Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protein kinase D (PKD) is a serine/threonine protein kinase that is directly stimulated in vitro by phorbol esters and diacylglycerol in the presence of phospholipids. Here, we examine the regulation of PKD in living cells. Our results demonstrate that tumour-promoting phorbol esters, membrane-permeant diacylglycerol and serum growth factors rapidly induced PKD activation in immortalized cell lines (e.g. Swiss 3T3 and Rat-1 cells), in secondary cultures of mouse embryo fibroblasts and in COS-7 cells transiently transfected with a PKD expression construct. PKD activation was maintained during cell disruption and immunopurification and was associated with an electrophoretic mobility shift and enhanced 32P incorporation into the enzyme, but was reversed by treatment with alkaline phosphatase. PKD was activated, deactivated and reactivated in response to consecutive cycles of addition and removal of PDB. PKD activation was completely abrogated by exposure of the cells to the protein kinase C inhibitors GF I and Ro 31-8220. In contrast, these compounds did not inhibit PKD activity when added directly in vitro. Co-transfection of PKD with constitutively activated mutants of PKCs showed that PKCepsilon and eta but not PKCzeta strongly induced PKD activation in COS-7 cells. Thus, our results indicate that PKD is activated in living cells through a PKC-dependent signal transduction pathway.
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PMID:Protein kinase D (PKD) activation in intact cells through a protein kinase C-dependent signal transduction pathway. 894 45

Metabotropic glutamate receptors (mGluRs) control intracellular signaling cascades through activation of G proteins. The inwardly rectifying K+ channel, GIRK, is activated by the beta gamma subunits of G proteins and is widely expressed in the brain. We investigated whether an interaction between mGluRs and GIRK is possible, using Xenopus oocytes expressing mGluRs and a cardiac/brain subunit of GIRK, GIRK1, with or without another brain subunit, GIRK2. mGluRs known to inhibit adenylyl cyclase (types 2, 3, 4, 6, and 7) activated the GIRK channel. The strongest response was observed with mGluR2; it was inhibited by pertussis toxin (PTX). This is consistent with the activation of GIRK by Gi/Go-coupled receptors. In contrast, mGluR1a and mGluR5 receptors known to activate phospholipase C, presumably via G proteins of the Gq class, inhibited the channel's activity. The inhibition was preceded by an initial weak activation, which was more prominent at higher levels of mGluR1a expression. The inhibition of GIRK activity by mGluR1a was suppressed by a broad-specificity protein kinase inhibitor, staurosporine, and by a specific protein kinase C (PKC) inhibitor, bis-indolylmaleimide, but not by PTX, Ca(2-)chelation, or calphostin C. Thus, mGluR1a inhibits the GIRK channel primarily via a pathway involving activation of a PTX-insensitive G protein and, eventually, of a subtype of PKC, possibly PKC-mu. In contrast, the initial activation of GIRK1 caused by mGluR1a was suppressed by PTX but not by the protein kinase inhibitors. Thus, this activation probably results from a promiscuous coupling of mGluR1a to a Gi/Go protein. The observed modulations may be involved in the mGluRs effects on neuronal excitability in the brain. Inhibition of GIRK by phospholipase C-activating mGluRs bears upon the problem of specificity of G protein (GIRK interaction) helping to explain why receptors coupled to Gq are inefficient in activating GIRK.
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PMID:Positive and negative coupling of the metabotropic glutamate receptors to a G protein-activated K+ channel, GIRK, in Xenopus oocytes. 910 6

Protein kinase D (PKD) is a serine/threonine protein kinase that is activated by phorbol esters via protein kinase C in intact cells. To assess the physiological significance of this putative pathway, we examined the regulation of PKD in living cells by mitogenic regulatory peptides and by platelet-derived growth factors (PDGF). Our results demonstrate that bombesin rapidly induces PKD activation in Swiss 3T3 cells, as shown by autophosphorylation and syntide-2 phosphorylation assays. Maximum PKD activation (14-fold above base-line levels) was obtained 90 s after bombesin stimulation. Bombesin also induced PKD activation in Rat-1 cells stably transfected with the bombesin/gastrin releasing peptide (GRP) receptor and in COS-7 cells transiently co-transfected with PKD and bombesin/GRP receptor expression constructs. No inducible kinase activity was demonstrated when COS-7 cells were transfected with a kinase-deficient PKD mutant. Bombesin-mediated PKD activation was prevented by treatment of Swiss 3T3 cells with the protein kinase C inhibitors GF 1092030X and Ro 31-8220. In contrast, these compounds did not inhibit PKD activity when added directly in vitro. Vasopressin, endothelin, and bradykinin also activated PKD in Swiss 3T3 cells through a PKC-dependent pathway. Platelet-derived growth factor-stimulated PKD activation in Swiss 3T3 cells and in porcine aortic endothelial cells stably transfected with PDGF-beta receptors. Treatment with GF 1092030X or Ro 31-8220 inhibited PKD activation induced by PDGF. Thus, our results indicate that PKD is activated by multiple signaling peptides through a protein kinase C-dependent signal transduction pathway in a variety of cell types.
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PMID:Bombesin, vasopressin, endothelin, bradykinin, and platelet-derived growth factor rapidly activate protein kinase D through a protein kinase C-dependent signal transduction pathway. 929 46

Protein kinase D (PKD) is a serine/threonine protein kinase that contains a cysteine-rich repeat sequence homologous to that seen in the regulatory domain of protein kinase C (PKC) and a catalytic domain with only a low degree of sequence similarity to PKCs. PKD also contains a pleckstrin homology (PH) domain inserted between the cysteine-rich motifs and the catalytic domain that is not present in any of the PKCs. To investigate the function of the PH domain in the regulation of PKD activity, we determined the kinase activity of several PKD PH domain mutants immunoprecipitated from lysates of transiently transfected COS-7 cells. Deletion of the entire PH domain (amino acids 429-557) markedly increased the basal activity of the enzyme as assessed by autophosphorylation ( approximately 16-fold) and exogenous syntide-2 peptide substrate phosphorylation assays (approximately 12-fold). Mutant PKD proteins with partial deletions or single amino acid substitutions within the PH domain (e. g. R447C and W538A) also exhibited increased basal kinase activity. These constitutive active mutants of PKD were only slightly further stimulated by phorbol-12,13-dibutyrate treatment of intact cells. Our results demonstrate, for the first time, that the PKD PH domain plays a negative role in the regulation of enzyme activity.
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PMID:Protein kinase D activation by mutations within its pleckstrin homology domain. 941 97

Protein kinase D (PKD)/protein kinase Cmicro (PKCmicro, a serine/threonine protein kinase with distinct structural and enzymological properties, is rapidly activated in intact cells via PKC. The amino-terminal region of PKD contains a cysteine-rich domain (CRD) that directly binds phorbol esters with a high affinity. Here, we show that treatment of transfected RBL 2H3 cells with phorbol 12,13-dibutyrate (PDB) induces a striking CRD-dependent translocation of PKD from the cytosol to the plasma membrane, as shown by real time visualization of a functional green fluorescent protein (GFP)-PKD fusion protein. A single amino acid substitution in the second cysteine-rich motif of PKD (P287G) prevented PDB-induced membrane translocation but did not affect PKD activation. Our results indicate that PKD translocation and activation are distinct processes that operate in parallel to regulate the activity and localization of this enzyme in intact cells.
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PMID:Dynamic re-distribution of protein kinase D (PKD) as revealed by a GFP-PKD fusion protein: dissociation from PKD activation. 1047 40

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.
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PMID:Oxidative stress induces protein kinase D activation in intact cells. Involvement of Src and dependence on protein kinase C. 1074 11

Protein kinase D (PKD; also known as PKCmicro) is a serine/threonine kinase activated by diacylglycerol signalling pathways in a variety of cells. PKD has been described previously as Golgi-localized, but herein we show that it is present within the cytosol of quiescent B cells and mast cells and moves rapidly to the plasma membrane after antigen receptor triggering. The membrane redistribution of PKD requires the diacylglycerol-binding domain of the enzyme, but is independent of its catalytic activity and does not require the integrity of the pleckstrin homology domain. Antigen receptor signalling initiates in glycosphingolipid-enriched microdomains, but membrane-associated PKD does not co-localize with these specialized structures. Membrane targeting of PKD is transient, the enzyme returns to the cytosol within 10 min of antigen receptor engagement. Strikingly, the membrane-recycled PKD remains active in the cytosol for several hours. The present work thus characterizes a sustained antigen receptor-induced signal transduction pathway and establishes PKD as a serine kinase that temporally and spatially disseminates antigen receptor signals away from the plasma membrane into the cytosol.
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PMID:Spatial and temporal regulation of protein kinase D (PKD). 1085 38

Here we show that human protein kinase C mu (PKC mu) activates the mitogen-activated protein kinase (MAPK). Transient expression of constitutive active PKC mu leads to an activation of Raf-1 kinase as demonstrated by in vitro phosphorylation of MAPK. PKC mu enhances transcriptional activity of a basal thymidine kinase promotor containing serum response elements (SREs) as shown by luciferase reporter gene assays. SRE driven gene activation by PKC mu is triggered by the Elk-1 ternary complex factor. PKC mu-mediated activation of SRE driven transcription can be inhibited by the MEK1 inhibitor PD98059. In contrast to the activation of the p42/ERK1 MAPK cascade, transient expression of constitutive active PKC mu does neither affect c-jun N-terminal kinase nor p38 MAPK.
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PMID:Protein kinase C mu selectively activates the mitogen-activated protein kinase (MAPK) p42 pathway. 1124 33

Protein kinase D (PKD)/protein kinase C (PKC) mu is a serine/threonine protein kinase that can be activated by physiological stimuli like growth factors, antigen-receptor engagement and G protein-coupled receptor (GPCR) agonists via a phosphorylation-dependent mechanism that requires PKC activity. In order to investigate the dynamic mechanisms associated with GPCR signaling, the intracellular translocation of a green fluorescent protein-tagged PKD was analyzed by real-time visualization in fibroblasts and epithelial cells stimulated with bombesin, a GPCR agonist. We found that bombesin induced a rapidly reversible plasma membrane translocation of green fluorescent protein-tagged PKD, an event that can be divided into two distinct mechanistic steps. The first step, which is exclusively mediated by the cysteine-rich domain in the N terminus of PKD, involved its translocation from the cytosol to the plasma membrane. The second step, i.e. the rapid reverse translocation of PKD from the plasma membrane to the cytosol, required its catalytic domain and surprisingly PKC activity. These findings provide evidence for a novel mechanism by which PKC coordinates the translocation and activation of PKD in response to bombesin-induced GPCR activation.
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PMID:Rapid protein kinase D translocation in response to G protein-coupled receptor activation. Dependence on protein kinase C. 1141 May 87

Protein kinase D (PKD)/protein kinase Cmu is a serine/threonine protein kinase that has been localized in the cytosol and in several intracellular compartments including Golgi, mitochondria and plasma membrane. Using real time imaging of fluorescent protein (GFP)-tagged PKD, we have found that the accumulation of PKD in the Golgi compartment, following a temperature shift from 37 to 20 degrees C, was mediated by the cysteine-rich domain (CRD) of PKD. The CRD of PKD also mediates its interaction with the plasma membrane, further supporting the conclusion that the CRD of PKD may act as a subcellular localization signal.
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PMID:Protein kinase D interacts with Golgi via its cysteine-rich domain. 1154 47


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