Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

Protein phosphorylation has been recognized as a major mechanism by which cellular functions are controlled by neurotransmitters and hormones. In this review, applications of molecular biological techniques to the analyses of regulatory mechanisms of protein phosphorylation by four major second messengers, cAMP, cGMP, diacylglycerol, and Ca2+, are described. 1) Complementary DNA of the regulatory subunit of the cAMP-dependent protein kinase was cloned and expressed in E. coli. Point mutations were introduced in order to analyze functional domains of the subunit. 2) The soluble isoform of guanylate cyclase was purified, and a cDNA of its 70-KD subunit was cloned. Cyclic GMP binding to purified cGMP-dependent protein kinase was characterized using a rapid filtration assay. 3) Primary structure of the catalytic subunit of calmodulin-dependent protein phosphatase (calcineurin A) was determined and the presence of the second isoform of the enzyme was shown by the cDNA cloning technique. 4) The regulatory domain of the protein kinase C was expressed in E. coli. Analysis using site-directed mutagenesis revealed that a "zinc finger"-like structure is responsible for the binding of phorbol esters. In these studies, the molecular biological approach has proven to be useful for clarifying the molecular mechanisms of cellular signal transduction related to second messengers and protein phosphorylation.
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PMID:[Second messengers and protein phosphorylation in cellular signal transduction]. 222 19

KT5926, (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-14-n-propoxy-2,3 ,9, 10-tetrahydro-8,11-epoxy, 1H,8H, 11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde] trinden-1-one, was found to be a potent and selective inhibitor of myosin light chain kinase. The compound inhibited both Ca2+/calmodulin-dependent and -independent smooth muscle myosin light chain kinases to a similar extent. The inhibition was not affected by the concentration of calmodulin. Kinetic analyses showed that the mode of inhibition was of the competitive type with respect to ATP (Ki, 18 nM) and of the noncompetitive type with respect to myosin light chain (Ki, 12 nM). These results indicated that KT5926 directly interacted with the enzyme at the catalytic site. KT5926 also inhibited other protein kinases, but with relatively high Ki values; the values for protein kinase C, cAMP-dependent protein kinase, and cGMP-dependent protein kinase were 723, 1200, and 158 nM, respectively. Ca2(+)-ATPase, Na+/K(+)-ATPase, hexokinase, and 5'-nucleotidase were not inhibited by KT5926 at less than 10 microM. The effect of KT5926 on serotonin secretion and protein phosphorylation induced by platelet-activating factor or phorbol ester was examined in rabbit platelets. KT5926 inhibited the phosphorylation of a 20-kDa protein but had no effect on the phosphorylation of a 40-kDa protein, thereby indicating that the compound exerts its selective inhibition of myosin light chain kinase in intact cells. The compound inhibited serotonin secretion induced by platelet-activating factor, but its potency was significantly less than that of K-252a, (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9, 10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b, 11a-triazadibenzo[a,g]cycloocta [cde]trinden-1-one, which inhibited the phosphorylation of both the 20-kDa protein and the 40-kDa protein. Phorbol ester-induced secretion was not suppressed by KT5926. These results provide the evidence that both the 20-kDa protein phosphorylation by myosin light chain kinase and the 40-kDa protein phosphorylation by protein kinase C substantially contribute to the secretion response in platelets.
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PMID:KT5926, a potent and selective inhibitor of myosin light chain kinase. 232 35

ARPP-21 (cAMP-regulated phosphoprotein, Mr = 21,000 as determined by SDS/PAGE) is a major cytosolic substrate for cAMP-stimulated protein phosphorylation in dopamine-innervated regions of rat CNS (Walaas et al., 1983c). This acidic phosphoprotein has now been identified in bovine caudate nucleus cytosol and purified to homogeneity from this source. The purification procedure involved diethylaminoethyl-cellulose chromatography, ammonium sulfate fractionation, phenyl-Sepharose CL-4B chromatography, and fast protein liquid chromatography using Mono Q anion-exchange resin. Two isoforms of ARPP-21 (ARPP-21A and ARPP-21B) were obtained, which were present in approximately equal amounts in the starting material. ARPP-21A was purified 2610-fold with a final yield of 20% and ARPP-21B was purified 2940-fold with a final yield of 21%. The purified preparations of both isoforms were judged to be homogenous by SDS/PAGE. ARPP-21A and ARPP-21B yielded identical 2-dimensional thin-layer tryptic phosphopeptide maps, identical amino acid compositions and closely related, but distinct, reverse-phase high-pressure liquid chromatograms of tryptic digests. The amino acid composition of ARPP-21 showed a high content of glutamic acid/glutamine, and no methionine, tryptophan, tyrosine, phenylalanine, or histidine. ARPP-21 was stable to heat denaturation and to 50% (vol/vol) ethanol treatment and was partially soluble at pH 2. The Mr determined for ARPP-21 by SDS/PAGE was 21,000. The Stokes radius of ARPP-21 was 26.3 A, and the sedimentation coefficient of ARPP-21 was 1.3 S; these values yield a calculated molecular mass of 13,700 Da and a frictional ratio of 1.7, indicative of an elongated tertiary structure. ARPP-21 was an excellent substrate for cAMP-dependent protein kinase and was either not phosphorylated or only poorly phosphorylated by cGMP-dependent protein kinase, calcium/calmodulin-dependent protein kinase I, calcium/calmodulin-dependent protein kinase II, casein kinase II, or protein kinase C. The purified catalytic subunit of cAMP-dependent protein kinase catalyzed the incorporation of 1.2 mol phosphate/mol purified ARPP-21. Phosphorylation occurred exclusively on seryl residues. Phospho-ARPP-21 was dephosphorylated effectively by protein phosphatase-1 or -2A, but not by protein phosphatase-2B or -2C. Rabbit polyclonal and mouse monoclonal antibodies were prepared to purified ARPP-21. These antibodies specifically immunoprecipitated ARPP-21, which was found to be highly enriched in the caudate nucleus and putamen of monkey brain.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:ARPP-21, a cyclic AMP-regulated phosphoprotein enriched in dopamine-innervated brain regions. I. Purification and characterization of the protein from bovine caudate nucleus. 253 84

This paper reviews our work on the modulation of voltage-dependent Ca currents in identified snail neurons. Ca currents of snail neurones are enhanced or decreased by neurotransmitters. Serotonin and acetylcholine enhance the Ca current of identified neurons, the effect of serotonin being mediated by cGMP and cGMP-dependent protein kinase. Cholecystokinin (CCK8) and dopamine both decrease the Ca current of identified neurons. The effect of CCK8 is irreversible and involves the activation of protein kinase C. The dopamine-induced decrease in Ca current is reversible and involves an alpha 40 subunit of a snail G protein immunologically and functionally related to alpha o of mammalian brain.
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PMID:Intracellular mechanism of neurotransmitter-induced modulations of voltage-dependent Ca current in snail neurons. 257 63

Cyclic GMP depresses Ba2+ current through high-voltage-activated Ca2+ channels (ICa) in acutely isolated hippocampal neurons. The effect is produced by intra-, but not extracellular, cGMP or by 5' GMP. The membrane-permeant derivative, 8-Br-cGMP, produces a reversible suppression. The effect of 8-Br-cGMP is similar to phorbol ester-induced ICa depression, except that ICa depression due to 8-Br-cGMP is not blocked by protein kinase inhibitors H-8 or H-7, whereas phorbol ester effects are. The data suggest that cGMP depresses ICa by a cGMP-kinase- and protein kinase C (PKC)-independent mechanism. Cyclic AMP, which enhances ICa, and the cyclic nucleotide phosphodiesterase inhibitor, IBMX, both antagonize ICa depression induced by 8-Br-cGMP, but not that due to phorbol esters. Cyclic IMP, a more potent activator of phosphodiesterase than of cGMP-dependent protein kinase, is also a powerful depressant of ICa. We conclude that cGMP-induced depression of ICa is mediated by activation of cyclic nucleotide phosphodiesterase with consequent reduction of intracellular cAMP.
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PMID:Cyclic GMP depresses hippocampal Ca2+ current through a mechanism independent of cGMP-dependent protein kinase. 285 1

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 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

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

Phytohemagglutinin (PHA) and a tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) act synergistically to induce interleukin 2 (IL2) mRNA in human lymphocytes in vitro. The induction was inhibited by a potent inhibitor of protein kinase C (C-kinase), 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) at less than 10 microM. H-7 inhibited C-kinase activity itself in lymphocytes at the same range of the concentration but did not interfere with the translocation of C-kinase from the cytosol to the membrane fraction of the lymphocytes induced by TPA. H-7 is also known to inhibit cAMP-dependent protein kinase (A-kinase) and cGMP-dependent protein kinase (G-kinase). However, the lymphocytes cultured with dibutyryl cAMP or dibutyryl cGMP could not be activated to produce IL2 mRNA. These results show that activation of C-kinase but not A-kinase and G-kinase is necessary in signal transduction for IL2 gene expression. Prostaglandin E2, which is known to elevate intracellular cAMP level, also inhibited IL2 mRNA induction in the lymphocytes stimulated with PHA and TPA. Addition of alpha-methylornithine and methylglyoxal bis (guanyl hydrazone), which inhibit polyamine synthesis, did not affect the induction of IL2 mRNA in the lymphocytes stimulated with PHA and TPA, indicating that polyamine synthesis is not necessary for IL2 mRNA induction.
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PMID:Induction and regulation of human interleukin 2 gene expression: significance of protein kinase C activation. 302 5


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