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)

Cardiac sarcolemmae from guinea pig ventricles were purified and incubated with cGMP-dependent protein kinase. In the presence of the purified kinase plus 10(-5) M cGMP or 8-Br-cGMP, a protein of approximately 50 kD, (Kilodalton) was phosphorylated. This membrane-associated cGMP-dependent protein kinase substrate is similar in MW to the regulatory subunit of the cAMP-dependent protein kinase, which is known to be a substrate for the cGMP-dependent protein kinase. Thus, this substrate, the identity of which remains to be proven, may be a possible mediator of cGMP-mediated control of cardiac function.
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PMID:Cardiac sarcolemmal substrate of the cGMP-dependent protein kinase. 285 60

N-(6-Aminoethyl)-5-chloro-1-naphthalenesulfonamide (A-3), which is a shorter alkyl chain derivative of the calmodulin (CaM) antagonist, W-7, was found to inhibit smooth muscle myosin light chain kinase (MLC-kinase) through a mechanism different from that related to W-7. Both the holoenzyme and the catalytic fragment, which is active without CaM, were susceptible to A-3 with a similar concentration dependency, thereby indicating that the inhibitory effect is due to the direct interaction of the compound with the enzyme molecule and not with the enzyme activator. Naphthalenesulfonamides are both CaM antagonists and direct inhibitors of MLC-kinase, and these actions depend on the length of the alkyl chain (C2-C6). Although the potencies in inhibiting CaM functions increased, the direct effects on MLC-kinase decreased with extension of the carbon chain of the derivatives. Kinetic studies indicated that A-3 inhibited MLC-kinase competitively with respect to ATP and that the Ki value was 7.4 microM. A-3 was also a competitive inhibitor of cAMP-dependent protein kinase, cGMP-dependent protein kinase, protein kinase C, casein kinase I, and casein kinase II, with respect to ATP. The Ki values of naphthalenesulfonamides for these enzymes also increased with extension of the carbon chain of the derivatives. These results suggest that naphthalenesulfonamides inhibit protein phosphorylation not only by inhibition of the enzyme-activating process but also by inhibition of the catalytic process. The mode of interaction between the derivatives and protein kinases differs from the interaction between the derivatives and CaM.
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PMID:Naphthalenesulfonamides as calmodulin antagonists and protein kinase inhibitors. 287 89

The type II cAMP-dependent protein kinase (cAMP-PK-II) from cilia of Paramecium, purified free of type I cAMP-PK (cAMP-PK-I) and of cGMP-dependent protein kinase (cGMP-PK), phosphorylated several basic proteins and a heptapeptide containing serine (Kemptide). The enzyme was partially inhibited by the protein kinase inhibitor (Walsh inhibitor), but only at relatively high inhibitor concentrations. Half-maximal activation of cAMP-PK-II occurred at 15-25 nM cAMP. Several cAMP analogs were tested for ability to bind and activate the enzyme. 8-bromo-cGMP, a potent activator of Paramecium cGMP-PK, was a poor activator of Paramecium cAMP-PK-II. Activation of cAMP-PK-II was influenced by the phosphorylation assay buffer. Phosphate buffers provided increased activation by cAMP but decreased total activity relative to that measured in Mops-Tris buffer. The kinase was cAMP-independent when the pH of the assay buffer was high. Preincubation of cAMP-PK-II with histones also activated the enzyme in the absence of cAMP. The cAMP-PK-II bound cAMP with a Kd of 23 nM, and bound cAMP was released with a biphasic time course, suggesting two non-identical binding sites. The properties of the cAMP-PK of this ciliated protozoan appear to be closely similar to those of vertebrates.
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PMID:Cyclic AMP-dependent protein kinases of Paramecium. II. Catalytic and regulatory properties of type II kinase from cilia. 290 46

The cytosolic fraction of insulin-treated adipocytes exhibits a 2-fold increase in protein kinase activity when Kemptide is used as a substrate. The detection of insulin-stimulated kinase activity is critically dependent on the presence of phosphatase inhibitors such as fluoride and vanadate in the cell homogenization buffer. The cytosolic protein kinase activity exhibits high sensitivity (ED50 = 2 X 10(-10) M) and a rapid response (maximal after 2 min) to insulin. Kinetic analyses of the cytosolic kinase indicate that insulin increases the Vmax of Kemptide phosphorylation and ATP utilization without affecting the affinities of this enzyme toward the substrate or nucleotide. Upon chromatography on anion-exchange and gel filtration columns, the insulin-stimulated cytosolic kinase activity is resolved from the cAMP-dependent protein kinase and migrates as a single peak with an apparent Mr = 50,000-60,000. The partially purified kinase preferentially utilizes histones, Kemptide, multifunctional calmodulin-dependent protein kinase substrate peptide, ATP citrate-lyase, and acetyl-coenzyme A carboxylase as substrates but does not catalyze phosphorylation of ribosomal protein S6, casein, phosvitin, phosphorylase b, glycogen synthase, inhibitor II, and substrate peptides for casein kinase II, protein kinase C, and cGMP-dependent protein kinase. Phosphoamino acid analyses of the 32P-labeled substrates reveal that the insulin-stimulated cytosolic kinase is primarily serine-specific. The insulin-activated cytosolic kinase prefers Mn2+ to Mg2+ and is independent of Ca2+. Unlike ribosomal protein S6 kinase and protease-activated kinase II, the insulin-sensitive cytosolic kinase is fluoride-insensitive. Taken together, these results indicate that a novel cytosolic protein kinase activity is activated by insulin.
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PMID:Insulin stimulates a novel Mn2+-dependent cytosolic serine kinase in rat adipocytes. 296 Jun 79

cAMP modulates estrogen, hCG, and lactate syntheses by the human placenta. The major effects of cAMP are presumably mediated through the phosphorylation of specific regulatory phosphoproteins after cAMP activation of cAMP-dependent protein kinase. cAMP-dependent phosphoproteins have not been identified in the placenta. Homogenates and cytosol from term human placenta were subjected to an endogenous protein phosphorylation assay using [gamma-32P]ATP in the presence and absence of 1.0 microM cAMP. Protein phosphorylation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. cAMP stimulated 32P incorporation into proteins with mol wt of 25,000, 27,000, 39,000, 45,000, 52,000, 58,000, and 73,000 (P less than 0.02). Half-maximal 32P incorporation was observed with 1.0 X 10(-7) M cAMP, which was similar to the concentration required for half-maximal histone kinase activity (8.5 +/- 2.9 X 10(-8) M). cGMP induced 32P incorporation into the same placental proteins as cAMP, but 2 orders of magnitude greater cGMP concentrations were required to achieve phosphorylation levels similar to those caused by cAMP. cAMP-dependent protein kinase inhibitor completely blocked cGMP-induced phosphorylation, even when histone protein was added. Therefore, no evidence of a cGMP-dependent protein kinase or specific cGMP-dependent phosphoproteins were detected. CaCl2 (10(-8) - 10(-4) M) had no effect on cAMP-induced 32P incorporation into the seven cAMP-dependent phosphoproteins. However calcium induced 32P incorporation into four other proteins (mol wt, 97,000, 90,000, 20,000, and 19,000). Regulation of placental metabolism by catecholamines and other hormones known to mediate intracellular cAMP or calcium levels may be accomplished by phosphorylation of these phosphoproteins.
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PMID:Adenosine 3',5'-monophosphate-dependent phosphoproteins in human placenta. 298 Oct 67

Monomeric regulatory subunit (R) fragments of type II cAMP-dependent protein kinase were compared with the parent dimeric R. The monomeric fragments were generated by either endogenous proteolysis of rabbit muscle R or by trypsin treatment of bovine heart R in the holoenzyme form. During isolation of pure R from rabbit muscle, carboxyl-terminal fragments of Mr = 42,000 (42 K) and Mr = 37,000 by denaturing gels are generated by endogenous proteolysis. Although the autophosphorylation site is retained, the 42 K is not dimeric (as is its native 56 K precursor) but, in contrast to the monomeric 37 K product, actively reassociates with purified catalytic subunit (C). Several lines of evidence indicate a type II R origin of the 42 K. N-terminal sequence analysis of the 42 K shows some homology with known bovine RI, RII, and cGMP-dependent protein kinase sequences. Both cyclic nucleotide-binding sites (two/42 K or 37 K) and the site selectivity of cAMP analogs are retained in the monomeric fragments. When purified bovine heart holoenzyme, which contains a dimeric Mr = 56,000 R (denaturing gel analysis) and two C subunits, is treated with trypsin followed by separation procedures, the product is a fully recovered active enzyme with an unaltered ratio of cAMP binding to catalytic activity. From Mr considerations, the product is a dimer containing one intact C and a proteolyzed R of Mr = 48,000 on denaturing gels. This dimeric enzyme is not significantly different from the parent tetramer in cAMP concentration dependence (Hill constant = 1.63), [3H]cAMP dissociation behavior (both intrasubunit cAMP-binding sites are present), stimulation of [3H]cIMP binding by site-selective cAMP analogs, and synergism between two analogs in kinase activation. The data indicate that 1) proteolytic cleavage of the native R dimer can cause monomerization without appreciably affecting the inhibition of C and 2) essentially all of the cAMP binding cooperativity is an intrasubunit interaction.
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PMID:The regulatory subunit monomer of cAMP-dependent protein kinase retains the salient kinetic properties of the native dimeric subunit. 298 60

The present study was undertaken in order to identify the inhibitory site of the heat-stable inhibitor of cAMP-dependent protein kinase (PKI) and to synthesize a peptide that could serve as a useful inhibitor of the enzyme. Digestion of purified PKI by mast cell proteinase II yielded a peptide fragment that retained inhibitory activity. A sequence of 20 amino acids of the peptide, (sequence in text) revealed the presence of a "pseudosubstrate site" (Arg-Arg-Asn-Ala-Ile) for the cAMP-dependent protein kinase in which alanine replaces the seryl or threonyl residue that is normally phosphorylated. Digestion of PKI with various other proteinases implicated the involvement of arginyl and hydrophobic residues as determinants for the inhibitory activity. The assumption that this region is part of the inhibitory site was confirmed by the synthesis of a corresponding duodecapeptide that displayed strong inhibitory activity. Inhibition by the peptide was competitive with a Ki of 0.8 microM as measured against a number of protein substrates. The sequence of this fragment bears a strong resemblance to the autophosphorylation site in the type II regulatory subunit of cAMP-dependent protein kinase, a region also postulated to interact with the catalytic subunit, and the analogous region of type I regulatory subunit. Neither intact PKI nor the synthetic peptide inhibit the cGMP-dependent protein kinase, phosphorylase kinase, myosin light-chain kinase, casein kinase II, or protein kinase C.
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PMID:Identification of an inhibitory region of the heat-stable protein inhibitor of the cAMP-dependent protein kinase. 298 19

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

cGMP-dependent protein kinase (G-kinase) and the regulatory subunit of type I (RI) cAMP-dependent protein kinase (A-kinase) both contain a phosphorylation site located near the NH2 terminus of each enzyme. These sites can be utilized as convenient markers for the determination of the position of an amino acid residue susceptible to either chemical or enzymatic digestion. Using the tryptophan-specific reagent, N-chlorosuccinimide, the approximate location along the polypeptide chain of six reactive tryptophans in G-kinase and three reactive residues in RI were identified. Similarly, cleavage with cyanide was used to locate free and disulfide-bonded cysteines in both proteins. The approximate positions of nine cysteines in G-kinase were determined along with the location of the interchain disulfide bond and an intrachain disulfide bond. RI was found to contain three cyanide-reactive cysteines, two of which are involved in interchain disulfide bonding. A comparison of the positions of the cysteines and tryptophans determined by chemical cleavage in G-kinase and RI, with the positions of cysteine and tryptophan in the known sequence of the type II A-kinase, support the structural relationships between these enzymes. Comparison with subsequently reported primary sequences of all three enzymes indicates the limits of precision of this chemical cleavage procedure.
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PMID:A comparison of the cyclic nucleotide-dependent protein kinases using chemical cleavage at tryptophan and cysteine. 299 85

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


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