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

A purified bovine lung cGMP-binding cGMP-specific phosphodiesterase (cG-BPDE) was rapidly phosphorylated by purified bovine lung cGMP-dependent protein kinase (cGK). Within a physiological concentration range, cGK catalyzed phosphorylation of cG-BPDE at a rate approximately 10 times greater than did equimolar concentrations of purified catalytic subunit of cAMP-dependent protein kinase (cAK). cG-BPDE was a poor substrate for either purified protein kinase C or Ca2+/calmodulin-dependent protein kinase II. Binding of cGMP to the cG-BPDE binding site was required for phosphorylation since (a) phosphorylation of cG-BPDE by the catalytic subunit of cAK was cGMP-dependent, (b) phosphorylation of cG-BPDE in the presence of a cGMP analog specific for activation of cGK was cGMP-dependent, and (c) occupation of the cG-BPDE hydrolytic site with competitive inhibitors did not produce the cGMP-dependent effect. cGMP-dependent phosphorylation of cG-BPDE by both cGK and cAK occurred at serine. Proteolytic digestion of cG-BPDE phosphorylated by either cGK or cAK revealed the same phosphopeptide pattern, suggesting that phosphorylation by the two kinases occurred at the same or adjacent site(s). Tryptic digestion of cG-BPDE phosphorylated by cGK and [gamma-32P]ATP produced a single major phosphopeptide of approximately 2 kDa with the following amino-terminal sequence: Lys-Ile-Ser-Ala-Ser-Glu-Phe-Asp-Arg-Pro-Leu-Arg- Radioactivity was released during the third cycle of Edman degradation. cG-BPDE is one of few specific in vitro cGK substrates of known function to be identified. Elevation of intracellular cGMP may cause phosphorylation of cG-BPDE by modulating the substrate site availability as well as by activating cGK. Such regulation would greatly increase the selectivity of the phosphorylation of cG-BPDE and would represent a unique mechanism of action of a cyclic nucleotide or other second messenger.
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PMID:Substrate- and kinase-directed regulation of phosphorylation of a cGMP-binding phosphodiesterase by cGMP. 216 96

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

Two murine monoclonal antibodies (H5 and B6) generated against bovine heart type II regulatory subunit of cAMP-dependent protein kinase were shown to cross-react equally well with the homologous subunit from porcine heart. The antibodies demonstrated specificity for only the type II regulatory subunit and showed negligible cross-reactivity with the type I regulatory subunit, the catalytic subunit, and cGMP-dependent protein kinase. Following limited proteolysis of type II regulatory subunit with chymotrypsin, the H5 monoclonal antibody was shown to cross-react with the Mr = 37,000 cAMP-binding domain corresponding to the COOH-terminal region of the polypeptide chain. To more specifically localize the antigenic sites, the porcine type II regulatory subunit was carboxymethylated and cleaved with cyanogen bromide. Both monoclonal antibodies cross-reacted with the NH2-terminal CNBr peptide, and this peptide demonstrated affinities similar to native bovine type II regulatory subunit in competitive displacement radioimmunoassays. Tryptic cleavage of this CNBr fragment destroyed all antigenicity for both monoclonal antibodies, whereas antigenicity was retained following chymotryptic digestion. A single major immunoreactive chymotryptic fragment that cross-reacted with H5 was isolated by gel filtration and reverse phase high performance liquid chromatography. this peptide retained the complete antigenic site and had the following sequence: Asn-Pro-Asp-Glu-Glu-Glu-Glu-Asp-Thr-Asp-Pro-Arg-Val-Ile-His-Pro-Lys-Thr-Asp-Gl n. This antigenic site was localized just beyond the major site of autophosphorylation, approximately a third of the distance from the NH2-terminal end of the polypeptide chain.
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PMID:Monoclonal antibodies as structural probes of surface residues in the regulatory subunit of cAMP-dependent protein kinase II from porcine heart. 618 75

Interaction of cGMP-dependent protein kinase with histones H2A, H2B, H3, and H4, or poly(L-arginine) resulted in changes in enzyme conformation such that inactivation of cGMP binding and activation of basal catalytic activity (assayed without cGMP) occurred. Total kinase activity as determined by phosphorylation of exogenous substrates subsequently decreased, but autophosphorylation of the enzyme was enhanced. The reaction was specific for nucleosome core histones and poly(L-arginine); H1, troponin, and poly(L-lysine) had no effect. Inactivation of cyclic nucleotide binding sites followed pseudo-first order kinetics and, at various histone concentrations, exhibited saturation kinetics at low ionic strength (2 mM potassium phosphate, pH 6.8), but non-saturation kinetics at higher ionic strength (37.5 mM potassium phosphate, pH 6.8, 12.5 mM MgCl2). Saturation kinetics was observed with poly(L-arginine) at both low and high ionic strength. Kinetic parameters measured under saturation conditions were determined for each core histone and poly(L-arginine). Core histones and poly(L-arginine) were noncompetitive inhibitors of cGMP binding; core histones and poly(L-arginine) interacted competitively at an enzyme site designated as the poly(L-arginine) binding site. Regulatory subunits of cAMP-dependent protein kinase contain a similar poly(L-arginine) binding site. Modulator proteins bind to poly(L-arginine) or arginyl residues in histone to prevent interaction with the poly(L-arginine) binding site on the enzymes. Through this mechanism, modulator proteins maintain cyclic nucleotide dependency and full enzyme activity.
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PMID:Regulation of cyclic nucleotide-dependent protein kinase activity by histones and poly(L-arginine). 625 84

The amino acid sequence around the site of the regulatory subunit of type I cAMP-dependent protein kinase (RI) that is phosphorylated by cGMP-dependent protein kinase has been determined. This site was found to be located near the site on RI previously shown to be very sensitive to hydrolysis by trypsin (Potter, R. L., and Taylor, S. S. (1979) J. Biol. Chem. 254, 2413-2418). The primary sequence surrounding the site is as follows: -Lys-Ala-Gly-Ser-Arg-Ala-Asp-Ser-Arg-Glu-Asp-Glu-Ile-Ser-Pro-Pro-Pro-Pro-Asn-Pro-Val-Val-Lys-Gly-Arg-Arg-Arg-Arg-Gly-Ala-Ile-Ser(P)-Ala-Glu-Val-Tyr-Thr-Glu-Glu-Asp-Ala-Ala-Ser-Tyr-Val-Arg-Lys-Val-Ile-Pro-Lys-Asp-Tyr-Lys-Thr-. As described previously (Geahlen, R. L., and Krebs, E. G. (1980) J. Biol. Chem. 255, 1164-1169), this site is specific for cGMP-dependent protein kinase and is not phosphorylated by the catalytic subunit of cAMP-dependent protein kinase.
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PMID:Studies on the site in the regulatory subunit of type I cAMP-dependent protein kinase phosphorylated by cGMP-dependent protein kinase. 626 84

The amino acid sequence at the ATP-binding site on the cGMP-dependent protein kinase has been determined. For this determination the enzyme was labeled covalently by 5'-p-fluorosulfonyl[14C]benzoyladenosine and fragmented using cyanogen bromide or digested by trypsin after succinylation. The 14C-labeled peptides were purified by gel filtration and high performance liquid chromatography. The amino acid sequence around the site was found to be: -Val-Glu-Leu-Val-Gln-Leu-Lys-Ser-Glu-Glu-Ser-Lys-Thr-Phe-Ala-Met-*Lys-Ile-Leu-Lys--Lys-Arg-His-Ile-Val-Asp-Thr-Arg-Gln-Gln-Glu-His-Ile-Arg-Ser-Glu-Lys-, in which *Lys is the lysine residue that was modified by the affinity reagent. When this sequence was compared with that of the ATP-binding site of the catalytic subunit of cAMP-dependent protein kinase, a high degree of structural homology was observed for this site in the two proteins.
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PMID:Amino acid sequence at the ATP-binding site of cGMP-dependent protein kinase. 627 62

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

Chromosomal high mobility group (HMG) proteins have been examined as substrates for cGMP-dependent and cAMP-dependent protein kinases. Of the four HMG proteins only HMG 14 contained a major high affinity site which could be phosphorylated by both enzymes, preferentially by cGMP-dependent protein kinase. One mol of 32P was incorporated/mol of HMG 14. Kinetic analysis revealed apparent Km and Vmax of 40.5 microM and 14.7 mumol/min/mg, respectively, for cGMP-dependent protein kinase, and 123 microM and 11.1 mumol/min/mg, respectively, for cAMP-dependent protein kinase. Tryptic maps of 32P-labeled phosphopeptides of HMG 14 demonstrated phosphorylation of the same site by both enzymes. The tryptic fragment containing the major phosphorylation site was identified by amino acid composition and sequence as HMG 14 (residues 4-13): H-Lys-Val-Ser(P)-Ser-Ala-Glu-Gly-Ala-Ala-Lys-OH. HMG 14 and HMG 17 also contained minor sites which could be phosphorylated by cGMP-dependent protein kinase. Tryptic phosphopeptides mapping suggested that the same minor site was phosphorylated on both HMG 14 and 17. On the basis of amino acid composition, the tryptic peptides carrying the minor phosphorylation sites were identified as H-Leu-Ser(P)-Ala-Lys representing residues 23-26 and 27-30 of HMG 14 and HMG 17, respectively.
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PMID:Phosphorylation of high mobility group 14 protein by cyclic nucleotide-dependent protein kinases. 627 43

A monoclonal antibody (MAb-46-1) specifically recognizing a 46 kDa basic protein solubilized from brain membranes was used to prepare an affinity column, which allowed a one-step purification of the 46 kDa protein to homogeneity starting from solubilized cerebellar membranes. MAb-46-1 could also immunoprecipitate the 46 kDa protein from solubilized pig or rat cerebellar membranes. Microsequence analysis of affinity purified 46 kDa protein treated with Lys C demonstrated the identity of the 46 kDa protein as a myelin associated protein, i.e. 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP, EC 3.1.4.37). The amino acid sequences obtained for the porcine CNP were nearly identical with the known sequences of the bovine and human isoforms but only partially with those of rat and mouse CNP. In SDS PAGE the porcine CNP appeared as a doublet of 44.6 and 45.9 kDa. Both bands of the doublet were equally well recognized by MAb-46-1. Porcine CNP was rapidly and specifically phosphorylated by both protein kinase A and cGMP-dependent protein kinase.
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PMID:The myelin protein CNP (2',3'-cyclic nucleotide 3'-phosphodiesterase): immunoaffinity purification of CNP from pig and rat brain using a monoclonal antibody and phosphorylation of CNP by cyclic nucleotide-dependent protein kinases. 801 Nov 77


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