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)

We cloned the cDNA for human RGSZ1, the major Gz-selective GTPase-activating protein (GAP) in brain (Wang, J., Tu, Y., Woodson, J., Song, X., and Ross, E. M. (1997) J. Biol. Chem. 272, 5732-5740) and a member of the RGS family of G protein GAPs. Its sequence is 83% identical to RET-RGS1 (except its N-terminal extension) and 56% identical to GAIP. Purified, recombinant RGSZ1, RET-RGS1, and GAIP each accelerated the hydrolysis of Galphaz-GTP over 400-fold with Km values of approximately 2 nM. RGSZ1 was 100-fold selective for Galphaz over Galphai, unusually specific among RGS proteins. Other enzymological properties of RGSZ1, brain Gz GAP, and RET-RGS1 were identical; GAIP differed only in Mg2+ dependence and in its slightly lower selectivity for Galphaz. RGSZ1, RET-RGS1, and GAIP thus define a subfamily of Gz GAPs within the RGS proteins. RGSZ1 has no obvious membrane-spanning region but is tightly membrane-bound in brain. Its regulatory activity in membranes depends on stable bilayer association. When co-reconstituted into phospholipid vesicles with Gz and m2 muscarinic receptors, RGSZ1 increased agonist-stimulated GTPase >15-fold with EC50 <12 nM, but RGSZ1 added to the vesicle suspension was <0.1% as active. RGSZ1, RET-RGS1, and GAIP share a cysteine string sequence, perhaps targeting them to secretory vesicles and allowing them to participate in the proposed control of secretion by Gz. Phosphorylation of Galphaz by protein kinase C inhibited the GAP activity of RGSZ1 and other RGS proteins, providing a mechanism for potentiation of Gz signaling by protein kinase C.
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PMID:RGSZ1, a Gz-selective RGS protein in brain. Structure, membrane association, regulation by Galphaz phosphorylation, and relationship to a Gz gtpase-activating protein subfamily. 974 80

The effects of angiotensin II (Ang II) are mediated primarily by Ang II type 1 receptors, which in turn are coupled to heterotrimeric G proteins. After receptor activation, the G(alpha) and G(betagamma) subunits dissociate, contributing to the signaling cascades involving protein kinase C (PKC) activation. Regulators of G protein signaling (RGS proteins) comprise a class of proteins that have been shown to negatively regulate the G(alpha) subunit. We examined which RGS sequences were expressed in vascular smooth muscle cells and which of these were regulated by Ang II. Reverse transcription-polymerase chain reaction showed that of 16 RGS sequences screened, six RGS transcripts (RGS2, 3, 10, 11, and 12 and GAIP) were present. Northern blot analysis demonstrated that RGS3, 10, and 12 and GAIP were not regulated by Ang II at the mRNA level. In contrast, RGS2 mRNA was rapidly and dose dependently increased (395 +/- 24% peak, 45 min) by Ang II but returned to baseline level by 6 to 8 h. Phorbol-12-myristate-13-acetate, a PKC activator, robustly increased RGS2. This signal was attenuated by the PKC inhibitor GF 109203X (50 +/- 4%) and by phorbol-12, 13-dibutyrate-mediated down-regulation of PKC (48 +/- 13%). Tyrosine kinase inhibition and calcium deprivation did not affect the up-regulation of RGS2 mRNA after Ang II stimulation. Actinomycin D treatment inhibited both Ang II- and phorbol-12-myristate-13-acetate-stimulated RGS2 up-regulation, suggesting activation of transcription by these agonists. The stability of RGS2 mRNA did not appear to be affected by Ang II. Thus, RGS2 is a likely candidate for negative regulation of the G proteins coupled to the Ang II type 1 receptor in vascular smooth muscle cells. Regulation of this protein may be of critical importance in modulating the role of Ang II in vascular disease.
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PMID:Specific regulation of RGS2 messenger RNA by angiotensin II in cultured vascular smooth muscle cells. 1069 85

G protein regulated inward rectifying potassium channels (GIRKs) are activated by G protein coupled receptors (GPCRs) via the G protein betagamma subunits. However, little is known about the effects of different GPCRs on the deactivation kinetics of transmitter-mediated GIRK currents. In the present study we investigated the influence of different GPCRs in the presence and absence of RGS proteins on the deactivation kinetics of GIRK channels by coexpressing the recombinant protein subunits in Xenopus oocytes. The stimulation of both G(i/o)- and G(q)-coupled pathways accelerated GIRK deactivation. GIRK currents deactivated faster upon stimulation of G(i/o)- and G(q)-coupled pathways by P(2)Y(2) receptors (P(2)Y(2)Rs) than upon activation of the G(i/o)-coupled pathway alone via muscarinic acetylcholine receptor M2 (M(2) mAChRs). This acceleration was found to be dependent on phospholipase C (PLC) and protein kinase C (PKC) activities and intracellular calcium. With the assumption that RGS2 has a higher affinity for Galpha(q) than Galpha(i/o), we demonstrated that the deactivation kinetics of GIRK channels can be differentially regulated by the relative amount of RGS proteins. These data indicate that transmitter-mediated deactivation of GIRK currents is modulated by crosstalk between G(i/o)- and G(q)-coupled pathways.
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PMID:Regulation of GIRK channel deactivation by Galpha(q) and Galpha(i/o) pathways. 1097 20

RGS proteins (regulators of G protein signaling) attenuate heterotrimeric G protein signaling by functioning as both GTPase-activating proteins (GAPs) and inhibitors of G protein/effector interaction. RGS2 has been shown to regulate Galpha(q)-mediated inositol lipid signaling. Although purified RGS2 blocks PLC-beta activation by the nonhydrolyzable GTP analog guanosine 5'-O-thiophosphate (GTPgammaS), its capacity to regulate inositol lipid signaling under conditions where GTPase-promoted hydrolysis of GTP is operative has not been fully explored. Utilizing the turkey erythrocyte membrane model of inositol lipid signaling, we investigated regulation by RGS2 of both GTP and GTPgammaS-stimulated Galpha(11) signaling. Different inhibitory potencies of RGS2 were observed under conditions assessing its activity as a GAP versus as an effector antagonist; i.e. RGS2 was a 10-20-fold more potent inhibitor of aluminum fluoride and GTP-stimulated PLC-betat activity than of GTPgammaS-promoted PLC-betat activity. We also examined whether RGS2 was regulated by downstream components of the inositol lipid signaling pathway. RGS2 was phosphorylated by PKC in vitro to a stoichiometry of approximately unity by both a mixture of PKC isozymes and individual calcium and phospholipid-dependent PKC isoforms. Moreover, RGS2 was phosphorylated in intact COS7 cells in response to PKC activation by 4beta-phorbol 12beta-myristate 13alpha-acetate and, to a lesser extent, by the P2Y(2) receptor agonist UTP. In vitro phosphorylation of RGS2 by PKC decreased its capacity to attenuate both GTP and GTPgammaS-stimulated PLC-betat activation, with the extent of attenuation correlating with the level of RGS2 phosphorylation. A phosphorylation-dependent inhibition of RGS2 GAP activity was also observed in proteoliposomes reconstituted with purified P2Y(1) receptor and Galpha(q)betagamma. These results identify for the first time a phosphorylation-induced change in the activity of an RGS protein and suggest a mechanism for potentiation of inositol lipid signaling by PKC.
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PMID:Protein kinase C phosphorylates RGS2 and modulates its capacity for negative regulation of Galpha 11 signaling. 1106 46

The RGS (regulator of G-protein signalling) proteins are GTPase-activating proteins for activated Galpha subunits. We investigated the effects of protein kinase C (PKC) on RGS proteins in various T cell lines by treating them with PMA. mRNA levels of both RGS16 and tumour necrosis factor alpha (TNFalpha) were found to be up-regulated in CEM leukaemia cells in a PKC-dependent manner. Mezerein, a non-phorbol-ester activator of PKC, also elevated RGS16 and TNFalpha mRNA levels, while the specific PKC inhibitor Go6983 abrogated their expression. In view of the slower kinetics of PMA-induced RGS16 expression and the tight correlation between TNFalpha and RGS16 mRNA induction among the cell lines studied, we suggest that activation of PKC up-regulates RGS16 via TNFalpha. Indeed, addition of recombinant TNFalpha to CEM cells rapidly stimulated RGS16 mRNA expression independently of PKC. Furthermore, mobilization of calcium by A23187 and thapsigargin blocked the TNFalpha-mediated induction of RGS16, which was reversed by EGTA and by the immunosuppressants FK506 and cyclosporin A, suggesting that the calcineurin/NF-AT (nuclear factor of activated T cells) pathway may repress the up-regulation process. Our results demonstrate for the first time that activation of PKC induces RGS16 expression via TNFalpha in a calcium-sensitive manner, thereby implicating RGS16 in the regulation of T cell responses to inflammation.
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PMID:Specific induction of RGS16 (regulator of G-protein signalling 16) mRNA by protein kinase C in CEM leukaemia cells is mediated via tumour necrosis factor alpha in a calcium-sensitive manner. 1110 82

The intrinsic GTPase activity of Galpha q is low, and RGS proteins which activate GTPase are expressed in the heart; however, their functional relevance in vivo is unknown. Transgenic mice with cardiac-specific overexpression of Galpha q in myocardium exhibit cardiac hypertrophy, enhanced PKC xi membrane translocation, embryonic gene expression, and depressed cardiac contractility. We recently reported that transgenic mice with cardiac-specific expression of RGS4, a Galpha q and Galpha i GTPase activator, exhibit decreased left ventricular hypertrophy and ANF induction in response to pressure overload. To test the hypothesis that RGS4 can act as a Galpha q-specific GTPase activating protein (GAP) in the in vivo heart, dual transgenic Galpha q-40xRGS4 mice were generated to determine if RGS4 co-expression would ameliorate the Galpha q-40 phenotype. At age 4 weeks, percent fractional shortening was normalized in dual transgenic mice as was left ventricular internal dimension and posterior and septal wall thicknesses. PKC xi membrane translocation and ANF and alpha -skeletal actin mRNA levels were also normalized. Compound transgenic mice eventually developed depressed cardiac contractility that was evident by 9 weeks of age. These studies establish for the first time a role for RGS4 as a GAP for Galpha q in the in vivo heart, and demonstrate that its regulated expression can have pathophysiologic consequences.
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PMID:RGS4 reduces contractile dysfunction and hypertrophic gene induction in Galpha q overexpressing mice. 1116 25

Heterotrimeric G protein alpha,beta, and gamma subunits are subject to several kinds of co- and post-translational covalent modifications. Among those relevant to G protein-coupled receptor signaling in normal cell function are lipid modifications and phosphorylation. N-myristoylation is a co-translational modification occurring for members of the G(i) family of Galpha subunits, while palmitoylation is a post-translational modification that occurs for these and most other Galpha subunits. One or both modifications are required for plasma membrane targeting and contribute to regulating strength of interaction with the Gbetagamma heterodimer, effectors, and regulators of G protein signaling (RGS proteins). Galpha subunits, including those with transforming activity, are often inactive when unable to be modified with lipids. The reversible nature of palmitoylation is intriguing in this regard, as it lends itself to a regulation integrated with the activation state of the G protein. Several Galpha subunits are substrates for phosphorylation by protein kinase C and at least one is a substrate for phosphorylation by the p21-activated protein kinase. Phosphorylation in both instances inhibits the interactions of these subunits with the Gbetagamma heterodimer and RGS proteins. Several Galpha subunits are also substrates for tyrosine phosphorylation. A Ggamma subunit is phosphorylated by protein kinase C, with the consequence that it interacts more tightly with a Galpha subunit but less well with an effector.
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PMID:Regulation of G proteins by covalent modification. 1131 12

Regulator of G-protein signaling-2 (RGS-2) belongs to a novel family of GTPase-activating proteins that rapidly turn-off G-protein coupled receptor signaling. RGS proteins contain a characteristic RGS domain by which they interact with the alpha-subunit of G-proteins and drive them into their inactive GDP-bound forms. Previously, we have reported that RGS-2 mRNA is rapidly and transiently increased by PTH in rat bone and in osteoblast cultures in vitro. In this study, we further explored the molecular basis for the regulation of RGS-2 by cloning and functionally characterizing the RGS-2 gene promoter. We cloned 2.3- and 2.8-kb fragments of the 5'-flanking regions of the rat and mouse RGS-2 genes, respectively, and generated a stable clone of UMR106 osteoblastic cells containing the rat RGS-2 promoter driving the beta-gal reporter gene (p2.3RGS-2-beta-gal). Treatment of the stable clone with PTH resulted in a maximal 2.2- to 3.6-fold increase in promoter activity at 8 h, reminiscent of the early response observed with endogenous RGS-2 mRNA regulation. Further, PTH (1-38), (1-31), PTHrP (1-34), and forskolin, which elevate cAMP levels, stimulated the promoter, while PTH (3-34) and (7-34), which do not readily stimulate cAMP accumulation, and PMA that directly activates protein kinase C, had no effect on promoter activity. Taken together, these results implicate the involvement of the Galpha(s)-adenylate cyclase-protein kinase A pathway in stimulating RGS-2 expression. Maintenance of a hyperphosphorylated state via the inhibition of type 2A protein phosphatases by okadaic acid, resulted in a strong dose-dependent increase in transcriptional activity of the RGS-2 promoter as well as that of the endogenous RGS-2 gene. Furthermore, overexpression of the osteoblast-specific transcription factor Runx2 also led to a stimulation of RGS-2 promoter activity. Functional analysis using RGS-2 overexpression suggests the potential negative regulatory effects of RGS-2 on PTH- and forskolin-induced cAMP production in osteoblastic cells. In summary, our data suggest that PTH treatment results in a direct transcriptional stimulation of RGS-2 that in turn may play a role in modulating the duration/intensity of PTH receptor signaling.
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PMID:Analysis of regulator of G-protein signaling-2 (RGS-2) expression and function in osteoblastic cells. 1196 23

Parathyroid hormone (PTH) is a promising anabolic agent for the treatment of osteoporosis. However, PTH is also potently catabolic. To help delineate the molecular mediators of PTH's opposing effects on skeletal metabolism, we have examined PTH-induced regulator of G-protein signaling-2 (RGS-2) expression and function in murine osteoblasts. RGS proteins are GTPase-activating proteins (GAPs) that regulate GTP-binding protein-coupled receptor (GPCR) signaling by enhancing the intrinsic GTPase activity of Galpha subunits. We found that 10 nmol/L PTH maximally induced RGS-2 mRNA in murine MC3T3-E1 cells, rat Py1a and ROS-17/2.8 cells, primary mouse osteoblasts (MOB cells), and mouse calvariae organ culture at 1-2 h posttreatment. PTH signaling through its receptor, PTHR1, is coupled to cAMP-protein kinase A (PKA), protein kinase C (PKC), and calcium signaling pathways. We examined the effect of selective signaling agonists and antagonists on RGS-2 expression in MOB cells to determine which pathway(s) mediates PTH-induced RGS-2 expression. Although selective activation of all three pathways led to RGS-2 expression, cAMP-PKA activation with 10 nmol/L PTH and 10 micromol/L forskolin elicited the strongest induction. Similarly, RGS-2 mRNA expression was most strongly inhibited by the PKA inhibitor, H89 (10-30 micromol/L). The phorbol ester, PMA (1 micromol/L), which activates the PKC pathway, and ionomycin (1 micromol/L), which activates the calcium pathway, produced small but detectable elevations in RGS-2 mRNA levels. Overnight treatment with 1 micromol/L PMA to deplete PKC did not affect subsequent RGS-2 induction by PTH, but significantly inhibited PMA-induced RGS-2 expression. Treatment with 1-100 nmol/L PTH(3-34), which does not activate cAMP-PKA signaling, did not induce RGS-2 expression. MOB cells pretreated with 3 microg/mL cycloheximide produced sustained RGS-2 mRNA levels 2 h after 10 nmol/L PTH treatment. Actinomycin D (5 microg/mL) completely blocked 10 nmol/L PTH-induced RGS-2 expression. Finally, we tested the effect of RGS-2 overexpression on PTH- and fluprostenol-induced interleukin (IL)-6 promoter activity in MOB cells. PTH induces IL-6 through PKA activation, whereas fluprostenol induces IL-6 through PKC activation. We found that RGS-2 overexpression significantly inhibited IL-6 promoter activity following fluprostenol treatment, but not following PTH treatment. We conclude that RGS-2 is a PTH-induced primary response gene in murine osteoblasts that is induced mainly through the cAMP-PKA pathway and specifically inhibits Galphaq-coupled receptors.
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PMID:Parathyroid hormone induces RGS-2 expression by a cyclic adenosine 3',5'-monophosphate-mediated pathway in primary neonatal murine osteoblasts. 1199 4

G protein-coupled receptors (GPCRs) are widely expressed hepta-helical receptors with tightly regulated pleiotropic effects. ADP-Ribosylation Factor 6 (ARF6) plays an important role in GPCR trafficking and is the subject of intense research. However, the mechanisms underlying activation and regulation of ARF6 by GPCRs are poorly characterized. Here we report that Galpha(q) signaling leads to the activation of ARF6. Stimulation of the TPbeta receptor triggered ARF6 activation which was completely inhibited by the RGS domain of GRK2 known to specifically bind and sequester Galpha(q). Co-immunoprecipitation studies revealed that ARNO (a guanine nucleotide exchange factor for ARF6) and ARF6 formed complexes preferentially with activated Galpha(q) compared to non-activated Galpha(q). Formation of the Galpha(q) complexes with ARNO and ARF6 was detected early and was optimal after 30 min of receptor stimulation corresponding with the profile of ARF6 activation. Interestingly, binding experiments using purified proteins showed that Galpha(q) interacted directly with ARNO. Galpha(q)-dependent TPbeta receptor-mediated activation of ARF6 resulted in phosphoinositol-4,5-bisphosphate production which was potently inhibited by dominant negative mutants of ARNO and ARF6. Furthermore, our data show that the expression of ARNO and ARF6 promoted, whereas dominant negative mutants of these proteins inhibited the internalization of the TPbeta receptor. This further elucidates our previous data on the PLCbeta- and PKC-independent mechanism involved in Galpha(q)-mediated internalization of the TPbeta receptor. Taken altogether, our results support a novel model where activated Galpha(q) forms molecular complexes with ARNO and ARF6, possibly through a direct interaction with ARNO, leading to ARF6 activation.
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PMID:ARF6 activation by Galpha q signaling: Galpha q forms molecular complexes with ARNO and ARF6. 1665 Sep 66


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