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:2.7.11.11 (AMPK)
12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using computer simulation we have modeled the kinetics of cAMP-dependent protein kinase, type II, following transient pulses of cAMP. We show that under the appropriate physiological conditions, the kinase can remain activated 20 min or longer after the cessation of adenylate cyclase activation, in a process we term long-term activation. Long-term activation depends in part on the state of phosphorylation of the regulatory subunit, because phosphorylation of the regulatory subunit regulates the affinity of this subunit for the catalytic subunit. We have used our model to simulate experiments that have been performed on the kinetic and steady state activities of cAMP-dependent protein kinase and have found good agreement between the simulations and the experimental data. The effects of the activity of phosphodiesterase, adenylate cyclase, and protein phosphatase on the kinetics of cAMP-dependent protein kinase have been modeled, as have the effects of different ratios of regulatory subunit to catalytic subunit. We have also simulated the activation of the cAMP-dependent protein kinase in Drosophila learning and memory mutants having primary or secondary defects in the cAMP cascade. We make predictions regarding the behavior of different mutants, which are in line with the experimental data. The model corroborates the assumption that the cAMP cascade may play a role in learning and short-term memory.
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PMID:A quantitative model for the kinetics of cAMP-dependent protein kinase (type II) activity. Long-term activation of the kinase and its possible relevance to learning and memory. 272 37

A form of glycogen synthase kinase designated GSK-M3 was purified 4000-fold from rat skeletal muscle by phosphocellulose, Affi-Gel blue, Sephacryl S-300 and carboxymethyl-Sephadex column chromatography. Separation of GSK-M from the catalytic subunit of the cAMP-dependent protein kinase was facilitated by converting the catalytic subunit to the holoenzyme form by addition of the regulatory subunit prior to the gel filtration step. GSK-M had an apparent Mr 62,000 (based on gel filtration), an apparent Km of 11 microM for ATP, and an apparent Km of 4 microM for rat skeletal muscle glycogen synthase. The kinase had very little activity with 0.2 mM GTP as the phosphate donor. Kinase activity was not affected by the addition of cyclic nucleotides, EGTA, heparin, glucose 6-P, glycogen, or the heat-stable inhibitor of cAMP-dependent protein kinase. Phosphorylation of glycogen synthase from rat skeletal muscle by GSK-M reduced the activity ratio (activity in the absence of Glc-6-P/activity in the presence of Glc-6-P X 100) from 90 to 25% when approximately 1.2 mol of phosphate was incorporated per mole of glycogen synthase subunit. Phosphopeptide maps of glycogen synthase obtained after digestion with CNBr or trypsin showed that this kinase phosphorylated glycogen synthase in serine residues found in the peptides containing the sites known as site 2, which is located in the N-terminal CNBr peptide, and site 3, which is located in the C-terminal CNBr peptide of glycogen synthase. In addition to phosphorylating glycogen synthase, GSK-M phosphorylated inhibitor 2 and activated ATP-Mg-dependent protein phosphatase. Activation of the protein phosphatase by GSK-M was dependent on ATP and was virtually absent when ATP was replaced with GTP. GSK-M had minimal activity toward phosphorylase b, casein, phosvitin, and mixed histones. These data indicate that GSK-M, a major form of glycogen synthase kinase from rat skeletal muscle, differs from the known glycogen synthase kinases isolated from rabbit skeletal muscle.
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PMID:Characterization of GSK-M, a glycogen synthase kinase from rat skeletal muscle. 282 16

Phosphorylation of microtubule-associated protein 2 (MAP 2) by Ca2+-, calmodulin-dependent protein kinase II (protein kinase II) inhibited the actin filament cross-linking activity of MAP 2. This inhibition required the presence of ATP, Mg2+, Ca2+ and calmodulin. The minimal concentration of MAP 2 required for gel formation of actin filaments was increased with increasing amounts of phosphate incorporated into MAP 2, and the phosphorylated MAP 2, into which 10.3 mol of phosphate/mol of protein had been incorporated, did not cause actin filaments to gel under the experimental conditions used. The phosphorylation of MAP 2 by Ca2+-, phospholipid-dependent protein kinase (protein kinase C) and cAMP-dependent protein kinase also inhibited the actin filament cross-linking activity of MAP 2. The extent and rate of phosphorylation of MAP 2 by protein kinase II were higher than those of the phosphorylation by protein kinase C and cAMP-dependent protein kinase. The interaction of actin filaments with MAP 2 was inhibited more by the actions of protein kinase II and protein kinase C than by cAMP-dependent protein kinase. The actin filament cross-linking activity of MAP 2 phosphorylated either by protein kinase II, cAMP-dependent protein kinase or protein kinase C was retrieved when phosphorylated MAP 2 was treated by protein phosphatase. These results indicate that the interaction of actin filaments with MAP 2 is regulated by the phosphorylation-dephosphorylation of MAP 2.
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PMID:Regulation of the interaction of actin filaments with microtubule-associated protein 2 by calmodulin-dependent protein kinase II. 282 88

Protein phosphatase T from rat liver, so termed due to its activity toward [32P-Thr]casein and its marked preference for the phosphopeptide Arg-Arg-Ala-Thr(P)-Val-Ala over its phosphoseryl derivative (Donella Deana, A., Marchiori, F., Meggio, F. and Pinna, L.A. (1982) J. Biol. Chem. 257, 8565-8568), is shown here to belong to the family of type 2A protein phosphatase according to Cohen's nomenclature (Ingebritsen, T.S. and Cohen, P. (1983) Eur. J. Biochem. 132, 255-261). In particular, protein phosphatase T is endowed with phosphorylase phosphatase activity that is stimulated by protamine, histone H1 and heparin, it is inhibited by spermine, it does not bind to heparin-Sepharose and it readily dephosphorylates the phosphopeptide Arg-Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser reproducing the phosphorylation site of the alpha-subunit of phosphorylase kinase. The Mr of protein phosphatase T determined by gel filtration under non-denaturating conditions is about 150 kDa and its activity ratio toward histone H1 phosphorylated by protein kinase C versus histone H1 phosphorylated by cAMP-dependent protein kinase is unusually high. Some properties of protein phosphatase T, such as its weak binding to DEAE-cellulose and its high stimulation by protamine as compared to a relatively poor stimulation by histone H1, suggest that it may be similar to subtype 2Ao of protein phosphatase 2A.
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PMID:Identification of pseudo 'phosphothreonyl-specific' protein phosphatase T with a fraction of polycation-stimulated protein phosphatase 2A. 282 78

Studies were performed to determine if the Na+-H+ exchanger, solubilized from renal brush border membranes from the rabbit and assayed in reconstituted artificial proteoliposomes, could be regulated by cAMP-dependent protein kinase. Octyl glucoside solubilized renal apical membrane proteins from the rabbit kidney were phosphorylated by incubation with ATP and highly purified catalytic subunit of cAMP-dependent kinase. 22Na+ uptake was determined subsequently after reconstitution of the proteins into proteoliposomes. cAMP-dependent protein kinase resulted in sustained protein phosphorylation and a concentration-dependent decrease in the amiloride-sensitive component of pH gradient-stimulated sodium uptake. The inhibitory effect of cAMP-dependent protein kinase demonstrated an absolute requirement for ATP and was blocked by the specific protein inhibitor of this kinase. cAMP-dependent protein kinase also inhibited 22Na+ uptake in the absence of a pH gradient (pHin 6.0, pHout 6.0) and the inhibitory effect was blocked by the specific inhibitor of the kinase. Solubilized membrane proteins exhibited little endogenous protein kinase or protein phosphatase activity. These studies indicate that Na+-H+ exchange activity of proteoliposomes reconstituted with proteins from renal brush border membranes is inhibited by phosphorylation of selected proteins by cAMP-dependent protein kinase. These findings also indicate that the regulatory components of the Na+-H+ exchanger remain active during the process of solubilization and reconstitution of renal apical membrane proteins.
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PMID:Reconstitution of cAMP-dependent protein kinase regulated renal Na+-H+ exchanger. 283 85

A protein phosphatase which dephosphorylates phospholamban was purified from canine cardiac cytosol. Purification involved sequential chromatography on DEAE-Sephacel, polylysine-agarose, heparin-agarose, Mono Q HR 10/10, and Superose 6. The enzyme was composed of three subunits with Mr = 63,000, 55,000, and 38,000, and it could dephosphorylate the sites on phospholamban phosphorylated by either cAMP-dependent or calcium-calmodulin-dependent protein kinase. Phospholamban phosphatase activity was enhanced 12-, 9-, and 3-fold by the divalent cations Mg2+, Mn2+, and Ca2+, respectively. The phosphatase was inhibited by PPi, ATP, NaF, and Pi and the degree of inhibition was different with each compound. The substrate specificity of the purified phosphatase for cardiac phosphoproteins was determined using troponin I, phospholamban, and highly enriched sarcolemmal and sarcoplasmic reticulum preparations, phosphorylated by the cAMP-dependent protein kinase. The phosphatase exhibited the highest activity with phospholamban as substrate. Thus, dephosphorylation of phospholamban by this phosphatase may participate in regulation of sarcoplasmic reticulum function in cardiac muscle.
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PMID:Purification and characterization of phospholamban phosphatase from cardiac muscle. 284 19

The protein phosphatases in rat liver cytosol, active on rat liver acetyl-CoA carboxylase (ACC) phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase, have been partially purified by anion-exchange and gel filtration chromatography. The major phosphatase activities against all three substrates copurify through fractionation and appear to be identical to protein phosphatases 2A1 and 2A2. No unique protein phosphatase active on 32P-ACC phosphorylated by the casein kinases was identified.
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PMID:Protein phosphatases active on acetyl-CoA carboxylase phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase. 286 68

Acetyl-CoA carboxylase and its associated kinase have been purified to homogeneity from rat liver and, together with the catalytic subunit of liver protein phosphatase, used to study the effect of phosphorylation on the carboxylase activity. Phosphatase increases the carboxylase activity, whereas the kinase decreases it. A linear inverse relationship (correlation coefficient = 0.98) exists between phosphate incorporated by the kinase and the specific activity. The kinetics of activation by citrate show an increased Ka and a decreased Vmax for carboxylase preparations with increasing levels of phosphate. On this basis an enzymic test was devised for phosphate incorporated by the kinase. Thus the ratio of activities at 0 and 2 mM citrate is inversely proportional to the phosphate incorporated (correlation coefficient = -0.95), with 0.8 mol of P incorporated per mol of subunit decreasing the activity ratio from 0.5 to 0. This activity ratio method has an inherent internal control which makes it suitable for determining the level of protein-bound phosphate affecting the carboxylase activity in crude tissue extracts, and hence it should be useful for physiological studies. Tryptic maps of carboxylase labeled with radioactive phosphate by the carboxylase kinase indicate that the slightly less than 1 mol of P/mol of subunit is distributed equally between two peptides, whereas cAMP-dependent protein kinase phosphorylates these two sites and a third which may not affect activity.
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PMID:Phosphorylation state of acetyl-coenzyme A carboxylase. I. Linear inverse relationship to activity ratios at different citrate concentrations. 287 33

The meiotic maturation of Xenopus laevis oocytes is induced in vitro by progesterone which interacts at the cell surface level. A cell-free membrane preparation (P-10,000) incorporated 32P from [gamma-32P]ATP, mostly into two proteins, Mr approximately 56,000 and approximately 48,000 (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Progesterone, added in vitro, specifically inhibited the phosphorylation of the Mr approximately 48,000 protein (named p48). Half-maximal inhibition of p48 phosphorylation occurred with progesterone approximately 8 microM, in good correlation with hormone concentration inducing oocyte maturation. The effect was not due to stimulation of protein phosphatase activity. The potent maturation inducers testosterone and deoxycorticosterone also inhibited p48 phosphorylation, whereas biologically inactive steroids or cholesterol did not. p48 phosphorylation was not affected by cAMP, cGMP, polyamines, calmodulin, and phospholipids + diolein. EGTA had a stimulatory effect which was reversed by added Ca2+. The inhibitory effects of progesterone and Ca2+ were additive, suggesting two distinct sites of action. Phospho-p48 was not detected in yolk platelets, microsomes, and cytosol of oocytes. Contrary to p48 itself, the p48 kinase activity was loosely associated with P-10,000. Progesterone inhibited p48 phosphorylation produced by either cytosol or exogenous pure catalytic subunit of cAMP-dependent protein kinase. Conversely, phosphorylation of casein and histones by protein kinase activity present in P-10,000 was not modified by progesterone. It is then suggested that progesterone regulates p48 phosphorylation by affecting the protein substrate in the membrane, rather than by inhibiting the protein kinase enzyme itself. The data demonstrate a direct effect (not mediated by change of protein synthesis) of steroids on p48 phosphorylation in the plasma membrane, and they suggest that this protein could be implicated in the initial action of progesterone on oocyte maturation.
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PMID:Progesterone-inhibited phosphorylation of an unique Mr 48,000 protein in the plasma membrane of Xenopus laevis oocytes. 298 68

Calmodulin tryptic fragments 78-148, 107-148, and 1-77 coupled to Sepharose 4B were used to test the ability of different calmodulin-regulated enzymes to recognize different domains of calmodulin. Fragment 107-148, which contains a single Ca2+-binding domain, does not interact with any of the calmodulin binding proteins. Fragments 1-77 and 78-148, each of which contains two Ca2+-binding domains, have preserved their ability to interact with several calmodulin-dependent enzymes. Most of the calmodulin-regulated enzymes in brain extracts, such as cAMP phosphodiesterase, cAMP-dependent protein kinase, and the calmodulin-stimulated protein phosphatase (calcineurin) interact with fragment 78-148 in a Ca2+-dependent fashion. An ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-sensitive, calmodulin-independent, p-nitrophenyl phosphatase does not bind to the affinity column and is resolved from calcineurin at this step. Although calmodulin-stimulated protein kinase(s) can interact with fragment 78-148, their interaction is prevented by increased ionic strength even in the presence of Ca2+. Fragment 1-77 exhibits a higher degree of selectivity than fragment 78-148. Only cAMP-dependent protein kinase and cAMP phosphodiesterase bind to fragment 1-77. These results confirm the multiple modes of interaction of calmodulin with its target proteins and provide the basis for a selective purification of calmodulin-regulated enzymes by affinity chromatography on specific calmodulin fragments coupled to Sepharose.
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PMID:Selective affinity chromatography with calmodulin fragments coupled to sepharose. 298 37


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