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 underlying mechanism of Ca2+ uptake function of cardiac sarcoplasmic reticulum (SR) was investigated in the rat septic shock model produced by cecal ligation and puncture (CLP). The results are as follows. During the early phase of sepsis, the initial rate of ATP-dependent Ca2+ uptake by SR was decreased, while both the capacity of Ca2+ uptake and the activity of Ca(2+)-ATPase were unaffected. In the late sepsis, the impairment in SR function was even greater as the initial rate and the capacity of Ca2+ uptake by SR were significantly decreased, and this was paralleled by a reduction in Ca(2+)-ATPase activity. Although Ca2+ affinity (Km value) to calcium pump and the A0.5 values for Mg2+ and ATP activation on the Ca2+ uptake rate were unchanged, during sepsis the phosphorylation of SR vesicles by adding of catalytic subunit of the cAMP-dependent protein kinase (PKA), calmodulin, or the fragment of PKC into Ca2+ uptake buffer, failed to stimulate Ca2+ uptake activities of SR isolated from early or late septic rats. These data suggest that depression of cardiac SR function is aggravated as sepsis develops, the impairment of SR Ca2+ uptake is possibly based on a mechanism of defective phosphorylation of SR rather than the ionic and energic regulatory actions of Ca2+, Mg2+, ATP on cardiac SR.
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PMID:[Impaired calcium uptake by cardiac sarcoplasmic reticulum and its underlying mechanism during rat septic shock]. 748 74

We investigated the effects of the nonionic detergent octaethylene glycol monododecyl ether (C12E8) on the sarcoplasmic reticulum calcium pump in cardiac microsomes in view of its specific effects on different ATP-accelerated steps in the catalytic cycle of the Ca-ATPase in leaky fast skeletal muscle microsomes. At low concentrations of MgATP2- (< 2.5 microM), a nonsolubilizing concentration of added C12E8 (15 microM) increased apparent Vmax(MgATP) of oxalate-facilitated calcium uptake associated with MgATP2- binding to the high affinity catalytic site. An ATP induced acceleration of calcium uptake, attributable to regulatory nucleotide binding, was seen between 2 and 3 microM MgATP2- in both C12E8-treated and control microsomes. These effects of C12E8 are similar to those seen previously with trypsin treatment of microsomes [Lu, Y.-Z., Xu, Z.-C., & Kirchberger, M.A. (1993) Biochemistry 32, 3105-3111]. However, at a saturating Ca2+ between 3 and 10 microM MgATP2-, C12E8 produced a greater reduction in the magnitude of the ATP-induced acceleration of calcium uptake seen with trypsin. At 1 mM MgATP2-, C12E8 and trypsin as well as protein kinase A-catalyzed microsomal phosphorylation all increased the Ca2+ affinity of the pump, but only the latter two treatments significantly increased apparent Vmax(Ca). In fact in trypsin-treated and phosphorylated microsomes, C12E8 reduced Vmax(Ca) to close to the control values; it reduced Vmax(Ca) only slightly in control microsomes. Under our experimental conditions, comparable effects of 15 microM C12E8 on calcium uptake were absent in fast skeletal muscle microsomes, which lack phospholamban.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of a nonionic detergent on calcium uptake by cardiac microsomes. 817 81

The purpose of this study was to investigate the functional relationship between phospholamban and the nucleotide site of the calcium pump protein of the cardiac sarcoplasmic reticulum. We used control and trypsin-treated cardiac microsomes in which cleavage of the inhibitory cytoplasmic domain of phospholamban is associated with an activation of the calcium pump similar to that produced by protein kinase A catalyzed phospholamban phosphorylation. Phenylglyoxal was shown to inactivate the calcium pump in a pseudo-first-order reaction by binding to a single Arg at the nucleotide binding site. No differences upon trypsin treatment of microsomes were observed in the kinetics of phenylglyoxal inactivation or the ability of millimolar ATP to protect against inactivation. In subsequent kinetic studies, Ca-uptake rates measured at saturating Ca2+ and 5 microM-1 mM MgATP2- were increased 15-32% by trypsin treatment in each of three different microsome preparations. Double-reciprocal plots of the data showed marked downward curvature indicating an acceleratory effect associated with ligand binding to a lower affinity site. At 0.32 microM Ca2+, Ca-uptake rates were lower than at 11 microM Ca2+ but were stimulated to a greater extent by trypsin treatment; control microsomes showed reduced evidence of apparent negative cooperativity. At 0-2 microM MgATP2- and saturating Ca2+, there was a 50% increase in Vmax(app) when the Hill coefficient (N) was 1. At 0-10 microM MgATP2-, second-site binding was evident. At both 0-10 microM and 5 microM-1 mM MgATP2-, trypsin-treated microsomes showed greater activation of Ca uptake attributable to second-site binding than did control microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Evidence for an effect of phospholamban on the regulatory role of ATP in calcium uptake by the calcium pump of the cardiac sarcoplasmic reticulum. 838 87

5-Hydroxytryptamine (5-HT) stimulates corticosteroid secretion from adrenal cells through activation of 5-HT4 receptors positively coupled to adenylyl-cyclase. In the present study, we investigated in frog adrenocortical cells the effect of 5-HT4 receptor agonists on cytosolic calcium concentration ([Ca2+]i) and determined the sequence of events associated with 5-HT4 receptor agonist zacopride (10[-8] to 10[-5]M each in the vicinity of cultured adrenocortical cells caused a dose-dependent increase in [Ca2+]i. Preincubation of the cells with the selective 5-HT4 receptor antagonist [1-[2-(methylsulfonylamino)ethyl]-4- piperidinyl]methyl-1-methyl-1H-indole-3-carboxylate maleate totally blocked the 5-HT-induced stimulation of [Ca2+]i. Chelation of extracellular calcium with ethylene glycol bis (beta-aminoethyl ether)-N,N,N', N'-tetraacetic acid (10 MM) suppressed the stimulatory effect of 5-HT on [Ca2+]i. Conversely, thapsigargin, an inhibitor of calcium ATPase activity, had no effect on the [Ca2+]i rise. The calcium influx induced by 5-HT4 receptor agonists was not affected by nifedipine and omega-conotoxin GVIA but was totally blocked by pimozide, a T-type calcium channel antagonist. The [Ca2+]i response to zacopride was potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and markedly reduced by the protein kinase A inhibitor adenosine-3',5'-cyclic monophosphorothioate. We studied in perifused frog adrenal slices the involvement of [Ca2+]i rise and cAMP formation in the mechanism of action of 5-HT4 receptor agonists. Zacopride-induced steroidogenesis was significantly reduced in the presence of adenosine-3'5'-cyclic monophosphorothioate or after suppression of calcium in the perifusion medium. The stimulatory effect of zacopride on corticosteroid secretion was not affected by nifedipine and omega-conotoxin GVIA but was significantly inhibited by pimozide. Taken together, these data indicate that activation of 5-HT4 receptors in adrenocortical cells causes stimulation of adenylyl cyclase and subsequently increases calcium influx through a T-type calcium channel. Both the increased in cAMP formation and the calcium rise are involved in the stimulatory effect of 5-HT on corticosteroid secretion.
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PMID:Activation of 5-hydroxytryptamine4 receptors causes calcium influx in adrenocortical cells: involvement of calcium in 5-hydroxytryptamine-induced steroid secretion. 864 88

Regulation of calcium transport by sarcoplasmic reticulum provides increased cardiac contractility in response to beta-adrenergic stimulation. This is due to phosphorylation of phospholamban by cAMP-dependent protein kinase or by calcium/calmodulin-dependent protein kinase, which activates the calcium pump (Ca2+-ATPase). Recently, direct phosphorylation of Ca2+-ATPase by calcium/calmodulin-dependent protein kinase has been proposed to provide additional regulation. To investigate these effects in detail, we have purified Ca2+-ATPase from cardiac sarcoplasmic reticulum using affinity chromatography and reconstituted it with purified, recombinant phospholamban. The resulting proteoliposomes had high rates of calcium transport, which was tightly coupled to ATP hydrolysis (approximately 1.7 calcium ions transported per ATP molecule hydrolyzed). Co-reconstitution with phospholamban suppressed both calcium uptake and ATPase activities by approximately 50%, and this suppression was fully relieved by a phospholamban monoclonal antibody or by phosphorylation either with cAMP-dependent protein kinase or with calcium/calmodulin-dependent protein kinase. These effects were consistent with a change in the apparent calcium affinity of Ca2+-ATPase and not with a change in Vmax. Neither the purified, reconstituted cardiac Ca2+-ATPase nor the Ca2+-ATPase in longitudinal cardiac sarcoplasmic reticulum vesicles was a substrate for calcium/calmodulin-dependent protein kinase, and accordingly, we found no effect of calcium/calmodulin-dependent protein kinase phosphorylation on Vmax for calcium transport.
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PMID:Purified, reconstituted cardiac Ca2+-ATPase is regulated by phospholamban but not by direct phosphorylation with Ca2+/calmodulin-dependent protein kinase. 866 79

The report is a discussion of previously published and newly analyzed results concerning the association between heart diseases and alterations in the force-frequency relation (FFR). The optimum stimulation frequency of the FFR is measured and compared in isolated left ventricular myocardium from non-failing hearts with atrial septal defect, coronary artery disease (without and with insulin dependent diabetes mellitus) and from failing hearts with mitral regurgitation, or idiopathic dilated cardiomyopathy. Specifically, we examine the role of altered control of the excitation-contraction coupling system in blunting the force-frequency relation. We use the percent slope of the FFR as a measure of changes in the frequency sensitivity of this control. Our finding of a linear, direct relation between optimum stimulation frequency and % slope across all disease types suggests both parameters are coupled to the same underlying mechanism. To investigate the possible role of altered control of the calcium pump in this mechanism, we analyzed the detailed relation between isometric twitch relaxation kinetics and stimulation frequency in mitral regurgitation myocardium (MR). In the presence of 0.5 microM forskolin the depressed slope and optimum frequency of the FFR and the prolonged half-time of twitch relaxation were all restored to values found in non-failing myocardium. We use the kinetics of isometric twitch relaxation as an index of changes in pumping rate that occur in response to changes in stimulation frequency or in intracellular cyclic adenosine monophosphate concentration. A mathematical model based on the Hill relations for calcium pump uptake rate and for isometric tension as a function of intracellular pCa is developed to simulate isometric twitch relaxation in MR and non-failing myocardium. The success of this model in simulating non-failing and failing twitch relaxation supports a proposed mechanism for the prolonged relaxation time and depressed FFR in MR involving depressed protein kinase-A activity (due to lowered cAMP or to a defect in the Ser16 site of phospholamban) as a mechanism of altered control of the calcium pump in MR heart disease.
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PMID:Role of cAMP in modulating relaxation kinetics and the force-frequency relation in mitral regurgitation heart failure. 920 49

The plasma membrane Ca2+-ATPase (PMCA), the enzyme responsible for the maintenance of intracellular calcium homeostasis, is regulated by several independent mechanisms. In this paper we report that the protein kinases A and C differentially activate the Ca2+-ATPase purified from synaptosomal membranes of rat cortex, cerebellum and hippocampus. The effect of protein kinases was more pronounced for the cortical enzyme, whereas cerebellar and hippocampal Ca2+-ATPases were activated to a lesser degree. The preparation of Ca2+-ATPase contained the phosphoamino acids, i.e., P-Ser and P-Thr, indicating that the enzyme was purified in phosphorylated state. The phosphorylation of Ca2+-ATPase by PKA and PKC increased the amount of phosphoamino acids, but in a region-dependent manner. Using the specific antibodies against N-terminal portion of four main PMCA isoforms we have characterized the isoforms composition of Ca2+-ATPase purified from the nervous endings of examined brain areas. Our results indicate that the activity of calcium pump is related to its phosphorylated state, and that the phosphorylation is region-dependent. Moreover, the differences observed could be related to the composition of PMCA isoforms in the different brain areas. Phosphorylation of the plasma membrane Ca2+-ATPase appears to be a mechanism to control its activity. The results support also the possible involvement of PKA and PKC.
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PMID:Protein kinases A and C phosphorylate purified Ca2+-ATPase from rat cortex, cerebellum and hippocampus. 982 78

This study examined the effect of salmon calcitonin (sCT) on hypothalamic tyrosine hydroxylase (TH) activity and evaluated the cellular signaling mechanisms involved in the response. Fetal hypothalamic cells were cultured in a defined medium and treated with sCT and/or specific protein kinase inhibitors on day 14 in vitro. sCT (0.1-10 nM) increased both TH activity and cellular cAMP content in a concentration-dependent manner. sCT (10 nM) increased TH activity to 150-175% of control values and resulted in a 10-fold increase in cellular cAMP content. Both the C1a and C1b CT receptor isoforms were present in the cultures, as assessed by RT-PCR. Rp-adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS), a cAMP antagonist, and H-8, a cyclic nucleotide kinase inhibitor, blocked the sCT-induced increase in TH activity, with complete abolition of the response observed at concentrations of 1 mM and 5 microM, respectively. sCT (10 nM) increased radiolabeled phosphate incorporation into TH protein to 169% of control values and 1 mM Rp-cAMPS completely blocked this effect. In contrast, neither Calphostin C, a protein kinase C inhibitor, nor U-73122, a phospholipase C inhibitor, significantly altered the ability of sCT to increase TH activity. Likewise, the sCT-induced increase in TH activity was observed after pretreating the cells with either BAPTA/AM, an intracellular calcium chelator, or thapsigargin, an inhibitor of the endoplasmic reticulum calcium pump. These data indicate that sCT has a profound stimulatory effect on TH activity in fetal hypothalamic cells and that enhanced phosphorylation of TH coincides with the sCT-induced increase in enzyme activity. Moreover, CT receptors, which are linked to cAMP production, are expressed in the hypothalamic cells and a cAMP-dependent mechanism mediates the sCT-induced activation and phosphorylation of TH.
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PMID:3,5 cyclic adenosine monophosphate mediates the salmon calcitonin-induced increase in hypothalamic tyrosine hydroxylase activity. 1038 24

Quantitative immunoassays to discriminate and quantitate phospholamban and its phosphorylation states in heart homogenates were developed using known amounts of protein determined by amino acid analysis. Synthetic 1-52 phospholamban, the hydrophilic 1-25 peptide, and 1-25 phosphopeptides containing P-Ser(16), P-Thr(17), and dually phosphorylated (P-Ser(16), P-Thr(17)) were used to calibrate immunoblot systems. In addition, synthetic 1-52 peptide was phosphorylated using cAMP-dependent protein kinase (P-Ser(16)) or Ca(2+)-calmodulin protein kinase (P-Thr(17)) and then separated from unphosphorylated 1-52 by HPLC prior to quantitation. Further, canine cardiac sarcoplasmic reticulum was phosphorylated in vitro using [gamma-(32)P]-ATP with cAMP-dependent protein kinase and/or Ca(2+)-calmodulin-dependent protein kinase as well as sequential phosphorylation in both orders to assess the veracity of antibody recognition of phosphorylated forms. Western blots proved useful in characterizing the reactivity of the different antibodies to phospholamban and phosphorylated phospholamban, but were inefficient for accurate quantitation and problems with antibody recognition of dually phosphorylated phospholamban were found. mAb 1D11 recognized all forms of phospholamban, polyclonal antibodies 285 and PS-16 were highly selective for P-Ser(16) phospholamban but had diminished reactivity to diphosphorylated (P-Ser(16), P-Thr(17)) phospholamban, and polyclonal antibody PT-17, although selective for P-Thr(17) phospholamban, generated very weak signals on Western blots and reacted poorly with diphosphorylated phospholamban. Results in quantitative immunodot blot experiments were even more compelling. None of the phosphorylation specific antibodies reacted with the diphospho 1-25 phospholamban peptide. Transgenic mouse hearts expressing varying levels of PLB and ferret heart biopsy samples taken before and after isoproterenol perfusion were analyzed. In all samples containing phospholamban, a basal level of Ser(16) phosphorylation (about 4% of the total PLB population) and a lesser amount of Thr(17) phosphorylation was observed. Upon isoproterenol perfusion, Ser(16) phosphorylation increased only to 17% of the total phospholamban population with a similar change in Thr(17) phosphorylation. This suggests that phospholamban phosphorylation may serve as an electrostatic switch that dissociates inactive calcium pump complexes into catalytically active units. Thus, direct correlations between phospholamban phosphorylation state and contractile parameters may not be valid.
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PMID:Characterization and quantitation of phospholamban and its phosphorylation state using antibodies. 1062 71

Calreticulin (CRT) and calnexin (CLNX) are lectin chaperones that participate in protein folding in the endoplasmic reticulum (ER). CRT is a soluble ER lumenal protein, whereas CLNX is a transmembrane protein with a cytosolic domain that contains two consensus motifs for protein kinase (PK) C/proline- directed kinase (PDK) phosphorylation. Using confocal Ca(2+) imaging in Xenopus oocytes, we report here that coexpression of CLNX with sarco endoplasmic reticulum calcium ATPase (SERCA) 2b results in inhibition of intracellular Ca(2+) oscillations, suggesting a functional inhibition of the pump. By site-directed mutagenesis, we demonstrate that this interaction is regulated by a COOH-terminal serine residue (S562) in CLNX. Furthermore, inositol 1,4,5-trisphosphate- mediated Ca(2+) release results in a dephosphorylation of this residue. We also demonstrate by coimmunoprecipitation that CLNX physically interacts with the COOH terminus of SERCA2b and that after dephosphorylation treatment, this interaction is significantly reduced. Together, our results suggest that CRT is uniquely regulated by ER lumenal conditions, whereas CLNX is, in addition, regulated by the phosphorylation status of its cytosolic domain. The S562 residue in CLNX acts as a molecular switch that regulates the interaction of the chaperone with SERCA2b, thereby affecting Ca(2+) signaling and controlling Ca(2+)-sensitive chaperone functions in the ER.
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PMID:Cytosolic phosphorylation of calnexin controls intracellular Ca(2+) oscillations via an interaction with SERCA2b. 1085 Oct 21


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