Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.12 (PKG)
2,515 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In these studies we demonstrate that insulin stimulates both tyrosine and serine phosphorylation of the insulin receptor after its partial purification on wheat germ-agarose, and after affinity purification on insulin-agarose. Analysis of the serine phosphate incorporated into partially purified or highly purified insulin receptor suggests that an insulin-sensitive serine kinase (IRSK) copurifies with the insulin receptor. Following trypsin digestion, reversed-phase high pressure liquid chromatography (HPLC) analysis of the phosphorylated, affinity-purified insulin receptor preparation reveals phosphopeptide profiles similar to those of trypsin-digested receptors immunoprecipitated from 32P-labeled fibroblasts overexpressing the human insulin receptor. The major insulin-stimulated HPLC phosphopeptide peak from insulin receptors labeled in intact cells contains a hydrophilic phosphoserine-containing peptide which rapidly elutes from a C18 column. HPLC and two-dimensional separation indicate that the same phosphopeptide is obtained when affinity-purified insulin receptors are phosphorylated by IRSK. The serine containing tryptic peptide within the cytoplasmic domain of the human insulin receptor predicted to elute most rapidly upon HPLC had the sequence SSHCQR corresponding to residues 1293-1298. A synthetic peptide containing this sequence is phosphorylated by the insulin receptor/IRSK preparation. After alkylation and trypsin digestion, the synthetic phosphopeptide comigrates with the alkylated, tryptic phosphopeptide derived from insulin receptor phosphorylated in vitro by IRSK. We propose that serine 1293 or 1294 of the human insulin receptor is a major site(s) phosphorylated on the insulin receptor in intact cells and is phosphorylated by IRSK. Furthermore, insulin added directly to affinity-purified insulin receptor/IRSK preparations stimulates the phosphorylation of synthetic peptides corresponding to this receptor phosphorylation site and another containing threonine 1336. Kemptide phosphorylation is not stimulated by insulin under these conditions. No phosphorylation of peptide substrates for Ca2+/calmodulin-dependent protein kinase, protein kinase C, casein kinase II, or cGMP-dependent protein kinase by IRSK is detected. These data indicate that IRSK exhibits specificity for the insulin receptor and may be activated by the insulin receptor tyrosine kinase in an insulin-dependent manner.
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PMID:Insulin-sensitive phosphorylation of serine 1293/1294 on the human insulin receptor by a tightly associated serine kinase. 213 51

The cytosolic fraction of insulin-treated adipocytes exhibits a 2-fold increase in protein kinase activity when Kemptide is used as a substrate. The detection of insulin-stimulated kinase activity is critically dependent on the presence of phosphatase inhibitors such as fluoride and vanadate in the cell homogenization buffer. The cytosolic protein kinase activity exhibits high sensitivity (ED50 = 2 X 10(-10) M) and a rapid response (maximal after 2 min) to insulin. Kinetic analyses of the cytosolic kinase indicate that insulin increases the Vmax of Kemptide phosphorylation and ATP utilization without affecting the affinities of this enzyme toward the substrate or nucleotide. Upon chromatography on anion-exchange and gel filtration columns, the insulin-stimulated cytosolic kinase activity is resolved from the cAMP-dependent protein kinase and migrates as a single peak with an apparent Mr = 50,000-60,000. The partially purified kinase preferentially utilizes histones, Kemptide, multifunctional calmodulin-dependent protein kinase substrate peptide, ATP citrate-lyase, and acetyl-coenzyme A carboxylase as substrates but does not catalyze phosphorylation of ribosomal protein S6, casein, phosvitin, phosphorylase b, glycogen synthase, inhibitor II, and substrate peptides for casein kinase II, protein kinase C, and cGMP-dependent protein kinase. Phosphoamino acid analyses of the 32P-labeled substrates reveal that the insulin-stimulated cytosolic kinase is primarily serine-specific. The insulin-activated cytosolic kinase prefers Mn2+ to Mg2+ and is independent of Ca2+. Unlike ribosomal protein S6 kinase and protease-activated kinase II, the insulin-sensitive cytosolic kinase is fluoride-insensitive. Taken together, these results indicate that a novel cytosolic protein kinase activity is activated by insulin.
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PMID:Insulin stimulates a novel Mn2+-dependent cytosolic serine kinase in rat adipocytes. 296 Jun 79

cGMP-dependent protein kinase (cGMP kinase) has been implicated in the regulation of the cytosolic calcium level ([Ca2+]i). In Chinese hamster ovary (CHO) cells stably transfected with the cGMP kinase I alpha (CHO-cGK cells), cGMP kinase suppressed the thrombin-induced increase in inositol 1,4,5-trisphosphate and [Ca2+]i (Ruth, P., Wang, G.-X., Boekhoff, I., May, B., Pfeifer, A., Penner, R., Korth, M., Breer, H., and Hofmann, F. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2623-2627). Cholecystokinin activated intracellular calcium release via a pertussis toxin (PTX)-insensitive pathway in CHO-cGK cells. cGMP kinase did not attenuate the CCK-stimulated [Ca2+]i. In contrast, cGMP kinase suppressed calcium influx stimulated by insulin-like growth factors 1 and 2 (IGF-1 and IGF-2) via PTX-sensitive pathways. The effects of PTX and cGMP kinase on [Ca2+]i were not additive. 8-Bromo-cGMP had no effect on [Ca2+]i stimulated by IGF-1 or IGF-2 in wild type CHO cells. These results suggested that cGMP kinase inhibited the different signaling pathways by the phosphorylation of a PTX-sensitive G protein. cGMP kinase phosphorylated the alpha subunits of Gi1, Gi2, and Gi3 in vitro. Phosphorylation stoichiometry was 0.4 mol of phosphate/mol of G alpha i1 after reconstitution of heterotrimeric Gi1 in phospholipid vesicles. The alpha subunit of Gi was also phosphorylated in vivo. These results show that cGMP kinase blocks transduction of distinct hormone pathways that signal via PTX-sensitive Gi proteins.
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PMID:Cyclic GMP-dependent protein kinase blocks pertussis toxin-sensitive hormone receptor signaling pathways in Chinese hamster ovary cells. 772 18

Intracellular production of nitric oxide (NO) is thought to mediate the pancreatic B-cell-directed cytotoxicity of cytokines in insulin-dependent diabetes mellitus, and recent evidence has indicated that this may involve induction of apoptosis. A primary effect of NO is to activate soluble guanylyl cyclase leading to increased cGMP levels and this effect has been demonstrated in pancreatic B-cells, although no intracellular function has been defined for islet cGMP. Here we demonstrate that the NO donor, GSNO, induces apoptosis in the pancreatic B-cell line HIT-T15 in a dose- and time-dependent manner. This response was significantly attenuated by micromolar concentrations of a specific inhibitor of soluble guanylyl cyclase, ODQ, and both 8-bromo cGMP (100 microM) and dibutyryl cGMP (300 microM) were able to fully relieve this inhibition. In addition, incubation of HIT-T15 cells with each cGMP analogue directly promoted cell death in the absence of ODQ. KT5823, a potent and highly selective inhibitor of cGMP-dependent protein kinase (PKG), abolished the induction of cell death in HIT cells in response to either GSNO or cGMP analogues. This effect was dose-dependent over the concentration range of 10-250 nM. Overall, these data provide evidence that the activation of apoptosis in HIT-T15 cells by NO donors is secondary to a rise in cGMP and suggest that the pathway controlling cell death involves activation of PKG.
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PMID:Evidence for the involvement of cGMP and protein kinase G in nitric oxide-induced apoptosis in the pancreatic B-cell line, HIT-T15. 900 15

Biotin regulation of asialoglycoprotein receptor expression and insulin receptor activity has been established in two human hepatoblastoma cell lines, Hep G2 and HuH-7. Second messenger cGMP mimics the effect of biotin on asialoglycoprotein receptor expression at the translational level. Metabolic labeling and subsequent immunoprecipitation indicate that the loss of insulin receptor activity during biotin deprivation was due to suppression of receptor synthesis. Evidence for posttranscriptional regulation of insulin receptor synthesis was provided by rapid biotin induction of receptor synthesis without an increase in gene transcript number. Addition of a cGMP-dependent protein kinase (cGK) inhibitor prevented biotin induction of the insulin and asialoglycoprotein receptors, suggesting that protein phosphorylation propagates the cGMP signal transduction cascade. Coatomer protein COPI was recently identified as the trans-acting factor that regulates in vitro translation of the asialoglycoprotein receptor. Biotin repletion of the culture medium resulted in the hyperphosphorylation of alpha-COP, which was prevented by simultaneous addition of the cGK inhibitor. These findings suggest that the end point of this cGMP signal cascade is modulated by cGK and that a phosphorylation reaction governs the expression of both receptor proteins.
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PMID:Regulation of the insulin and asialoglycoprotein receptors via cGMP-dependent protein kinase. 1107 21

Glucose homeostasis in blood is mainly maintained by insulin released from beta-cells and glucagon released from alpha-cells, both integrated within the pancreatic islet of Langerhans. The secretory processes in both types of cells are triggered by a rise in intracellular calcium concentration ([Ca2+](i)). In this study, rapid effects of the natural hormone E2 on [Ca2+](i) were studied in both types of cells within intact islets using laser scanning confocal microscopy. alpha- And beta-cells showed opposite [Ca2+](i) responses when stimulated with physiological concentrations of 17beta-E2. Although the estrogen produced an increase in the frequency of glucose-induced [Ca2+](i) oscillations in insulin-releasing beta-cells, it prevented the low glucose-induced [Ca2+](i) oscillations in glucagon-releasing alpha-cells. The effects of 17beta-E2 on alpha-cells were mimicked by the cGMP permeable analog 8bromo-cGMP and blocked by the cGMP-dependent protein kinase (PKG) inhibitor KT5823. Evidence indicated that these were membrane actions mediated by a nonclassical ER. Both effects were rapid in onset and were reproduced by 17beta-E2 linked to horseradish peroxidase, a cell-impermeable molecule. Furthermore, these actions were not blocked by the specific ER blocker ICI 182,780. Competition studies performed with 17beta-E2 linked to horseradish peroxidase binding in alpha-cells supported the idea that the membrane receptor involved is neither ERalpha nor ERbeta. Additionally, the binding site was shared by the neurotransmitters epinephrine, norepinephrine, and dopamine and had the same pharmacological profile as the receptor previously described for beta-cells. Therefore, rapid estrogen actions in islet cells are initiated by a nonclassical estrogen membrane receptor.
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PMID:A nonclassical estrogen membrane receptor triggers rapid differential actions in the endocrine pancreas. 1187 8

In this study, we examined the role of insulin in the control of vascular smooth muscle cell (VSMC) migration in the normal vasculature. Platelet-derived growth factor (PDGF) increased VSMC migration, which was inhibited by pretreatment with insulin in a dose-dependent manner. Insulin also caused a 60% decrease in PDGF-stimulated mitogen-activated protein kinase (MAPK) phosphorylation and activation. Insulin inhibition of MAPK was accompanied by a rapid induction of MAPK phosphatase (MKP-1), which inactivates MAPKs by dephosphorylation. Pretreatment with inhibitors of the nitric oxide (NO)/cGMP pathway, blocked insulin-induced MKP-1 expression and restored PDGF-stimulated MAPK activation and migration. In contrast, adenoviral infection of VSMCs with MKP-1 or cGMP-dependent protein kinase Ialpha (cGK Ialpha), the downstream effector of cGMP signaling, blocked the activation of MAPK and prevented PDGF-directed VSMC migration. Expression of antisense MKP-1 RNA prevented insulin's inhibitory effect and restored PDGF-directed VSMC migration and MAPK phosphorylation. We conclude that insulin inhibition of VSMC migration may be mediated in part by NO/cGMP/cGK Ialpha induction of MKP-1 and consequent inactivation of MAPKs.
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PMID:Insulin inhibits PDGF-directed VSMC migration via NO/ cGMP increase of MKP-1 and its inactivation of MAPKs. 1217 27

In isolated rat pancreatic beta-cells, the nitric oxide (NO) donor NOC-7 at 1 microM reduced the amplitude of the oscillations of cytosolic Ca(2+) concentration ([Ca(2+)](c)) induced by 11.1 mM glucose, and at 10 microM terminated them. In the presence of N(G)-nitro-l-arginine (l-NNA), however, NOC-7 at 0.5 and 1 microM increased the amplitude of the [Ca(2+)](c) oscillations, although the NO donor at 10 microM still suppressed them. Aqueous NO solution also had a dual effect on the [Ca(2+)](c) oscillations. The soluble guanylate cyclase inhibitor LY-83583 and the cGMP-dependent protein kinase inhibitor KT5823 inhibited the stimulatory effect of NO, and 8-bromo-cGMP increased the amplitude of the [Ca(2+)](c) oscillations. Patch-clamp analyses in the perforated configuration showed that 8-bromo-cGMP inhibited whole cell ATP-sensitive K(+) currents in the isolated rat pancreatic beta-cells, suggesting that the inhibition by cGMP of ATP-sensitive K(+) channels is, at least in part, responsible for the stimulatory effect of NO on the [Ca(2+)](c) oscillations. In the presence of l-NNA, the glucose-induced insulin secretion from isolated islets was facilitated by 0.5 microM NOC-7, whereas it was suppressed by 10 microM NOC-7. These results suggest that NO facilitates glucose-induced [Ca(2+)](c) oscillations of beta-cells and insulin secretion at low concentrations, which effects are mediated by cGMP, whereas NO inhibits them in a cGMP-independent manner at high concentrations.
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PMID:Dual effect of nitric oxide on cytosolic Ca2+ concentration and insulin secretion in rat pancreatic beta-cells. 1252 41

Despite interest in understanding glucagon-like peptide-1 (GLP-1) production, the factors important for GLP-1 biosynthesis remain poorly understood. We examined control of human proglucagon gene expression in NCI-H716 cells, a cell line that secretes GLP-1 in a regulated manner. Insulin, phorbol myristate acetate, or forskolin, known regulators of rodent proglucagon gene expression, had no effect, whereas sodium butyrate decreased levels of NCI-H716 proglucagon mRNA transcripts. The inhibitory effect of sodium butyrate was mimicked by trichostatin A but was not detected with sodium acetate or isobutyrate. The actions of butyrate were not diminished by the ERK1/2 inhibitor PD98059, p38 inhibitor SB203580, or soluble guanylate cyclase inhibitor LY83583 or following treatment of cells with KT5823, a selective inhibitor of cGMP-dependent protein kinase. NCI-H716 cells expressed multiple proglucagon gene transcription factors including isl-1, pax-6, pax-2, cdx-2/3, pax-4, hepatocyte nuclear factor (HNF)-3 alpha, HNF-3beta, HNF-3 gamma, and Nkx2.2. Nevertheless, the butyrate-dependent inhibition of proglucagon gene expression was not associated with coordinate changes in transcription factor expression and both the human and rat transfected proglucagon promoters were transcriptionally inactive in NCI-H716 cells. Hence, NCI-H716 cells may not be a physiologically optimal model for studies of human enteroendocrine proglucagon gene transcription.
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PMID:Aberrant regulation of human intestinal proglucagon gene expression in the NCI-H716 cell line. 1269 11

Insulin-like growth factor (IGF-1) is critical for normal development and maintenance of cartilage, however arthritic cartilage responds poorly to IGF-1; part of this insensitivity is mediated by nitric oxide (NO). These studies test if cGMP is responsible for NO dependent insensitivity to IGF-1 in chondrocytes in situ in organ culture and in monolayer culture. Lapine cartilage and chondrocytes in monolayer culture and cartilage from osteoarthritic human knees were used. Tissues were exposed to NO from iNOS induced by IL-1, and proteoglycan synthesis in response to IGF-1 was evaluated in the presence and absence of cGMP dependent protein kinase (PKG) inhibitors. PKG activators inhibited IGF-1 responses in cartilage but not chondrocytes in monolayer. IL-1 stimulated cGMP synthesis in both monolayer and organ cultures. However, PKG inhibitors in cartilage slices but not in monolayer cultures restored response to IGF-1. PKG activity was detected in both fresh and monolayer chondrocytes, confirming this part of the cGMP signal cascade is intact in both of the preparations evaluated. Arthritic cartilage response to IGF-1 was restored by both N(G)-monomethyl-L-arginine inhibition of NO synthesis and PKG inhibitors. The data suggests that cGMP mediated effects are critical to NO actions on chondrocytes in situ in the cartilage matrix and supports a role for cGMP in the pathophysiologic effects of NO in osteoarthritis.
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PMID:Nitric oxide inhibition of IGF-1 stimulated proteoglycan synthesis: role of cGMP. 1291 81


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