plays an essential role in angiogenesis during development and in disease largely mediated by signalling events initiated by binding of VEGF to its receptor, VEGFR2 (VEGF receptor 2)/KDR (kinase insert domain receptor). Recent studies indicate that VEGF activates PKD (protein kinase D) in endothelial cells to regulate a variety of cellular functions, including signalling events, proliferation, migration and angiogenesis. To better understand the role of PKD in VEGF-mediated endothelial function, we characterized the effects of a novel pyrazine benzamide PKD inhibitor CRT5 in HUVECs (human umbilical vein endothelial cells). The activity of the isoforms PKD1 and PKD2 were blocked by this inhibitor as indicated by reduced phosphorylation, at Ser916 and Ser876 respectively, after VEGF stimulation. The VEGF-induced phosphorylation of three PKD substrates, histone deacetylase 5, CREB (cAMP-response-element-binding protein) and HSP27 (heat-shock protein 27) at Ser82, was also inhibited by CRT5. In contrast, CRT6, an inactive analogue of CRT5, had no effect on PKD or HSP27 Ser82 phosphorylation. Furthermore, phosphorylation of HSP27 at Ser78, which occurs solely via the p38 MAPK (mitogen-activated protein kinase) pathway, was also unaffected by CRT5. In vitro kinase assays show that CRT5 did not significantly inhibit several PKC isoforms expressed in endothelial cells. CRT5 also decreased VEGF-induced endothelial migration, proliferation and tubulogenesis, similar to effects seen when the cells were transfected with PKD siRNA (small interfering RNA). CRT5, a novel specific PKD inhibitor, will greatly facilitate the study of the role of PKD signalling mechanisms in angiogenesis.
Proteomic analysis identified HSP27 phosphorylation as a major change in protein phosphorylation stimulated by Vascular Endothelial Growth Factor (VEGF) in Human Umbilical Vein Endothelial Cells (HUVEC). VEGF-induced HSP27 phosphorylation at serines 15, 78 and 82, but whereas HSP27 phosphorylation induced by H2O2 and TNFalpha was completely blocked by the p38 kinase inhibitor, SB203580, VEGF-stimulated serine 82 phosphorylation was resistant to SB203580 and small interfering(si)RNA-mediated knockdown of p38 kinase and MAPKAPK2. The PKC inhibitor, GF109203X, partially reduced VEGF-induced HSP27 serine 82 phosphorylation, and SB203580 plus GF109203X abolished phosphorylation. VEGF activated Protein Kinase D (PKD) via PKC, and siRNAs targeted to PKD1 and PKD2 inhibited VEGF-induced HSP27 serine 82 phosphorylation. Furthermore recombinant PKD selectively phosphorylated HSP27 at serine 82 in vitro, and PKD2 activated by VEGF in HUVECs also phosphorylated HSP27 selectively at this site. Knockdown of HSP27 and PKDs markedly inhibited VEGF-induced HUVEC migration and tubulogenesis, whereas inhibition of the p38 kinase pathway using either SB203580 or siRNAs against p38alpha or MAPKAPK2, had no significant effect on the chemotactic response to VEGF. These findings identify a novel pathway for VEGF-induced HSP27 serine 82 phosphorylation via PKC-mediated PKD activation and direct phosphorylation of HSP27 by PKD, and show that PKDs and HSP27 play major roles in the angiogenic response to VEGF.
Evidence
4:
Inferred from Mutant PhenotypeUniProtKB
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 process that is carried out at the cellular level which results in the assembly, arrangement of constituent parts, or disassembly of the Golgi apparatus.
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
Protein kinase D (PKD) is known to participate in various cellular functions, including secretory vesicle fission from the Golgi and plasma membrane-directed transport. Here, we report on expression and function of PKD in hippocampal neurons. Expression of an enhanced green fluorescent protein (EGFP)-tagged PKD activity reporter in mouse embryonal hippocampal neurons revealed high endogenous PKD activity at the Golgi complex and in the dendrites, whereas PKD activity was excluded from the axon in parallel with axonal maturation. Expression of fluorescently tagged wild-type PKD1 and constitutively active PKD1(S738/742E) (caPKD1) in neurons revealed that both proteins were slightly enriched at the trans-Golgi network (TGN) and did not interfere with its thread-like morphology. By contrast, expression of dominant-negative kinase inactive PKD1(K612W) (kdPKD1) led to the disruption of the neuronal Golgi complex, with kdPKD1 strongly localized to the TGN fragments. Similar findings were obtained from transgenic mice with inducible, neuron-specific expression of kdPKD1-EGFP. As a prominent consequence of kdPKD1 expression, the dendritic tree of transfected neurons was reduced, whereas caPKD1 increased dendritic arborization. Our results thus provide direct evidence that PKD activity is selectively involved in the maintenance of dendritic arborization and Golgi structure of hippocampal neurons.
The immediate defensive reaction (by vertebrate tissue) to infection or injury caused by chemical or physical agents. The process is characterized by local vasodilation, extravasation of plasma into intercellular spaces and accumulation of white blood cells and macrophages.
A series of molecular signals initiated by the binding of extracellular ligand to an integrin on the surface of a target cell, and ending with regulation of a downstream cellular process, e.g. transcription.
The Protein Kinase D (PKD) family comprises diacylglycerol stimulated serine/threonine protein kinases that participate in many key signaling pathways in a diverse range of cells. Recent studies show that PKD isoforms 1 and 2 play critical roles in vascular biology and angiogenesis and there has been considerable progress in determining some of the key angiogenic signaling pathways mediated by PKD in endothelial cells. Less is currently known regarding the specific roles of PKD isoforms in endothelial cells and the role of PKD in smooth muscle cells. PKD is also emerging as a potentially important mediator of tumor growth and tumor angiogenesis and there is growing interest in PKD as a novel therapeutic target in cancer.
A series of reactions in which a signal is passed on to downstream proteins within the cell by sequential protein phosphorylation and activation of the cascade components.
Evidence
1:
Inferred from Genetic InteractionBHF-UCL
VEGF (vascular endothelial growth factor) plays an essential role in angiogenesis during development and in disease largely mediated by signalling events initiated by binding of VEGF to its receptor, VEGFR2 (VEGF receptor 2)/KDR (kinase insert domain receptor). Recent studies indicate that VEGF activates PKD (protein kinase D) in endothelial cells to regulate a variety of cellular functions, including signalling events, proliferation, migration and angiogenesis. To better understand the role of PKD in VEGF-mediated endothelial function, we characterized the effects of a novel pyrazine benzamide PKD inhibitor CRT5 in HUVECs (human umbilical vein endothelial cells). The activity of the isoforms PKD1 and PKD2 were blocked by this inhibitor as indicated by reduced phosphorylation, at Ser916 and Ser876 respectively, after VEGF stimulation. The VEGF-induced phosphorylation of three PKD substrates, histone deacetylase 5, CREB (cAMP-response-element-binding protein) and HSP27 (heat-shock protein 27) at Ser82, was also inhibited by CRT5. In contrast, CRT6, an inactive analogue of CRT5, had no effect on PKD or HSP27 Ser82 phosphorylation. Furthermore, phosphorylation of HSP27 at Ser78, which occurs solely via the p38 MAPK (mitogen-activated protein kinase) pathway, was also unaffected by CRT5. In vitro kinase assays show that CRT5 did not significantly inhibit several PKC isoforms expressed in endothelial cells. CRT5 also decreased VEGF-induced endothelial migration, proliferation and tubulogenesis, similar to effects seen when the cells were transfected with PKD siRNA (small interfering RNA). CRT5, a novel specific PKD inhibitor, will greatly facilitate the study of the role of PKD signalling mechanisms in angiogenesis.
Proteomic analysis identified HSP27 phosphorylation as a major change in protein phosphorylation stimulated by Vascular Endothelial Growth Factor (VEGF) in Human Umbilical Vein Endothelial Cells (HUVEC). VEGF-induced HSP27 phosphorylation at serines 15, 78 and 82, but whereas HSP27 phosphorylation induced by H2O2 and TNFalpha was completely blocked by the p38 kinase inhibitor, SB203580, VEGF-stimulated serine 82 phosphorylation was resistant to SB203580 and small interfering(si)RNA-mediated knockdown of p38 kinase and MAPKAPK2. The PKC inhibitor, GF109203X, partially reduced VEGF-induced HSP27 serine 82 phosphorylation, and SB203580 plus GF109203X abolished phosphorylation. VEGF activated Protein Kinase D (PKD) via PKC, and siRNAs targeted to PKD1 and PKD2 inhibited VEGF-induced HSP27 serine 82 phosphorylation. Furthermore recombinant PKD selectively phosphorylated HSP27 at serine 82 in vitro, and PKD2 activated by VEGF in HUVECs also phosphorylated HSP27 selectively at this site. Knockdown of HSP27 and PKDs markedly inhibited VEGF-induced HUVEC migration and tubulogenesis, whereas inhibition of the p38 kinase pathway using either SB203580 or siRNAs against p38alpha or MAPKAPK2, had no significant effect on the chemotactic response to VEGF. These findings identify a novel pathway for VEGF-induced HSP27 serine 82 phosphorylation via PKC-mediated PKD activation and direct phosphorylation of HSP27 by PKD, and show that PKDs and HSP27 play major roles in the angiogenic response to VEGF.
Early growth response 3 (Egr3) is a member of a zinc-finger transcription factor subfamily, which we previously found to be strongly upregulated by vascular endothelial growth factor (VEGF)-A in an oligonucleotide microarray screen of endothelial cells. Here, we show that Egr3 is the predominant Egr family member upregulated by VEGF in endothelial cells at 45 min, and that VEGF induced a rapid increase in Egr-dependent transcriptional activation mediated via its major signalling receptor, VEGFR2/KDR, and the protein kinase C (PKC) pathway. VEGF-induced Egr3 gene expression was also mediated in part via a PKC-dependent activation of protein kinase D. Inhibition of Egr3 gene expression by RNA interference was effective in inhibiting basal and VEGF-induced Egr3 gene expression, and it also inhibited VEGF-mediated endothelial cell proliferation, migration and tubulogenesis. These findings indicate that Egr3 has an essential downstream role in VEGF-mediated endothelial functions leading to angiogenesis and may have particular relevance for adult angiogenic processes involved in vascular repair and neovascular disease.
Any process that decreases the rate or frequency of cell death. Cell death is the specific activation or halting of processes within a cell so that its vital functions markedly cease, rather than simply deteriorating gradually over time, which culminates in cell death.
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
The activation of the transcription factor NF-kappaB is critical for a number of physiological responses. Here, we provide evidence for a signaling pathway that mediates NF-kappaB activation in response to oxidative stress. We show that tyrosine phosphorylation of protein kinase D (PKD) at Y463 in the Pleckstrin Homology (PH) domain is mediated by the Src and Abl tyrosine kinase signaling pathway, and that this is both necessary and sufficient to activate NF-kappaB in response to oxidative stress. PKD activates NF-kappaB through the IKK complex and more specifically, IKKbeta, leading to I(kappa)B(alpha) degradation. We also present evidence that this pathway is required for increased cellular survival in response to oxidative stress. We propose a model in which protection from oxidative stress-induced cell death requires the tyrosine phosphorylation of PKD leading to the activation of the transcription factor NF-kappaB.
The Protein Kinase D (PKD) family comprises diacylglycerol stimulated serine/threonine protein kinases that participate in many key signaling pathways in a diverse range of cells. Recent studies show that PKD isoforms 1 and 2 play critical roles in vascular biology and angiogenesis and there has been considerable progress in determining some of the key angiogenic signaling pathways mediated by PKD in endothelial cells. Less is currently known regarding the specific roles of PKD isoforms in endothelial cells and the role of PKD in smooth muscle cells. PKD is also emerging as a potentially important mediator of tumor growth and tumor angiogenesis and there is growing interest in PKD as a novel therapeutic target in cancer.
Proteomic analysis identified HSP27 phosphorylation as a major change in protein phosphorylation stimulated by Vascular Endothelial Growth Factor (VEGF) in Human Umbilical Vein Endothelial Cells (HUVEC). VEGF-induced HSP27 phosphorylation at serines 15, 78 and 82, but whereas HSP27 phosphorylation induced by H2O2 and TNFalpha was completely blocked by the p38 kinase inhibitor, SB203580, VEGF-stimulated serine 82 phosphorylation was resistant to SB203580 and small interfering(si)RNA-mediated knockdown of p38 kinase and MAPKAPK2. The PKC inhibitor, GF109203X, partially reduced VEGF-induced HSP27 serine 82 phosphorylation, and SB203580 plus GF109203X abolished phosphorylation. VEGF activated Protein Kinase D (PKD) via PKC, and siRNAs targeted to PKD1 and PKD2 inhibited VEGF-induced HSP27 serine 82 phosphorylation. Furthermore recombinant PKD selectively phosphorylated HSP27 at serine 82 in vitro, and PKD2 activated by VEGF in HUVECs also phosphorylated HSP27 selectively at this site. Knockdown of HSP27 and PKDs markedly inhibited VEGF-induced HUVEC migration and tubulogenesis, whereas inhibition of the p38 kinase pathway using either SB203580 or siRNAs against p38alpha or MAPKAPK2, had no significant effect on the chemotactic response to VEGF. These findings identify a novel pathway for VEGF-induced HSP27 serine 82 phosphorylation via PKC-mediated PKD activation and direct phosphorylation of HSP27 by PKD, and show that PKDs and HSP27 play major roles in the angiogenic response to VEGF.
Proteomic analysis identified HSP27 phosphorylation as a major change in protein phosphorylation stimulated by Vascular Endothelial Growth Factor (VEGF) in Human Umbilical Vein Endothelial Cells (HUVEC). VEGF-induced HSP27 phosphorylation at serines 15, 78 and 82, but whereas HSP27 phosphorylation induced by H2O2 and TNFalpha was completely blocked by the p38 kinase inhibitor, SB203580, VEGF-stimulated serine 82 phosphorylation was resistant to SB203580 and small interfering(si)RNA-mediated knockdown of p38 kinase and MAPKAPK2. The PKC inhibitor, GF109203X, partially reduced VEGF-induced HSP27 serine 82 phosphorylation, and SB203580 plus GF109203X abolished phosphorylation. VEGF activated Protein Kinase D (PKD) via PKC, and siRNAs targeted to PKD1 and PKD2 inhibited VEGF-induced HSP27 serine 82 phosphorylation. Furthermore recombinant PKD selectively phosphorylated HSP27 at serine 82 in vitro, and PKD2 activated by VEGF in HUVECs also phosphorylated HSP27 selectively at this site. Knockdown of HSP27 and PKDs markedly inhibited VEGF-induced HUVEC migration and tubulogenesis, whereas inhibition of the p38 kinase pathway using either SB203580 or siRNAs against p38alpha or MAPKAPK2, had no significant effect on the chemotactic response to VEGF. These findings identify a novel pathway for VEGF-induced HSP27 serine 82 phosphorylation via PKC-mediated PKD activation and direct phosphorylation of HSP27 by PKD, and show that PKDs and HSP27 play major roles in the angiogenic response to VEGF.
Evidence
2:
Inferred from Genetic InteractionBHF-UCL
VEGF (vascular endothelial growth factor) plays an essential role in angiogenesis during development and in disease largely mediated by signalling events initiated by binding of VEGF to its receptor, VEGFR2 (VEGF receptor 2)/KDR (kinase insert domain receptor). Recent studies indicate that VEGF activates PKD (protein kinase D) in endothelial cells to regulate a variety of cellular functions, including signalling events, proliferation, migration and angiogenesis. To better understand the role of PKD in VEGF-mediated endothelial function, we characterized the effects of a novel pyrazine benzamide PKD inhibitor CRT5 in HUVECs (human umbilical vein endothelial cells). The activity of the isoforms PKD1 and PKD2 were blocked by this inhibitor as indicated by reduced phosphorylation, at Ser916 and Ser876 respectively, after VEGF stimulation. The VEGF-induced phosphorylation of three PKD substrates, histone deacetylase 5, CREB (cAMP-response-element-binding protein) and HSP27 (heat-shock protein 27) at Ser82, was also inhibited by CRT5. In contrast, CRT6, an inactive analogue of CRT5, had no effect on PKD or HSP27 Ser82 phosphorylation. Furthermore, phosphorylation of HSP27 at Ser78, which occurs solely via the p38 MAPK (mitogen-activated protein kinase) pathway, was also unaffected by CRT5. In vitro kinase assays show that CRT5 did not significantly inhibit several PKC isoforms expressed in endothelial cells. CRT5 also decreased VEGF-induced endothelial migration, proliferation and tubulogenesis, similar to effects seen when the cells were transfected with PKD siRNA (small interfering RNA). CRT5, a novel specific PKD inhibitor, will greatly facilitate the study of the role of PKD signalling mechanisms in angiogenesis.
Evidence
3:
Inferred from Mutant PhenotypeUniProtKB
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.
VEGF (vascular endothelial growth factor) plays an essential role in angiogenesis during development and in disease largely mediated by signalling events initiated by binding of VEGF to its receptor, VEGFR2 (VEGF receptor 2)/KDR (kinase insert domain receptor). Recent studies indicate that VEGF activates PKD (protein kinase D) in endothelial cells to regulate a variety of cellular functions, including signalling events, proliferation, migration and angiogenesis. To better understand the role of PKD in VEGF-mediated endothelial function, we characterized the effects of a novel pyrazine benzamide PKD inhibitor CRT5 in HUVECs (human umbilical vein endothelial cells). The activity of the isoforms PKD1 and PKD2 were blocked by this inhibitor as indicated by reduced phosphorylation, at Ser916 and Ser876 respectively, after VEGF stimulation. The VEGF-induced phosphorylation of three PKD substrates, histone deacetylase 5, CREB (cAMP-response-element-binding protein) and HSP27 (heat-shock protein 27) at Ser82, was also inhibited by CRT5. In contrast, CRT6, an inactive analogue of CRT5, had no effect on PKD or HSP27 Ser82 phosphorylation. Furthermore, phosphorylation of HSP27 at Ser78, which occurs solely via the p38 MAPK (mitogen-activated protein kinase) pathway, was also unaffected by CRT5. In vitro kinase assays show that CRT5 did not significantly inhibit several PKC isoforms expressed in endothelial cells. CRT5 also decreased VEGF-induced endothelial migration, proliferation and tubulogenesis, similar to effects seen when the cells were transfected with PKD siRNA (small interfering RNA). CRT5, a novel specific PKD inhibitor, will greatly facilitate the study of the role of PKD signalling mechanisms in angiogenesis.
Proteomic analysis identified HSP27 phosphorylation as a major change in protein phosphorylation stimulated by Vascular Endothelial Growth Factor (VEGF) in Human Umbilical Vein Endothelial Cells (HUVEC). VEGF-induced HSP27 phosphorylation at serines 15, 78 and 82, but whereas HSP27 phosphorylation induced by H2O2 and TNFalpha was completely blocked by the p38 kinase inhibitor, SB203580, VEGF-stimulated serine 82 phosphorylation was resistant to SB203580 and small interfering(si)RNA-mediated knockdown of p38 kinase and MAPKAPK2. The PKC inhibitor, GF109203X, partially reduced VEGF-induced HSP27 serine 82 phosphorylation, and SB203580 plus GF109203X abolished phosphorylation. VEGF activated Protein Kinase D (PKD) via PKC, and siRNAs targeted to PKD1 and PKD2 inhibited VEGF-induced HSP27 serine 82 phosphorylation. Furthermore recombinant PKD selectively phosphorylated HSP27 at serine 82 in vitro, and PKD2 activated by VEGF in HUVECs also phosphorylated HSP27 selectively at this site. Knockdown of HSP27 and PKDs markedly inhibited VEGF-induced HUVEC migration and tubulogenesis, whereas inhibition of the p38 kinase pathway using either SB203580 or siRNAs against p38alpha or MAPKAPK2, had no significant effect on the chemotactic response to VEGF. These findings identify a novel pathway for VEGF-induced HSP27 serine 82 phosphorylation via PKC-mediated PKD activation and direct phosphorylation of HSP27 by PKD, and show that PKDs and HSP27 play major roles in the angiogenic response to VEGF.
VEGF (vascular endothelial growth factor) plays an essential role in angiogenesis during development and in disease largely mediated by signalling events initiated by binding of VEGF to its receptor, VEGFR2 (VEGF receptor 2)/KDR (kinase insert domain receptor). Recent studies indicate that VEGF activates PKD (protein kinase D) in endothelial cells to regulate a variety of cellular functions, including signalling events, proliferation, migration and angiogenesis. To better understand the role of PKD in VEGF-mediated endothelial function, we characterized the effects of a novel pyrazine benzamide PKD inhibitor CRT5 in HUVECs (human umbilical vein endothelial cells). The activity of the isoforms PKD1 and PKD2 were blocked by this inhibitor as indicated by reduced phosphorylation, at Ser916 and Ser876 respectively, after VEGF stimulation. The VEGF-induced phosphorylation of three PKD substrates, histone deacetylase 5, CREB (cAMP-response-element-binding protein) and HSP27 (heat-shock protein 27) at Ser82, was also inhibited by CRT5. In contrast, CRT6, an inactive analogue of CRT5, had no effect on PKD or HSP27 Ser82 phosphorylation. Furthermore, phosphorylation of HSP27 at Ser78, which occurs solely via the p38 MAPK (mitogen-activated protein kinase) pathway, was also unaffected by CRT5. In vitro kinase assays show that CRT5 did not significantly inhibit several PKC isoforms expressed in endothelial cells. CRT5 also decreased VEGF-induced endothelial migration, proliferation and tubulogenesis, similar to effects seen when the cells were transfected with PKD siRNA (small interfering RNA). CRT5, a novel specific PKD inhibitor, will greatly facilitate the study of the role of PKD signalling mechanisms in angiogenesis.
Proteomic analysis identified HSP27 phosphorylation as a major change in protein phosphorylation stimulated by Vascular Endothelial Growth Factor (VEGF) in Human Umbilical Vein Endothelial Cells (HUVEC). VEGF-induced HSP27 phosphorylation at serines 15, 78 and 82, but whereas HSP27 phosphorylation induced by H2O2 and TNFalpha was completely blocked by the p38 kinase inhibitor, SB203580, VEGF-stimulated serine 82 phosphorylation was resistant to SB203580 and small interfering(si)RNA-mediated knockdown of p38 kinase and MAPKAPK2. The PKC inhibitor, GF109203X, partially reduced VEGF-induced HSP27 serine 82 phosphorylation, and SB203580 plus GF109203X abolished phosphorylation. VEGF activated Protein Kinase D (PKD) via PKC, and siRNAs targeted to PKD1 and PKD2 inhibited VEGF-induced HSP27 serine 82 phosphorylation. Furthermore recombinant PKD selectively phosphorylated HSP27 at serine 82 in vitro, and PKD2 activated by VEGF in HUVECs also phosphorylated HSP27 selectively at this site. Knockdown of HSP27 and PKDs markedly inhibited VEGF-induced HUVEC migration and tubulogenesis, whereas inhibition of the p38 kinase pathway using either SB203580 or siRNAs against p38alpha or MAPKAPK2, had no significant effect on the chemotactic response to VEGF. These findings identify a novel pathway for VEGF-induced HSP27 serine 82 phosphorylation via PKC-mediated PKD activation and direct phosphorylation of HSP27 by PKD, and show that PKDs and HSP27 play major roles in the angiogenic response to VEGF.
Positive regulation of endothelial cell chemotaxis by VEGF-activated vascular endothelial growth factor receptor signaling pathwaydefinition[GO:0038033]
A series of molecular signals initiated by the binding of a vascular endothelial growth factor (VEGF) to a VEGFR on the surface of a cell, which activates or increases the frequency, rate or extent of endothelial cell chemotaxis.
Evidence
1:
Inferred from Genetic InteractionBHF-UCL
VEGF (vascular endothelial growth factor) plays an essential role in angiogenesis during development and in disease largely mediated by signalling events initiated by binding of VEGF to its receptor, VEGFR2 (VEGF receptor 2)/KDR (kinase insert domain receptor). Recent studies indicate that VEGF activates PKD (protein kinase D) in endothelial cells to regulate a variety of cellular functions, including signalling events, proliferation, migration and angiogenesis. To better understand the role of PKD in VEGF-mediated endothelial function, we characterized the effects of a novel pyrazine benzamide PKD inhibitor CRT5 in HUVECs (human umbilical vein endothelial cells). The activity of the isoforms PKD1 and PKD2 were blocked by this inhibitor as indicated by reduced phosphorylation, at Ser916 and Ser876 respectively, after VEGF stimulation. The VEGF-induced phosphorylation of three PKD substrates, histone deacetylase 5, CREB (cAMP-response-element-binding protein) and HSP27 (heat-shock protein 27) at Ser82, was also inhibited by CRT5. In contrast, CRT6, an inactive analogue of CRT5, had no effect on PKD or HSP27 Ser82 phosphorylation. Furthermore, phosphorylation of HSP27 at Ser78, which occurs solely via the p38 MAPK (mitogen-activated protein kinase) pathway, was also unaffected by CRT5. In vitro kinase assays show that CRT5 did not significantly inhibit several PKC isoforms expressed in endothelial cells. CRT5 also decreased VEGF-induced endothelial migration, proliferation and tubulogenesis, similar to effects seen when the cells were transfected with PKD siRNA (small interfering RNA). CRT5, a novel specific PKD inhibitor, will greatly facilitate the study of the role of PKD signalling mechanisms in angiogenesis.
Proteomic analysis identified HSP27 phosphorylation as a major change in protein phosphorylation stimulated by Vascular Endothelial Growth Factor (VEGF) in Human Umbilical Vein Endothelial Cells (HUVEC). VEGF-induced HSP27 phosphorylation at serines 15, 78 and 82, but whereas HSP27 phosphorylation induced by H2O2 and TNFalpha was completely blocked by the p38 kinase inhibitor, SB203580, VEGF-stimulated serine 82 phosphorylation was resistant to SB203580 and small interfering(si)RNA-mediated knockdown of p38 kinase and MAPKAPK2. The PKC inhibitor, GF109203X, partially reduced VEGF-induced HSP27 serine 82 phosphorylation, and SB203580 plus GF109203X abolished phosphorylation. VEGF activated Protein Kinase D (PKD) via PKC, and siRNAs targeted to PKD1 and PKD2 inhibited VEGF-induced HSP27 serine 82 phosphorylation. Furthermore recombinant PKD selectively phosphorylated HSP27 at serine 82 in vitro, and PKD2 activated by VEGF in HUVECs also phosphorylated HSP27 selectively at this site. Knockdown of HSP27 and PKDs markedly inhibited VEGF-induced HUVEC migration and tubulogenesis, whereas inhibition of the p38 kinase pathway using either SB203580 or siRNAs against p38alpha or MAPKAPK2, had no significant effect on the chemotactic response to VEGF. These findings identify a novel pathway for VEGF-induced HSP27 serine 82 phosphorylation via PKC-mediated PKD activation and direct phosphorylation of HSP27 by PKD, and show that PKDs and HSP27 play major roles in the angiogenic response to VEGF.
Any process that increases the rate, frequency, or extent of the orderly movement of an endothelial cell into the extracellular matrix to form an endothelium.
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
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.
VEGF (vascular endothelial growth factor) plays an essential role in angiogenesis during development and in disease largely mediated by signalling events initiated by binding of VEGF to its receptor, VEGFR2 (VEGF receptor 2)/KDR (kinase insert domain receptor). Recent studies indicate that VEGF activates PKD (protein kinase D) in endothelial cells to regulate a variety of cellular functions, including signalling events, proliferation, migration and angiogenesis. To better understand the role of PKD in VEGF-mediated endothelial function, we characterized the effects of a novel pyrazine benzamide PKD inhibitor CRT5 in HUVECs (human umbilical vein endothelial cells). The activity of the isoforms PKD1 and PKD2 were blocked by this inhibitor as indicated by reduced phosphorylation, at Ser916 and Ser876 respectively, after VEGF stimulation. The VEGF-induced phosphorylation of three PKD substrates, histone deacetylase 5, CREB (cAMP-response-element-binding protein) and HSP27 (heat-shock protein 27) at Ser82, was also inhibited by CRT5. In contrast, CRT6, an inactive analogue of CRT5, had no effect on PKD or HSP27 Ser82 phosphorylation. Furthermore, phosphorylation of HSP27 at Ser78, which occurs solely via the p38 MAPK (mitogen-activated protein kinase) pathway, was also unaffected by CRT5. In vitro kinase assays show that CRT5 did not significantly inhibit several PKC isoforms expressed in endothelial cells. CRT5 also decreased VEGF-induced endothelial migration, proliferation and tubulogenesis, similar to effects seen when the cells were transfected with PKD siRNA (small interfering RNA). CRT5, a novel specific PKD inhibitor, will greatly facilitate the study of the role of PKD signalling mechanisms in angiogenesis.
VEGF (vascular endothelial growth factor) plays an essential role in angiogenesis during development and in disease largely mediated by signalling events initiated by binding of VEGF to its receptor, VEGFR2 (VEGF receptor 2)/KDR (kinase insert domain receptor). Recent studies indicate that VEGF activates PKD (protein kinase D) in endothelial cells to regulate a variety of cellular functions, including signalling events, proliferation, migration and angiogenesis. To better understand the role of PKD in VEGF-mediated endothelial function, we characterized the effects of a novel pyrazine benzamide PKD inhibitor CRT5 in HUVECs (human umbilical vein endothelial cells). The activity of the isoforms PKD1 and PKD2 were blocked by this inhibitor as indicated by reduced phosphorylation, at Ser916 and Ser876 respectively, after VEGF stimulation. The VEGF-induced phosphorylation of three PKD substrates, histone deacetylase 5, CREB (cAMP-response-element-binding protein) and HSP27 (heat-shock protein 27) at Ser82, was also inhibited by CRT5. In contrast, CRT6, an inactive analogue of CRT5, had no effect on PKD or HSP27 Ser82 phosphorylation. Furthermore, phosphorylation of HSP27 at Ser78, which occurs solely via the p38 MAPK (mitogen-activated protein kinase) pathway, was also unaffected by CRT5. In vitro kinase assays show that CRT5 did not significantly inhibit several PKC isoforms expressed in endothelial cells. CRT5 also decreased VEGF-induced endothelial migration, proliferation and tubulogenesis, similar to effects seen when the cells were transfected with PKD siRNA (small interfering RNA). CRT5, a novel specific PKD inhibitor, will greatly facilitate the study of the role of PKD signalling mechanisms in angiogenesis.
The activation of the transcription factor NF-kappaB is critical for a number of physiological responses. Here, we provide evidence for a signaling pathway that mediates NF-kappaB activation in response to oxidative stress. We show that tyrosine phosphorylation of protein kinase D (PKD) at Y463 in the Pleckstrin Homology (PH) domain is mediated by the Src and Abl tyrosine kinase signaling pathway, and that this is both necessary and sufficient to activate NF-kappaB in response to oxidative stress. PKD activates NF-kappaB through the IKK complex and more specifically, IKKbeta, leading to I(kappa)B(alpha) degradation. We also present evidence that this pathway is required for increased cellular survival in response to oxidative stress. We propose a model in which protection from oxidative stress-induced cell death requires the tyrosine phosphorylation of PKD leading to the activation of the transcription factor NF-kappaB.
Any process that increases the rate, frequency or extent of neuron projection development. Neuron projection development is the process whose specific outcome is the progression of a neuron projection over time, from its formation to the mature structure. A neuron projection is any process extending from a neural cell, such as axons or dendrites (collectively called neurites).
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
Protein kinase D (PKD) is known to participate in various cellular functions, including secretory vesicle fission from the Golgi and plasma membrane-directed transport. Here, we report on expression and function of PKD in hippocampal neurons. Expression of an enhanced green fluorescent protein (EGFP)-tagged PKD activity reporter in mouse embryonal hippocampal neurons revealed high endogenous PKD activity at the Golgi complex and in the dendrites, whereas PKD activity was excluded from the axon in parallel with axonal maturation. Expression of fluorescently tagged wild-type PKD1 and constitutively active PKD1(S738/742E) (caPKD1) in neurons revealed that both proteins were slightly enriched at the trans-Golgi network (TGN) and did not interfere with its thread-like morphology. By contrast, expression of dominant-negative kinase inactive PKD1(K612W) (kdPKD1) led to the disruption of the neuronal Golgi complex, with kdPKD1 strongly localized to the TGN fragments. Similar findings were obtained from transgenic mice with inducible, neuron-specific expression of kdPKD1-EGFP. As a prominent consequence of kdPKD1 expression, the dendritic tree of transfected neurons was reduced, whereas caPKD1 increased dendritic arborization. Our results thus provide direct evidence that PKD activity is selectively involved in the maintenance of dendritic arborization and Golgi structure of hippocampal neurons.
The activation of the transcription factor NF-kappaB is critical for a number of physiological responses. Here, we provide evidence for a signaling pathway that mediates NF-kappaB activation in response to oxidative stress. We show that tyrosine phosphorylation of protein kinase D (PKD) at Y463 in the Pleckstrin Homology (PH) domain is mediated by the Src and Abl tyrosine kinase signaling pathway, and that this is both necessary and sufficient to activate NF-kappaB in response to oxidative stress. PKD activates NF-kappaB through the IKK complex and more specifically, IKKbeta, leading to I(kappa)B(alpha) degradation. We also present evidence that this pathway is required for increased cellular survival in response to oxidative stress. We propose a model in which protection from oxidative stress-induced cell death requires the tyrosine phosphorylation of PKD leading to the activation of the transcription factor NF-kappaB.
VEGF (vascular endothelial growth factor) plays an essential role in angiogenesis during development and in disease largely mediated by signalling events initiated by binding of VEGF to its receptor, VEGFR2 (VEGF receptor 2)/KDR (kinase insert domain receptor). Recent studies indicate that VEGF activates PKD (protein kinase D) in endothelial cells to regulate a variety of cellular functions, including signalling events, proliferation, migration and angiogenesis. To better understand the role of PKD in VEGF-mediated endothelial function, we characterized the effects of a novel pyrazine benzamide PKD inhibitor CRT5 in HUVECs (human umbilical vein endothelial cells). The activity of the isoforms PKD1 and PKD2 were blocked by this inhibitor as indicated by reduced phosphorylation, at Ser916 and Ser876 respectively, after VEGF stimulation. The VEGF-induced phosphorylation of three PKD substrates, histone deacetylase 5, CREB (cAMP-response-element-binding protein) and HSP27 (heat-shock protein 27) at Ser82, was also inhibited by CRT5. In contrast, CRT6, an inactive analogue of CRT5, had no effect on PKD or HSP27 Ser82 phosphorylation. Furthermore, phosphorylation of HSP27 at Ser78, which occurs solely via the p38 MAPK (mitogen-activated protein kinase) pathway, was also unaffected by CRT5. In vitro kinase assays show that CRT5 did not significantly inhibit several PKC isoforms expressed in endothelial cells. CRT5 also decreased VEGF-induced endothelial migration, proliferation and tubulogenesis, similar to effects seen when the cells were transfected with PKD siRNA (small interfering RNA). CRT5, a novel specific PKD inhibitor, will greatly facilitate the study of the role of PKD signalling mechanisms in angiogenesis.
Early growth response 3 (Egr3) is a member of a zinc-finger transcription factor subfamily, which we previously found to be strongly upregulated by vascular endothelial growth factor (VEGF)-A in an oligonucleotide microarray screen of endothelial cells. Here, we show that Egr3 is the predominant Egr family member upregulated by VEGF in endothelial cells at 45 min, and that VEGF induced a rapid increase in Egr-dependent transcriptional activation mediated via its major signalling receptor, VEGFR2/KDR, and the protein kinase C (PKC) pathway. VEGF-induced Egr3 gene expression was also mediated in part via a PKC-dependent activation of protein kinase D. Inhibition of Egr3 gene expression by RNA interference was effective in inhibiting basal and VEGF-induced Egr3 gene expression, and it also inhibited VEGF-mediated endothelial cell proliferation, migration and tubulogenesis. These findings indicate that Egr3 has an essential downstream role in VEGF-mediated endothelial functions leading to angiogenesis and may have particular relevance for adult angiogenic processes involved in vascular repair and neovascular disease.
The Protein Kinase D (PKD) family comprises diacylglycerol stimulated serine/threonine protein kinases that participate in many key signaling pathways in a diverse range of cells. Recent studies show that PKD isoforms 1 and 2 play critical roles in vascular biology and angiogenesis and there has been considerable progress in determining some of the key angiogenic signaling pathways mediated by PKD in endothelial cells. Less is currently known regarding the specific roles of PKD isoforms in endothelial cells and the role of PKD in smooth muscle cells. PKD is also emerging as a potentially important mediator of tumor growth and tumor angiogenesis and there is growing interest in PKD as a novel therapeutic target in cancer.
The Protein Kinase D (PKD) family comprises diacylglycerol stimulated serine/threonine protein kinases that participate in many key signaling pathways in a diverse range of cells. Recent studies show that PKD isoforms 1 and 2 play critical roles in vascular biology and angiogenesis and there has been considerable progress in determining some of the key angiogenic signaling pathways mediated by PKD in endothelial cells. Less is currently known regarding the specific roles of PKD isoforms in endothelial cells and the role of PKD in smooth muscle cells. PKD is also emerging as a potentially important mediator of tumor growth and tumor angiogenesis and there is growing interest in PKD as a novel therapeutic target in cancer.
Any process that modulates the rate, frequency or extent of keratinocyte proliferation. Keratinocyte proliferation is the multiplication or reproduction of keratinocytes, resulting in the expansion of a cell population.
The cellular process in which a signal is conveyed to trigger a change in the activity or state of a cell. Signal transduction begins with reception of a signal (e.g. a ligand binding to a receptor or receptor activation by a stimulus such as light), or for signal transduction in the absence of ligand, signal-withdrawal or the activity of a constitutively active receptor. Signal transduction ends with regulation of a downstream cellular process, e.g. regulation of transcription or regulation of a metabolic process. Signal transduction covers signaling from receptors located on the surface of the cell and signaling via molecules located within the cell. For signaling between cells, signal transduction is restricted to events at and within the receiving cell.
J. Biol. Chem. 269, 6140-6148 (1994)[PubMed:8119958]
We have isolated the full-length cDNA of a novel human serine/threonine protein kinase gene. The deduced protein sequence shows strong homology to conserved domains of members of the protein kinase C (PKC) subfamily. Homologies reside in the duplex zinc-finger-like cysteine-rich motif and in the protein kinase domain. The lack of the C2 domain of the Ca(2+)-dependent PKCs and the presence of a unique NH2-terminal sequence with a potential signal peptide and a transmembrane domain suggest that PKC mu is a novel member of the subgroup of atypical PKCs. An open reading frame coding for 912 amino acids directs an in vitro translation product with an apparent M(r) of 115,000. In vitro phorbol ester binding studies and kinase assays with lysates of cells overexpressing PKC mu showed phorbol ester-independent kinase activity, autophosphorylation, and, in normal rat kidney (NRK) cells, predominant phosphorylation of a 30-kDa protein at serine residues. Southern analysis revealed that PKC mu is a single copy gene located on human chromosome 21. There is constitutive low level expression of the human PKC mu gene in normal tissues with a single transcript of 3.8 kilobases and elevated expression levels in selected tumor cell lines. These data suggest a role of PKC mu in signal transduction pathways related to growth control.
Any series of molecular signals initiated by the binding of an extracellular ligand to a vascular endothelial growth factor receptor (VEGFR) located on the surface of the receiving cell, and ending with regulation of a downstream cellular process, e.g. transcription.
Early growth response 3 (Egr3) is a member of a zinc-finger transcription factor subfamily, which we previously found to be strongly upregulated by vascular endothelial growth factor (VEGF)-A in an oligonucleotide microarray screen of endothelial cells. Here, we show that Egr3 is the predominant Egr family member upregulated by VEGF in endothelial cells at 45 min, and that VEGF induced a rapid increase in Egr-dependent transcriptional activation mediated via its major signalling receptor, VEGFR2/KDR, and the protein kinase C (PKC) pathway. VEGF-induced Egr3 gene expression was also mediated in part via a PKC-dependent activation of protein kinase D. Inhibition of Egr3 gene expression by RNA interference was effective in inhibiting basal and VEGF-induced Egr3 gene expression, and it also inhibited VEGF-mediated endothelial cell proliferation, migration and tubulogenesis. These findings indicate that Egr3 has an essential downstream role in VEGF-mediated endothelial functions leading to angiogenesis and may have particular relevance for adult angiogenic processes involved in vascular repair and neovascular disease.
Activated by DAG and phorbol esters. Phorbol-ester/DAG-type domain 1 binds DAG with high affinity and appears to play the dominant role in mediating translocation to the cell membrane and trans-Golgi network. Phorbol-ester/DAG-type domain 2 binds phorbol ester with higher affinity. Autophosphorylation of Ser-742 and phosphorylation of Ser-738 by PKC relieves auto-inhibition by the PH domain. Phosphorylation on Tyr-463 by the SRC-ABL1 pathway in response to oxidative stress, is also required for activation. Activated by DAPK1 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.
The PKD (protein kinase D) family are novel DAG (diacylglycerol) receptors. The twin C1 domains of PKD, designated C1a and C1b, have been shown to bind DAG or phorbol esters. However, their ligand-binding activities and selectivities have not been fully characterized. Here, binding activities of isolated C1a, C1b and intact C1a-C1b domains to DAG and phorbol esters were analysed. The isolated C1b domains of PKD isoforms bind [(3)H]PDBu ([20-(3)H]phorbol 12, 13-dibutyrate) with similar high affinities, while they exhibit weaker affinities towards a synthetic DAG analogue, DOG (1,2-dioctanoyl-sn-glycerol), as compared to the control. Mutating a conserved lysine residue at position 22 to tryptophan in C1b of PKD3 fully restores its affinity to DOG, indicating that this residue accounts for its weaker affinity to DOG. In contrast, the non-consensus residues in the isolated C1a domain of PKD mainly contribute to maintaining the protein's structural fold, since converting these residues in C1a of PKD3 to those in PKD1 or PKD2 drastically reduces the maximal number of active receptors, while only minimally impacting ligand-binding activities. Moreover, ligand-binding activities of C1a and C1b are sensitive to the structural context in an intact C1a-C1b domain and exhibit unique patterns of ligand selectivity. C1a and C1b in the intact C1a-C1b of PKD1 are opposite in selectivity for PDBu and DOG. In contrast, C1a of PKD3 exhibits 48-fold higher affinity to DOG as compared to C1b, although both domains bind PDBu with equivalent affinities. Accordingly, mutating C1a of a full-length PKD3-GFP greatly reduces DOG-induced plasma membrane translocation, but does not affect that induced by PMA. In summary, individual C1 domains of PKD isoforms differ in ligand-binding activity and selectivity, implying isoform-selective regulation of PKD by phorbol esters and DAG.
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 involved in angiogenesis, the sprouting or splitting of capillaries from pre-existing vasculature. Angiogenesis plays an important role for example during embryonic development, normal growth of tissues and maintenance of the normal vasculature, wound healing, tumor growth and metastasis.
Protein involved in apoptotic programmed cell death. Apoptosis is characterized by cell morphological changes, including blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation and chromosomal DNA fragmentation, and eventually death. Unlike necrosis, apoptosis produces cell fragments, called apoptotic bodies, that phagocytic cells are able to engulf and quickly remove before the contents of the cell can spill out onto surrounding cells and cause damage. In general, apoptosis confers advantages during an organism's life cycle.
Protein involved in differentiation, the developmental process of a multicellular organism by which cells become specialized for particular functions. Differentiation requires selective expression of the genome; the fully differentiated state may be preceded by a stage in which the cell is already programmed for differentiation but is not yet expressing the characteristic phenotype determination. Also used for fungal conidiation proteins, and for some bacteria that present specialization of function in cell types, such as Caulobacter crescentus.
Protein involved in immunity, any immune system process that functions in the response of an organism to a potential internal or invasive threat. The vertebrate immune system is formed by the innate immune system (composed of phagocytes, complement, antimicrobial peptides, etc) and by the adaptive immune system which consists of T- and B- lymphocytes.
Protein involved in the localized protective response to tissue damage, microbial infection, or the presence of foreign matter. It is characterized by swelling, redness, heat and pain and involves a complex series of events including vascular changes and accumulation of blood cells, such as neutrophil leucocytes and mononuclear phagocytes, at the site of injury.
Protein involved in innate immunity, an inborn defense mechanism used by organisms to defend themselves against invasion by pathogens (bacteria, fungi, viruses, etc.). Initially discovered in insects which are devoid of an adaptive immune system and rely only on innate immune reactions for their defense, this immediate response accomplishes many activities including recognition and effector functions. Recognition is mediated by broad specificity, pattern recognition, receptors which recognize many related molecular structures (e.g. polysaccharides, polynucleotides) present in microorganisms but not found in the host. The innate responses include the release of antimicrobial peptides, production of cytokines, acute- phase proteins, complement. Although many different innate immune mechanisms are deployed for host defence, a unifying theme of innate immunity is the use of germline-encoded pattern recognition receptors for pathogens or damaged self components, such as the Toll-like receptors, nucleotide-binding domain leucine-rich repeat (LRR)- containing receptors, retinoic acid-inducible gene I-like RNA helicases and C-type lectin receptors.
Protein which catalyzes the phosphorylation of serine or threonine residues on target proteins by using ATP as phosphate donor. Such phosphorylation may cause changes in the function of the target protein. Protein kinases share a conserved catalytic core common to both serine/ threonine and tyrosine protein kinases.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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