Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.12 (PKG)
 2,515 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bovine lung cGMP-binding cGMP-specific phosphodiesterase (cG-BPDE) is a potent and relatively specific substrate for cGMP-dependent protein kinase (cGK) as compared to cAMP-dependent protein kinase (cAK) (Thomas, M. K., Francis, S. H., and Corbin, J. D. (1990) J. Biol. Chem. 265, 14971-14978). A synthetic peptide, RKISASEFDRPLR (BPDEtide), was synthesized corresponding to the sequence surrounding the phosphorylation site in cG-BPDE. BPDEtide retained the cGK/cAK kinase specificity demonstrated by native cG-BPDE: the apparent Km of BPDEtide for cGK was 5-fold lower than that for cAK (Km = 68 and 320 microM, respectively). Vmax values were 11 mumol/min/mg for cGK and 3.2 mumol/min/mg for cAK. The peptide was not phosphorylated to a measurable extent by protein kinase C or by calcium/calmodulin-dependent protein kinase II. Thus, the primary amino acid sequence of the peptide substrate was sufficient to confer kinase specificity. Studies in crude tissue extracts indicated that BPDEtide was the most selective peptide substrate documented for measuring cGK activity. Peptide analogs of BPDEtide were synthesized to determine the contribution of specific residues to cGK or cAK substrate specificity. Substitution of a Lys for the amino-terminal Arg did not reduce cGK/cAK specificity; neither did the exchange of an Ala for the non-phosphorylated Ser nor the removal of the 3 carboxyl-terminal residues. A truncated BPDEtide (RKISASE) served equally well as substrate (Km approximately 90 microM) for both kinases. However, restoration of the Phe, to yield RKISASEF, reproduced the original cGK/cAK specificity for BPDEtide (Km = 120 and 480 microM, respectively), primarily by decreasing the affinity of cAK. Addition of a carboxyl-terminal Phe to the peptide RKRSRAE (derived from the sequence of the cGK phosphorylation site in histone H2B) or to the peptide LRRASLG (derived from the sequence of the cAK phosphorylation site in pyruvate kinase) also improved the cGK/cAK specificity by decreasing the affinity of cAK. These data suggested that the Phe in each substrate tested is a negative determinant for cAK.
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PMID:A phenylalanine in peptide substrates provides for selectivity between cGMP- and cAMP-dependent protein kinases. 131 60

Analogues of a synthetic heptapeptide substrate corresponding to the sequence around a phosphorylation site in histone H2B [Glass, D. B. & Krebs, E. G. (1982) J. Biol. Chem. 257, 1196-1200] were used to assess interactions between the peptide substrate and the ATP binding sites of cGMP-dependent protein kinase and the catalytic subunit of cAMP-dependent protein kinase. The affinity of each protein kinase for lin-benzo-ADP was determined in the absence and presence of substrate peptide by fluorescence anisotropy titrations [Bhatnagar, D., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 6310-6317]. The Kd values of cGMP-dependent protein kinase for lin-benzo-ADP in the absence and presence of cGMP were 7.6 and 9.7 microM, respectively. Histone H2B(29-35) (Arg-Lys-Arg-Ser-Arg-Lys-Glu) had no effect on nucleotide affinity in either the absence or presence of cGMP. However, when lysine-34 located two residues after the phosphorylatable serine is replaced with an alanyl residue, the resulting [Ala34]histone H2B(29-35) and its analogue peptides interact with cGMP-dependent protein kinase and/or the nucleotide in a fashion that decreases nucleotide binding affinity approximately 3-fold. This amino acid replacement had previously been shown to cause an increase in Vmax and a decrease in the pH optimum for the phosphotransferase reaction. Replacement of positively charged residues at positions 30 and 31 of the peptide also decreased nucleotide affinity. Other analogues of histone H2B(29-35) failed to affect binding of lin-benzo-ADP to the active site of the cGMP-dependent enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Synthetic peptide analogues differentially alter the binding affinities of cyclic nucleotide dependent protein kinases for nucleotide substrates. 283 78

Using the activated cGMP-dependent protein kinase in the presence of the phosphorylatable peptide [[Ala34]histone H2B-(29-35)], we found that lin-benzoadenosine 5'-diphosphate (lin-benzo-ADP) was a competitive inhibitor of the enzyme with respect to ATP with a Ki (22 microM) similar to the Kd (20 microM) determined by fluorescence polarization titrations. The Kd for lin-benzo-ADP determined in the absence of the phosphorylatable peptide, however, was only 12 microM. ADP bound with lower affinity (Ki = 169 microM; Kd = 114 microM). With [Ala34]histone H2B-(29-35) as phosphoryl acceptor, the Km for lin-benzo-ATP was 29 microM, and that for ATP was 32 microM. The Vmax with lin-benzo-ATP, however, was only 0.06% of that with ATP as substrate [0.00623 +/- 0.00035 vs. 11.1 +/- 0.17 mumol (min.mg)-1]. Binding of lin-benzo-ADP to the kinase was dependent upon a divalent cation. Fluorescence polarization revealed that Mg2+, Mn2+, Co2+, Ni2+, Ca2+, Sr2+, and Ba2+ supported nucleotide binding to the enzyme; Ca2+, Sr2+, and Ba2+, however, did not support any measurable phosphotransferase activity. The rank order of metal ion effectiveness in mediating phosphotransferase activity was Mg2+ greater than Ni2+ greater than Co2+ greater than Mn2+. Although these results were similar to those observed with the cAMP-dependent protein kinase [Hartl, F. T., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 2347], major differences in the Vmax with lin-benzo-ATP as substrate and the effect of peptide substrates on nucleotide (both lin-benzo-ADP and ADP) binding were observed.
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PMID:Interaction of guanosine cyclic 3',5'-phosphate dependent protein kinase with lin-benzoadenine nucleotides. 300 44

Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein of cAMP-dependent protein kinase (Cheng, H.-C., Kemp, B. E., Pearson, R. B., Smith, A. J., Misconi, L., Van Patten, S. M., and Walsh, D. A. (1986) J. Biol. Chem. 261, 989-992) were tested as inhibitors of cGMP-dependent protein kinase. The peptides themselves were not substrates. cGMP-dependent protein kinase activity was assayed using histone H2B and two synthetic peptide substrates. Consistent with previous observations of other peptide inhibitors of this enzyme (Glass, D. B. (1983) Biochem. J. 213, 159-164), the inhibitory peptides had no effect on the phosphorylation of histone H2B, but they competitively inhibited cGMP-dependent phosphorylation of the two peptide substrates. The parent inhibitor peptide, PKI(5-24)amide, and a series of analogs had Ki (or IC50) values for cGMP-dependent protein kinase in the range of 15-190 microM. In contrast to their effects on the cAMP-dependent protein kinase, the inhibitory peptides were substantially less potent with cGMP-dependent protein kinase, and potency was reduced by the presence of the NH2-terminal residues (residues 5-13). We conclude that the two protein kinases share a recognition of the basic amino acid cluster within the pseudosubstrate region of the peptide, but that the cGMP-dependent protein kinase does not recognize additional NH2-terminal determinants that make the inhibitor protein extremely potent toward the cAMP-dependent enzyme. Even- when tested at high concentrations and with peptide substrates, the native inhibitor protein did not inhibit cGMP-dependent protein kinase under assay conditions in which the peptides derived from it were inhibitory. Thus, the native inhibitor protein appears to have structural features which block interaction with the cGMP-dependent enzyme and enhance its selectivity for cAMP-dependent protein kinase.
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PMID:Differential and common recognition of the catalytic sites of the cGMP-dependent and cAMP-dependent protein kinases by inhibitory peptides derived from the heat-stable inhibitor protein. 301 64

The regulatory subunit of the type I cAMP-dependent protein kinase (Rt) serves as a substrate for the phosphotransferase reaction catalyzed by cGMP-dependent protein kinase (Km = 2.2 microM). The reaction is stimulated by cGMP when RI . cAMP is the substrate, but not when nucleotide-free RI is used. The cGMP-dependent protein kinase catalyzes the incorporation of 2 mol of phosphate/mol of RI dimer in the presence of cAMP and a self-phosphorylation reaction to the extent of 4 mol of phosphate/mol of enzyme dimer. In the absence of cAMP, RI is a competitive inhibitor of the phosphorylation of histone H2B (Ki = 0.25 microM) and of the synthetic peptide substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly (Ki = 0.15 microM) by the cGMP-dependent enzyme. Nucleotide-free RI also inhibits the intramolecular self-phosphorylation of cGMP-dependent protein kinase. The inhibition of the phosphorylation reactions are reversed by cAMP. The catalytic subunit of cAMP-dependent protein kinase does not catalyze the phosphorylation of RIand does not significantly alter the ability of RI to serve as a substrate or an inhibitor of cGMP-dependent protein kinase. These observations are consistent with the concept that the cGMP- and cAMP-dependent protein kinases are closely related proteins whose functional domains may interact.
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PMID:Regulatory subunit of the type I cAMP-dependent protein kinase as an inhibitor and substrate of the cGMP-dependent protein kinase. 624 94

Analogs of a synthetic heptapeptide substrate corresponding to the sequence around a phosphorylation site in histone H2B were used to assess the substrate specificity of cGMP-dependent protein kinase. cGMP-dependent kinase phosphorylated the oligopeptide Arg-Lys-Arg-Ser32-Arg-Lys-Glu with favorable kinetic parameters as compared to those for cAMP-dependent kinase (Glass, D. B., and Krebs, E. G. (1979) J. Biol. Chem. 254, 9728-9738). The contribution of each amino acid to the ability of the peptide to be phosphorylated by cGMP-dependent or cAMP-dependent kinase was studied by replacement of individual residues and evaluation of the kinetic constants of the substituted peptides. Peptides containing acetylated lysine residues or nitroarginine residues were poor substrates for both kinases. Substitution of either arginine 29 or lysine 30 with alanine increased the Km values and decreased the Vmax values for both kinases. Substitution of lysine 34 with alanine increased the Vmax values for both kinases but did not affect the Km values for either enzyme. Substitution of the phosphorylatable serine with a threonine residue greatly depressed the Vmax for both kinases. Peptides in which arginine 31 or arginine 33 were replaced by an alanine residue revealed several apparent differences in the specificity requirements between cGMP-dependent and cAMP-dependent kinases.
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PMID:Phosphorylation by guanosine 3':5'-monophosphate-dependent protein kinase of synthetic peptide analogs of a site phosphorylated in histone H2B. 627 76

The protein encoded by the Drosophila cGMP-dependent protein kinase gene, DG1, was expressed in Sf9 cells. cGMP (10 microM) stimulated histone H2B phosphorylation by the DG1 protein kinase 20-fold. Maximal activity was observed at 40-50 mM Mg2+. The concentrations of cGMP, cAMP, cIMP, 8-bromo-cGMP, and 8-bromo-cAMP that gave 50% activation were 0.19 +/- 0.06, 11.7 +/- 2.8, 5.3 +/- 1.5, 0.04 +/- 0. 01, and 0.62 +/- 0.06 microM, respectively. cGMP activation was cooperative with a Hill coefficient (nH) of 1.28 +/- 0.10, whereas activation by cAMP was not cooperative. DG1 kinase expressed in Sf9 cells was found to be a dimer with an amino-terminal dimerization domain. It also autophosphorylated in a reaction stimulated by cGMP and cAMP. Immunoadsorbed DG1 protein from fly extracts was also capable of autophosphorylation, and this assay was used to quantitate the DG1 kinase in extracts from heads and bodies of adults and whole embryos. Activity was highest in heads of either sex and male bodies, intermediate in female bodies, and lowest in embryos. These results were in accord with DG1 mRNA abundance. Tissue distribution of the DG1 kinase was investigated by immunohistochemistry. In embryos, specific immunoreactivity was observed in large cells scattered along the anterior-posterior axis at stage 13. Prominent staining of adult heads was restricted to the proximal level of the lamina cortex.
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PMID:Biochemical properties and cellular localization of the Drosophila DG1 cGMP-dependent protein kinase. 879 33