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)

Intact rat fat cells exposed to 12.5 microM [gamma-32P]ATP incorporate label into specific proteins within minutes. By solubilizing the reaction mixture with SDS which by passes the subcellular fractionation steps, the labeled proteins can be identified in autoradiographs of SDS-PAGE gels. The most prominently labeled protein has an Mr of 42,000. Localization of this component to the cell surface can be made on the basis of inhibition of phosphorylation by addition of a protein derived from the rat brain with protein kinase inhibitory property, susceptibility of the phosphorylated protein to tryptic digestion, whereas the unphosphorylated protein is unaffected by digestion with trypsin (15 min), inhibition of phosphorylation of this protein after brief exposure to melittin, and the consistent observation that more label is associated with the 42,000 Mr band in homogenates and permeabilized cells than in comparable numbers of intact cells exposed to the same amount of label. A 42,000 Mr phosphoprotein is also present in mitochondria which is most likely the alpha subunit of pyruvate dehydrogenase. To rule out the possibility that the cell surface protein might be a mitochondrial contaminant from broken cells, 32Pi-labeled and [gamma-32P]ATP-labeled cells were solubilized with Triton and chromatographed on a rabbit anti-pyruvate dehydrogenase antibody-Sepharose 4B column. A single labeled peak was detected upon elution of the bound fraction only in the 32Pi-labeled sample, and not in the [gamma-32P]ATP-labeled sample. Subcellular fractionation studies of intact cells labeled with [gamma-32P]ATP showed differences in the recovery of phosphoproteins of 42,000 Mr depending on whether a continuous sucrose gradient (27.6-54.1%, g/ml) or a discontinuous sucrose gradient (16, 35 and 48%, g/ml) was used. Phosphoproteins of 42,000 Mr were located in the mitochondrial and membrane fractions collected by discontinuous sucrose gradient separation, whereas a phosphoprotein of 42,000 Mr was found primarily in the mitochondrial fraction after continuous sucrose gradient separation. By 5'-nucleotidase activity measurements, the latter approach appears to result in the isolation of a heavy fragment of the plasma membrane with the mitochondrial light fraction which is 42,000 in Mr and labeled. Finally, comparison of the autoradiographs of two-dimensional (2D) gels (isoelectric focusing followed by 10% SDS-PAGE) show different isoelectric points for 42,000 Mr components in [gamma-32P]ATP- and 32Pi-labeled cells.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Characterization of the major phosphoprotein and its kinase on the surface of the rat adipocyte. 377 93

Stimulation of bovine chromaffin cell in culture changed (increased or decreased) the phosphorylation state of several proteins as examined by 32P incorporation. Enhanced phosphorylation of 22 protein bands as well as increased dephosphorylation of a 20.4 kilodaltons protein band was observed when extracts of cultured chromaffin cells stimulated by either acetylcholine or high K+ were subjected to mono-dimensional gel electrophoresis. For several protein bands, the degree of phosphorylation was larger in cells stimulated by acetylcholine than in those challenged by a depolarizing concentration of K+. The most affected phosphoproteins have apparent molecular weights of 14,800, 29,000, 33,000, 57,000 (tubulin subunit), 63,000 (tyrosine hydroxylase subunit) and 94,000. The presence of a low extracellular calcium concentration (0.5 mM Ca2+ plus 15 mM Mg2+) in the incubation medium inhibited (38-100%) the acetylcholine-evoked increases in protein phosphorylation observed previously for 18 protein bands. Trifluoperazine at the concentration required for 50% inhibition of acetylcholine-induced catecholamine release decreases (33-100%) the stimulation-induced phosphorylation in all polypeptides, with the exception of the 14.8 kilodaltons and the dephosphorylated 20.4 kilodaltons components which were not affected. Two-dimensional gel electrophoresis analysis revealed that exposure of chromaffin cells to acetylcholine produced two types of effect on protein phosphorylation: activation of protein kinase activities affecting about 30 polypeptides; activation of protein phosphatase activities resulting in the dephosphorylation of about 40 polypeptides, most of them appearing as minor phosphoproteins, with the exception of the alpha-subunit of pyruvate dehydrogenase and the 20.4 kilodaltons polypeptide. On the basis of their molecular properties (molecular weight and pI) and their abundance in chromaffin cells, the 80 kilodaltons phosphoprotein which focused at pI 4.8 and the 117.5 kilodaltons phosphoprotein which focused at pI 5.0 were identified as chromogranins A and B, respectively. The relationship between acetylcholine-induced protein phosphorylation (or dephosphorylation) and catecholamine secretion was also investigated. The time course of protein phosphorylation (or dephosphorylation) paralleled or preceded [3H]noradrenaline release for 16 phosphoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Phosphorylation and dephosphorylation of chromaffin cell proteins in response to stimulation. 377 57

Mitochondria from bovine hearts were fractionated by three different procedures and the fractions were characterized by marker enzymes. Highly purified outer membranes, membrane vesicles, and inner membranes, as well as two high-speed soluble fractions, were obtained. Azide (or oligomycin) resistant ATPase was not found to be a marker for outer membranes. The data were consistent with the association of the protein kinase activity with the soluble matrix of the mitochondria. Activity was highest with histone H2B as the substrate, with histone H1 next in preference. In contrast to the mitochondrial protein kinases studied previously, protamine, casein, and phosvitin were very poor substrates and there was no detectable phosphorylation of pyruvate dehydrogenase. Activity was stimulated by cAMP but not by cGMP, calmodulin, or phosphatidylserine--diolein, with or without Ca2+. Two cAMP-dependent isozymes were separated from the soluble fraction of the mitochondria by chromatography on DE-52 columns. Phosphorylation of histone H2B by the isozymes was inhibited by 98% by Kemptide.
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PMID:cAMP-dependent protein kinase isozymes with preference for histone H2B as substrate in mitochondria of bovine heart. 382 13

The pyruvate dehydrogenase complex has been demonstrated in high speed pellet preparations from sonicated ribbed mussel gill mitochondria. The activity of the complex is inhibited by low chloride (less than 100 mM) concentrations, EDTA (1 mM), succinate, ATP, and NAD/NADH ratios below 4. Inhibition by EDTA is relieved by addition of 10 mM MgCl2-1 mM CaCl2. ATP inhibition was enhanced by NaF and reversed by high Mg++ concentrations in the absence of NaF. Pyruvate and thiamine pyrophosphate inhibited the inactivation by ATP. The nonhydrolyzable ATP analog AMP-PNP caused inhibition of the overall catalytic activity that was identical to ATP. Factors involved in the ATP inhibition and Mg++ reversal are lost with freezing or cold storage. Preliminary results using gamma-32P-ATP indicate that a protein kinase that phosphorylates the alpha subunit of E1 (pyruvate dehydrogenase) from the mammalian PDC is associated with the gill PDC. The activity of the complex may be regulated by a phosphorylation/dephosphorylation mechanism and by the relative levels of substrates, products, and other metabolites in the mitochondria.
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PMID:Pyruvate dehydrogenase complex from ribbed mussel gill mitochondria. 408 84

We examined the patterns of cyclic AMP-dependent protein phosphorylation in membranes prepared from rat cortical synaptosomes following gel electrophoresis and autoradiography. We determined the optimum pH (6.2), time (20 s), Mg2+ concentration (10 mM) and cyclic AMP concentration (5 microM) for the reaction. We also found that the detergents Triton X-100 and gramicidin S enhanced cyclic AMP-dependent protein phosphorylation. Inhibitors of the Na+, K+ ATPase (ouabain, NaF, vanadate) enhanced protein phosphorylation. This effect occurred in the presence but not in the absence of detergent. The addition of purified bovine brain cyclic AMP-dependent protein kinase catalytic subunit enhanced membrane protein phosphorylation. The addition of homogeneous neural (bovine brain) and non-neural (bovine skeletal muscle) cyclic AMP-dependent protein kinase type II regulatory subunit partially inhibited protein phosphorylation. Both neural and non-neural regulatory subunits behaved similarly. In addition to cyclic AMP-dependent phosphorylation, the alpha-subunit of pyruvate dehydrogenase (Mr = 41,000) is phosphorylated in a cyclic AMP-independent fashion. We also examined the phosphorylation pattern of membranes prepared from rat heart and found that the number of acceptor substrates was much less than that from the nervous system.
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PMID:Characterization of cyclic AMP-dependent phosphorylation of neuronal membrane proteins. 608 38

Insulin stimulates fatty acid synthesis in white and brown fat cells as well as in liver and mammary tissue. Hormones that increase cellular cyclic AMP concentrations inhibit fatty acid synthesis, at least in white adipose tissue and liver. These changes in fatty acid synthesis occur within minutes. In white fat cells, they are brought about not only by changes in glucose transport but also changes in the activities of pyruvate kinase, pyruvate dehydrogenase and acetyl-CoA carboxylase. The basis of the alterations in pyruvate kinase activity in fat cells is not understood. Unlike the liver isoenzyme, the isoenzyme present in fat cells does not appear to be phosphorylated either in the absence or presence of hormones. The changes in pyruvate dehydrogenase activity in fat cells are undoubtedly due to changes in phosphorylation of the alpha subunits. Insulin appears to act by causing the parallel dephosphorylation of all three sites. The persistence of the effect of insulin during the preparation and subsequent incubation of mitochondria has allowed the demonstration that insulin acts mainly by stimulating pyruvate dehydrogenase phosphatase rather than inhibiting the kinase. Acetyl-CoA carboxylase within fat cells is phosphorylated on a number of different sites. The exposure of cells to insulin leads to activation of the enzyme and this is associated with increased phosphorylation of a specific site on the enzyme. Exposure to adrenalin, which results in a marked diminution in activity, also causes a small increase in the overall level of phosphorylation, but this increase is due to an enhanced phosphorylation of different sites; probably those phosphorylated by cyclic-AMP-dependent protein kinase. Acetyl-CoA carboxylase is one of a number of proteins in fat cells that exhibit increased phosphorylation with insulin. Others include ATP-citrate lyase, the ribosomal protein S6, the beta subunit of the insulin receptor and a heat and acid stable protein of Mr 22000. Changes in phosphorylation of ATP-citrate lyase do not appear to result in any appreciable changes in catalytic activity. A central aspect of insulin action may be the activation and perhaps release of a membrane-associated protein kinase. Plasma membranes from fat cells have been shown to contain a cyclic-nucleotide-independent kinase able to phosphorylate and activate acetyl-CoA carboxylase. Furthermore, high-speed supernatant fractions from cells previously exposed to insulin contain elevated levels of the same or similar kinase activity capable of phosphorylating both ATP-citrate lyase and acetyl-CoA carboxylase.
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PMID:The role of phosphorylation in the regulation of fatty acid synthesis by insulin and other hormones. 613 7

A method was devised to purify branched-chain oxo acid dehydrogenase (BCOAD) from rat kidney which retains endogenous kinase activity. Incorporation of 32P into purified enzyme parallels the time course of enzyme inhibition by ATP. Phosphorylation occurs on a serine residue(s) of the 46000-mol.wt. subunit of the enzyme complex. Endogenous phosphatase activity is not present after purification, and added pyruvate dehydrogenase phosphate phosphatase does not re-activate BCOAD or liberate 32P from previously labelled enzyme. These results demonstrate that BCOAD can be regulated by an endogenous protein kinase and that the phosphorylation-cycle enzymes regulating BCOAD appear to be distinct from those associated with pyruvate dehydrogenase complex.
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PMID:Purification of rat kidney branched-chain oxo acid dehydrogenase complex with endogenous kinase activity. 628 17

A fraction of chromaffin granule membranes contained a number of substrates for endogenous protein kinase activity as well as endogenous phosphatase activity. The major 32P-labelled polypeptide of molecular weight 43,000 appeared to be the alpha-subunit of pyruvate dehydrogenase of residual mitochondria. Several polypeptides showed cyclic AMP stimulation of phosphorylation of which the major polypeptide of molecular weight 59,000 shows half-maximal phosphorylation with 0.49 microM cyclic AMP. The phosphorylation of several other polypeptides is inhibited at high cyclic AMP concentrations. From studies with immunoprecipitation and two-dimensional electrophoresis it was found that alpha- and beta-tubulin and actin were absent from the granule membranes. However 32P labelling of a proportion of the copies of dopamine-beta-hydroxylase was demonstrated. The majority of the substrates for endogenous protein kinase activity are probably on the cytoplasmic side of the granule membrane.
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PMID:Phosphoproteins of the adrenal chromaffin granule membrane. 628 73

An insulin mediator which inhibits cAMP-dependent protein kinase has been purified approximately 1000-2000-fold from skeletal muscle. Following heat treatment, charcoal adsorption and Sephadex G-25 sieving, Sephadex G-15 sieving and HPLC over an anion exchange column were performed. The mediator has characteristics of a relatively low molecular weight peptide or derivatized peptide which acts on cAMP-dependent protein kinase but not on mitochondrial pyruvate dehydrogenase.
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PMID:Purification and partial characterization of a putative mediator of insulin action on cyclic AMP-dependent protein kinase. 637 44

Insulin action on [32P]-phosphate incorporation into brain membranes was determined. Hippocampal homogenate tissue was phosphorylated with [32P]-ATP, and insulin was introduced at various times before or after ATP addition. With 50 microM Mg++ in the medium, insulin selectively stimulated the phosphorylation of a 47kD phosphoprotein, Protein F1. This effect required the prior presence of ATP. No effect of insulin on other phosphoproteins, or on [32P]-phosphate incorporation into TCA-precipitated material, was observed under these conditions. At 1 mM Mg++, insulin selectively decreased the phosphorylation of the alpha-subunit of pyruvate dehydrogenase. Insulin had no effect on other phosphoproteins, or on [32P]-phosphate incorporation into TCA-precipitated material under these conditions. The present study suggests a role for insulin in the modulation of brain protein phosphorylation. Since Protein F1 is phosphorylated by exogenous C kinase, and is likely the CNS-specific B-50 protein, these data also indicate a brain-specific function for insulin, possibly by action on a Ca++/phospholipid protein kinase.
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PMID:Brain protein phosphorylation in vitro: selective substrate action of insulin. 638 12


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