Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The enhanced phosphorylations via cAMP, Ca2+ mobilization, and diacyl glycerol formation via the activation of the respective kinases is now classical. The decreased phosphorylation via inhibition of adenylate cyclase via the alpha adrenergic receptor is also becoming understood. What the insulin studies on the control of glycogen synthesis have taught us is that the rate limiting enzyme glycogen synthase is regulated by multiple covalent phosphorylation in an elegant but complex manner. The overall pattern of dephosphorylation is influenced by effecting both phosphatase and kinase activities in a set of interrelated mechanisms. In the presence of glucose, in muscle, fat, and liver under physiological conditions G-6-P acts as a signal to stimulate the phosphatase. An additional stimulation could occur via a novel insulin phosphatase stimulatory mediator. The phosphatase is also stimulated by at least three covalent mechanisms involving altered phosphorylation state. In one there is a decreased phosphorylation of the phosphatase inhibitor 1 potentially related to decreased cAMP-dependent protein kinase activity. In the second, there is decreased phosphorylation of the deinhibitor also potentially related to decreased cAMP-dependent protein kinase phosphorylation. In the third, an increased activity of casein kinase 2 could activate the ATP-Mg dependent phosphatase by an increased phosphorylation of phosphatase inhibitor 2 (modulatory subunit). In the liver, allosteric control of the phosphatase by G-6-P and nucleotides is of great importance. Insulin also stimulates the phosphatase in long-term experiments via increased protein synthesis. It is clear that future work will be required to determine which species of the various classes of phosphatases are regulated in short-term and long-term regulation by insulin. In terms of kinases, the effects of insulin to inactivate and desensitize the cAMP-dependent protein kinase are established. The molecular mechanisms of this effect remain to be worked out. The enhanced activity of MAP and S-6 kinase would appear to be part of a cascade of reactions perhaps originating in the autophosphorylation and activation of the insulin receptor tyrosine kinase. The mechanism of the short-term activation of casein kinase 2 remains to be elucidated. A cAMP-dependent protein kinase inhibitory mediator, which also inhibits adenylate cyclase is an important element in the regulation of kinase and adenylate cyclase activity by insulin. Its physiological significance must be established in the future, in terms of its control of glycogen synthase activation by insulin. Clearly this kinase inhibitor as well as the phosphatase stimulator are potential regulators of glycogen synthase activity by insulin.
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PMID:Insulin and the stimulation of glycogen synthesis. The road from glycogen structure to glycogen synthase to cyclic AMP-dependent protein kinase to insulin mediators. 215 10

The effect of insulin on hepatic glucose production has been studied in anesthetized rats in the postabsorptive state. Insulin decreases significantly hepatic glucose production within 5-10 min. It also increases the level of fructose 2,6-bisphosphate, via an increase in the Vmax of 6-phosphofructo-2-kinase and concomitantly decreased the activity of fructose-2,6-bisphosphatase, resulting in a 5-fold increase in the ratio of kinase/phosphatase. Insulin also increased the apparent Kd of pyruvate kinase for phosphoenolpyruvate. The changes in the activity of 6-phosphofructo-2-kinase and pyruvate kinase were measured after separation from possible modulators, and suggest a decrease in their phosphorylation state which cannot be attributed to a decrease in the level of cAMP or in the activity of cAMP-dependent protein kinase since these two parameters were not modified by insulin. In addition, neither the activity of phosphorylase a nor that of glycogen synthase were modified. The data strongly suggest that the increase in the glycolytic rate plays a role in the effect of insulin on hepatic glucose production and that insulin mediates its effect on the activity of these enzymes via one or more phosphatases.
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PMID:Insulin activates 6-phosphofructo-2-kinase and pyruvate kinase in the liver. Indirect evidence for an action via a phosphatase. 215 92

Isolated rat ventricular cardiomyocytes were used to study the effects of insulin on glycogen metabolism in cells treated with various agents that activate adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. Incubation of myocytes with isoproterenol produced a rapid concentration-dependent increase in cAMP concentration, cAMP-dependent protein kinase activity, and phosphorylase activity and a simultaneous decrease in the glycogen synthase activity ratio. Various cAMP analogues also produced a concentration-dependent increase in phosphorylase activity and a decline in the glycogen synthase activity ratio. Incubation of cells with insulin produced no change in basal phosphorylase activity but produced a rapid 40% increase in the glycogen synthase activity ratio. Inclusion of insulin in cell incubations containing increasing concentrations of isoproterenol did not modify the increases in cAMP concentration, protein kinase activity, or phosphorylase activity. Insulin also did not antagonize the ability of any of the cAMP analogues tested to activate phosphorylase, irrespective of the suitability of the particular cAMP analogue as a substrate for cAMP phosphodiesterases. The failure of insulin to antagonize the glycogenolytic effects of isoproterenol or cAMP analogues was paralleled by its failure to activate low-Km phosphodiesterase activity, but the cAMP analogue, 8-parachlorophenylthio-cAMP produced a small reproducible activation of the low-Km enzyme. In contrast to hepatocytes and adipocytes, where some effects of insulin appear to be due to activation of the phosphodiesterase and hydrolysis of cAMP, the effects in cardiomyocytes appear to be independent of an insulin-sensitive phosphodiesterase or of the effects on other components of the cAMP cascade.
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PMID:Failure of insulin to antagonize cAMP-mediated glycogenolysis in rat ventricular cardiomyocytes. 215 37

Relaxin is a member of the insulin family of polypeptide hormones and is known to exert its biological effects on various parts of the mammalian reproductive system. Biologically active human relaxin has been chemically synthesized based on the nucleotide sequence obtained from an ovarian cDNA clone. In the present study synthetic human relaxin was radiolabled by phosphorylation with cAMP-dependent protein kinase and [gamma-32P]ATP to a specific activity of 5000 Ci/mmol. The phosphorylated relaxin was purified on cation exchange high performance liquid chromatography and was shown to co-migrate with relaxin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mass spectrometry revealed a single phosphorylated site on the B chain of relaxin. The 32P-relaxin was able to bind to a goat anti-relaxin antibody, and this binding could be displaced by unlabeled relaxin in a concentration-dependent manner. A comparison of the concentration responses of cellular cAMP production stimulated by relaxin and phosphorylated relaxin in a primary human uterine cell line showed that phosphorylation did not affect the in vitro biological efficacy of relaxin. This made it suitable for in situ autoradiographic localization of relaxin binding sites in rat uterine, cervical, and brain tissue sections. Displacement of the binding of 100 pM 32P-relaxin by 100, 10, and 3 nM unlabeled relaxin, but not by 100 nM insulin, insulin-like growth factor-I, and an insulin-like growth factor-I analog, demonstrated the high affinity and specificity of such binding. We conclude that 32P-labeled human relaxin is biologically and immunologically active and that this novel probe binds reversibly and with high affinity to classical (e.g. uterus) and unpredicted (e.g. brain) tissues.
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PMID:Preparation of biologically active 32P-labeled human relaxin. Displaceable binding to rat uterus, cervix, and brain. 216 Sep 76

Hormonal regulation of hepatic gluconeogenic pathway flux is brought about by phosphorylation/dephosphorylation and control of gene expression of several key regulatory enzymes. Regulation by cAMP-dependent phosphorylation occurs at the level of pyruvate kinase and 6-phosphofructo-2-kinase (6PF-1-K)/fructose-2,6-bisphosphatase (Fru-2,6-P2ase). The latter is a unique bifunctional enzyme that catalyzes both the synthesis and degradation of fructose-2,6-bisphosphate (Fru-2,6-P2), which is an activator of 6PF-1-K and an inhibitor of Fru-1,6-P2ase. The bifunctional enzyme is a homodimer whose activities are regulated by cAMP-dependent protein kinase-catalyzed phosphorylation at a single NH2-terminal seryl residue/subunit, which results in activation of the Fru-2,6-P2ase and inhibition of the PF-1-K reactions. Hormone-mediated changes in the phosphorylation state of the bifunctional enzyme are responsible for acute regulation of Fru-2,6-P2 levels. 6PF-2-K/Fru-2,6-P2ase thus provides a switching mechanism between glycolysis and gluconeogenesis in mammalian liver. Pyruvate kinase is regulated by both phosphorylation and allosteric effectors. Fru-1,6-P2, an allosteric activator, also inhibits cAMP-dependent enzyme phosphorylation, and its steady-state concentration is indirectly determined by the level of Fru-2,6-P2. Therefore, acute regulation of both pyruvate kinase and the bifunctional enzyme provide coordinated control at both the pyruvate/phosphoenolpyruvate and Fru-6-P/Fru-1,6-P2 substrate cycles. The Fru-2,6-P2 system is also subject to complex multihormonal long-term control through regulation of 6 PF-2-K/Fru-2,6-P2ase gene expression. Glucocorticoids are the major factor in turning on this gene in liver, but insulin is also a positive effector. cAMP prevents the effects of glucocorticoids and insulin. Although Fru-2,6-P2 plays a key role in the regulation of carbon flux in the gluconeogenic pathway, the regulation of this flux depends on several factors and regulation of other key enzymes whose importance varies depending on the dietary and hormonal status of the animal. Molecular cloning of the cDNA encoding PF-2-K/Fru-2,6-P2ase has elucidated its structure and permitted analysis of its evolutionary origin as well as its tissue distribution and control of its gene expression. The rat liver and skeletal muscle isoforms arose by alternative splicing of a single gene. The muscle form differs from the liver form only at the NH2-terminal and does not have a cAMP-dependent protein kinase phosphorylation site. The hepatic enzyme subunit consists of 470 amino acids.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Fructose-2,6-bisphosphate in control of hepatic gluconeogenesis. From metabolites to molecular genetics. 216 55

In isolated, 32Pi-loaded, rat adipocytes, we have examined phosphorylation of the major cAMP-dependent protein kinase (A-kinase) substrate, a protein that appears to be associated with the lipid storage droplet and migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 65-67-kDa doublet. In control cells, a strong phosphorylation signal is detected as the (+/- cAMP) A-kinase activity ratio ranges from approximately 0.1 to approximately 0.3-0.4 with increasing isoproterenol concentrations. By contrast, insulin-treated cells exhibiting A-kinase activity ratios over the range of 0.1-0.25 contain less 32P in the 65-67-kDa protein than control cells exhibiting identical A-kinase activity ratios. At higher activity ratios (greater than 0.3), this reduction in phosphorylation of the 65-67-kDa protein by insulin disappears. It is concluded that insulin stimulates a phosphatase activity that acts on the 65-67-kDa protein. Insulin actions aside, these studies reveal two interesting phenomena. 1) Whereas elevated, steady-state A-kinase activities are established rapidly (1-2 min) upon isoproterenol stimulation, phosphorylation of the 65-67-kDa substrate proceeds through a burst, followed by a decline to a steady-state level by 10-12 min. An "adaptation" mechanism, providing for a constant response to a constant stimulus, may underlie this lack of parallelism between the time course of phosphorylation and A-kinase activity. 2) Removal of [32Pi] orthophosphate immediately before isoproterenol stimulation leads to a rapid (t approximately 10 min) loss in labeling of the 65-67-kDa protein, whereas the phosphorylation state of other phosphoproteins are not changed. These data suggest that elevation of A-kinase activity leads to a rapid exchange of external Pi with an ATP pool that is used by A-kinase.
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PMID:Control of endogenous phosphorylation of the major cAMP-dependent protein kinase substrate in adipocytes by insulin and beta-adrenergic stimulation. 217 32

Phosphorylation of the insulin-regulatable glucose transporter (IRGT) is increased by incubating rat adipocytes with isoproterenol or by incubating microsomal membranes with cAMP-dependent protein kinase. To attempt to locate the sites of phosphorylation, the IRGT (apparent Mr = 46,000) was immunoprecipitated from 32P-labeled adipocytes and cleaved with CNBr or trypsin. Essentially all of the 32P could be recovered in a single CNBr fragment, denoted CB-T (Mr = 8,000), which bound a polyclonal antibody (R820) against a peptide having the sequence of the last 12 amino acids in the COOH terminus of the IRGT. 32P-Labeling of the IRGT was also confined to CB-T when membranes were incubated with [gamma-32P]ATP and cAMP-dependent protein kinase. Isoproterenol increased phosphorylation of CB-T, but insulin was without effect. To resolve phosphorylation sites further, IRGT from 32P-labeled cells was subjected to exhaustive proteolysis with trypsin. Samples were applied to a C-18 column, and 32P-labeled fragments were resolved into three peak fractions by elution with an increasing gradient of acetonitrile. [32P]Phosphoserine was the only phosphoamino acid detected in any of the peaks. Peak III contained approximately 80% of the 32P and was increased by isoproterenol. Almost all of the 32P introduced by cAMP-dependent protein kinase in vitro eluted in Peak III. In all cases, the 32P-labeled species in Peak III were quantitatively immunoprecipitated by R820. Digesting the peptide(s) in Peak III with V8 protease generated a single peak of 32P which eluted at lower acetonitrile than Peak III and contained 32P-labeled species that did not interact with R820. Automated Edman degradation indicated that the serine residue in Peak III phosphorylated by cAMP-dependent protein kinase was the 3rd or 4th residue from the NH2 terminus of the peptide. These findings indicate that phosphorylation of the IRGT is restricted to the presumed intracellular domain at the COOH terminus and that Ser488 is a major site phosphorylated both by cAMP-dependent protein kinase in vitro and in response to isoproterenol in vivo.
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PMID:Phosphorylation of the glucose transporter in rat adipocytes. Identification of the intracellular domain at the carboxyl terminus as a target for phosphorylation in intact-cells and in vitro. 240 83

Incubating 32P-labeled fat cells with insulin increased by as much as 80-fold the amount of 32Pi in a soluble species of apparent Mr 62,000. This species, designated isp62, was specifically immunoprecipitated from cellular extracts with a monoclonal antibody against the type II regulatory subunit (RII) of cAMP-dependent protein kinase. Fat-cell RII, purified from extracts with cAMP-Sepharose or labeled with 8-azido [32P]cAMP, had an apparent Mr 51,000. Peptide mapping indicated that isp62 and adipocyte RII were different proteins. When cells were metabolically labeled with [35S]methionine, insulin stimulated the appearance of 35S-labeled isp62, indicating that the hormonal effect involves generation of the protein. The insulin-induced increase in isp62 could be observed within 1 min, occurred with physiological concentrations of the hormone, and was rapidly reversible. The increase in isp62 was unaffected by cycloheximide, indicating that insulin stimulates the posttranslational processing of a precursor, rather than de novo synthesis of the protein.
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PMID:Insulin stimulates the generation of an adipocyte phosphoprotein that is isolated with a monoclonal antibody against the regulatory subunit of bovine heart cAMP-dependent protein kinase. 242 9

P19, a group of 19,000 mol wt cytosolic proteins, with apparent isoelectric points of pI 5.9, pI 5.7, and pI 5.4, respectively, was identified in three peptide hormone-producing cell types: AtT20 mouse pituitary tumor cells, RIN-1122 rat insulinoma cells, and hamster insulinoma cells. Secretagogue-dependent phosphorylation of P19 was analyzed in 32P-labeled cells by two-dimensional electrophoresis and autoradiography. The results were quantitated by computer-assisted densitometry. Cellular levels of cAMP and hormone release were measured in parallel incubations. In addition to stimulating ACTH release, CRF raised the cellular level of cAMP and increased the 32P labeling of all three 19,000 mol wt proteins in AtT20 cells. Other agents known to act through cAMP, which included isoproterenol, forskolin, and 8-bromo-cAMP, mimicked the effect of CRF on both ACTH release and phosphorylation of P19. 12-O-Tetra-decanoylphorbol-13-acetate, a tumor-promoting phorbol ester, also stimulated both ACTH release and phosphorylation of P19. In contrast, although 40 mM K+ promoted ACTH release, it did not affect the phosphorylation of P19. Analogous findings were observed in insulinoma cells. Glucagon stimulated insulin release, increased cellular cAMP and promoted phosphorylation of P19 in RIN 1122 cells. 12-O-Tetradecanoylphorbol-13-acetate also enhanced insulin release and the phosphorylation of P19 in these cells. The results obtained with hamster insulinoma cells closely resembled the observations in RIN-1122 cells. In conclusion, P19, an apparently homologous set of cytosolic proteins, undergoes phosphorylation in three peptide hormone-producing cells in response to two groups of secretagogues, the effect of which is probably mediated, in one case, by cAMP-dependent protein kinase and, in the other, by protein kinase C. The data suggest the possibility that P19 participates in a secretory pathway activated by these two effector systems.
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PMID:P19, a hormonally regulated phosphoprotein of peptide hormone-producing cells: secretagogue-induced phosphorylation in AtT-20 mouse pituitary tumor cells and in rat and hamster insulinoma cells. 242 97

Ca2+ pump activity of skeletal muscle microsomes containing fragments of sarcoplasmic reticulum was examined in rats 8 wk after the induction of chronic diabetes by an intravenous injection of streptozotocin (65 mg/kg). In comparison with the control values, both ATP-dependent Ca2+ uptake and Ca2+-stimulated ATPase activities were increased in the microsomal fraction from diabetic rats. These changes were seen as early as 7 days after streptozotocin injection and were apparent at various times of incubation (1-10 min) as well as at different concentrations of free Ca2+ (10(-7)-5 X 10(-5) M Ca2+). Insulin administration to diabetic animals for 2 wk reversed Ca2+ uptake and ATPase activities to control levels. The increase in microsomal ATPase activity of the diabetic preparation due to cAMP-dependent protein kinase or calmodulin was greater than in the control microsomes and the depression by a specific inhibitor of protein kinase, but not of calmodulin, was greater in diabetic muscle. The enhanced Ca2+ pump activity was associated with altered phospholipid composition and protein profile of the diabetic preparations. The rate of Ca2+ release from microsomal vesicles was unaffected by the diabetic condition. Isometric contractile force development as well as positive dF/dt and negative dF/dt of the skeletal muscle from diabetic animals were higher at different pulse strengths (0.5-100 V) and at different Ca2+ concentrations (0.25-2.5 mM). These results suggest that diabetes is associated with enhanced sarcoplasmic reticular Ca2+ pump activity, and this may account for the hyperfunction of skeletal muscle in this disease.
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PMID:Calcium pump activity of sarcoplasmic reticulum in diabetic rat skeletal muscle. 243 Apr 66


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