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)

Homogeneous preparations of fructose-1,6-bisphosphatase from mouse, man, rabbit, pig, and rat were tested as substrates for cyclic AMP-dependent protein kinase. Up to 1 mol of [32P]phosphate per mole enzyme subunit was incorporated into fructose-1,6-bisphosphatase from pig and rabbit liver, which should be compared with 2.6 mol of phosphate per mole enzyme subunit in the case of the rat liver enzyme. The phosphorylation of fructose-1,6-bisphosphatase from the livers of man and mouse was negligible. Phosphorylation of pig and rabbit fructose-1,6-bisphosphatase decreased the apparent Km for fructose-1,6-bisphosphate, but in contrast to the case of the rat liver enzyme it did not change the inhibition constants for AMP and fructose-2,6-bisphosphate. The phosphorylation sites in rabbit and pig liver fructose-1,6-bisphosphatase were located close to the carboxyterminal of the polypeptide chains, since trypsin treatment of the phosphorylated enzyme quantitatively removed all of the protein-bound radioactivity without significantly altering the subunit molecular weight and with a maintained neutral pH optimum.
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PMID:In vitro phosphorylation of fructose-1,6-bisphosphatase from rabbit and pig liver with cyclic AMP-dependent protein kinase. 283 70

The phosphorylation of the cytoplasmic domain of band 3 by the human erythrocyte membrane kinase and casein kinase A has been investigated. The cytoplasmic domain of band 3 was released from erythrocyte vesicles by treatment with alpha-chymotrypsin and isolated as a 43,000-Da peptide. Both the membrane kinase and casein kinase A catalyzed the incorporation of about 1 mol of phosphate per mole of the band 3 fragment. The phosphorylation of the band 3 fragment by both kinases was not additive, suggesting that the two enzymes might recognize the same phosphorylation sites. Also in support of this notion was the observation that the phosphopeptide maps of the band 3 fragment phosphorylated by the two kinases were identical. Phosphoamino acid analysis of the band 3 fragment phosphorylated by casein kinase A revealed the presence of approximately equal amounts of phosphoserine and phosphothreonine and, to a lesser extent, phosphotyrosine. The interaction between the 43,000-Da peptide with ankyrin and the effect of phosphorylation on this interaction have been examined. The band 3 fragment was found to form two different types of complexes, termed C1 and C2, with ankyrin in a saturable manner. The C1 and C2 complexes contained about 1.7 and 0.43 mol of band 3 fragment per mole of ankyrin, respectively. Interestingly, these binding stoichiometries were found to be reduced by half by the phosphorylation of ankyrin but not by the phosphorylation of the band 3 fragment. The results suggest that the structure and dynamics of the erythrocyte membrane cytoskeletal network may be regulated by phosphorylation.
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PMID:Analysis of band 3 cytoplasmic domain phosphorylation and association with ankyrin. 295 9

Reconstituted porcine cAMP-dependent protein kinase type I was labeled with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) to study cyclic nucleotide binding and to identify amino acid residues that are either in or in close proximity to the cAMP binding sites. The photoaffinity analogue 8-N3cAMP behaved as cAMP itself with respect to cyclic nucleotide binding. For both cAMP and 8-N3cAMP, 2 mol of nucleotide was bound per mole of type I regulatory subunit monomer (RI), the apparent Kd's observed were approximately 10-17 nM on the basis of either Millipore filtration assays, equilibrium dialysis, or ammonium sulfate precipitation, Scatchard plots showed positive cooperativity, and (4) the Hill coefficients were approximately 1.5-1.6. After photolysis and addition of an excess of cAMP, approximately 1 mol of 8-N3cAMP/mol of RI monomer was covalently incorporated. Tryptic digestion of the labeled protein revealed that two unique tryptic peptides were modified. Proline-271 and tyrosine-371 were identified as the two residues that were covalently modified by 8-N3cAMP in RI. These results contrast with the type II regulatory subunit (RII) where 8-N3cAMP modified covalently a single tyrosine residue [Kerlavage, A. R., & Taylor, S. S. (1980) J. Biol. Chem. 255, 8483-8488]. RI contains two adjacent regions of sequence homology in the COOH-terminal fragment that binds two molecules of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Covalent modification of both cAMP binding sites in cAMP-dependent protein kinase I by 8-azidoadenosine 3',5'-monophosphate. 298 89

Bovine serum albumin (BSA) was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase under general protein phosphorylation conditions. The optimal pH for this phosphorylation was 9.0. The K0.5 (the concentration required for 50% of maximal phosphorylation) for BSA at pH 7.5 was 15 microM. One mole of phosphate was incorporated per mole of BSA, and only one phosphopeptide fragment was obtained after extensive proteolysis with trypsin. BSA phosphorylation required dithiothreitol or GSH, but GSH was only one-fiftieth as effective as dithiothreitol. GSSG counteracted the effect of dithiothreitol and GSH. Phosphorylation increased in a time-dependent and dithiothreitol concentration-dependent manner when BSA was preincubated with dithiothreitol. The increase in the incorporation of 32P correlated with the appearance of up to six free sulfhydryl groups. The effect of dithiothreitol on BSA appeared reversible, since reoxidation of reduced BSA decreased its susceptibility to phosphorylation. These experiments showed that this in vitro phosphorylation is dependent on the sulfhydryl-disulfide state of BSA. The possible implications of the sulfhydryl-disulfide state of proteins in the regulation of phosphorylation are discussed.
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PMID:Effect of sulfhydryl-disulfide state on protein phosphorylation: phosphorylation of bovine serum albumin. 298 43

Two proteins, termed P85gag-mos and P58gag, are encoded by the temperature-sensitive transformation mutant, ts110 Moloney murine sarcoma virus (MuSV). Based on temperature-shift studies, P85gag-mos is believed to be important for the transforming potential of ts110 MuSV and has been found to be associated with a thermolabile kinase activity that phosphorylates both P85gag-mos and P58gag in immune complexes. Modifications of the original kinase assay conditions are reported here that have allowed a 30-fold increase in the specific activity of P85gag-mos phosphorylated in vitro. The in vitro P85gag-mos-phosphorylating activity was found to be unresponsive to 10 microM-cAMP or 10 microM-cGMP. Addition of 1 mM-pyrophosphate, a known phosphatase inhibitor, to the reaction mixture resulted in an increased yield of phosphorylated P85gag-mos and P58gag; the molar phosphate incorporation per mole of P85gag-mos increased from 0.032 to 0.9, whereas the specific activity of in vitro-phosphorylated P58gag increased 18-fold, from 0.013 to 0.234. pH curves of the in vitro kinase reaction further confirmed the presence of phosphatase activity; in the absence of pyrophosphate, a sharp optimum at pH 4 to 5 was observed, whereas it shifted broadly to pH 7.0 in the presence of pyrophosphate. Under the latter conditions, several experiments were performed in order to determine if the kinase was associated with either gag or mos sequences of P85gag-mos. Antisera directed against p15, p12 and p30 sequences of the gag protein region of P85gag-mos yielded immune complexes that allowed phosphorylation in vitro of P85gag-mos. No phosphorylating activity was detected in immune complexes containing MuSV-124-encoded P62gag. An anti-mos serum generated against a synthetic peptide representing the predicted v-mos amino acid residues 37 to 55 recognizes P85gag-mos and allowed phosphorylation of P85gag-mos in vitro in the absence of P58gag. Peptide mapping of both phosphorylated P85gag-mos and P58gag, by using a combination of Cleveland and Western/immunoperoxidase techniques, demonstrated that P85gag-mos became phosphorylated not only on gag sequences, but also at the N-terminal portion of v-mos. Phosphoamino acid analyses of P85gag-mos and P58gag phosphorylated in vitro under these modified conditions yielded predominantly phosphoserine and lesser amounts of phosphothreonine. Metabolically 32P-labelled P85gag-mos and P58gag were also found to contain phosphoserine and phosphothreonine. Based on these results, we conclude that a cAMP-independent, serine/threonine protein kinase activity is associated with the mos sequences of P85gag-mos.
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PMID:A cAMP-independent serine/threonine kinase activity is associated with the mos sequences of ts110 Moloney murine sarcoma virus-encoded P85gag-mos. 299 51

The enzyme, myosin light chain kinase, has been purified to homogeneity from bovine aortic vascular smooth muscle. Approximately 10 mg of enzyme could be obtained from 1 kg of fresh aortas with an overall yield of 26% of the original activity. The vascular myosin light chain kinase has a molecular weight of 160 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Antiserum raised to the aortic myosin light chain kinase in rabbits strongly inhibited phosphotransferase activity. In addition, the antiserum was used to identify myosin kinase in a crude homogenate of vascular smooth muscle by radioimmunoblotting. A single species of the enzyme (Mr = 160 000) was identified. The bovine aortic myosin kinase could be phosphorylated by both cyclic AMP- and GMP-dependent protein kinases. Approximately 2 mols PO4/mole of enzyme could be incorporated by the cyclic AMP-dependent protein kinase in the absence of calmodulin. If Ca2+ and calmodulin were included in the reaction mixture, phosphate incorporation by the cyclic AMP-dependent protein kinase was reduced to 1 mol and phosphorylation by cyclic GMP-dependent protein kinase was completely inhibited. These results were confirmed by tryptic peptide mapping. Two distinct phosphopeptides were identified: site-1 and site-2. Both could be phosphorylated by the cyclic AMP-dependent protein kinase but only site-1 was phosphorylated by the cyclic GMP-dependent enzyme. In the presence of Ca2+ and calmodulin, phosphorylation by cAMP-dependent protein kinase was restricted to site-1. The effect of phosphorylation on myosin light chain kinase activity was determined. Only phosphorylation by cyclic AMP-dependent protein kinase was found to alter the requirement of myosin kinase for calmodulin. The K0.5 (i.e. the concentration of calmodulin required for half-maximal enzyme activation) for calmodulin was 5 nM for the unphosphorylated myosin kinase. With 2 mol PO4/mol myosin kinase incorporated, the K0.5 for calmodulin was increased to 82 nM. When only 1 mol PO4/mol myosin kinase was incorporated, no effect on calmodulin requirement was observed. Moreover, single site phosphorylation had no effect on other activity parameters, including Km for ATP and for light chains. Our studies suggest that cyclic AMP-dependent protein kinase may play an important role in the regulation of vascular myosin kinase activity. Moreover, our results indicate that cyclic GMP-dependent protein kinase does not affect calmodulin-activation of myosin kinase or several other activity parameters.
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PMID:Phosphorylation of myosin light chain kinase from vascular smooth muscle by cAMP- and cGMP-dependent protein kinases. 299 88

Guanosine cyclic 3',5'-monophosphate (cGMP) dependent protein kinase is inactivated by o-phthalaldehyde. The loss of phosphotransferase activity following treatment with o-phthalaldehyde was rapid, and the second-order rate constant at 25 degrees C and pH 7.3 was 35 M-1 s-1. The inactivation reaction did not follow saturation kinetics. The cGMP-dependent protein kinase was protected from inactivation by its substrates, MgATP and Ser-peptide. Fluorescence excitation and emission spectroscopic data showed that an isoindole derivative was formed following the reaction between cGMP-dependent protein kinase and o-phthalaldehyde. Four moles of isoindole per mole of the cGMP-dependent protein kinase dimer was formed following complete inactivation by o-phthalaldehyde. In the absence of cGMP, the protein kinase lost only 50% of its cGMP binding activity while there was almost a complete loss of its phosphotransferase activity. Studies in the presence of 20 microM cGMP, however, showed that about 2 mol of isoindole groups per mole of the protein kinase dimer was formed following complete inactivation by o-phthalaldehyde. The second-order rate constant for inactivation of cGMP-dependent protein kinase by o-phthalaldehyde in the presence of 20 microM cGMP was 40 M-1 s-1. Fluorescence measurements of samples containing inactivated, iodoacetamide-modified, or 5'-[p-(fluorosulfonyl)benzoyl]adenosine-modified, cGMP-dependent protein kinase and o-phthalaldehyde showed that the intensity of fluorescence in each case was about 50% of that obtained from unmodified, active cGMP-dependent protein kinase and o-phthalaldehyde.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Inactivation of guanosine cyclic 3',5'-monophosphate dependent protein kinase from bovine lung by o-phthalaldehyde. 300 44

The calmodulin-dependent protein phosphatase was shown to be phosphorylated by the Ca2+, phospholipid-dependent protein kinase (protein kinase C). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the 61 kDa catalytic subunit was phosphorylated. Phosphorylation by protein kinase C was stimulated up to 15-fold by addition of phosphatidyl-L-serine and between 0.5 to 1.0 mole of phosphate was incorporated per mole of phosphatase. It is possible that protein kinase C is involved in the regulation of the calmodulin-dependent protein phosphatase via this novel phosphorylation of the enzyme.
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PMID:Phosphorylation of the calmodulin-dependent protein phosphatase by protein kinase C. 301 38

The 36kDa subunit of protein I (p36) is a major substrate of several tyrosine protein kinases. Here we demonstrate that protein kinase C catalyzes the incorporation of 1.7 moles of phosphate per mole of protein I. Phosphorylation is absolutely dependent on the presence of both calcium and phospholipid, and is specific for serine and threonine residues. Phosphorylation of protein I by the c-AMP dependent protein kinase, phosphorylase kinase, casein kinase I, and casein kinase II was not observed. The in vivo significance of protein kinase C dependent phosphorylation of p36 is discussed.
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PMID:Phosphorylation of p36 in vitro by protein kinase C. 308 58

We have tested the hypothesis that the mechanism of platelet-derived growth factor (PDGF) and phorbol diester action to decrease the apparent affinity of the epidermal growth factor (EGF) receptor is the phosphorylation of the EGF receptor at the Ca2+/phospholipid-dependent protein kinase (protein kinase C) phosphorylation site, threonine 654. Protein kinase C-deficient cells were prepared by prolonged incubation of human fibroblasts with phorbol diester. Addition of phorbol diesters to these cells fails to regulate EGF receptor affinity or threonine 654 phosphorylation. In contrast, PDGF treatment of both control and protein kinase C-deficient fibroblasts causes a decrease in the apparent affinity of the EGF receptor and an increase in threonine 654 phosphorylation. Thus, the ability of PDGF or phorbol diester to modulate EGF receptor affinity occurs only when threonine 654 phosphorylation is increased. The stoichiometry of threonine 654 phosphorylation associated with a 50% decrease in the binding of 125I-EGF to high affinity sites was 0.15 versus 0.3 mol of phosphate per mole of EGF receptor when 32P-labeled fibroblasts are treated with PDGF or phorbol diester, respectively. It is concluded that EGF receptor phosphorylation at threonine 654 can be regulated by PDGF independently of protein kinase C, substoichiometric phosphorylation of the total EGF receptor pool at threonine 654 is caused by maximally effective concentrations of PDGF, and different extents of phosphorylation of EGF receptors at threonine 654 are observed for maximally effective concentrations of PDGF and phorbol diester, respectively. The data are consistent with the hypothesis that a specific subpopulation of EGF receptors that exhibit high affinity for EGF are regulated by threonine 654 phosphorylation.
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PMID:Stimulation of epidermal growth factor receptor threonine 654 phosphorylation by platelet-derived growth factor in protein kinase C-deficient human fibroblasts. 310 61


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