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)

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

The binding of [3H]cGMP to purified beef lung cGMP-dependent protein kinase (cG kinase) was examined using two methods of membrane filtration which avoided loss of bound [3H]cGMP. The enzyme bound 1.6-2.0 mol of [3H]cGMP/mol of monomer. If the kinase was saturated with [3H]cGMP and then excess unlabeled cGMP was added, [3H]cGMP dissociated from the enzyme as two approximately equal components (Sites 1 and 2). When 8-bromo-cGMP or cIMP was added to the [3H]cGMP-binding reaction at a concentration sufficient to competitively inhibit binding by greater than 50%, the relative amount of the slower or faster component, respectively, of [3H]cGMP dissociation decreased during the cGMP chase. The data indicated that the cG kinase, like its cAMP-dependent protein kinase homologue, possesses two highly conserved intrachain cyclic nucleotide-binding sites which have different dissociation rates and analog specificity. The Ka of the kinase for cGMP was about 20-fold lower using histone instead of heptapeptide as substrate. Aging of the enzyme caused conversion to a higher Ka form of the kinase and an apparent increase in the Site 1 cGMP dissociation rate. Using fresh enzyme and heptapeptide as substrate, Site 1 occupation occurred at lower concentrations of cGMP than did Site 2 occupation, and was associated with an increase in protein kinase activity. However, kinase activity appeared to correlate better with total cGMP binding than with binding to either of the two sites, and the activation by cGMP exhibited positive cooperativity (n = 1.57). It is suggested that both intrachain sites are involved in protein kinase activation. E2 + 4 cGMP in equilibrium E2 . cGMP4 The cG kinase could be photoaffinity-labeled using 8-azido-[32P]cAMP. When the labeled cG kinase was trypsin-treated followed by sodium dodecyl sulfate-slab gel electrophoresis, a single major peptide of approximate Mr = 12,000 was resolved.
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PMID:Studies of two different intrachain cGMP-binding sites of cGMP-dependent protein kinase. 630 46

The vasodilator-stimulated phosphoprotein (VASP) is a major substrate for cAMP-dependent- (cAK) and cGMP-dependent protein kinase (cGK) in human platelets and other cardiovascular cells. To identify the VASP phosphorylation sites, purified VASP was phosphorylated by either protein kinase and subjected to trypsin, V8 and Lys-C proteolysis. The phosphorylated proteolytic fragments obtained were separated by reversed phase high performance liquid chromatography. Sequence analysis of the phosphorylated peptides and 32P measurement of the released 32P-labeled amino acids revealed three phosphorylation sites: a serine 1-containing site (LRKVSKQEEA), a serine 2-containing site (HIERRVSNAG), and a threonine-containing site (MNAVLARRRKATQVGE). Additional experiments with purified VASP demonstrated that both cAK and cGK phosphorylated serine 2 rapidly and the threonine residue slowly, whereas cGK phosphorylated the serine 1 residue more rapidly than the cAK. These differences in the phosphorylation rates of VASP by the two protein kinases were also observed with synthetic peptides corresponding to the sequences of the three identified phosphorylation sites. These experiments also established the synthetic peptide serine 1 as one of the best in vitro cGK substrates and the serine 2-containing site as the site responsible for the phosphorylation-induced mobility shift of VASP in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Experiments with 32P-labeled platelets provided evidence that VASP is phosphorylated at the same three identified sites also in intact cells and that selective activation of cAK or cGK primarily increased the phosphorylation of both serine 2 and serine 1 but not threonine. Our results demonstrated overlapping substrate specificities of cAK and cGK in vitro and in intact cells. However, important quantitative and qualitative differences between cAK- and cGK-mediated phosphorylation of the focal adhesion protein VASP in human platelets were also observed, suggesting distinct functions of the two types of cyclic nucleotide-mediated VASP phosphorylation.
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PMID:cAMP- and cGMP-dependent protein kinase phosphorylation sites of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) in vitro and in intact human platelets. 818 57

The aim of the present study was to determine the phosphorylation of the purified ryanodine receptor-calcium release channel (RyR) of rabbit skeletal muscle sarcoplasmic reticulum by the cAMP-dependent protein kinase (PK-A), cGMP-dependent protein kinase (PK-G) and Ca(2+)-, CaM-dependent protein kinase (PK-CaM) and the localization of phosphorylation sites. Phosphorylation was highest with PK-A (about 0.9 mol phosphate/mol receptor subunit), between one-half to two-thirds with PK-G and between one-third and more than two-thirds with PK-CaM. Phosphoamino acid analysis revealed solely labeled phosphoserine with PK-A and PK-G and phosphoserine and phosphothreonine with PK-CaM. Reverse-phase high-performance liquid chromatography (HPLC) of cyanogen bromide/trypsin digests of the phosphorylated RyR (purified by gel permeation HPLC) and two-dimensional peptide maps revealed one major phosphopeptide by PK-A and PK-G phosphorylation and several labeled peaks by PK-CaM phosphorylation. Automated Edman sequence analysis of the major phosphopeptide obtained from PK-A and PK-G phosphorylation and one phosphopeptide obtained from PK-CaM phosphorylation yielded the sequence KISQTAQTYDPR (residues 2841-2852) with serine 2843 as phosphorylation site (corresponding to the consensus sequence RKIS), demonstrating that all three protein kinases phosphorylate the same serine residue in the center of the receptor subunit, a region proposed to contain the modulator binding sites of the calcium release channel.
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PMID:Phosphorylation of serine 2843 in ryanodine receptor-calcium release channel of skeletal muscle by cAMP-, cGMP- and CaM-dependent protein kinase. 838 Mar 42

The RIalpha subunit of cAMP-dependent protein kinase is maintained as an asymmetric dimer by a dimerization motif at the N terminus. Based on resistance to proteolysis and expression as a discrete domain in Escherichia coli, this motif is defined as residues 12-61. This motif is chemically, kinetically, and thermally stable. The two endogenous interchain disulfide bonds between Cys16 and Cys37 in RIalpha are extremely resistant to reduction even in 8 M urea, indicating that they are well shielded from the reducing environment of the cell. The disulfide bonds were present in recombinant RIalpha as well as when the dimerization domain alone was expressed in E. coli, emphasizing the unusual stability of this motif and the disulfide bonds. Although 100 mM dithiothreitol was sufficient to reduce the disulfide bonds, it did not abolish dimerization. In addition, a stable dimer also still formed when Cys37 was replaced with His, confirming unambiguously the original antiparallel alignment of the disulfide bonds. Thus, both in vitro and in vivo, disulfide bonds are not required for dimerization. Circular dichroism of the dimerization domain indicated a high content of a thermostable alpha-helix. Based on the CD data, trypsin resistance of the fragment, location of the disulfide bonds, and amphipathic helix predictions, potential models are discussed. A new alignment of the dimerization domains of RI, RII, and cGMP-dependent protein kinase elucidates fundamental similarities as well as significant differences among these three domains.
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PMID:A stable alpha-helical domain at the N terminus of the RIalpha subunits of cAMP-dependent protein kinase is a novel dimerization/docking motif. 935 2

The molecular mechanism of cGMP-dependent protein kinase activation by its allosteric regulator cyclic-3',5'-guanosine monophosphate (cGMP) has been intensely studied. However, the structural as well as thermodynamic changes upon binding of cGMP to type I cGMP-dependent protein kinase are not fully understood. Here we report a cGMP-induced shift of Gibbs free enthalpy (DeltaDeltaGD) of 2.5 kJ.mol-1 as determined from changes in tryptophan fluorescence using urea-induced unfolding for bovine PKG Ialpha. However, this apparent increase in overall stability specifically excluded the N-terminal region of the kinase. Analyses of tryptic cleavage patterns using liquid chromatography-coupled ESI-TOF mass spectrometry and SDS/PAGE revealed that cGMP binding destabilizes the N-terminus at the hinge region, centered around residue 77, while the C-terminus was protected from degradation. Furthermore, two recombinantly expressed mutants: the deletion fragment Delta1-77 and the trypsin resistant mutant Arg77Leu (R77L) revealed that the labile nature of the N-terminus is primarily associated with the hinge region. The R77L mutation not only stabilized the N-terminus but extended a stabilizing effect on the remaining domains of the enzyme as well. These findings support the concept that the hinge region of PKG acts as a stability switch.
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PMID:The hinge region operates as a stability switch in cGMP-dependent protein kinase I alpha. 1740 45


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