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)

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

p34 kinase, the product of the CDC2 gene, is a cell-cycle regulated protein kinase that is most active during mitosis. In HeLa cells, p34 kinase has previously been shown to exist in both a low- and a high-molecular-mass form, the latter of which is only found in cells in the G2/M phase of the cell cycle and contains a 62-kDa subunit. Here we show that although each form of the kinase phosphorylates casein in vitro, only the high-molecular-mass form uses histone H1 as substrate. The high-molecular-mass form of p34 kinase from nocodazole-treated HeLa cells was purified 6700-fold. The apparent molecular mass of the mitotic CDC2-encoded protein kinase complex was 220 kDa. The purified enzyme phosphorylated not only its endogenous 62-kDa subunit but also phosphorylated histone H1 with a Km of 3 microM and used ATP 40 times more efficiently than GTP (Km 54 microM and 2 mM, respectively). The enzyme activity was unaffected by cAMP, calcium/calmodulin, or by the heat-stable inhibitor of cAMP-dependent protein kinase. These characteristics are typical of growth-associated histone H1 kinase from different organisms. These results suggest that CDC2 protein may be activated as an M-phase-specific protein kinase in part by its association with the p62 subunit.
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PMID:Activation of human CDC2 protein as a histone H1 kinase is associated with complex formation with the p62 subunit. 254 71

The molecular basis of opioid receptor mechanisms was studied in reconstitution experiments using purified or membrane-bound opioid receptors and purified GTP-binding proteins (G-proteins). mu-Opioid receptor exclusively purified from rat brains was reconstituted with G-proteins in lipid vesicles. The mu-agonist stimulated the G-protein activity in both G1 or Go-reconstituted vesicles. The stoichiometry revealed that one molecule of mu-receptor is functionally coupled to plural numbers of Gi or Go molecules and that mu-receptor exists in at least two different subtypes, mu i and mu o, separately coupled to Gi and Go, respectively. In addition, when the mu-receptor was phosphorylated by cAMP-dependent protein kinase, the mu-agonist-stimulation of G-protein activity disappeared, while the guanine nucleotide-sensitivity of agonist binding was unchanged. These findings suggest that there are independent domains in the receptor which are related to functional coupling to G-protein and to the agonist-binding modulation by G-protein. kappa-Opioid receptor agonist inhibited the G-protein activity in guinea pig cerebellar membranes. Further experiments revealed that the kappa-opioid receptor is functionally coupled to an inhibition of phospholipase C activity via an inhibition of Gi-activity. Such a receptor-mediated inhibition of G-protein activity may be the first demonstration of a signal transduction mechanism. The delta-opioid receptor agonist showed no effect on G-protein activity in guinea pig striatal and rat cortical membranes, while it stimulated it in NG108-15 cells. In all these membranes, the delta-agonist binding was markedly reduced by GTP gamma S in the presence of MgCl2. These findings suggest that delta-receptors in the brain might be coupled to G-protein without signal transduction.
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PMID:[Molecular pharmacology of opioid receptor mechanisms]. 255 62

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

The phosphorylation of DNA topoisomerase II in Drosophila Kc tissue culture cells was characterized by in vivo labeling studies and in vitro studies that examined the modification of exogenous enzyme in total homogenates of these embryonic cells. Several lines of evidence identified casein kinase II as the kinase primarily responsible for phosphorylating DNA topoisomerase II. First, the only amino acyl residue modified in the enzyme was serine. Second, partial proteolytic maps of topoisomerase II which had been labeled with [32P]phosphate by Drosophila cells in vivo, by cell homogenates in vitro, or by purified casein kinase II were indistinguishable from one another. Third, phosphorylation in cell homogenates was inhibited by micrograms/ml concentrations of heparin, micromolar concentrations of nonradioactive GTP, or anti-Drosophila casein kinase II antiserum. Fourth, cell homogenates were able to employ [gamma-32P]GTP as a phosphate donor nearly as well as [gamma-32P]ATP. Although topoisomerase II was phosphorylated in homogenates under conditions that specifically stimulate protein kinase C, calcium/calmodulin-dependent protein kinase, or cAMP-dependent protein kinase, modification was always sensitive to anti-casein kinase II antiserum or heparin. Thus, under a variety of conditions, topoisomerase II appears to be phosphorylated primarily by casein kinase II in the Drosophila embryonic Kc cell system.
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PMID:Phosphorylation of DNA topoisomerase II in vivo and in total homogenates of Drosophila Kc cells. The role of casein kinase II. 284 38

A study has been made of the action of 5-hydroxytryptamine (5-HT) on the radio-sodium efflux from single barnacle muscle fibres. (i) Stimulation of the Na efflux by external application of 5-HT is seen in both unpoisoned and ouabain-poisoned fibres. (ii) Concentrations of 5-HT as low as 10(-9)M are effective. (iii) Characteristically, the response to 5-HT is prompt in onset, reaches a peak within 20 min and then decays rather rapidly. Fibres from certain barnacle specimens are sometimes unresponsive to 5-HT. Such fibres, however, can be rendered responsive by preinjecting into them the non-hydrolysable GTP analogue, Gpp(NH)p. The response of the ouabain-insensitive Na efflux to 5-HT depends on external Ca2+ and, to a certain extent, on external Na+. (i) The response to 5-HT is unaffected by prior external application of Ca2+ antagonists, viz. verapamil, Cd2+ and WB-4101. (ii) The calmodulin antagonist, trifluoperazine (10(-5)M), completely abolishes the response to 5-HT, even in fibres preinjected with Gpp(NH)p. (iii) Diphenylhydantoin is less effective than trifluoperazine (TFP). Whereas the receptor antagonist methysergide is ineffective, cyproheptadine is very effective. (i) Prior application of the phosphodiesterase inhibitor 1-propyl-3-methyl-7-(5-hydroxyhexyl)-xanthine (PMX) or the inhibitor 1-isoamyl-3-isobutyl-xanthine (IAX) augments the size of the response to 5-HT, but fails to stop the response from decaying. (ii) Augmentation of the response to 5-HT by IAX is seen despite the presence of 10(-5) M-TFP. Prior injection of Mg2+ or protein kinase inhibitor (PKI) leads to abolition or reduction of the response to 5-HT. These results demonstrate that barnacle fibres are a useful preparation for investigation the natriferic action of 5-HT. They also support the view that the response to 5-HT involves a receptor-adenylate cyclase complex and is the result of activation by newly formed cAMP of cAMP-dependent protein kinase.
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PMID:The modulatory action of 5-hydroxytryptamine on sodium efflux: the barnacle muscle fibre as a model system. 286 Oct 30

The photoaffinity analog [32P]8-N3 cAMP (8-azido adenosine 3',5'-monophosphate was used to analyze the membrane sidedness of rat sperm cAMP binding proteins during epididymal maturation. Evidence is presented here which supports the hypothesis that 35-45% of the regulatory subunits of the Type I and Type II cAMP-dependent protein kinases are readily available to externally added cyclic nucleotide. It was observed by sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) and autoradiography that only two rat sperm proteins (Mr = 49K and 55K) were photolabeled which comigrated on gels with partially purified Type I and Type II regulatory subunits, respectively. Both of these photolabeled epididymal sperm proteins were saturated at physiological titers of [32P]8-N3cAMP and photoincorporation of [32P]8-N3 cAMP was specific since other SDS-resolvable sperm proteins did not photoincorporate the analog. Caput and cauda sperm protein photoincorporation could be effectively blocked by low levels of cAMP, but not by cGMP, ATP or GTP. Sperm epididymal maturation coincided with changes in the cAMP-dependent protein kinase subunits since cauda sperm contained more available Type II than did caput sperm. A subcellular analysis of cAMP-dependent protein kinase regulatory subunit in head and tail fractions was done for caput and cauda sperm and demonstrated that the tail fractions showed more photo-labeling of both Type I and II regulatory subunits than did the head fractions.
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PMID:A study of rat epididymal sperm adenosine 3',5'-monophosphate-dependent protein kinases: maturation differences and cellular location. 298 32

Preincubation of pigeon erythrocyte plasma membranes with the catalytic subunit of cAMP-dependent protein kinase results in the desensitization of erythrocyte adenylate cyclase. The adenylate cyclase activity measured in the presence of 10 microM isoproterenol and 50 microM GTP-gamma-S decreases by 40% after 10 min incubation; that in the presence of 50 microM GTP-gamma-S by 35% (20 min). The decrease of the adenylate cyclase activity is due to the prolongation of the lag phase of the enzyme activation in the presence of a hydrolysis-resistant GTP analog and to the drop in activity in the steady state of the activation. The heterologous desensitization of adenylate cyclase induced by cAMP-dependent protein kinase is also coupled with the decrease of the number of beta-adrenoreceptors capable of acquiring a high affinity for the agonists in the absence of guanyl nucleotides. The effect of the catalytic subunit on adenylate cyclase is fully compatible with the process of the enzyme desensitization in erythrocytes treated with isoproterenol or cAMP.
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PMID:[Heterologous desensitization of pigeon erythrocyte adenylate cyclase by the catalytic subunit of cAMP-dependent protein kinase]. 298 33

Sequences termed v-abl, which encode the protein-tyrosine kinase activity of Abelson murine leukemia virus, have been expressed in Escherichia coli as a fusion product (ptabl50 kinase). This fusion protein contains 80 amino acids of SV40 small t and the 403 amino acid protein kinase domain of v-abl. We report here the purification and characterization of this kinase. The purified material contains two proteins (Mr = 59,800 and 57,200), both of which possess sequences derived from v-abl. Overall purification was 3,750-fold, with a 31% yield, such that 117 micrograms of kinase could be obtained from 40 g of E. coli within 6-7 days. The specific kinase activity is over 170 mumol of phosphate min-1 mumol-1, comparable to the most active protein-serine kinases. Kinase activity is insensitive to K+, Na+, Ca2+, Ca2+-calmodulin, cAMP, or cAMP-dependent protein kinase inhibitor. The Km for ATP is dependent on the concentration of the second substrate. GTP can also be used as a phosphate donor. The enzyme can phosphorylate peptides consisting of as few as two amino acids and, at a very low rate, free tyrosine. Incubation of the kinase with [gamma-32P]ATP results in incorporation of 1.0 mol of phosphate/mol of protein. This reaction, however, cannot be blocked by prior incubation with unlabeled ATP. Incubation of 32P-labeled kinase with either ADP or ATP results in the synthesis of [32P]ATP. This suggests the phosphotyrosine residue on the Abelson kinase contains a high energy phosphate bond.
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PMID:Purification and characterization of a protein-tyrosine kinase encoded by the Abelson murine leukemia virus. 298 75

We have shown by gel filtration on Sepharose 4B at low ionic strength that casein kinases S (type 1), heparin-insensitive, and TS (type 2), heparin-inhibited, of rat liver cytosol participate in two distinct multimolecular systems, Ve/Vo = 1.25 and Ve/Vo = 1.90, respectively, both less retarded than the peak of cAMP-dependent protein kinase activity (Ve/Vo = 2.04). Both casein kinase I and casein kinase II complexes are unstable in 0.5 M NaCl, giving rise by gel filtration under these conditions to the free forms of casein kinase S (Ve/Vo = 2.37, Mr 34 000) and casein kinase TS (Ve/Vo = 2.10, Mr 130 000), respectively. In contrast, the elution volume of cAMP-dependent protein kinase activity is always the same irrespective of the ionic strength of the medium. Casein kinase I, accounting for the whole casein kinase S activity of cytosol, also contains a phosphorylatable 31-kDa protein (p31) which is a substrate of casein kinase S, since its phosphorylation is insensitive to heparin, the heat-stable inhibitor and trifluoperazine, but it is prevented by beryllium. Casein kinase II, on the other hand, apparently results from the association of the whole casein kinase TS (type 2) of rat liver cytosol with a 90-kDa protein substrate (p90) which is distinct from glycogen synthase according to their different peptide mappings. The radiolabelling of p90 is inhibited by heparin, unlabeled GTP and polyglutamates, while it is dramatically and specifically enhanced by polylysine. At least three more protein bands of Mr 58 000, 52 000 and 37 000 are phosphorylated by casein kinase TS in the casein kinase II fraction: their co-elution with casein kinase TS, however, seems to be accidental and their radiolabeling in the presence of polylysine is almost negligible compared to that of p90. It is concluded that p31 and p90 may represent specific targets of casein kinase S and casein kinase TS, respectively, whose intimate association with the enzymes could be functionally significant.
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PMID:Casein kinases and their protein substrates in rat liver cytosol: evidence for their participation in multimolecular systems. 299 5


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