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)

Phosphoenolpyruvate PyrP carboxylase (PyrPC) and PyrPC kinase were copurified from dark-adapted leaves of the common ice plant Mesembryanthemum crystallinum L. with crassulacean-acid metabolism (CAM). Purification by (NH4)2SO4 fractionation, chromatography on Fractogel-DEAE and hydroxylapatite resulted in a PyrPC preparation with a specific activity of 23-25 U/mg protein and a protein kinase activity of 255 mumol Pi.mol-1 PyrPC.s-1. After in vitro phosphorylation, the most prominently phosphorylated polypeptide was identified as PyrPC by immunoblotting and sequencing. Phosphorylation of PyrPC in vitro by incubation with 400 microM MgATP decreased its sensitivity towards malate. When purified in the absence of the protease inhibitor chymostatin, PyrPC lost an N-terminal sequence of 128 amino acids. Although the carboxylation reaction was unaffected, the truncated PyrPC could neither be phosphorylated in vitro nor inhibited by malate. This result and data obtained by limited proteolysis concur with the hypothesis [Jiao, J.A. & Chollet, R. (1989) Arch. Biochem. Biophys. 283, 300-305] that Ser11 is the phosphorylation site of the CAM PyrPC of M. crystallinum. At pH 7.0, the Km for ATP of the protein kinase was 25 microM; phosphorylation of PyrPC was maximal after 30 min at pH 7.0. The kinase showed also activity with histone III-S but not with dephosphorylated casein. It was inhibited by malate. The results show, that reversible protein phosphorylation is an important factor in the regulation of PyrPC in the facultative CAM plant M. crystallinum, similar to C4 and constitutive CAM plants.
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PMID:Regulatory protein phosphorylation of phosphoenolpyruvate carboxylase in the facultative crassulacean-acid-metabolism plant Mesembryanthemum crystallinum L. 139 23

We have examined the synthesis of the protease inhibitors alpha 1-antitrypsin (alpha 1-AT) and alpha 1-antichymotrypsin (alpha 1-ACHY) by variants of the MCF-7 human breast cancer cell line. Spent medium from MCF-7 203P cells, grown in the absence of serum, was found to contain immunoreactive alpha 1-AT and alpha 1-ACHY by Western blotting. In the presence of 10(-8) M estradiol, levels of both inhibitors were increased 3- to 6-fold. Incubation of spent medium with [125I]trypsin or [125I]chymotrypsin resulted in the formation of stable 75- and 90-kDa complexes identical to the complexes formed between these proteases and the protease inhibitors in plasma, showing the release of active protease inhibitors by MCF-7 cells in culture. Immunoprecipitation of 35S-labeled proteins from the medium of cells grown in the presence of [35S]methionine yielded comparable results, confirming hormonally sensitive synthesis of both protease inhibitors. Northern blot analysis suggests that stimulation of estradiol occurs at the level of transcription. Tetradecanoyl phorbol acetate (50 ng/ml) also stimulated alpha 1-AT and alpha 1-ACHY synthesis 2- to 4-fold, suggesting the involvement of protein kinase-C. Comparison studies with MCF-7 cell sublines ML, BK, 203P, and 300P (a variant spontaneously appearing after 100 passages of 203P) show a wide variation in synthesis of alpha 1-AT and alpha 1-ACHY proteins; sublines 203P and 300P synthesize both inhibitors, the ML subline synthesizes detectable amounts only of alpha 1-ACHY, while no detectable synthesis of either inhibitor was seen in the BK subline. Similar results were obtained for protease inhibitor mRNA transcription by Northern blotting, although low levels of alpha 1-AT mRNA transcription by the ML subline and of alpha 1-AT and alpha 1-ACHY mRNA transcription by the BK subline could be detected.
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PMID:Regulation of antitrypsin and antichymotrypsin synthesis by MCF-7 breast cancer cell sublines. 220 35

GH exerts a number of metabolic effects on adipose tissue. Depending on the circumstances, it may increase or decrease glucose metabolism and lipolysis. These effects appear to be mediated by a single class of receptors, which bind GH with high affinity. Incubation of isolated rat adipocytes with a variety of lipolytic agents, including catecholamines, forskolin, or (Bu)2cAMP, decreased the specific binding of [125I]human (h) GH within 10 min. In the presence of 10 microM forskolin, GH binding declined to less than 20% of the control value within 50 min. Cholera and pertussis toxins, which increase cAMP secondary to ADP ribosylation of guanine nucleotide-binding proteins associated with hormone receptors, also decreased the binding of GH. None of these agents affected the rate of loss of cell-associated 125I when added to cells that had previously equilibrated with [125I]hGH. The inhibitory effects of forskolin and (Bu)2cAMP were at least as great when binding was measured in the presence of the protease inhibitor leupeptin, suggesting that increased rates of internalization and processing of bound hormone could not account for the decline in binding. Scatchard plots of data obtained in the presence of forskolin or (Bu)2cAMP were linear and parallel to control plots, indicating that the decline in binding could be accounted for by a decrease in the number of binding sites, with no change in affinity. To determine whether phosphorylation affected binding to receptors already present in the membrane or modified the turnover of receptors, we studied adipocyte ghosts, whose cellular apparatus for receptor turnover is disrupted. Incubation of adipocyte ghosts with cAMP-dependent protein kinase decreased the binding of [125I]hGH by 25%. The data suggest that cAMP-dependent phosphorylation of the GH receptor or a closely associated membrane protein renders the receptor incapable of binding GH.
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PMID:Adenosine 3',5'-monophosphate-dependent loss of growth hormone binding in rat adipocytes. 283 58

Tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of catecholamines, was reversibly inactivated by incubation with antipain, which is known as a microbial protease inhibitor. The inactivation was a time-dependent reaction and it was prominent when the enzyme was assayed at a neutral pH but not when assayed at an acidic pH. The inactivated enzyme was markedly activated by cyclic AMP-dependent protein kinase. Other microbial protease inhibitors such as leupeptin, chymostatin, and pepstatin did not induce such as inactivation of the enzyme.
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PMID:Reversible conversion of tyrosine hydroxylase to an inactive form by antipain. 288 68

Production of cAMP- and Ca2+-independent protein kinase was stimulated when rat liver plasma membrane was incubated with increasing concentrations of NaCl. This protein kinase release was diminished by addition of protease inhibitor. The molecular mass of this enzyme was approx. 50 kDa and a high concentration of Mg2+ was required for whole histone phosphorylation. These properties are similar to those of the protease-activated form of protein kinase C. The NaCl effect could be replaced by other salts such as LiCl and NaHCO3. These results suggest that membrane-bound protein kinase C is activated by limited proteolysis corresponding to an increase in ionic strength.
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PMID:Effect of ionic strength on production of cAMP- and Ca2+-independent protein kinase from rat liver plasma membrane. 300 29

Soluble tyrosine hydroxylase (TH) prepared from rat striatum by sonication, centrifugation, and gel filtration on Sephadex G-25 was activated by preincubation with Ca2+, ATP, and Mg2+. Activation occurred with micromolar concentrations of Ca2+ and required the presence of both ATP and Mg2+. The activation was reversible and was characterized by a large decrease of apparent Km for the pteridine cofactor and a small increase of Vmax. Ca2+-induced activation was small when TH activity was assayed at pH values near the optimum, but the magnitude of the activation increased with increasing assay pH. The activation apparently did not involve Ca2+-activated protease because it was not affected by the protease inhibitor leupeptin. Nor did it involve cyclic AMP-dependent protein kinase, as evidenced by the failure of the heat-stable inhibitor of this kinase to decrease Ca2+-induced TH activation. Furthermore, the activation of TH evoked by Ca2+ and that produced by cyclic AMP was additive. These experiments indicate that striatal TH can be activated in vitro by an endogenous Ca2+-dependent mechanism. The similarity of the Ca2+-induced activation of TH to that elicited by increased neuronal activity and terminal depolarization is discussed.
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PMID:Calcium, ATP, and magnesium activate soluble tyrosine hydroxylase from rat striatum. 614 58

Transport of sugars across the cytoplasmic membranes of gram-positive bacteria appears to be regulated by the action of a metabolite-activated, ATP-dependent protein kinase that phosphorylates a seryl residue in the phosphocarrier protein of the phosphotransferase system, HPr. We have developed a quantitative assay for measuring the activity of this enzyme from Streptococcus pyogenes. The product of the in vitro protein kinase-catalyzed reaction was shown to be phosphoseryl-HPr by several independent criteria (rates of hydrolysis in the presence of various agents, detection of serine-phosphate in acid hydrolysates, immunological assay, and electrophoretic migration rates). HPrs isolated from four different gram-positive bacteria (S. pyogenes, Streptococcus faecalis, Staphylococcus aureus, and Bacillus subtilis) were shown to be phosphorylated by the kinase from S. pyogenes. In contrast, Escherichia coli HPr was not a substrate of this enzyme. The soluble kinase released from the particulate fraction of the cells with high salt in the presence of a protease inhibitor was shown to have an approximate molecular weight of 60,000 as estimated by gel filtration. Its activity was dependent on divalent cations, with Mg2+ and Mn2+ being most active. EDTA, Pi, and high concentrations of salt were strongly inhibitory. The enzyme was optimally active at pH 7.0, exhibited high affinity for its substrates, and was dependent on the presence of one of several metabolites. Of these compounds, fructose 1-6-diphosphate was most active, with gluconate 6-phosphate, 2-phosphoglycerate, 2,3-diphosphoglycerate, phosphoenolpyruvate, and pyruvate exhibiting moderate to low stimulatory activities. Other compounds tested, including a variety of sugar phosphates, pyridine nucleotides, and other metabolites were without effect. The ATP-dependent phosphorylation of HPr on the seryl residue was strongly inhibited by phosphoenolpyruvate-dependent phosphorylation of the active histidyl residue of this protein. Treatment of the kinase with diethyl pyrocarbonate strongly inhibited the ATP-dependent phosphorylation activity, although the sulfhydryl reagents N-ethylmaleimide, p-chloromercuribenzoate, and iodoacetate were without effect. These results serve to characterize the HPr (serine) kinase, which apparently regulates the rates of carbohydrate transport in streptococcal cells via the phosphotransferase system. A primary role of this kinase in the control of cellular inducer levels and carbohydrate metabolic rates is proposed.
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PMID:Properties of ATP-dependent protein kinase from Streptococcus pyogenes that phosphorylates a seryl residue in HPr, a phosphocarrier protein of the phosphotransferase system. 643 22

Two isoenzymes of cAMP-dependent protein kinases were found in MCF-7 cytosol. The regulatory (cAMP-binding) subunit of protein kinase I (predominant form) has an apparent molecular weight (MW) of 49,000 and the two forms of regulatory subunits of protein kinase II have MWs of 52,000 and 54,000. Substantial amounts of the 49,000 protein cochromatographed on DEAE cellulose with protein kinase II. The quantities of protein kinase holoenzyme activity are strongly influenced by extraction procedures: the use of EDTA and of the protease inhibitor benzamidine can lead to extensive dissociation. On the other hand, high yields of cAMP-dependent protein kinase holoenzyme activity were consistently obtained with 150 mM KCl.
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PMID:Cyclic AMP-dependent protein kinases and cAMP-binding proteins in human mammary tumor MCF-7 cells. 673 88

A scintillation proximity assay (SPA) using 33phosphorous is described for human cytomegalovirus (HCMV) UL80 protease. This is the first demonstration that 33phosphorous is compatible with the SPA system. The peptide substrate used in the assay contains an HCMV protease cleavage site and is biotinylated at its amino terminus. The peptide also contains a site for protein kinase A, enabling radiolabeling at its carboxy terminus with [gamma-33P]ATP. Peptide is incubated with protease, followed by binding to streptavidin-coated SPA beads via biotin. Cleavage of the peptide by the protease results in a decrease in the radioactive signal, which is prevented by the presence of a protease inhibitor. This methodology is applicable to other proteases whose cleavage site is known.
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PMID:Development of a scintillation proximity assay for human cytomegalovirus protease using 33phosphorous. 866 May 47

Clotting factor XII (Hageman factor) contains epidermal growth factor (EGF)-homologous domains and is reported to be a potent mitogen for human hepatoma (HepG2) cells. In this study, we tested whether factor XII exhibits growth factor activity on several other EGF-sensitive target cells, including fetal hepatocytes, endothelial cells, alveolar type II cells, and aortic smooth muscle cells. We found that factor XII significantly enhanced [3H]thymidine incorporation in aortic smooth muscle cells (SMCs) and all other cells tested. Tyrphostin, a growth factor receptor/tyrosine kinase antagonist, inhibited both EGF- and factor XII-induced responses. However, differences in the levels of magnitude of DNA synthesis, the observed synergism between EGF and factor XII, and the differential sensitivity to tyrphostin suggest that the EGF receptor and the factor XII receptor may be nonidentical. The factor XII-induced mitogenic response was achieved at concentrations that were 1/10th the physiologic range for the circulating factor and was reduced by popcorn inhibitor, a specific factor XII protease inhibitor. Treatment of aortic SMCs with factor XII, as well as activated factor XII, resulted in a rapid and transient activation of a mitogen-activated/extracellular signal-regulated protein kinase with peak activity/tyrosine phosphorylation observed at 5 to 10 min of exposure. Taken together, these data (i) confirm that clotting factor XII functions as a mitogenic growth factor and (ii) demonstrate that factor XII activates a signal transduction pathway, which includes a mitogen-activated protein kinase.
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PMID:Factor XII-induced mitogenesis is mediated via a distinct signal transduction pathway that activates a mitogen-activated protein kinase. 870 Sep 4


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