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)

Activation of glycolysis by insulin in cultured rat hepatocytes is preceded by an activation of phosphofructokinase 2 (PFK 2) and subsequent rise of the fructose 2,6-bisphosphate [Fru(2,6)P2] level. Extracellular addition of ATP or puromycin prevented the hormonal effect on glycolysis. The mechanism through which the purines abolished glycolytic stimulation was investigated. 1. 50 microM ATP completely prevented the 3-5-fold insulin-dependent increase of glycolysis, irrespective of whether the cells initially possessed a low or a high Fru(2,6)P2 content. 50 microM puromycin prevented the stimulation of glycolysis by insulin only in cells whose initial Fru(2,6)P2 levels were low and had to be increased by insulin prior to the increase in glycolysis. It did not antagonize the action of insulin cells with initial high Fru(2,6)P2 content. 2. ATP exerted effects on its own; it decreased initially high Fru(2,6)P2 levels by 95% within 10 min and decreased the basal glycolytic rate by 60%. Half-maximal effects on the Fru(2,6)P2 level were obtained with about 25 microM ATP or 15 microM adenosine 5'[beta, gamma-methylene]triphosphate. ADP and adenosine-5-[gamma-thio]triphosphate were as effective as ATP, whereas 100 microM adenosine 5'[alpha, beta-methylene]triphosphate elicited no effect. Puromycin neither decreased high Fru(2,6)P2 levels nor inhibited basal glycolysis. 3. Extracellular ATP (100 microM) led to inhibition of the active form of PFK 2. Intracellular levels of Glc6P, citrate, ATP, ADP and AMP were increased by extracellular ATP, the phosphoenolpyruvate content was decreased, Fru6P and glycerol 3-phosphate levels stayed constant. Puromycin did not inhibit PFK 2. 4. Both puromycin and ATP prevented the insulin-dependent rise of the Fru(2,6)P2 level, they abolished the activation of PFK 2 by the hormone. Puromycin did not block the accumulation of Fru(2,6)P2 provoked by glucose addition; ATP also antagonized the glucose-dependent increase. 5. 100 microM ATP elevated the cAMP-dependent protein kinase activity ratio from 0.1 to 0.38 and increased the level of inositol trisphosphate by 16-fold within 5 min, whereas puromycin was without effect on either level. It is concluded that the two purines block the insulin effect on glycolysis by preventing the hormone increasing the Fru(2,6)P2 level. The mode of action, however, seems to be different: ATP antagonizes insulin action in that it leads to increased inhibition of PFK 2 whereas puromycin prevents the activation of PFK 2 by insulin.
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PMID:Stimulation by insulin of glycolysis in cultured hepatocytes is attenuated by extracellular ATP and puromycin through purine-dependent inhibition of phosphofructokinase 2 activation. 252 68

The Ca2+ dependence of the Ca2+-pumping ATPase of bovine cardiac sarcolemma was studied for four states of activation: (a) unactivated, (b) cAMP-dependent protein kinase (cAMP protein kinase C-subunit)-activated, (c) calmodulin (CAM)-activated, and (d) CAM plus cAMP protein kinase C-subunit-activated. Analysis of the Ca2+ dependence of active transport gave the following Vmax (nanomoles Ca2+/(mg x min], Km (nM) for Ca2+, and Hill coefficient values for the four states at pH 7.4, 37 degrees C: (a) 1.7 +/- 0.3, 1800 +/- 100, 1.6 +/- 0.1; (b) 3.1 +/- 0.5, 1100 +/- 100, 1.7 +/- 0.1; (c) 15.0 +/- 2.5, 64 +/- 1.4, 3.7 +/- 0.2; and (d) 36.0 +/- 6.5, 63 +/- 1.7, 3.7 +/- 0.1. CAM has the most dramatic effect, increasing the apparent Ca2+ affinity by a factor of 28, increasing the Hill coefficient 2.0 units to a value approaching 4 and increasing the Vmax by a factor of 9 or 12. The effective Ca2+ concentration (EC50) for the Ca2+-induced activation of the enzyme in the presence of 5 microM calmodulin is close to the Km for Ca2+ for the CAM-activated state (64 nM). Activation by cAMP protein kinase C-subunit had only minor effects on the Km and Hill coefficient, but increased the Vmax of both the unactivated and the CAM-activated forms of the pump by factor of 1.8 and 2.4, respectively. Analysis suggests that CAM activation is the result of direct binding of Ca2-CAM or high complexes, conferring higher Ca2+ affinity to the enzyme. Analysis suggests that regulatory phosphorylation (cAMP protein kinase C-subunit) increases the rates of processes subsequent to or distinct from Ca2+ binding. The CAM-activated form of the pump was further characterized. Unexpectedly, this form of the enzyme is stimulated a factor of 1.9 by ADP, with half-maximal stimulation between 0.4 and 0.7 mM. Analysis of the progress curves for uptake show that the CAM-activated enzyme is highly resistant to inhibition by transported Ca2+, with an IC50 of 32 mM. The implications of these findings for the pump mechanism and for its role in the regulation of cardiac contractility are discussed.
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PMID:Kinetic characterization of the Ca2+-pumping ATPase of cardia sarcolemma in four states of activation. 252 31

Phosphoinositide-specific phospholipase C (PLC) activity of human platelet membranes was activated by the nonhydrolyzable guanine nucleotide GTP gamma S. This activation did not occur in either membranes prepared from dibutyryl cyclic AMP-pretreated platelets (A-membranes) or those prepared from untreated cells and subsequently incubated with cyclic AMP (cAMP) (B-membranes). This cAMP-mediated inhibition was abolished in the presence of inhibitors of cAMP-dependent protein kinase (A-kinase), suggesting that the inhibition was due to phosphorylation of (a) protein component(s). No significant differences were observed in the basal PLC activity and the extent of pertussis toxin-catalyzed ADP-ribosylation among control membranes and the two types of phosphorylated membranes (A- and B-membranes). GTP-binding activities of Gs, Gi and GTP-binding proteins of lower molecular masses were not altered by the phosphorylation of the membranes. These findings suggest that a GTP-binding protein is involved in the GTP gamma S-mediated activation of PLC and that cAMP (plus A-kinase) inhibits this activation by phosphorylating a membrane protein (probably a 240-kDa protein), rather than the GTP-binding protein or PLC itself. It is likely that this phosphorylation uncouples the GTP-binding protein from PLC.
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PMID:Inhibition by cyclic AMP of guanine nucleotide-induced activation of phosphoinositide-specific phospholipase C in human platelets. 253 21

Cholera toxin stimulates adenylyl cyclase by catalyzing ADP-ribosylation of the alpha chain (alpha s) of Gs, a guanine nucleotide binding regulatory protein. In a rat pituitary cell line, GH3, the toxin-induced increase in GTP-dependent adenylyl cyclase activity is maximal at 1 h; adenylyl cyclase remains elevated for at least 32 h. Surprisingly, cholera toxin also induces a 74-95% decrease in the amount of immunoreactive alpha s in the same cells, as assessed on immunoblots probed with either of two antisera directed against separate alpha s peptide sequences. The decrease in immunoreactive alpha s, which begins after 1 h of toxin treatment and is complete by 8 h, is accompanied by a comparable decrease in the amount of biochemically active alpha s, as assessed by its ability to complement the biochemical defect of alpha s-deficient S49 cyc- membranes. Cholera toxin induces similar decreases in alpha s in wild type S49 lymphoma cells, in S49 kin- mutants, which lack cAMP-dependent protein kinase, and in S49 H21 a mutants, in which alpha s is unable to assume an active conformation upon binding GTP. The toxin-induced decrease in alpha s is somewhat temperature-dependent, but is not blocked by agents that increase lysosomal pH or by colchicine, which promotes breakdown of microtubules. alpha s in detergent-solubilized GH3 membranes is susceptible to proteolysis by an endogenous protease; this susceptibility is markedly increased in membranes from cells previously exposed to cholera toxin for 1 h. Taken together, these results suggest that cholera toxin-induced covalent modification of alpha s marks the protein for accelerated degradation. In addition, the persistence of elevated GTP-dependent adenylyl cyclase activity despite loss of a substantial fraction of alpha s suggests that the amount of alpha s membranes is greater than the amount necessary for maximal activation of cAMP synthesis by cholera toxin.
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PMID:Cholera toxin induces cAMP-independent degradation of Gs. 253 15

The purified human placenta alpha 2 beta 2 heterotetrameric insulin receptor was reduced and dissociated into a functional alpha beta heterodimeric complex by a combination of alkaline pH and dithiothreitol treatment. In the presence of Mn/MgATP, insulin binding to the isolated alpha beta heterodimeric insulin receptor was found to induce the formation of a covalent disulfide-linked alpha 2 beta 2 heterotetrameric complex. In the absence of insulin, a noncovalent association of the alpha beta heterodimeric insulin receptor complex into an alpha 2 beta 2 heterotetrameric state required the continuous presence of both a divalent metal ion (Mn or Mg) and an adenine nucleotide (ATP, ADP, or AMPPCP). Thus, Mn/MgATP binding and not insulin receptor autophosphorylation was responsible for the noncovalent association into the alpha 2 beta 2 heterotetrameric state. However, the divalent metal ions or NaATP separately was ineffective in inducing the noncovalent association between the alpha beta heterodimers. The specific sulfhydryl agent iodoacetamide (IAN) was observed to inhibit the insulin-dependent covalent association of the alpha beta heterodimers without affecting the Mn/MgATP-induced noncovalent association into the alpha 2 beta 2 heterotetrameric state. Insulin treatment of the isolated alpha beta heterodimeric complex in the presence of IAN demonstrated that the Mn/MgATP-induce noncovalent association into the alpha 2 beta 2 heterotetrameric state was sufficient for insulin stimulation of beta-subunit autophosphorylation and exogenous substrate protein kinase activity. These data indicate that although interaction between the individual insulin receptor alpha beta heterodimers is necessary for insulin stimulation of protein kinase activity it does not require covalent disulfide bond formation.
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PMID:Relationship between insulin receptor subunit association and protein kinase activation: insulin-dependent covalent and Mn/MgATP-dependent noncovalent association of alpha beta heterodimeric insulin receptors into an alpha 2 beta 2 heterotetrameric state. 254 Aug 6

Polymorphonuclear leukocytes (PMNL) release superoxide anions formed by a membrane-bound NADPH oxidase induced by stimulations. Properties of the inducers and their antagonists indicate that Ca2+, GTP-binding protein (G-protein), phospholipase C and Ca2+, phospholipid-dependent protein kinase (C-kinase) are mainly associated with the stimulation of receptors. Low concentrations of ATP induce the oxidase accompanied by the increase in the intracellular Ca2+ due to the flux from the medium and the storage site. ATP-gamma-S, UTP and ITP are effective but mononucleotides, dinucleotides, GTP and CTP are not. Leukotriene B4 (LTB4) which acts as a chemotactic agent and the inducer of the NADPH oxidase is catabolized. It is hydroxylated by a specific cytochrome P450 and then oxidized to a carboxy derivative by a cytosolic alcohol dehydrogenase and a microsomal aldehyde dehydrogenase in PMNL. Active NADPH oxidase was obtained by incubating membrane and cytosolic components of resting PMNL in the presence of sodium dodecyl sulfate (SDS). Two cytosolic components were obtained by an affinity chromatography on 2',5'-ADP Sepharose. One component is active in the presence of GTP or GTP-gamma-S and the other component in the presence of another cytosolic fraction.
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PMID:Metabolism of stimulated polymorphonuclear leukocytes. 254 77

We have examined the cell-free heterologous desensitization of adenylyl cyclase in plasma membrane preparations from S49 wild-type (WT) and kin- cells (which lack cAMP-dependent protein kinase) incubated with purified catalytic subunit of cAMP-dependent protein kinase (cA.PKc). cA.PKc caused a rapid (t1/2 = 40 s) decrease in the hormone responsiveness of adenylyl cyclase in the WT membrane preparations that mimicked the intact cell heterologous desensitization; that is, there was an increase in the Kact for both epinephrine and prostaglandin E1 (PGE1) stimulations of adenylyl cyclase induced at the receptor level because neither forskolin- nor NaF-stimulated activity was affected. The desensitization was independent of agonist occupancy of the receptor, and the effects were blocked both by the active fragment (amino acids 5-22) of the specific inhibitor of cA.PK and by p[NH]ppA. cA.PKc treatment of kin- membranes resulted in a heterologous desensitization that resembled the effects of WT adenylyl cyclase, with the exception that forskolin-stimulated activity was also reproducibly decreased by 24%. cA.PKc had no effect on WT membranes isolated from cells that had previously undergone maximal heterologous desensitization during treatment with 10 microM forskolin. In contrast, cA.PKc-induced heterologous desensitization of kin- membranes was additive with the epinephrine-induced homologous desensitization of intact cells. Cell-free desensitizations were reversed by incubation of membranes with cA.PKc and ADP, conditions that drive the kinase reaction backward. The similarities of our cell-free cA.PKc-mediated heterologous desensitization of adenylyl cyclase with the intact cell desensitization support our hypothesis that heterologous desensitization of the WT lymphoma cells is mediated by cA.PK via a mechanism independent of homologous desensitization.
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PMID:Cell-free heterologous desensitization of adenylyl cyclase in S49 lymphoma cell membranes mediated by cAMP-dependent protein kinase. 254 97

Nuclear DNA topoisomerase II activity in quail oviduct tissue was found to increase by about 70% with age. This age-dependent increase was observed with both the enzyme in whole nuclear extract and nuclear matrix-associated topoisomerase II. Both purified topoisomerase II and the nuclear matrix-bound enzyme were found to be modifiable by phosphorylation and poly(ADP-ribosyl)ation. Phosphorylation of the purified enzyme by isolated nuclear protein kinase NII or protein kinase C resulted in a 2- to 3-fold increase in specific activity, while poly(ADP-ribosyl)ation by soluble poly(ADP-ribose) synthetase caused a 50% inhibition of the enzyme. Using immunoprecipitation and immunoblotting procedures, phosphorylation and poly(ADP-ribosyl)ation could also be demonstrated to occur with the nuclear matrix-associated enzyme. The nuclear matrix-associated NII-like protein kinase activity, assumed to be involved in post-translational modification of topoisomerase II, displayed a 1.4- to 1.6-fold increase in old animals compared to mature ones, while the matrix-bound poly(ADP-ribose) synthetase activity decreased by about 50%. It is suggested that age-correlated enhancement of DNA topoisomerase II activity, possibly due to age-dependent changes in activities of nuclear protein kinases and poly(ADP-ribose) synthetase, may result in alterations in the topological state of DNA, possibly affecting DNA replication, transcription and repair with age.
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PMID:Age-dependent increase of DNA topoisomerase II activity in quail oviduct; modulation of the nuclear matrix-associated enzyme activity by protein phosphorylation and poly(ADP-ribosyl)ation. 255 26

Aggregation and autophosphorylation of the insulin receptor-protein kinase, from cultured 3T3-L1 adipocytes, were studied in the presence of cationic polyamino acids. Poly-L-lysine and poly-L-arginine produced the following effects with the purified receptor: first, the autophosphorylation rate was increased by polycations. Half-maximal stimulation was proportional to polymer length. The rate enhancement was greater at lower ATP concentrations. Second, near-endpoint (equilibrium) autophosphorylation was greater in the presence of the polycations. Polycations inhibited the reverse reaction: ADP + phosphoreceptor yielding ATP + aporeceptor. Third, the [32P]phosphopeptides generated by trypsin digestion of the 32P-beta-subunit, showed that no new autophosphorylation sites resulted from the presence of polycations. Fourth, the polycations, but not insulin, promoted receptor aggregation, and phosphoreceptor aggregated more readily than aporeceptor. Insulin receptor enriched through the wheat germ agglutinin eluate step was compared with purified receptor. Higher concentrations of poly-L-arginine were required to stimulate autophosphorylation and to promote aggregation. Finally, several polycation-dependent substrates present in the wheat germ agglutinin eluate co-aggregated with the insulin receptor. Polycation-stimulated receptor autophosphorylation is linked to a lower KM,app for ATP, but substrate phosphorylation may require the aggregation.
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PMID:Insulin receptor aggregation and autophosphorylation in the presence of cationic polyamino acids. 259 62

Translocation of Ca2+/phospholipid-dependent protein kinase (PKC) activity from cytosolic to membrane fractions was assessed in washed human platelet suspensions. Phorbol myristate acetate (PMA) induced a rapid loss of PKC activity from the cytosolic compartment in stirred platelets, which was not accompanied by measurable increases in membrane-associated activity, but was paralleled by a decrease in total cellular enzyme activity (cytosol plus membrane). When platelet aggregation was prevented by not stirring, (i) cytosolic activity was decreased by PMA, (ii) significant and maintained (1-15 min with PMA) increases in membrane-bound PKC were detected, and (iii) the decline in total enzyme activity was markedly slower. In stirred platelets, total and specific inhibition of PMA-induced aggregation by a fibrinogen-derived peptide (RGDS, i.e. Arg-Gly-Asp-Ser) promoted maximal increases in membrane-associated PKC in the presence of PMA and completely prevented the loss in cellular activity. Thrombin and collagen both induced a decrease in cytosolic PKC and a loss of total activity, but a significant rise in membrane activity was seen only with collagen; ADP had no detectable effect on enzyme distribution. These results demonstrate an agonist-induced redistribution of PKC and indicate that platelet aggregation may play an important role in the proteolysis, and hence persistence, of membrane-associated PKC. This observation has implications for the potency and duration of PKC-mediated responses induced by agonists and exogenous PKC activators.
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PMID:Receptor- and phorbol-ester-mediated redistribution of protein kinase C in human platelets. Evidence that aggregation promotes degradation of protein kinase C. 259 39


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