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 minimal structure in the heat-stable inhibitor protein of cAMP-dependent protein kinase required for a low nanomolar potency of inhibition is the peptide Thr6-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-+ ++Ile22-NH2 (PKI-(6-22)-amide). While primary structural determinants for interaction with the protein kinase are distributed throughout the 17 residues of this peptide, we have previously shown that phenylalanine 10 in the NH2-terminal portion is a particularly important determinant for high affinity binding (Glass, D. B., Cheng, H.-C., Mende-Mueller, L., Reed, J., and Walsh, D. A. (1989) J. Biol. Chem. 264, 8802-8810). To investigate this requirement further, peptide analogs of PKI-(6-22)-amide in which various natural and nonstandard amino acids are substituted for phenylalanine 10 have been synthesized and tested for inhibitory potency against the catalytic subunit of the protein kinase. Consistent with the importance of the hydrophobicity of phenylalanine, an alanine 10 substitution analog exhibited a 270-fold decrease in inhibitory potency, whereas the leucine 10 analog lost only 33-fold in activity as compared to the parent peptide PKI-(6-22)-amide. Peptides containing the spatial conformation analogs D-phenylalanine, homophenylalanine, or phenylglycine were 60-120-fold less potent than the parent peptide. Peptides containing various para-substituted phenylalanines at position 10 were only 5-11-fold less potent. One exception to this was (4'-azidophenylalanine 10)PKI-(6-22)-amide, which was nearly equipotent with the parent inhibitor. The most potent analogs were those peptides containing highly aromatic residues at position 10. The 2'-thienylalanine 10, tryptophan (formyl) 10, tryptophan 10, and the 1'-naphthylalanine 10 analogs were 3-fold less potent, equipotent, slightly more potent, and 4-fold more potent than the parent peptide inhibitor, respectively. We conclude that phenylalanine 10 in PKI-(6-22)-amide, and presumably in the native protein inhibitor, interacts through specific hydrophobic and/or aromatic binding to a hydrophobic pocket or cleft near the active site of the protein kinase.
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PMID:Protein kinase inhibitor-(6-22)-amide peptide analogs with standard and nonstandard amino acid substitutions for phenylalanine 10. Inhibition of cAMP-dependent protein kinase. 276 75

Four mutants with amino acid substitution(s) at or near the putative phosphorylation site (Arg142 Arg143 Thr144 Ser145) of the regulatory subunit of cAMP-dependent protein kinase were obtained by site-directed mutagenesis. Three mutants, BCY1A1a145 (Ser145 to Ala), BCY1His143 (Arg143 to His) and BCY1Asn144, Ala145 (Thr144 to Asn and Ser145 to Ala) complemented a bcy1 mutant, whereas BCY1Gly143 (Arg143 to Gly) did not. In addition, mutant, BCY1Asn144, Ala145 exhibited a dominant cold-sensitive phenotype, which can be most easily explained by the functional alteration of the regulatory subunit of cAMP-dependent protein kinase by the mutations. Analyses of these mutant genes revealed that phosphorylation of the regulatory subunit is not a prerequisite for the regulation of the cAMP-dependent protein kinase activity in responding to the cAMP level.
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PMID:Mutant regulatory subunit of 3',5'-cAMP-dependent protein kinase of yeast Saccharomyces cerevisiae. 282 90

A combination of metabolic labeling and chemical or enzymatic modification was employed to isolate and biochemically characterize a set of glycosyl-phosphatidylinositol (gly-PI) molecules synthesized by T lymphocytes. Gly-PI displayed unique patterns of synthesis following mitogen activation relative to the phosphoinositides and major structural lipids. The increase with time in gly-PI was paralleled by the appearance of insulin receptors. Gly-PI molecules were sensitive to hydrolysis by a PI-specific phospholipase C and were rapidly (15 sec) degraded in response to insulin binding. The product of this hydrolysis is believed to be a novel inositol phosphate-glycan (IP-gly) that was shown to inhibit the activity of a cAMP-dependent protein kinase. These results demonstrate that T cells contain a structurally related set of gly-PI molecules, at least one of which is sensitive to insulin and may function as a second messenger of hormone action.
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PMID:Regulation and function of an insulin-sensitive glycosyl-phosphatidylinositol during T lymphocyte activation. 283 76

The type I regulatory subunit (R-I) of rat brain cAMP-dependent protein kinase was expressed in E. coli and site-directed mutagenesis was used to substitute amino acids in the putative cAMP-binding sites. The wild-type recombinant R-I bound 2 mol of cAMP/mol subunit, while two mutant R-Is with a single amino acid substitution in one of the two intrachain cAMP-binding sites (clone N153:a glutamate for Gly-200, and clone C254:an aspartate for Gly-324) bound 1 mol of cAMP/mol subunit. When these two substitutions were made in one mutant, cAMP did not bind to this mutant, indicating that binding of cAMP to N153 or C254 was to their nonmutated sites. Competition experiments with site-selective analogs and dissociation of bound cAMP from mutant R-Is provided evidence for strong intrachain interactions between the two classes of cAMP-binding sites in R-I.
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PMID:Site-directed mutagenesis of the cAMP-binding sites of the recombinant type I regulatory subunit of cAMP-dependent protein kinase. 283 71

cGMP-dependent protein kinase contains four cGMP-binding sites which are homologous to the four cAMP-binding sites of cAMP-dependent protein kinase. The interaction of the diastereomers of adenosine 3',5'-thionophosphate, (PS)-cAMP[S] and (PR)-cAMP[S], with cGMP-dependent protein kinase has been studied. Autophosphorylation of cGMP-dependent protein kinase is stimulated by cAMP and (PS)-cAMP[S] with apparent KA values of 7 microM and 94 microM, respectively. cAMP-stimulated autophosphorylation is inhibited competitively by (PR)-cAMP[S] with a Ki value of 15 microM. The phosphorylation of the peptide substrate (Leu-Arg-Arg-Ala-Ser-Leu-Gly) is stimulated by cGMP (approx. KA 1 microM) and cAMP (approx. KA 98 microM) but neither by the (PR) nor (PS) stereoisomer of cAMP[S]. (PR)-cAMP[S] and (PS)-cAMP[S] inhibit competitively cAMP-or cGMP-stimulated phosphorylation of the peptide substrate with Ki values of 52 microM and 73 microM, respectively. (PS)-cAMP[S] stimulates the phosphorylation of the peptide substrate by an autophosphorylated enzyme. Binding of [3H]cGMP to cGMP-dependent protein kinase is inhibited by (PS)-cAMP[S] and (PR)-cAMP[S] with IC50 values of 200 microM and 15 microM, respectively. These results show that both diastereomers of cAMP[S] bind to cGMP-dependent protein kinase. (PR)-cAMP[S] has properties of a pure antagonist whereas (PS)-cAMP[S] has properties of a partial agonist. The results provide further evidence that autophosphorylation of the enzyme affects the interaction between the cGMP-binding sites and the catalytic center of the enzyme by facilitating the activation of the phosphotransferase reaction.
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PMID:Diastereomers of adenosine 3',5'-monothionophosphate (cAMP[S]) antagonize the activation of cGMP-dependent protein kinase. 299 Sep 28

Release of surfactant from pulmonary type II epithelial cells was stimulated by the beta-adrenergic agonist terbutaline and the diterpene forskolin. Cytosolic cyclic adenosine monophosphate (cAMP) concentrations increased significantly following exposure to terbutaline or forskolin and reached maximal levels within 5 min after treatment. Terbutaline and forskolin had a synergistic effect on cytosolic cAMP levels when added simultaneously. cAMP-dependent protein kinase activity was identified in cytosolic preparations of type II pneumocytes by phosphorylation of the peptide substrate Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) and binding of 3H-cAMP to the regulatory components of cAMP-dependent protein kinase. Type I and type II regulatory subunits of the cANP-dependent kinase were present in approximately equal concentrations in type II cell cytosol. Activation ratio of cAMP-dependent protein kinase in cultured type II cells increased significantly in the presence of terbutaline, forskolin, or terbutaline plus forskolin. Activation ratios increased from 0.45 +/- 0.03 for control cells to 0.96 +/- 0.06 for cells exposed to terbutaline (10 microM) plus forskolin (5 microM) for 20 min. Release of 3H-phosphatidylcholine was also stimulated by terbutaline and forskolin. Effects of terbutaline and forskolin on surfactant release were approximately additive. Our results demonstrated increased cytosolic cAMP levels, increased cAMP-dependent protein kinase activation ratios, and subsequent augmented surfactant release from isolated type II pneumocytes in response to terbutaline and forskolin. These data support a role for activation of cAMP-dependent protein kinase as a mediator of surfactant release and document the utility of forskolin for study of cAMP-mediated effects in isolated type II cells.
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PMID:Activation of cAMP dependent protein kinase during surfactant release from type II pneumocytes. 299 42

The specificities of cAMP-dependent and cGMP-dependent protein kinases were studied using synthetic peptides corresponding to the phosphorylation site in 6-phosphofructo-2-kinase/Fru-2,6-P2ase (Murray, K.J., El-Maghrabi, M.R., Kountz, P.D., Lukas, T.J., Soderling, T.R., and Pilkis, S.J. (1984) J. Biol. Chem. 259, 7673-7681) as substrates. The peptide Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser-Ser-Ile-Pro-Gln was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase on predominantly the first of its 2 seryl residues. The Km (4 microM) and Vmax (14 mumol/min/mg) values were comparable to those for the phosphorylation of this site within native 6-phosphofructo-2-kinase/Fru-2,6-P2ase. An analog peptide containing only two arginines was phosphorylated with poorer kinetic constants than was the parent peptide. These results suggest that the amino acid sequence at its site of phosphorylation is a major determinant that makes 6-phosphofructo-2-kinase/Fru-2,6-P2ase an excellent substrate for cAMP-dependent protein kinase. Although 6-phosphofructo-2-kinase/Fru-2,6-P2ase was not phosphorylated by cGMP-dependent protein kinase, the synthetic peptide corresponding to the cAMP-dependent phosphorylation site was a relatively good substrate (Km = 33 microM, Vmax = 1 mumol/min/mg). Thus, structures other than the primary sequence at the phosphorylation site must be responsible for the inability of cGMP-dependent protein kinase to phosphorylate native 6-phosphofructo-2-kinase/Fru-2,6-P2ase. Peptides containing either a -Ser-Ser- or -Thr-Ser- moiety were all phosphorylated by cGMP-dependent kinase to 1.0 mol of phosphate/mol of peptide, but the phosphate was distributed between the two hydroxyamino acids. Substitution of a proline in place of the glycine between the three arginines and these phosphorylatable amino acids caused the protein kinase selectively to phosphorylate the threonyl or first seryl residue and also enhanced the Vmax values by 4-6-fold. These results are consistent with a role for proline in allowing an adjacent threonyl residue to be readily phosphorylated by cGMP-dependent protein kinase.
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PMID:Synthetic peptides corresponding to the site phosphorylated in 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as substrates of cyclic nucleotide-dependent protein kinases. 300 75

Fructose-1,6-bisphosphatase from the yeast Saccharomyces cerevisiae has properties similar to other gluconeogenic fructose-1,6-bisphosphatases, but an unusual characteristic of the yeast enzyme is that it can be phosphorylated in vitro by cAMP-dependent protein kinase. Phosphorylation also occurs in vivo, presumably as part of a signalling mechanism for the enzyme's degradation. To probe the structural basis for the phosphorylation of yeast fructose-1,6-bisphosphatase, we have developed an improved procedure for the purification of the enzyme and then performed sequence studies with the in vitro-phosphorylated protein as well as with tryptic and chymotryptic peptides containing the phosphorylation site. As a result of these studies, we have determined that yeast fructose-1,6-bisphosphatase has the following 24-residue NH2-terminal amino acid sequence: Pro-Thr-Leu-Val-Asn-Gly-Pro-Arg-Arg-Asp-Ser-Thr-Glu-Gly- Phe-Asp-Thr-Asp-Ile-Ile-Thr-Leu-Pro-Arg. The site of phosphorylation is located at Ser-11 in the above sequence. The amino acid sequence around the site of phosphorylation contains the sequence - Arg-Arg-X-Ser- associated with many of the better substrates of cAMP-dependent protein kinase. The sequence of residues 15-24 above is highly homologous with the sequence of residues 6-15 of pig kidney fructose-1,6-bisphosphatase, showing 7 out of 10 residues in identical positions. The yeast enzyme, however, has a dissimilar NH2-terminal region which extends beyond the NH2 terminus of mammalian fructose-1,6-bisphosphatases and contains a unique phosphorylation site.
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PMID:Amino acid sequence of the phosphorylation site of yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase. 300 13

The amino acid sequences surrounding three major phosphorylation sites in rat and bovine synapsin I have been determined by employing automated gas-phase sequencing and manual Edman degradation of purified phosphopeptide fragments. Site 1 is a serine residue phosphorylated by cAMP-dependent protein kinase and by calcium/calmodulin-dependent protein kinase I. The sequence around site 1 was derived from tryptic/chymotryptic phosphopeptides and overlapping cyanogen bromide cleavage fragments. This sequence, identical in rat and bovine synapsin I, is Asn-Tyr-Leu-Arg-Arg-Arg-Leu-Ser(P)-Asp-Ser-Asn-Phe-Met. Site 1 is located at the NH2 terminus of the protein, within the collagenase-resistant head region. Sites 2 and 3 are serine residues phosphorylated by calcium/calmodulin-dependent protein kinase II. The sequences surrounding bovine site 2 and site 3 were derived from tryptic phosphopeptides and overlapping fragments generated by cleavage with chymotrypsin, collagenase, and endoproteinase Lys-C. The sequence around bovine site 2 is Thr-Arg-Gln-Thr-Ser(P)-Val-Ser-Gly-Gln-Ala-Pro-Pro-Lys, and the sequence around bovine site 3 is Thr-Arg-Gln-Ala-Ser(P)-Gln-Ala-Gly-Pro-Met-Pro-Arg. Sites 2 and 3 are located within the COOH-terminal, collagenase-sensitive tail region of the molecule, separated by 36 amino acids. The sequences surrounding rat site 2 and site 3 were derived from tryptic phosphopeptides. The sequence around rat site 2 is Gln-Ala-Ser(P)-Ile-Ser-Gly-Pro-Ala-Pro-Pro-Lys, and the sequence around rat site 3 is Gln-Ala-Ser(P)-Gln-Ala-Gly-Pro-Gly-Pro-Arg. Thus, the sequences surrounding the four sites that are phosphorylated by calcium/calmodulin-dependent protein kinase II, namely sites 2 and 3 in rat and bovine synapsin I, exhibit a high degree of homology.
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PMID:Amino acid sequences surrounding the cAMP-dependent and calcium/calmodulin-dependent phosphorylation sites in rat and bovine synapsin I. 311 71

Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) is a good substrate for cholera toxin in comparison with the angiotensin peptides. Because kemptide contains two potential ADP-ribosylation sites and, is also a good substrate for cAMP-dependent protein kinase, it was possible to gain some insight into factors influencing the specificity of cholera toxin and to study the relationship between phosphorylation and ADP-ribosylation. The ADP-ribosylated products of kemptide were purified by high-performance liquid chromatography and characterized by peptide sequence analysis, trypsin digestion, and fast-atom bombardment mass spectrometry. The major product is mono(ADP-ribosyl)ated preferentially on the first arginyl residue and some mono(ADP-ribosyl)ation was observed to occur on the second arginine. The minor product is di(ADP-ribosyl)ated. The Km and Vmax for mono(ADP-ribosyl)ation of kemptide are approximately 4.3 +/- 1.2 mM and 38.1 +/- 5.5 nmol min-1 mg-1, respectively. Phosphorylated seryl residue of kemptide suppresses ADP-ribosylation of the arginyl residues by cholera toxin. Mono(ADP-ribosyl)ated kemptide is a poor substrate for the cAMP-dependent protein kinase in comparison with kemptide. Di(ADP-ribosyl)ated kemptide is not phosphorylated at all. These results suggest that a mere exposure of an arginyl residue in peptides is not a sufficient condition for effective ADP-ribosylation and that a relationship exists between ADP-ribosylation and phosphorylation.
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PMID:Relationship of phosphorylation and ADP-ribosylation using a synthetic peptide as a model substrate. 312 96


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