Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A density gradient-purified microsomal membrane preparation from rabbit fundic gastric mucosa was used for a detailed study of the K+-stimulated ATPase and associated intermediate reactions. Membranes incubated with gamma-[32P]ATP show the rapid incorporation of 32P into phosphoprotein. Phosphoprotein levels were markedly reduced (1) when ATP hydrolysis went to completion or (2) upon addition of unlabeled ATP, thus suggesting the participation of a rapid turnover phosphorylated intermediate in the gastric microsomal ATPase. Addition of K+, Rb+ or Tl+ greatly reduced the level of the intermediate while stimulating ATPase activity; the observed affinities of these cations were similar for the effects on both ATPase and intermediate levels, with Tl+ greater than K+ greater than Rb+. Neither ATPase nor intermediate were stimulated by Na+, and ouabain was without effect on the reactions, thus differentiating this system from the (Na+ + K+)-ATPase. Addition of various inhibitors showed differential effects on the partial reactions of the gastric ATPase system. N-ethylmaleimide and Zn2+ showed characteristics of completely abolishing the K+-stimulated component of ATPase as well as the effects of K+ in reducing the level of intermediate, thus suggesting that these agents exert their inhibitory effect on a phosphoprotein phosphatase partial reaction. F- abolished the K+-stimulated ATPase, but its more complex effects on the intermediate suggested an additional reaction step within the domain of the phosphorylated intermediate. Results are consistent with a model system for the gastric microsomal ATPase involving a Mg2+-dependent protein kinase, a phosphorylated intermediate(s), and a K+-stimulated phosphoprotein phosphatase.
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PMID:Studies on the phosphorylated intermediates of a K+-stimulated ATPase from rabbit gastric mucosa. 0 43

cGMP-dependent protein kinase from bovine lung has been purified to homogeneity using 8-(2-aminoethyl)-amino adenosine 3':5'-monophosphate/Sepharose. Conditions for adsorption of holoenzyme to the affinity chromatography media followed by competitive ligand elution with cGMP have been determined. The holoenzyme of 150,000 molecular weight is composed of two 74,000 molecular weight subunits which are linked in part by disulfide bridges. Two moles of cGMP are bound per mol of holoenzyme compatible with 1 mol of cGMP/monomer. Dissociation of subunits does not occur upon cGMP binding and protein kinase activation. cGMP-dependent protein kinase has an isoelectric point of 5.4 and a Stokes radius of 50 A. The enzyme is asymmetric with an f/f0 of 1.42 and an axial ratio of 7.4. Determination of enzyme activity at varying concentrations of ATP revealed that cGMP increased the Vmax for ATP without significant effect on the Km. The purified enzyme was maximally active at 5 mM Mg2+; other divalent cations could not substitute for Mg2+. In the presence of Mg2+, strong inhibitory effects of other cations were observed with Mn2+, greater than Zn2+, greater than Co2+ greater than Ca2+. Although maximal cGMP-dependence was observed at pH 5.7 to 7.0, basal activity rose at higher pH values to approach activity observed with cGMP. A molecular model comparing cGMP-dependent protein kinase with cAMP-dependnet protein kinase is presented.
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PMID:Guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung. Subunit structure and characterization of the purified enzyme. 19 91

Plasma membrane fractions I and II isolated from bovine corpus luteum contain phosphoprotein phosphatases. Enzyme activities associated with both membrane fractions showed pH optima in the neutral range and were most active with phosphoprotamine as the exogenous substrate. The enzyme activity was partially inhibited by Co2+, Zn2+ and Fe2+. Dithioerythritol, glutathione (reduced) and 2-mercaptoethanol stimulated the enzyme activity, whereas N-ethylmaleimide and N-phenylmaleimide were inhibitory. Similarly, various cyclic nucleotides and nuclsoside triphosphates also inhibited phosphoprotein phosphatase activities. The phosphatase activity was also observed with endogenous phosphorylated membrane proteins as substrate. The endogenous phosphorylation of membranes was rapid and attained a maximal level after 15--20 min of incubation. Initially endogenous dephosphorylation was also very rapid, but did not reach completion. In addition to phosphoprotein phosphatase, membrane preparations also possessed very active cyclic-AMP-dependent protein kinase activity. Phosphoprotein phosphatase activity from plasma membranes was solubilized by ionic and nonionic detergents. Optimal solubilization was achieved with 0.1% sodium deoxycholate. Sucrose density gradient centrifugation of deoxycholate-solubilized fraction I and fraction II membranes resolved phosphoprotein phosphatase activity into two species with apparent sedimentation coefficients of 6.7 S (Mr 130000) and 4.8 S (Mr 90000). Cyclic-AMPstimulated protein kinase activity sedimented as a broad peak with a sedimentation coefficient of 5.5 S (Mr 110000).
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PMID:Solubilization and characterization of phosphoprotein phosphatase(s) from bovine corpus-luteum plasma membranes. 24 Jun 98

Prostaglandin E2 (PGE2) stimulated in vitro invasion, migration, and adherence to reconstituted basement membrane by metastatic Lewis lung carcinoma (LLC) clones, but this stimulation was blocked by protein kinase A (PKA) inhibitors and by expression of a transfected mutant RI alpha which blocks PKA activation. In vitro migration and adherence of metastatic LLC transfectants was heightened by expression of a transfected C alpha gene and further heightened by Zn2+ induction of the expression vector. Nonmetastatic LLC were not migratory nor invasive. However, nonmetastatic LLC that were stably transfected with a C alpha gene were both migratory and invasive, particularly when C alpha expression was further induced with Zn2+. The results of these in vitro studies show that PKA can enhance the metastatic characteristics of LLC.
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PMID:Activation of protein kinase A increases the in vitro invasion, migration, and adherence to reconstituted basement membrane by Lewis lung carcinoma tumor cells. 129 38

The phosphorylation of endogenous proteins was investigated in subcellular fractions prepared from isolated rabbit parietal cells incubated with either cimetidine (unstimulated) or a combination of histamine and forskolin (maximally stimulated). Phosphorylation of endogenous proteins in subfractions was then assessed in a post hoc assay using [gamma-32P]ATP as a phosphate donor in vitro. The Mg(2+)-dependent incorporation of [32P]phosphate into a 52-kDa protein (pp52M) was observed in the 4,000 g membrane fraction from stimulated but not unstimulated cells. The pp52M protein was identified as the type II regulatory subunit of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (RII) by isoelectric focusing, comigration with cAMP-binding proteins, and immunoprecipitation. Incorporation of [32P]phosphate into RII in the in vitro assay in the presence of Zn2+ was apparent in the 4,000 g membrane from stimulated but not unstimulated cells. The results thus suggested that, on stimulation, RII in membrane was dephosphorylated. Incorporation of [32P]phosphate into membrane-associated RII was completely abolished in the presence of 10 microM cAMP. The decrease in RII phosphorylation in membrane from stimulated cells assayed in the presence of cAMP was due to a phosphoprotein phosphatase activity that was completely inhibited by okadaic acid (1 microM). The results indicate that stimulation of parietal cells with histamine and forskolin results in the dephosphorylation of membrane bound RII by a protein phosphatase that is also membrane associated. Furthermore, okadaic acid inhibited histamine-stimulated accumulation of [14C]aminopyrine into isolated parietal cells without altering stimulated increases in cAMP. Thus protein phosphatase may be a significant regulator of parietal cell function.
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PMID:Dephosphorylation of cAMP-dependent protein kinase regulatory subunit in stimulated parietal cells. 131

Exposure of mouse colliculi neurons to selective 5-hydroxytryptamine (5-HT)4 agonists was accompanied by a rapid desensitization of the receptor-stimulated adenylyl cyclase response. Half-maximal desensitization occurred after 2 min. Only exposure of neurons to selective 5-HT4 agonists led to a potent desensitization of the 5-HT4-mediated response. Neurons exposed to other agents, like isoproterenol, vasoactive intestinal peptide, or forskolin, that increase cAMP levels did not undergo any desensitization of 5-HT4 receptors. Activation of protein kinase A with either 8-bromo-cAMP or dibutyryl-cAMP or application of inhibitors of protein kinase A-dependent phosphorylation did not change the rate of 5-HT4-induced desensitization. No shift to lower potency of 5-HT4 agonists in the concentration-response curve was observed. These results suggest that 5-HT4 receptor agonists induced homologous but not cAMP-mediated heterologous desensitization. A good correlation was found between the affinities of nine 5-HT4 agonists and their abilities to desensitize the adenylyl cyclase response. This may indicate that homologous desensitization is a function of the mean occupancy time of the receptors by agonists. When permeabilized neurons were loaded with heparin, an inhibitor of the beta-adrenergic receptor kinase (beta ARK), 5-HT4 receptor desensitization was reduced by 30-40%. Interestingly, Zn2+, an other inhibitor of beta ARK, totally prevented 5-HT4-induced desensitization. Pretreatment of neurons with concanavalin A, reported to inhibit sequestration of beta-adrenergic receptors from the cell surface, reduced the desensitization process by 70%. These data suggest that both sequestration and phosphorylation by beta ARK, or another specific agonist-dependent receptor kinase, are involved in homologous desensitization of 5-HT4 receptors coupled to adenylyl cyclase.
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PMID:Characterization of homologous 5-hydroxytryptamine4 receptor desensitization in colliculi neurons. 133 63

There are at least three isozymes (C alpha, C beta, and C gamma) of the mammalian catalytic (C) subunit of cAMP-dependent protein kinase (PKA) (Beebe, S., Oyen, O., Sandberg, M., Froysa, A., Hansson, V., and Jahnsen, T. (1990) Mol. Endocrinol. 4, 465-475). To compare the C gamma and C alpha isozymes, the respective cDNAs were expressed in permanently transformed Kin-8 PKA-deficient Y1 adrenal cells using the mouse metallothionein promoter. The recombinant C subunits were characterized as immunoreactive, zinc-inducible, cAMP-dependent kinase activities. In contrast to C alpha, histone was a better substrate than Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) for C gamma. Furthermore, C gamma histone kinase activity was not inhibited by the protein kinase inhibitor peptide (5-24 amide), which has been widely used as a PKA-specific inhibitor. The major C gamma peak (type I) eluted from DEAE-Sepharose at a higher NaCl concentration (120 mM) than the C alpha type I eluted (70 mM). C gamma and C alpha type II eluted between 220 and 240 mM NaCl. C gamma required higher concentrations of cAMP than C alpha did for dissociation from the mutant type I holoenzyme. These differences provided a basis for the separation of the mutant RI-associated isozymes on DEAE-Sepharose. Both C alpha (41-42 kDa) and C gamma (39-40 kDa) were identified by a C subunit antibody after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis. Zinc induced the PKA-mediated rounding phenotype in C gamma and C alpha clones, thereby restoring the cells to the parent Y1 adrenal cell phenotype. Collectively, these data indicate that C gamma is an active PKA C subunit but suggest that C gamma and C alpha have different protein and peptide recognition determinants.
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PMID:The C gamma subunit is a unique isozyme of the cAMP-dependent protein kinase. 133 96

1. A protein kinase type II was purified from calf thymus chromatin using ammonium sulphate fractionation, ion exchange chromatography on DEAE and phosphocellulose and affinity chromatography on phosvitin- and casein-sepharose columns. 2. The enzyme moves as a single band in non-denaturing gel electrophoresis at pH 8.3, which coincides with the enzyme activity assayed on gel slices. 3. Sodium dodecyl sulphate gel electrophoresis shows three separate polypeptide chains having M(r) of 40,000, 38,000 and 25,000, respectively. The native M(r) was about 130,000, as measured by HPLC on Superose 12 column, suggesting a subunit structure of alpha, alpha', beta 2 type. The enzyme incubated with [gamma 32P]ATP or [gamma 32P]GTP as phosphoryl donors undergoes autophosphorylation in the M(r) = 25,000 subunit. 4. The enzyme phosphorylates casein (Km = 7 microM) and phosvitin (Km = 5 microM) but not histones and was strongly deactivated by Zn2+ ions (I50 = 0.05 mM) and heparin (I50 = 0.1 micrograms/ml). 5. The enzyme seems to be the major phosphorylating system present in the 0.35 M NaCl chromatin extract of calf thymus. The RNA polymerase II from calf thymus and RNA polymerase from E. coli are both phosphorylated by protein kinase NII. The effect of phosphorylation, which causes a remarkable increase of DNA transcription rate, was studied in vitro and extensively discussed.
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PMID:Protein kinase NII from calf thymus chromatin. Isolation, characterization and some functional properties. 145 14

A new protein kinase C (PKC)-related cDNA with unique tissue distribution has been isolated and characterized. This cDNA encodes a protein, nPKC theta, which consists of 707 amino acid residues and showed the highest sequence similarity to nPKC delta (67.0% in total). nPKC theta has a zinc-finger-like cysteine-rich sequence (C1 region) and a protein kinase domain sequence (C3 region), both of which are common in all PKC family members. However, nPKC theta lacks a putative Ca2+ binding region (C2 region) that is seen only in the conventional PKC subfamily (cPKC alpha, -beta I, -beta II, and -gamma) but not in the novel PKC subfamily (nPKC delta, -epsilon, -zeta, and -eta). Northern (RNA) blot analyses revealed that the mRNA for nPKC theta is expressed predominantly in skeletal muscle. Furthermore, nPKC theta mRNA is the most abundantly expressed PKC isoform in skeletal muscle among the nine PKC family members. nPKC theta expressed in COS1 cells serves as a phorbol ester receptor. By the use of an antipeptide antibody specific to the D2-D3 region of the nPKC theta sequence, nPKC theta was recognized as a 79-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis in mouse skeletal muscle extract and also in an extract from COS1 cells transfected with an nPKC theta cDNA expression plasmid. Autophosphorylation of immunoprecipitated nPKC theta was observed; it was enhanced by phosphatidylserine and 12-O-tetradecanoylphorbol-13-acetate but attenuated by the addition of Ca2+. These results clearly demonstrate that nPKC theta should be considered a member of the PKC family of proteins that play crucial roles in the signal transduction pathway.
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PMID:A new member of the protein kinase C family, nPKC theta, predominantly expressed in skeletal muscle. 150 94

During chemically induced differentiation of murine erythroleukemia (MEL) cells, cAMP-dependent protein kinase activity increases, and the enzyme's isozyme pattern changes. To examine the enzyme's role during MEL cell differentiation, we stably transfected MEL cells with recombinant plasmids in which the mouse metallothionein I promoter controlled expression of either a mutant form of the type I regulatory subunit of cAMP-dependent protein kinase (RI) or the enzyme's specific peptide inhibitor (PKI); expressing either sequence rendered cells cAMP-dependent protein kinase-deficient. Chemically induced differentiation of MEL cells as assessed by beta-globin mRNA and hemoglobin accumulation was inhibited in RI mutant and PKI transfectants; adding zinc further inhibited differentiation in the transfectants but had no effect on parental MEL cells. The inhibition of differentiation correlated with the amount of RI mutant mRNA and protein in the RI mutant transfectants and with the cells' degree of cAMP-dependent protein kinase deficiency in both the RI mutant and PKI transfectants. Overexpression of wild type RI did not interfere with differentiation or enzyme activity. We conclude that cAMP-dependent protein kinase activity is important for chemically induced differentiation of MEL cells and that the down-regulation of RI protein which occurs during MEL cell differentiation is not essential for differentiation to proceed.
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PMID:Chemically induced murine erythroleukemia cell differentiation is severely impaired when cAMP-dependent protein kinase activity is repressed by transfected genes. 164 3


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