Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Melanophore pigment dispersion is a sensitive bioassay for activation of the adenylyl cyclase and phospholipase C second-messenger pathways. The necessity of protein kinase activation in causing pigment dispersion was confirmed for eight agonists of endogenous melanophore receptors and for two transfected receptors. All agonists and receptors previously shown to elevate intracellular cAMP in melanophores--melanocyte stimulating hormone, light, (-) norepinephrine, 5-hydroxytrptamine, and the beta2-adrenergic receptor--were able to stimulate pigment dispersion in the presence of Ro31-8220, a potent inhibitor of protein kinase C, but were blocked in the presence of H89, an inhibitor of cAMP-dependent protein kinase. The bombesin receptor, which elevates intracellular IP3 in melanophores, was unable to stimulate pigment dispersion in the presence of Ro31-8220 or H89. Agonists whose mechanism of activation of pigment dispersion are unknown were also tested. Endothelin 3 responses were blocked by both H89 and Ro31-8220, predicting coupling to phospholipase C. Vasoactive intestinal polypeptide, oxytocin, and calcitonin gene-related peptide beta responses were blocked only by H89, predicting coupling to adenylyl cyclase.
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PMID:Melanophore pigment dispersion responses to agonists show two patterns of sensitivity to inhibitors of cAMP-dependent protein kinase and protein kinase C. 869 26

Adenosine exerts a mitogenic effect on human endothelial cells via stimulation of the A2A-adenosine receptor. This effect can also be elicited by the beta2-adrenergic receptor but is not mimicked by elevation of intracellular cAMP levels. In the present work, we report that stimulation of the A2A-adenosine receptor and of the beta2-adrenergic receptor activates mitogen-activated protein kinase (MAP kinase) in human endothelial cells based on the following criteria: adenosine analogues and beta-adrenergic agonists cause an (i) increase in tyrosine phosphorylation of the p42 isoform and to a lesser extent of the p44 isoform of MAP kinase and (ii) stimulate the phosphorylation of myelin basic protein by MAP kinase; (iii) this is accompanied by a redistribution of the enzyme to the perinuclear region. Pretreatment of the cells with cholera toxin (to down-regulate Gsalpha) abolishes activation of MAP kinase by isoproterenol but not that induced by adenosine analogues. In addition, MAP kinase stimulation via the A2A-adenosine receptor is neither impaired following pretreatment of the cells with pertussis toxin (to block Gi-dependent pathways) nor affected by GF109203X (1 microM; to inhibit typical protein kinase C isoforms) nor by a monoclonal antibody, which blocks epidermal growth factor-dependent signaling. In contrast, MAP kinase activation is blocked by PD 098059, an inhibitor of MAP kinase kinase 1 (MEK1) activation, which also blunts the A2A-adenosine receptor-mediated increase in [3H]thymidine incorporation. Activation of the A2A-adenosine receptor is associated with increased levels of GTP-bound p21(ras). Thus, our experiments define stimulation of MAP kinase as the candidate cellular target mediating the mitogenic action of the A2A-adenosine receptor on primary human endothelial cells; the signaling pathway operates via p21(ras) and MEK1 but is independent of Gi, Gs, and the typical protein kinase C isoforms. This implies an additional G protein which links this prototypical Gs-coupled receptor to the MAP kinase cascade.
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PMID:Stimulation of the mitogen-activated protein kinase via the A2A-adenosine receptor in primary human endothelial cells. 903 93

The molecular mechanisms involved in the regulation of muscarinic receptor gene expression are poorly understood. In an effort to gain a better understanding of the regulation of M2 receptors, we have investigated homologous and heterologous regulation of M2 muscarinic receptor protein and gene expression in human embryonic lung fibroblasts (HEL 299 cells). HEL 299 cells constitutively express m2 receptors, with no evidence of other muscarinic receptor subtypes. We have shown that M2 receptors in these cells can be down-regulated by muscarinic and beta2-adrenergic receptor agonists. Unlike the down-regulation mediated by muscarinic and beta-adrenergic stimulation, activation of PKC with PDBu was mediated through changes in m2 muscarinic receptor mRNA through reduced gene transcription. Because of the inflammatory nature of asthma, we have focused on delineating the interactions between cytokines and M2 receptors in an attempt to define potential endogenous modulators of M2 receptor expression. We have shown that the multi-functional cytokine, transforming growth factor beta1 (TGF-beta1), which is involved in several inflammatory conditions induces desensitization and down-regulation of M2 muscarinic receptor protein and gene expression that was mediated through a reduction in the rate of m2 receptor gene transcription. Other cytokines of interest are tumor necrosis factor alpha (TNF-alpha) and interleukin 1beta (IL-1beta) which are elevated in asthma. We have demonstrated that TNF-alpha and IL-1beta synergise to induce down-regulation of M2 muscarinic receptor protein and mRNA which was associated with functional desensitization of the receptor protein. The M2 receptor mRNA down-regulation appeared to be mediated through a reduction in the rate of m2 receptor gene transcription which may be dependent on the transcription and translation of unknown protein factor(s). Moreover, a role of PKA and ceramide pathways in M2 receptor regulation is suggested. Collectively, our work provides a mechanistic explanation of previous reports indicating altered function of M2 receptors in asthma. Ours results also suggest that the expression of this receptor subtype may be under the control of a cytokine network at the airways.
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PMID:Regulation of muscarinic M2 receptors. 912 42

The mechanisms for regulating platelet HDL3 binding sites were investigated. HDL3 binding was rapid (T(1/2) association=4 minutes) and completely reversible (T(1/2) dissociation=14.5 minutes) at 4 degrees C, 22 degrees C, and 37 degrees C, and kinetic analysis yielded forward and reverse constants of 7.3x10(-4) x s(-1) and 7.13x10(3) x s(-1) x M(-1), respectively. Nevertheless, neither inhibitors of binding sites recycling or of pinocytosis, such as ammonium chloride, chloroquine, monensin, colchicine, and sodium azide, modified the binding characteristics. Moreover, when platelets were loaded with cholesterol, binding sites were not regulated (up or down). However, when exposed to high concentrations of HDL3 (1.5 g/L), apoE-free HDL (1.5 g/L), HDL2 (0.5 g/L), apoE-rich HDL (0.5 g/L), and VLDL (0.3 g/L) there was rapid downregulation of the number of binding sites in isolated permeabilized platelets, as shown by the reduction of Bmax to 66%, 58%, 45%, 53%, and 51%, respectively. Downregulation was rapid, reversible, and dose and time dependent. In contrast, LDL (up to 2.0 g/L), IDL (up to 0.1 g/L), and chylomicrons (up to 0.5 g/L) had no effect. Protein kinase C inhibitors (150 nmol/L staurosporine, 100 micromol/L H-7, and 10 nmol/L bisindolylmaleimide) inhibited downregulation up to 62% (as average value). The role of the PKC activation in regulating the activity of HDL3 binding sites also was analyzed by determining the cytosol-to-membrane translocation of enzymatic activity. Downregulation mediated by HDL3 rapidly translocated PKC activity (21% +/- 11 of total PKC activity was membrane-associated in control platelets vs. 55+/-8% in downregulated platelets, mean+/-SEM, n=3). However, agents that block sequestration (0.30 g/L, concanavalin A), and other protein kinase inhibitors, such a cAMP-dependent protein kinase inhibitors (1 micromol/L, PKI), and beta2-adrenergic receptor kinase inhibitors (100 nmol/L, heparin) had no effect. The results show that neither endocytotic response nor cholesterol-dependent mechanisms participate in the modulation of platelet HDL3 binding sites. However, a new regulatory mechanism that involves PKC-dependent downregulation of the number of binding sites may be an important pathway to regulate the thrombogenicity of lipoproteins and their effects on platelet reactivity.
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PMID:Mechanisms for regulating platelet high density lipoprotein type3 binding sites: evidence that binding sites are downregulated by a protein kinase C-dependent mechanism. 1021 79

Previously we demonstrated that the histamine H2 receptor can activate both the adenylate cyclase and phosphoinositide/protein kinase (PKC) signaling pathways. Although dual coupling occurs via separate GTP-dependent mechanisms the structural components of the H2 receptor directing differential signaling have not been established. We explored this question by attempting to confer to the beta2-adrenergic receptor (betaAR), which is known to stimulate cAMP formation, the ability to activate PKC through the construction of beta2/H2 chimeric receptors. Intracytoplasmic domains of the human beta2 adrenergic receptor were substituted with the corresponding sequences of the human H2 receptor and stably expressed in HEK-293 cells. Binding of [(3)H]-CGP to chimeric wild type beta2 receptors was comparable. Substitution of the second intracellular loop (2i) of the betaAR led to a significant decrease in coupling to adenylate cyclase while leading to a 139.5 +/- 9.4% control increase in epinephrine mediated PKC activation. Introduction of the H2 receptor 3i also led to a decrease in betaAR mediated cAMP generation but provided the latter with the ability to stimulate PKC (182.2 +/- 8% of control). Concomitant expression of both 2i and 3i led to a substantial increase in epinephrine mediated PKC activation (201.8 +/- 10.5% of control). Addition of the carboxyl terminal tail did not facilitate stimulation of PKC. In summary, the third intracellular loop of the H2 receptor plays an essential role in activating PKC with maximal efficiency conferred by the second intracellular domain.
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PMID:Histamine H2 receptor mediated dual signaling: mapping of structural requirements using beta2 adrenergic chimeric receptors. 1102 10

To investigate the molecular mechanism(s) of action of catecholamines on the expression of the angiotensinogen (ANG) gene in kidney proximal tubular cells, we used opossum kidney (OK) cells with a fusion gene containing the 5'-flanking regulatory sequence of the rat ANG gene fused with a human growth hormone (hGH) gene as a reporter, pOGH (rANG N-1498/+18), permanently integrated into their genomes. The level of expression of the ANG-GH fusion gene was quantified by the amount of immunoreactive-hGH (IR-hGH) secreted into the medium. The addition of norepinephrine (NE), isoproterenol (a beta1/beta2-adrenergic receptor (AR) agonist) and iodoclonidine (an alpha2-AR agonist) stimulated the expression of the ANG-GH fusion gene in a dose-dependent manner, whereas the addition of epinephrine and phenylephrine (alpha1-AR agonist) had no effect. The stimulatory effect of NE was blocked by the presence of propranolol (beta-AR blocker), atenolol (beta1-AR blocker), yohimbine (alpha2-AR blocker), Rp-cAMP (an inhibitor of cAMP-dependent protein kinase AI & AII) and staurosporine (an inhibitor of protein kinase C), but was not blocked by ICI 118, 551 (beta2-AR blocker) and prazosin (alpha1-AR blocker). The addition of a combination of isoproterenol and iodoclonidine or a combination of 8-Bromo-cAMP (8-Br-cAMP) and phorbol 12-myristate (PMA) synergistically stimulated the expression of the ANG-GH fusion gene as compared to the addition of isoproterenol, iodoclonidine, 8-Br-cAMP or PMA alone. Furthermore, the addition of NE, 8-Br-cAMP or PMA stimulated the expression of pOGH (rANG N-806/-779/-53/+18), a fusion gene containing the putative cAMP responsive element (CRE, ANG N-806/-779) upstream of the ANG promoter (ANG N-53/+18) in OK cells, but had no effect on the expression of fusion genes containing the mutant of the CRE. Gel mobility shift assays revealed that the ANG-CRE binds with the DNA-binding domain (bZIP254-327) of the cAMP-responsive binding protein (CREB). The binding of the labeled ANG-CRE to CREB (bZIP254-327) was displaced by unlabeled ANG-CRE and the CRE of the somatostatin gene but not by the mutants of the ANG-CRE. Finally, NE stimulated the phosphorylation of CREB in OK cells. These studies demonstrate that the molecular mechanism(s) of NE action on the expression of the ANG gene in OK cells may be mediated via both the PKA and PKC signalling pathways and via the phosphorylation of CREB. The phosphorylated CREB then interacts with the CRE in the 5'-flanking region of the ANG gene and subsequently stimulates the gene expression.
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PMID:Catecholamines and angiotensinogen gene expression in kidney proximal tubular cells. 1110 38

We present evidence of a link between low-density lipoprotein (LDL) receptor binding and activation of a platelet G-coupled protein. LDL stimulation induced cytosolic [Ca2+]i mobilization, increase in inositol 1,4,5-triphosphate (IP3) formation and a rapid cytosol-to-membrane translocation of protein kinase C (PKC) enzymatic activity. Pertussis toxin inhibited all the stimulatory effects, whereas cholera toxin had no effect. Using ligand-binding assays, we demonstrated that exposing platelet LDL receptors to high concentrations of LDL (1.5 g/l) caused a rapid down-regulation and desensitization, as shown by the reduction in the Bmax, intracellular [Ca2+]i mobilization and IP3 formation to 65, 73 and 63%, respectively. The inhibitory effects were reversible and dose and time dependent. Furthermore, VLDL (0.2 g/l) and IDL (0.07 g/l) induced similar desensitization effects. However, HDL3 (up to 1.5 g/l), chylomicrons (up to 0.5 g/l) and cyclohexandione-modified LDL (which does not bind to platelets) had no significant effects. Protein kinase C inhibitors (150 nmol/l staurosporine, 100 micromol/l H-7, and 10 nmol/l bisindolylmaleimide) inhibited desensitization to 71%, on average. Sequestration blocking agents (0.30 g/l, concanavalin A) had no significant effect if phosphorylation was operative. However, there was a complete blockade with the concurrent inhibition of both pathways. In contrast, cAMP-dependent protein kinase inhibitors (PKI, 1 micromol/l) or beta2-adrenergic receptor kinase inhibitors (100 nmol/l, heparin), had no effect. Overall results indicate that LDL binds to a pertussis sensitive G-protein coupled receptor and that high levels of lipoproteins down-regulate the number of receptors and desensitize its mediated response by a mechanism that involves PKC-phosphorylation and sequestration of binding sites. This new regulatory mechanism may have implications for the thrombogenicity in hyperlipidemia and for effects of lipid lowering therapy.
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PMID:Low-density lipoprotein (LDL) binds to a G-protein coupled receptor in human platelets. Evidence that the proaggregatory effect induced by LDL is modulated by down-regulation of binding sites and desensitization of its mediated signaling. 1122 31

G protein-coupled kinase 2 (GRK2) has a key role in regulating signaling activities of a variety of G protein-coupled receptors (GPCRs). Several recent studies have directly implicated GRK2 phosphorylation in desensitization of GPCRs. In addition, binding by G(betagamma) or phosphorylation by PKC or c-Src [corrected] has been shown to activate or enhance GRK2 activity, respectively. Conversely, the calcium binding protein calmodulin or the serine/threonine kinase ERK has been implicated in inhibiting GRK2 activity. However, with the exception of a recent report indicating that activation of beta2-adrenergic receptor results in the ubiquitination and rapid degradation of GRK2, very little is known about cellular mechanisms that alter the protein levels of GRK2 [corrected]. Here, we report a novel serendipitous observation regarding alteration of GRK2 [corrected] protein levels. Exposure of CHO cells stably expressing the m1 muscarinic acetylcholine receptor (mAChR) to transient hypoxia caused near ablation of the GRK2 protein. In contrast, GRK2 protein levels remained unchanged in the parental CHO cells or in CHO cells stably expressing the m2 mAChR when exposed to transient hypoxia. The present study reports a novel observation that is unveiled by transient hypoxia in which GRK2 protein levels are altered by cellular mechanisms involving the m1 mAChR.
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PMID:Transient hypoxia differentially decreases GRK2 protein levels in CHO cells stably expressing the m1 mAChR. 1152 75

SSeCKS and its human orthologue, Gravin, are large scaffolding proteins that are thought to facilitate mitogenic control by anchoring key signal mediators such as protein kinase (PK) C, PKA, the plasma membrane associated isoform of alpha-1,4-galactosyltransferase (GalTase), beta2-adrenergic receptor, and cyclins. SSeCKS is also a major PKC substrate and phosphatidylserine-dependent PKC binding protein whose phosphorylation sites shares homology with a site in the MARCKS protein that encodes phosphorylation-sensitive calmodulin (CaM) binding activity. In the present study, we mapped the in vitro binding sites for CaM and cyclins on SSeCKS. Four CaM binding sites were identified by binding assays that conform to the so-called 1-5-10 motif. Notably, CaM binding was antagonized by prephosphorylation of SSeCKS by PKC. We also identified two major cyclin binding (CY) sites that overlap a major PKC phosphorylation site in SSeCKS (Ser(507/515)), and showed that cyclin D binding is attenuated if SSeCKS is prephosphorylated by PKC. These data suggest that the scaffolding activities of SSeCKS are modulated by mitogenically stimulated kinases such as PKC.
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PMID:Calmodulin and cyclin D anchoring sites on the Src-suppressed C kinase substrate, SSeCKS. 1182 Jul 72

G protein-coupled receptor kinases are well characterized for their ability to phosphorylate and desensitize G protein-coupled receptors (GPCRs). In addition to phosphorylating the beta2-adrenergic receptor (beta2AR) and other receptors, G protein-coupled receptor kinase 2 (GRK2) can also phosphorylate tubulin, a nonreceptor substrate. To identify novel nonreceptor substrates of GRK2, we used two-dimensional gel electrophoresis to find cellular proteins that were phosphorylated upon agonist-stimulation of the beta2AR in a GRK2-dependent manner. The ribosomal protein P2 was identified as an endogenous HEK-293 cell protein whose phosphorylation was increased following agonist stimulation of the beta2AR under conditions where tyrosine kinases, PKC and PKA, were inhibited. P2 along with its other family members, P0 and P1, constitutes a part of the elongation factor-binding site connected to the GTPase center in the 60S ribosomal subunit. Phosphorylation of P2 is known to regulate protein synthesis in vitro. Further, P2 and P1 are shown to be good in vitro substrates for GRK2 with K(M) values approximating 1 microM. The phosphorylation sites in GRK2-phosphorylated P2 are identified (S102 and S105) and are identical to the sites known to regulate P2 activity. When the 60S subunit deprived of endogenous P1 and P2 is reconstituted with GRK2-phosphorylated P2 and unphosphorylated P1, translational activity is greatly enhanced. These findings suggest a previously unrecognized relationship between GPCR activation and the translational control of gene expression mediated by GRK2 activation and P2 phosphorylation and represent a potential novel signaling pathway responsible for P2 phosphorylation in mammals.
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PMID:Beta 2-adrenergic receptor stimulated, G protein-coupled receptor kinase 2 mediated, phosphorylation of ribosomal protein P2. 1237 28


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