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)

Previously, two forms of cAMP-dependent protein kinase catalytic subunit generated by mutually exclusive use of two internal exon cassettes (A1 and A2) were demonstrated in Aplysia neurons. Here, it is shown that there also exist catalytic subunits with alternative N termini derived from two exons, N1 and N2, expressed in combination with either of the internal cassettes. Processed transcripts including N1 or N2 sequences are of about equal abundance in the nervous system, arise through alternative promoter use, and encode catalytically active polypeptides. The N2 amino acid sequence is 21 residues longer than the N1 sequence and is homologous to the nonmyristoylated N terminus of the TPK1 gene product, a yeast catalytic subunit homolog. These data support the view that cAMP-dependent protein kinase activity in Aplysia neurons is produced by a complex array of regulatory and catalytic subunits that generate multiple holoenzymes with a spectrum of properties.
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PMID:Catalytic subunits of Aplysia neuronal cAMP-dependent protein kinase with two different N termini. 154 55

Evidence is presented that demonstrates that phosphatidylserine synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) from Saccharomyces cerevisiae is phosphorylated in vivo and in vitro by cAMP-dependent protein kinase. Phosphatidylserine synthase activity in cell extracts was reduced in the bcy1 mutant (which has high cAMP-dependent protein kinase activity) and elevated in the cyr1 mutant (which has low cAMP-dependent protein kinase activity) when compared with wild-type cells. The reduced phosphatidylserine synthase activity in the bcy1 mutant correlated with elevated levels of a phosphorylated form of the phosphatidylserine synthase Mr 23,000 subunit. The elevated phosphatidylserine synthase activity in the cyr1 mutant correlated with reduced levels of the phosphorylated form of the enzyme. There was negligible phosphorylation of the phosphatidylserine synthase Mr 23,000 subunit from stationary-phase cells. Pure phosphatidylserine synthase was phosphorylated by the cAMP-dependent protein kinase catalytic subunit, which resulted in a 60-70% reduction in phosphatidylserine synthase activity. The cAMP-dependent protein kinase catalytic subunit catalyzed the incorporation of 0.7 mol of phosphate per mol of phosphatidylserine synthase Mr 23,000 subunit. The specific cAMP-dependent protein kinase inhibitor prevented the phosphorylation of phosphatidylserine synthase and the inhibition of its activity by the catalytic subunit. Analysis of peptides derived from protease-treated labeled phosphatidylserine synthase showed only one labeled peptide. Phospho amino acid analysis of labeled phosphatidylserine synthase showed that the enzyme was phosphorylated at a serine residue.
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PMID:Phosphorylation of yeast phosphatidylserine synthase in vivo and in vitro by cyclic AMP-dependent protein kinase. 284 49

A peptide-based photoaffinity label for the catalytic subunit of the cAMP-dependent protein kinase was prepared from the amino acid p-benzoyl-L-phenylalanine [L-Phe(pBz)]. By using solid-phase peptide synthesis methodology, DL-Phe(pBz) was incorporated into the cAMP-dependent protein kinase substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly in place of the phosphorylatable serine. The diastereomeric peptides were separated by reverse-phase HPLC. The peptide substrate analog containing L-Phe(pBz) had a Ki of approximately 110 microM at pH 7.5. When photolyzed at 350 nm in the presence of the enzyme, this peptide caused time- and concentration-dependent inactivation. Radioactive acetylated L-Phe(pBz) peptide was used to establish the binding stoichiometry of peptide to enzyme; these results, together with protection experiments, showed the photoaffinity labeling to be specific (approximately 1:1). To identify the residues that were modified on the catalytic subunit, the photoinactivated enzyme was cleaved with CNBr and V8 protease (Staphylococcus aureus). The resulting peptide fragments were purified by HPLC and were sequenced; these experiments identified the modified residues as Gly-125 and Met-127. This region of the cAMP-dependent protein kinase catalytic subunit contains many residues that are conserved in serine- and tyrosine-protein kinases.
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PMID:Probing the peptide binding site of the cAMP-dependent protein kinase by using a peptide-based photoaffinity label. 339 99

A heat-stable protein inhibitor of cAMP-dependent protein kinase has been isolated from pig brain tissue. During gel filtration the protein is eluted in three peaks corresponding to the tetramer, dimer and monomer. The monomer fraction was purified 609-fold. The molecular mass of the monomeric form as determined by gel filtration and electrophoresis is equal to 11,000 Da and 8000 Da, respectively. The inhibition of the phosphotransferase reaction with respect to ATP occurs via a non-competitive mechanism, while that for histone--via a competitive mechanism. A formal kinetic analysis of various modes of the inhibitor binding to different protein kinase forms, e. g., the cAMP-dependent protein kinase catalytic subunit, protein kinase holoenzyme in the presence and absence of cAMP as well as of holoenzyme preparations modified by dimethylsuberimidate and cupric 1,10-phenanthroline, has been carried out. It was demonstrated that 3-4 inhibitor molecules are involved in the interaction with protein kinase.
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PMID:[A thermostable protein inhibitor of protein kinase from the swine brain. Isolation of the inhibitor and interaction with the enzyme]. 344 Jan 12

An attempt was made to activate the capillary-bound fraction of lipoprotein lipase (LPL) with cAMP-dependent protein kinase catalytic subunit (PKC). Following a 30s washout period, hearts were perfused for 1 min with buffer containing heparin. Medium was collected during the second 30s of heparin perfusion. Addition of PKC+Mg-ATP to this capillary bed perfusate increased LPL activity from 6.84 +/- 0.72 nmol/ml/min to 13.76 +/- 1.12 nmol/ml/min (P less than 0.001). A similar 2-fold increase in activity was observed when results were expressed on a mg protein basis. Removal of serum from, or addition of 1.0M NaCl to, the assay system inhibited PKC-stimulated LPL activity approximately 85%. These results indicate that capillary alkaline LPL can be activated by PKC assayed under experimental conditions free of other TG lipases. Moreover, these findings suggest that the intracellular fraction of LPL can be activated by cAMP and that this activation is mediated through protein phosphorylation by cAMP-dependent protein kinase.
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PMID:Protein kinase activation of heparin-releasable lipoprotein lipase in rat heart. 395 70

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is activated following phosphorylation by the cAMP-dependent protein kinase (largely by decreasing the Km of the enzyme for its pterin co-substrate). Following its phosphorylation activation in rat striatal homogenates, we find that tyrosine hydroxylase is inactivated by two distinct processes. Because cAMP is hydrolyzed in crude extracts by a phosphodiesterase, cAMP-dependent protein kinase activity declines following a single addition of cAMP. When tyrosine hydroxylase is activated under these transient phosphorylation conditions, inactivation is accompanied by a reversion of the activated kinetic form (low apparent Km for pterin co-substrate, less than or equal to 0.2 mM) to the kinetic form characteristic of the untreated enzyme (high apparent Km, greater than or equal to 1.0 mM). This inactivation is readily reversed by the subsequent addition of cAMP. When striatal tyrosine hydroxylase is activated under constant phosphorylation conditions (incubated with purified cAMP-dependent protein kinase catalytic subunit), however, it is also inactivated. This second inactivation process is irreversible and is characterized kinetically by a decreasing apparent Vmax with no change in the low apparent Km for pterin co-substrate (0.2 mM). The latter inactivation process is greatly attenuated by gel filtration which resolves a low-molecular-weight inactivating factor(s) from the tyrosine hydroxylase. These results are consistent with a regulatory mechanism for tyrosine hydroxylase involving two processes: in the first case, reversible phosphorylation and dephosphorylation and, in the second case, an irreversible loss of activity of the phosphorylated form of tyrosine hydroxylase.
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PMID:Tyrosine hydroxylase inactivation following cAMP-dependent phosphorylation activation. 613 15

Glycogen synthase I, purified from bovine heart, had a specific activity of 33 units/mg and gave a single band on sodium dodecyl sulfate gel electrophoresis with a subunit molecular weight of 86,000. The enzyme was phosphorylated with cAMP-dependent protein kinase catalytic subunit, also isolated from heart. With 10 microM ATP, only one phosphate group was incorporated per subunit of glycogen synthase. The phosphorylation decreased the per cent of glycogen synthase I from 0.95 to 0.50 when activity was determined by assays with Na2SO4 and glucose 6-phosphate. Glycogen synthase containing one phosphate per subunit was designated GS-1. One additional phosphate was incorporated per synthase subunit when ATP was increased to 0.5 mM and the percent glycogen synthase I decreased from 0.50 to < 0.05. This enzyme form was designated GS-1,2. Conversion of GS-1 to Gs-1,2 gave cooperative kinetics with ATP concentration and a half-maximal stimulation at approximately 40 microM. Phosphorylation of GS-1 could also be achieved by adding other non-substrate nucleotide triphosphates such as ITP and UTP along with 10 microM ATP. Glucose-6-P and Na2SO4 were without effect on this phosphorylation reaction. Two separate peptides were obtained after CNBr cleavage of 32P-labeled GS-1,2 and only one from GS-1. Both enzyme forms contained a single phosphorylated peptide in common. Thus, heart glycogen synthase may be phosphorylated specifically in either of two different sites using appropriate concentrations of ATP. ATP acts as a substrate for the protein kinase and also affects the availability of a second site to phosphorylation by cAMP-dependent protein kinase.
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PMID:Phosphorylation of heart glycogen synthase by cAMP-dependent protein kinase. Regulatory effects of ATP. 625 72

In order to investigate the structure of the active site of the cAMP-dependent protein kinase catalytic subunit a synthesis of several previously unknown adenosine-5'-triphosphate (ATP) derivatives containing substituents of various nature at N(1), N(C6) and C(8) positions of the purine base was carried out. The interaction of these derivatives with a homogeneous preparation of the catalytic subunit of rabbit skeletal muscle cAMP-dependent protein kinase was investigated. All the nucleotide analogs were found to inhibit the enzyme activity; the inhibition was competitive with respect to ATP. It was assumed that the adenine moiety of the ATP molecule is bound to the active site of protein kinase by the hydrophobic interaction with the aromatic amino acid residues and by formation of the hydrogen bond between the exo-NH2-group of the substrate and a corresponding group of the enzyme. The "correct" binding of ATP to the enzyme active center is defined by the anti-conformation of the nucleotide.
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PMID:[Interaction of N1-, N6- and C8-substituted derivatives of adenosine-5'-triphosphate with the catalytic subunit of cAMP-dependent protein kinase from rabbit skeletal muscles]. 629 13

Epidermal growth factor (EGF)-dependent transfer of radiolabeled phosphate from [gamma-32P]ATP to 160-kDa EGF receptor solubilized from human epidermoid carcinoma A431 cell surface membranes was stimulated up to 3-fold by addition of 3',5'-cAMP and purified cAMP-dependent protein kinase. Phosphorylation of EGF receptors was stimulated to the same extent when cAMP-dependent protein kinase catalytic subunit was substituted for 3',5'-cAMP and cAMP-dependent protein kinase. Phosphoamino acid analysis revealed that the extent of phosphorylation of EGF receptor at tyrosine residues was the same regardless of whether cAMP-dependent protein kinase catalytic subunit was present in or omitted from the system. Increased EGF receptor phosphorylation occurring in response to cAMP-dependent protein kinase catalytic subunit was accounted for by phosphorylation at serine or threonine residues. In samples phosphorylated in the presence of cAMP-dependent protein kinase catalytic subunit, phosphate was present in tyrosine, serine, and threonine in a ratio of 32:60:8. Two-dimensional mapping of radiolabeled phosphopeptides produced from EGF receptors by digestion with trypsin revealed the generation of one additional major phosphoserine-containing peptide when cAMP-dependent protein kinase was present with EGF in the EGF receptor kinase system. Degradation of 160-kDa EGF receptors to a 145-kDa form by purified Ca2+-activated neutral protease produced a 145-kDa fragment with phosphoserine content increased over that present initially in the 160-kDa precursor.
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PMID:cAMP-dependent protein kinase stimulates epidermal growth factor-dependent phosphorylation of epidermal growth factor receptors. 632 45

The Ca2+ channel subunits alpha 1C-a and alpha 1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba2+ current (IBa) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 microM cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 microM PKA and 1 microM okadaic acid. The activity of the alpha 1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 microM H 89 and was not increased by superfusion with 5 microM forskolin plus 20 microM isobutyl-methylxanthine (IBMX). The alpha 1C-a.beta 2.alpha 2/delta complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 microM H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the alpha 1C-a.beta 2.alpha 2/delta channel with 10 microM PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca2+ current (ICa) of cardiac myocytes increased threefold during internal dialysis with 5 microM PKA or 25 microM microcystin and during external superfusion with 0.1 microM isoproterenol or 5 microM forskolin plus 50 microM IBMX. These results indicate that the L-type Ca2+ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.
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PMID:On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation. 749 Dec 57


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