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)

The characteristics of neutral cholesteryl ester hydrolase activities found in the microsomal and cytosolic subcellular fractions of rat lactating mammary tissue were investigated. The enzymes were assayed using cholesteryl oleate dispersed as a mixed micelle with phosphatidylcholine and sodium taurocholate (molar ratio 1:4:2) as substrate. This method gave activities approx. 20-fold higher than those seen when cholesteryl oleate was added in ethanol. Addition of phosphatidylcholine and sodium taurocholate to the assays using the ethanol-dissolved substrate did not increase the activities observed. When the cholesteryl oleate was dispersed with phosphatidylcholine only (molar ratio, 1:4) the activity of the two neutral cholesteryl ester hydrolases was also decreased considerably compared to that found with mixed micelles. In this case, however, approx. 60% of the cytosolic, but only 10% of the microsomal activity, was restored by separate addition of sodium taurocholate. The activities of both the microsomal and the cytosolic neutral cholesteryl ester hydrolases were inhibited by MgCl2, and this inhibition was almost completely reversed by the addition of an equimolar concentration of ATP. At a fixed concentration of MgCl2 increasing concentrations of ATP increased the enzyme activities in a dose-dependent way. The activity of the microsomal, but not the cytosolic enzyme was enhanced by a cyclic AMP-dependent protein kinase and both activities were inhibited by alkaline phosphatase (bovine milk). These results provide evidence for the regulation of neutral cholesteryl ester hydrolases in the rat lactating mammary gland by mechanisms involving phosphorylation-dephosphorylation and therefore suggest that these enzymes may be under hormonal control.
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PMID:Neutral cholesteryl ester hydrolase in the rat lactating mammary gland: regulation by phosphorylation-dephosphorylation. 217 66

Two characteristic responses of plant cells to fungal elicitors, induction of phenylalanine ammonia-lyase activity and of ethylene biosynthesis, were studied in suspension-cultured tomato cells. Induction of both responses was completely blocked by 500 nM K-252a, a known inhibitor of mammalian protein kinases. About 100 nM K-252a caused half-maximal inhibition. In vitro, K-252a inhibited protein kinase activity in microsomal preparations from tomato cells. Inhibition was competitive with respect to ATP and had a Ki of about 15 nM. Thus, protein kinases sensitive to K-252a occur in plants and might be important for the plant's response to fungal elicitors.
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PMID:K-252a inhibits the response of tomato cells to fungal elicitors in vivo and their microsomal protein kinase in vitro. 226 87

Calmodulin-dependent multiprotein kinase and protein kinase C phosphorylate and inactivate both intact, microsomal HMG-CoA reductase, and the purified 53 kDa catalytic fragment. Isolation of the single phosphopeptide produced by combined cleavage with cyanogen bromide and Lys-C proteinase reveals that this is due to phosphorylation of a single serine residue near the C-terminus, corresponding to serine-872 in the human enzyme. This is identical with the single serine phosphorylated by the AMP-activated protein kinase. The nature of the protein kinase responsible for phosphorylation of this site in vivo is discussed.
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PMID:Calmodulin-dependent multiprotein kinase and protein kinase C phosphorylate the same site on HMG-CoA reductase as the AMP-activated protein kinase. 238 4

Phosphorylation of the insulin-regulatable glucose transporter (IRGT) is increased by incubating rat adipocytes with isoproterenol or by incubating microsomal membranes with cAMP-dependent protein kinase. To attempt to locate the sites of phosphorylation, the IRGT (apparent Mr = 46,000) was immunoprecipitated from 32P-labeled adipocytes and cleaved with CNBr or trypsin. Essentially all of the 32P could be recovered in a single CNBr fragment, denoted CB-T (Mr = 8,000), which bound a polyclonal antibody (R820) against a peptide having the sequence of the last 12 amino acids in the COOH terminus of the IRGT. 32P-Labeling of the IRGT was also confined to CB-T when membranes were incubated with [gamma-32P]ATP and cAMP-dependent protein kinase. Isoproterenol increased phosphorylation of CB-T, but insulin was without effect. To resolve phosphorylation sites further, IRGT from 32P-labeled cells was subjected to exhaustive proteolysis with trypsin. Samples were applied to a C-18 column, and 32P-labeled fragments were resolved into three peak fractions by elution with an increasing gradient of acetonitrile. [32P]Phosphoserine was the only phosphoamino acid detected in any of the peaks. Peak III contained approximately 80% of the 32P and was increased by isoproterenol. Almost all of the 32P introduced by cAMP-dependent protein kinase in vitro eluted in Peak III. In all cases, the 32P-labeled species in Peak III were quantitatively immunoprecipitated by R820. Digesting the peptide(s) in Peak III with V8 protease generated a single peak of 32P which eluted at lower acetonitrile than Peak III and contained 32P-labeled species that did not interact with R820. Automated Edman degradation indicated that the serine residue in Peak III phosphorylated by cAMP-dependent protein kinase was the 3rd or 4th residue from the NH2 terminus of the peptide. These findings indicate that phosphorylation of the IRGT is restricted to the presumed intracellular domain at the COOH terminus and that Ser488 is a major site phosphorylated both by cAMP-dependent protein kinase in vitro and in response to isoproterenol in vivo.
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PMID:Phosphorylation of the glucose transporter in rat adipocytes. Identification of the intracellular domain at the carboxyl terminus as a target for phosphorylation in intact-cells and in vitro. 240 83

Ca2+ pump activity of skeletal muscle microsomes containing fragments of sarcoplasmic reticulum was examined in rats 8 wk after the induction of chronic diabetes by an intravenous injection of streptozotocin (65 mg/kg). In comparison with the control values, both ATP-dependent Ca2+ uptake and Ca2+-stimulated ATPase activities were increased in the microsomal fraction from diabetic rats. These changes were seen as early as 7 days after streptozotocin injection and were apparent at various times of incubation (1-10 min) as well as at different concentrations of free Ca2+ (10(-7)-5 X 10(-5) M Ca2+). Insulin administration to diabetic animals for 2 wk reversed Ca2+ uptake and ATPase activities to control levels. The increase in microsomal ATPase activity of the diabetic preparation due to cAMP-dependent protein kinase or calmodulin was greater than in the control microsomes and the depression by a specific inhibitor of protein kinase, but not of calmodulin, was greater in diabetic muscle. The enhanced Ca2+ pump activity was associated with altered phospholipid composition and protein profile of the diabetic preparations. The rate of Ca2+ release from microsomal vesicles was unaffected by the diabetic condition. Isometric contractile force development as well as positive dF/dt and negative dF/dt of the skeletal muscle from diabetic animals were higher at different pulse strengths (0.5-100 V) and at different Ca2+ concentrations (0.25-2.5 mM). These results suggest that diabetes is associated with enhanced sarcoplasmic reticular Ca2+ pump activity, and this may account for the hyperfunction of skeletal muscle in this disease.
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PMID:Calcium pump activity of sarcoplasmic reticulum in diabetic rat skeletal muscle. 243 Apr 66

The rat CNS contains high levels of tyrosine-specific protein kinases that specifically phosphorylate the tyrosine-containing synthetic peptide poly(Glu80,Tyr20). The phosphorylation of this peptide is rapid and occurs with normal Michaelis-Menten kinetics. Using this peptide to assay for enzyme activity, we have measured the protein tyrosine kinase activity in homogenates from various regions of rat CNS. A marked regional distribution pattern was observed, with high activity present in cerebellum, hippocampus, olfactory bulb, and pyriform cortex, and low activity in the pons/medulla and spinal cord. The distribution of protein tyrosine kinase activity was examined in various subcellular fractions of rat forebrain. The majority of the activity was associated with the particulate fractions, with enrichment in the crude microsomal (P3) and crude synaptic vesicle (LP2) fractions. Moreover, the subcellular distribution of pp60csrc, a well-characterized protein tyrosine kinase, was examined by immunoblot analysis using an affinity-purified antibody specific for pp60csrc. The subcellular distribution of pp60csrc paralleled the overall protein tyrosine kinase activity. In addition, using an antibody specific for phosphotyrosine, endogenous substrates for protein tyrosine kinases were demonstrated on immunoblots of homogenates from the various regions and the subcellular fractions. The immunoblots revealed numerous phosphotyrosine-containing proteins that were present in many of the CNS regions examined and were associated with specific subcellular fractions. The differences in tyrosine-specific protein kinase activity, and in phosphotyrosine-containing proteins, observed in various regional areas and subcellular fractions may reflect specific functional roles for protein tyrosine kinase activity in mammalian brain.
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PMID:Protein tyrosine kinase activity and its endogenous substrates in rat brain: a subcellular and regional survey. 245 35

Calcium uptake and (Ca2+ + Mg2+)-ATPase activity in canine cardiac microsomes were found to be stimulated by heparin and various other polyanions. Prior treatment of the microsomes with the ionophores alamethicin or A23187 produced no change in the extent of stimulation of the ATPase activity by heparin yet eliminated net calcium uptake. This finding and a lack of change in the stoichiometric ratio of mol of calcium transported/mol of ATP hydrolyzed (calcium:ATP) suggest that the effect of heparin is on the calcium pump rather than on a parallel calcium efflux pathway. Certain polycationic compounds including poly-L-arginine and histone inhibited both cardiac and fast skeletal muscle microsomal calcium uptake and also produced no change in the stoichiometric ratio of calcium to ATP. Several lines of evidence indicate that the polyanionic compounds tested stimulate calcium uptake by interacting with phospholamban, the putative phosphorylatable regulator of the cardiac sarcoplasmic reticulum calcium pump, whereas polycationic compounds appear to interact with the pump. (i) Heparin stimulated calcium uptake to the same extent as protein kinase A or trypsin, whereas prior phosphorylation or tryptic cleavage of phospholamban from the membrane abolished the stimulatory effect of heparin. (ii) Calcium uptake and (Ca2+ + Mg2+)-ATPase activity in fast skeletal muscle microsomes, which lack phospholamban, were unaffected by heparin. (iii) Purified cardiac (Ca2+ + Mg2+)-ATPase activity was no longer stimulated by heparin yet was still inhibited by polycationic compounds. The heparin-induced stimulation of calcium uptake was dependent on the pH and ionic strength of the heparin-containing preincubation medium, hence electrostatic interactions appear to play a significant role in heparin's stimulatory action. The data are consistent with an inhibitory role of the positively charged cytoplasmic domain of phospholamban with respect to calcium pump activity and the relief of the inhibition upon reduction in phospholamban's positive charge by phosphorylation or binding of polyanions.
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PMID:Modulation by polyelectrolytes of canine cardiac microsomal calcium uptake and the possible relationship to phospholamban. 247 44

1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM), an intracellular calcium [( Ca2+]i) chelator, was used to investigate the role of [Ca2+]i in acid secretory activity and protein phosphorylation in parietal cells from rabbit. Chelation of extracellular calcium [( Ca2+]o) with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid did not prevent the initial carbachol-induced elevation of [Ca2+]i as measured with the fluorescent Ca2+ probe, fura-2, and only partially inhibited [14C]-aminopyrine (AP) accumulation, an indirect indicator of acid secretory activity. [Ca2+]i chelation with BAPTA/AM eliminated carbachol-stimulated increases in [Ca2+]i and AP accumulation but only transiently reduced histamine stimulation of AP accumulation. Carbachol increased phosphorylation of a 36-kDa, pI approximately 7 protein (pp36) and transient phosphorylation of a 28-kDa, pI approximately 5 protein (pp28), whereas histamine increased phosphorylation of 40-kDa, pI approximately 6.5 (pp40) and 27-kDa, pI approximately 6.2 (pp27) proteins. Phosphorylation of pp36 and pp28 were mimicked by the protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA) and the calcium ionophore, ionomycin, respectively. Two other phosphoproteins with molecular weights of 66,000 and pIs of 5.7 and 5.9 were also phosphorylated in response to TPA and carbachol. Chelation of [Ca2+]i and [Ca2+]o blocked carbachol-induced phosphorylation of pp28 and pp36 and ionomycin phosphorylation of pp28 but not TPA-stimulated phosphorylation of pp36 or the two pp66s or histamine-stimulated phosphorylation of pp27 or pp40. Chelation of [Ca2+]i alone did not block increases in [Ca2+]i or phosphorylation of pp28 in response to ionomycin. Both pp28 and pp36 were localized in both microsomal and cytosolic fractions of cells, which suggests involvement in cytoskeleton-membrane interactions. These phosphoproteins could be common elements of Ca2+-dependent stimulus-secretion coupling as similar proteins were phosphorylated by carbachol and cholecystokinin (CCK) in chief cells. Based on data with TPA and ionomycin, both protein kinase C and an as yet unidentified Ca2+-dependent protein kinase(s) appear to be activated upon stimulation with cholinergic agonists and CCK.
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PMID:Carbachol-induced protein phosphorylation in parietal cells: regulation by [Ca2+]i. 250 25

Tyrosine protein kinases (TPKs) have been implicated in mitotic signalling in a wide range of cells including lymphocytes. We describe here the partial characterization of a heat stable TPK inhibitor from both normal and malignant human lymphoid cells. Inhibitory activity was not attributable to contaminating ATPase, protease or phosphatase activities or to the Ca2+-binding protein S100. Preparations of the TPK inhibitor did not reduce the activity of cAMP-dependent protein kinase. While the inhibitor decreased the activity of TPKs towards an exogenous peptide substrate, it did not affect the autophosphorylation of microsomal TPKs. These results raise the possibility that the activity of TPKs in lymphoid cells may be regulated by an inhibitor protein.
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PMID:An endogenous inhibitor of the protein tyrosine kinase activity of normal and malignant human lymphoid cells. 252 67

The role of phosphorylation/dephosphorylation in the regulation of CTP:phosphocholine cytidylyltransferase activity was investigated. Incubation of post mitochondrial supernatant with cAMP-dependent protein kinase (50 units) led to an increased (28%) recovery of the cytidylyltransferase in the cytosolic fraction, while incubation with an intestinal alkaline phosphatase (20 units) led to an increased (61%) recovery in the microsomal fraction. When pure cytidylyltransferase was incubated with washed microsomes in the presence of cAMP-dependent protein kinase (133 units), the enzyme associated with the supernatant fraction increased (3.12 +/- 0.02 to 3.77 +/- 0.03 nmol/min/ml) while that of the microsomal fraction decreased (1.36 +/- 0.01 to 0.56 +/- 0.05 nmol/min/ml) by 2.5-fold. The increase in the cytidylyltransferase activity in the supernatant corresponded to an increase in 32P incorporation into the cytidylyltransferase. Treatment with alkaline phosphatase (40 units) decreased the cytidylyltransferase activity in the supernatant (3.61 +/- 0.08 to 2.88 +/- 0.07 nmol/min/ml) while the activity in the microsomal fraction increased (0.56 +/- 0.08 to 1.16 +/- 0.06 nmol/min/ml) by 2-fold. The decrease in the cytidylyltransferase activity in the supernatant corresponded to a decrease in 32P incorporation into the cytidylyltransferase. Incubation of cytidylyltransferase with phosphatidylcholine vesicles in the presence of cAMP-dependent protein kinase (110 units) decreased the cytidylyltransferase activity by 30%. The decrease in cytidylyltransferase activity corresponded to an increase in 32P incorporation into the cytidylyltransferase. Treatment with alkaline phosphatase (20 units) resulted in a 41% increase in the cytidylyltransferase activity. The increase in cytidylyltransferase activity corresponded to a decrease in 32P incorporation into the cytidylyltransferase. Incubation of the cytidylyltransferase with [gamma-32P] ATP and cAMP-dependent protein kinase led to incorporation of 32P into the serine residues of cytidylyltransferase. If the cytidylyltransferase were preincubated with alkaline phosphatase prior to incubation with cAMP-dependent protein kinase, 2-fold more 32P (0.2 mol P/mol cytidylyltransferase) was incorporated into the cytidylyltransferase. Collectively, this data is in agreement with a role for reversible phosphorylation in the regulation of cytidylyltransferase.
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PMID:CTP:phosphocholine cytidylyltransferase is a substrate for cAMP-dependent protein kinase in vitro. 253 19


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