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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In rat pancreatic islets perifused in the presence of 2-cyclohexene-1-one (CHX; 1.0 mM), the secretory response to either D-glucose or 2-ketoisocaproate, but not that evoked by the association of L-leucine and L-glutamine, was severely decreased. This coincided with a decreased stimulation of [45Ca] efflux from prelabelled islets, whereas the inhibitory action of D-glucose or 2-ketoisocaproate upon both [86Rb] and [45Ca] efflux appeared little or not affected. In the presence of D-glucose, the islets exposed to CHX were virtually unresponsive to either forskolin, theophylline or cytochalasin B. A severe decrease in the secretory response to forskolin was also observed in CHX-treated islets exposed to L-leucine and L-glutamine. Except for a somewhat lower sensitivity to NaF, no major change in adenylate cyclase activity or cyclic AMP production was observed in CHX-treated islets. The activity of protein kinase A was decreased in such islets but its responsiveness to cyclic AMP appeared unaltered. Transglutaminase activity was severely decreased in homogenates derived from CHX-treated islets. These findings suggest that CHX, possibly by lowering the GSH content of islet cells, impairs the functional capacity of the effector system for insulin release, in addition to and independently of any effect that it may exert upon nutrient catabolism and cationic fluxes in the islet cells.
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PMID:The coupling of metabolic to secretory events in pancreatic islets: inhibition by 2-cyclohexene-1-one of the secretory response to cyclic AMP and cytochalasin B. 287 68

Studies were undertaken to determine whether factors which affect insulin secretion may exert their effects by altering the activity of an islet-cell plasma membrane Ca2+ extrusion pump. The insulin secretagogue, D-glucose, and a variety of phosphorylated hexoses, glucose 6-P, glucose 1,6-P, fructose 6-P, and fructose 2,6-P, were evaluated for their effect on an islet-cell plasma membrane (Ca2+ + Mg2+)-ATPase and were found to be ineffective in altering enzyme activity. D-Glucose also did not alter the rate of ATP-dependent Ca2+ uptake into plasma membrane vesicles. Similarly, cAMP, the catalytic subunit of cAMP-dependent protein kinase, arachidonic acid, or prostaglandin E2 did not affect either the plasma membrane (Ca2+ + Mg2+)-ATPase or the rate of ATP-dependent Ca2+ uptake into plasma membrane vesicles. Whereas previous studies have suggested that D-glucose and/or cAMP may inhibit ATPase activities in islets, these results indicate that the agents, i.e., D-glucose and cAMP, which stimulate and/or potentiate insulin secretion from the islet cell, do not modify Ca2+ fluxes by directly regulating the islet-cell plasma membrane (Ca2+ + Mg2+)-ATPase. In contrast, the acidic phospholipids, phosphatidic acid and phosphatidylserine, stimulated the enzyme activity in a concentration-dependent manner whereas phosphatidylcholine had only a minimal effect. The diacylglycerol, dilinolein, stimulated the (Ca2+ + Mg2+)-ATPase activity in the presence of phosphatidylserine, but not in the absence of phospholipids. These effects were independent of phospholipid-stimulated protein phosphorylation in the islet-cell plasma membrane under the conditions of the ATPase assay.
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PMID:Effect of insulin secretagogues and potential modulators of secretion on a plasma membrane (Ca2+ + Mg2+)-ATPase activity in islets of Langerhans. 298 94

Histidine-containing protein (HPr) of gram-positive bacteria was found to be phosphorylated at a seryl residue (P-ser-HPr) in an ATP-dependent reaction catalyzed by a protein kinase (J. Deutscher and M. H. Saier, Jr., Proc. Natl. Acad. Sci. U.S.A. 80:6790-6794, 1983). Here we describe the purification and characterization of a soluble enzyme of Streptococcus faecalis which splits the phosphoryl bond in P-ser-HPr. The enzyme has a molecular weight of ca. 7.5 X 10(4), as determined by its migration behavior on a Sephacryl S-200 column. On native polyacrylamide gels the purified enzyme produced only one protein band. On sodium dodecyl sulfate-polyacrylamide gels we found one major protein band of molecular weight 2.9 X 10(4) and two minor protein bands of molecular weights 2.3 X 10(4) and 7 X 10(4). Fructose 1,6-diphosphate, which stimulated the ATP-dependent, protein kinase-catalyzed phosphorylation of HPr, had no effect on the phosphatase activity. Other glycolytic intermediates also had no effect. However, inorganic phosphate, which inhibited the ATP-dependent HPr kinase, stimulated the P-ser-HPr phosphatase. EDTA at a concentration of 0.1 mM completely inhibited the phosphatase. Divalent cations like Mg2+, Mn2+, and Co2+ overcame the inhibition by EDTA. Fe2+, Zn2+, and Cu2+ had no effect, whereas Ca2+ slightly inhibited the phosphatase. ATP was also found to inhibit the phosphatase. Under conditions in which ATP severely inhibited the phosphatase, ADP was found to have no effect on the enzyme activity. Besides P-ser-HPr of S. faecalis, the phosphatase was also able to hydrolyze the phosphoryl bond in P-ser-HPr of Streptococcus lactis, Staphylococcus aureus, Bacillus subtilis, Streptococcus pyogenes, and Lactobacillus casei. Phosphoenolpyruvate-dependent o-nitrophenyl-beta-D-galactopyranoside phosphorylation, catalyzed by the S. aureus phosphoenolpyruvate:lactose phosphotransferase system, was about 150-fold decreased in the presence of P-ser-HPr of S. aureus, as compared with HPr. However, when P-ser-HPr was first incubated with P-ser-HPr phosphatase to allow complete hydrolysis of the phosphoryl bond, it had the same activity as HPr. Besides this cytoplasmic phosphoprotein phosphatase, we detected a membrane-bound phosphatase which also hydrolyzed the phosphoryl bond in P-ser-HPr.
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PMID:Streptococcal phosphoenolpyruvate: sugar phosphotransferase system: purification and characterization of a phosphoprotein phosphatase which hydrolyzes the phosphoryl bond in seryl-phosphorylated histidine-containing protein. 299 39

Postsynaptic densities (PSDs) were prepared by the aqueous two-phase extraction of synaptic membranes in the presence of n-octyl glucoside. Incubation of postsynaptic densities with [gamma-32P]ATP resulted in the incorporation of 32P into a range of proteins. Isolation of glycoproteins from 32P-labelled PSDs by affinity chromatography on concanavalin A-agarose identified the postsynaptic glycoprotein of apparent Mr 180,000 (gp180) as a substrate for endogenous protein kinase(s). When the phosphorylation reaction was performed in the presence of Ca2+ and calmodulin, there was an overall 13-fold increase in the phosphorylation of PSD proteins. The largest effects of calmodulin were associated with two proteins of molecular weights 51,000 and 60,000, which showed average calmodulin-dependent increases in phosphorylation of 68-fold. The phosphorylation of gp180 was increased 7.5-fold in the presence of calmodulin. Fifty percent of maximum phosphorylation of proteins and glycoproteins occurred with a free Ca2+ concentration of 0.3 X 10(-6) M. The amounts 12.6 micrograms/ml and 9.1 micrograms/ml of calmodulin were required for 50% of maximum phosphorylation of proteins and glycoproteins, respectively. Peptide mapping experiments identified three major phosphorylation sites in gp180. The phosphorylation of all three sites was increased in the presence of calmodulin. Phosphoamino acid analysis of gp180 revealed that [32P]phosphoserine and [32P]phosphothreonine were both produced during the phosphorylation reaction, with phosphoserine being the predominant product. The phosphorylation of both amino acids was increased in the presence of calmodulin. [32P]phosphotyrosine was also identified as a product of the phosphorylation of gp180.
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PMID:Phosphorylation of the postsynaptic density glycoprotein gp180 by Ca2+/calmodulin-dependent protein kinase. 299 21

This paper examines the modulation of insulin-stimulated glucose transport activity in rat adipose cells by ligands for receptors (R) that mediate stimulation (Rs; lipolytic) or inhibition (Ri; antilipolytic) of adenylate cyclase. The changes in glucose transport activity and cAMP, as assessed by 3-O-methylglucose uptake and (-/+) cAMP-dependent protein kinase (A-kinase) activity ratios, respectively, were monitored under conditions that maintain steady-state A-kinase activity ratios (Honnor, R. C., Dhillon, G. S., and Londos, C. (1985) J. Biol. Chem. 260, 15122-15129). Removal of endogenous adenosine with adenosine deaminase decreased insulin-stimulated glucose transport activity by approximately 30%, which was prevented or restored with Ri agonists such as phenylisopropyladenosine, nicotinic acid, and prostaglandin E1. These changes in transport activity were not accompanied by changes in A-kinase activity ratios, indicating that Ri-mediated effects on transport are independent of cAMP changes. Addition of an Rs ligand, isoproterenol, in the presence of adenosine increased kinase activity but did not change glucose transport activity. Conversely, upon removal of adenosine, addition of Rs ligands such as isoproterenol, adrenocorticotropic hormone, or glucagon strongly inhibited transport (approximately 50%) and stimulated kinase activity. However, subsequent addition of phenylisopropyladenosine nearly restored transport activity without alteration of A-kinase activity. These data and additional kinetic experiments suggest that Rs-mediated glucose transport modulations are also independent of cAMP. The interchangeability of ligands for both Rs and Ri receptors in modulating transport activity suggests that these cAMP-independent effects are mediated by the stimulatory (Ns) and inhibitory (Ni) guanyl nucleotide-binding regulatory proteins of adenylate cyclase. All Rs-and Ri-induced changes in transport activity occurred without a change in glucose transporter distribution, as assessed by D-glucose-inhibitable cytochalasin B binding, suggesting that Rs and Ri ligands modulate the intrinsic activity of the glucose transporter present in the plasma membrane.
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PMID:Regulation of insulin-stimulated glucose transport in the isolated rat adipocyte. cAMP-independent effects of lipolytic and antilipolytic agents. 302 4

The addition of beta-D-glucose (final concentration, 50 mM) to a cell suspension of Saccharomyces cerevisiae in stationary phase caused a rapid 4-fold increase in the concentration of cAMP, while a 2-fold increase of cAMP was observed by the addition of alpha-D-glucose. beta -D-Glucose was also more effective than alpha-D-glucose in the inactivation of fructose 1,6-bisphosphatase and the activation of trehalase. These results, taken together with the previous report that alpha-D-glucose is transported more rapidly than beta-D-glucose in Saccharomyces cerevisiae, do not support the view currently proposed by some investigators that cotransport of D-glucose with protons causes the depolarization of the cell membrane, resulting in the activation of adenylate cyclase. The present data, however, provides supporting evidence for the view that cAMP-dependent protein kinase is implicated in the inactivation of fructose 1,6-bisphosphatase and the activation of trehalase.
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PMID:Anomeric specificity of glucose effect on cAMP, fructose 1,6-bisphosphatase, and trehalase in yeast. 303 Mar 16

Plasmids encoding the amino terminal portion of an influenza virus hemagglutinin (HA) fused to polyoma virus middle T (mT) or large T (lT) sequences have been constructed. Stable expression of the chimeric proteins was obtained in established rat embryo fibroblasts following plasmid co-transfection and selection for G418 resistance. The synthesis and localization of the proteins was followed by metabolic labeling with [35S]methionine and [3H]mannose, cell fractionation, and immunoprecipitation with anti-polyoma T antibody. The HA leader and amino terminal peptide direct the synthesis of the lT and mT proteins into the endoplasmic reticulum where they undergo glycosylation, but this occurs with a very low efficiency. Most of the HA-mT and HA-lT fusion protein molecules do not enter completely into the endoplasmic reticulum, but rather achieve their normal locations in the cell as slightly higher molecular weight proteins, presumably due to the extra sequences derived from HA at their amino termini. HA-mT fusion protein is found to have associated tyrosine-specific protein kinase activity precipitable with anti-src as well as anti-T antibody, and cells expressing this fusion protein have a transformed phenotype.
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PMID:Expression of influenza hemagglutinin-polyoma T-antigen fusion proteins in a rat embryo fibroblast cell line. 303 93

Labeling with [3H]galactose was employed to isolate a glycosylphosphatidylinositol from rat hepatocytes which might be involved in the action of insulin. The polar head group of this glycosylphosphatidylinositol was generated by phosphodiesterase hydrolysis with a phosphatidylinositol-specific phospholipase C from Bacillus cereus. By Dowex AG1 x 8 chromatography the polar head group could be separated into three radioactive peaks eluting at 100 mM (peak I), 200 mM (peak II) and 500 mM (peak III) ammonium formate, respectively. Peak III was the most active as an inhibitor of the cAMP-dependent protein kinase. Treatment of peak III with alkaline phosphatase markedly reduced its activity on cAMP-dependent protein kinase. When peaks I, II or III were treated with alkaline phosphatase and analyzed again by Dowex AG1 x 8 chromatography, the radioactivity eluted with the aqueous fraction. The above results indicate that the polar head group of the insulin-sensitive glycosylphosphatidylinositol from rat hepatocytes exists in three different phosphorylated forms and that the biological activity of this molecule depends on its phosphorylation state.
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PMID:Different phosphorylated forms of an insulin-sensitive glycosylphosphatidylinositol from rat hepatocytes. 304 67

A glycophospholipid has been purified from rat liver membranes and shown to copurify with an insulin-sensitive glycophospholipid isolated from H35 hepatoma cells. The polar head group of this glycophospholipid is a phospho-oligosaccharide generated by treatment with phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. It has been proposed that this phospho-oligosaccharide, which is also generated in response to insulin, may play a role in insulin action. Incubation of the catalytic subunit of cyclic AMP-dependent protein kinase with this phospho-oligosaccharide inhibited the activity of the kinase to phosphorylate histone IIA, a purified preparation of phospholipid methyltransferase and kemptide, a phosphate-accepting peptide. Inhibition of kinase activity was dose-dependent and 50% inhibition of histone phosphorylation was demonstrated with a concentration of phospho-oligosaccharide of around 2 microM. This effect was demonstrated in the presence of ATP at concentrations up to 1 mM, indicating that the phospho-oligosaccharide acts at physiological concentrations of ATP and that it does not compete with this nucleotide for the same binding site in the kinase. Inhibition by the phospho-oligosaccharide of kinase activity could be reversed by dilution or dialysis and was not reproduced by up to 50 microM myo-inositol, glucosamine, galactose, myo-inositol 1-phosphate, glucosamine 1-phosphate, galactose 1-phosphate or phosphorylcholine. The inhibitory activity was resistant to mild acid treatment but was labile to treatment with alkali, exposure to nitrous acid or incubation with sodium periodate. The phospho-oligosaccharide had no effect on the phosphorylation of lysine-rich histone by rat brain protein kinase C and on the binding of cyclic AMP to a cyclic AMP-dependent protein kinase. In conclusion, the data in this study suggested that a phospho-oligosaccharide generated from an insulin-sensitive glycophospholipid may play a role in insulin action by modulating cyclic AMP-dependent protein kinase activity.
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PMID:Inhibition of cyclic AMP-dependent protein kinase by the polar head group of an insulin-sensitive glycophospholipid. 333 45

Insulin stimulates hexose transport and phosphorylation of the insulin receptor in monolayer cultures of intact 3T3-L1 adipocytes. To assess the phosphorylation state of the receptor in situ, cells were equilibrated with [32P]orthophosphate and then disrupted under denaturing conditions which preserved the phosphorylation state of the receptor established in the cell. The insulin receptor, isolated by lectin adsorption and two-dimensional nonreducing/reducing polyacrylamide gel electrophoresis, occurred as a single oligomeric species with an apparent alpha 2 beta 2 subunit composition. This oligomeric structure was not altered by treating cells with insulin. Only the beta-subunit of the receptor was phosphorylated; [32P]phosphoserine and [32P] phosphotyrosine were both identified in the beta-subunit from cells in the unstimulated state, but only [32P] phosphotyrosine increased in cells stimulated with insulin. Neither insulin-like growth factors I nor II stimulated insulin receptor beta-subunit phosphorylation, although both activated hexose transport. Upon the addition of insulin, [32P]orthophosphate incorporated into the beta-subunit increased 4.5-fold (7-fold with respect to [32P]tyrosine) and was complete within 1 min (t1/2 = 8 s). Following the removal of insulin from the monolayers, [32P]beta-subunit fell to the basal level (t1/2 = 2.5 min); there was no lag phase before either transition. The tyrosine protein kinase activity, measured in vitro with a model substrate, was higher with immunoaffinity-purified insulin receptor from insulin-stimulated cells than from cells in the basal state. Hexose transport rate, measured using 3-O-[methyl-14C]glucose, was half-maximally stimulated at 2 nM insulin. A 1-min latency period followed insulin addition, after which a 7-fold increase in the steady-state rate of hexose uptake was achieved within 5 min. Upon the removal of insulin, hexose transport continued at the stimulated steady-state rate for 2.5 min and then declined to the basal rate with a half-time of 8 min. These kinetic experiments in situ and protein kinase activity measurements in vitro support the hypothesis that beta-subunit phosphorylation is an intermediate step linking insulin binding to the increased glucose transport rate.
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PMID:Insulin-dependent phosphorylation of the insulin receptor-protein kinase and activation of glucose transport in 3T3-L1 adipocytes. 352 54


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