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:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Microsomal Ca(2+)-ATPase inhibitors such as thapsigargin (THG), cyclopiazonic acid (CPA) and 2,5-di-(tert-butyl)-1,4-hydroquinone (DBHQ) have been shown to inhibit Ca2+ reuptake by the intracellular stores and increase cytosolic free Ca2+ ([Ca2+]i). DBHQ is a commercially available non-toxic synthetic compound chemically unrelated to THG and CPA. In this study, we tested the feasibility of utilizing DBHQ to improve Cl- secretion via the Ca(2+)-dependent pathway, in the cystic fibrosis (CF)-derived pancreatic epithelial cell line CFPAC-1. DBHQ stimulated 125I efflux and mobilized intracellular free Ca2+ in a dose-dependent manner. The maximal effects were seen at concentrations of 25-50 microM. DBHQ (25 microM) caused a short-term rise in [Ca2+]i in the absence of ambient Ca2+, and a sustained elevation of [Ca2+]i in cell monolayers bathed in the efflux solution (1.2 mM Ca2+), which was largely attenuated by Ni2+ (5 mM). Bath-application of DBHQ induced an outwardly-rectifying whole-cell Cl- current, which was abolished by pipette addition of BAPTA (5 mM) or CaMK [273-302] (20 microM), an inhibitory peptide of multifunctional Ca2+/calmodulin-dependent protein kinase (CaMKII). Pretreatment of monolayers of CFPAC-1 cells with DBHQ for 4-5 min significantly increased the Ca(2+)-independent or autonomous activity of CaMKII assayed in the cell homogenates. Thus, DBHQ appears to enhance Cl- channel activity via a Ca(2+)-dependent mechanism involving CaMKII. Pretreatment of CFPAC-1 cells with up to 50 microM DBHQ for 6 h did not cause any detectable change in cell viability and did not significantly affect the cell proliferation rate. These results suggest that appropriate selective microsomal Ca(2+)-ATPase inhibitors may be therapeutically useful in improving Cl- secretion in CF epithelial cells.
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PMID:Calcium- and CaMKII-dependent chloride secretion induced by the microsomal Ca(2+)-ATPase inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone in cystic fibrosis pancreatic epithelial cells. 756 71

Phospholamban is the regulator of the Ca(2+)-ATPase in cardiac sarcoplasmic reticulum (SR). It is phosphorylated by a Ca2+/calmodulin-dependent protein kinase (SRCaM kinase) which is closely associated with cardiac SR membrane preparations. We found that, upon renaturation of pig cardiac SR proteins, blotted onto PVDF membrane, two polypeptides of 54 and 52 kDa showed Ca2+/calmodulin-dependent autophosphorylation. In Western blots of SR proteins, the 54/52 kDa polypeptides were recognized by an antibody specific for the delta-CaM kinase isoforms, but not by an anti-alpha-CaM kinase. The two polypeptides were selectively immunoprecipitated from solubilized SR vesicles with the anti-delta-CaM kinase. The CaM kinase inhibitors KN-62 and peptide CaMK-(281-302) inhibited the activity of the SRCaM kinase with IC50 values in the same range with those obtained for the brain isozyme. In addition, initial autophosphorylation (Ca(2+)-dependent) produced a partially Ca(2+)-independent enzyme while further autophosphorylation (Ca(2+)-independent) made the enzyme completely Ca(2+)-independent. Based on these results we suggest that the SRCaM kinase is a distinct delta-CaM kinase isozyme.
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PMID:The cardiac sarcoplasmic reticulum phospholamban kinase is a distinct delta-CaM kinase isozyme. 758 37

In both cardiac and slow-twitch skeletal muscle sarcoplasmic reticulum (SR) there are several systems involved in the regulation of Ca(2+)-ATPase function. These include substrate level regulation, covalent modification via phosphorylation-dephosphorylation of phospholamban by both cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase (CaM kinase) as well as direct CaM kinase phosphorylation of the Ca(2+)-ATPase. Studies comparing the effects of PKA and CaM kinase on cardiac Ca(2+)-ATPase function have yielded differing results; similar studies have not been performed in slow-twitch skeletal muscle. It has been suggested recently, however, that phospholamban is not tightly coupled to the Ca(2+)-ATPase in SR vesicles from slow-twitch skeletal muscle. Our results indicate that assay conditions strongly influence the extent of CaM kinase-dependent Ca(2+)-ATPase stimulation seen in both cardiac and slow-twitch skeletal muscle. Addition of calmodulin (0.2 microM) directly to the Ca2+ transport assay medium results in minimal (approximately 112-130% of control) stimulation of Ca2+ uptake activity when the Ca2+ uptake reaction is initiated by the addition or either ATP or Ca2+/EGTA. On the other hand, prephosphorylation of the SR by the endogenous CaM kinase and subsequent transfer of the membranes to the Ca2+ transport assay medium results in stimulation of Ca2+ uptake activity (202% of control). These effects are observable in both cardiac and slow-twitch skeletal muscle SR. PKA stimulates Ca2+ uptake markedly (215% of control) when the Ca2+ uptake reaction is initiated by the addition of prephosphorylated SR membranes or by Ca2+/EGTA but minimally (130% of control) when the Ca2+ uptake reaction is initiated by the addition of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Comparison of the effects of the membrane-associated Ca2+/calmodulin-dependent protein kinase on Ca(2+)-ATPase function in cardiac and slow-twitch skeletal muscle sarcoplasmic reticulum. 777 65

In previous studies (Xu, A., Hawkins, C., and Narayanan, N. (1993) J. Biol. Chem. 268, 8394-8397), the Ca(2+)-ATPase of cardiac muscle sarcoplasmic reticulum (SERCA2) was shown to be phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase) on a serine residue, likely to be either Ser38, Ser167, or Ser531. SERCA2 and SERCA2 mutants S38A, S167A, and S531A were expressed in HEK-293 cells and tested for phosphorylation with CaM kinase. Mutant S38A was not phosphorylated, while mutants S167A and S531A were phosphorylated, suggesting that Ser38 is the site of CaM kinase phosphorylation in SERCA2. This conclusion was supported by the observation that phosphorylation of SERCA2 and mutants S167A and S531A by CaM kinase increased the Vmax for Ca2+ transport, while the Vmax for Ca2+ transport by mutant S38A was unaffected by exposure to a phosphorylation reaction mix. SERCA1, containing a potential CaM kinase phosphorylation site at Ser167 and two SERCA1 mutants, K35R plus H38S and T532S, in which potential CaM kinase sites were created, were not phosphorylated by CaM kinase, and Vmax for Ca2+ transport was unaffected by exposure to a phosphorylation reaction mix. Thus phosphorylation of Ser38 in SERCA2 results in a unique activation of Vmax for Ca2+ transport, providing a potential regulatory mechanism for Ca2+ removal from cardiac and other tissues in which SERCA2 is expressed.
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PMID:Identification of Ser38 as the site in cardiac sarcoplasmic reticulum Ca(2+)-ATPase that is phosphorylated by Ca2+/calmodulin-dependent protein kinase. 792 71

We have demonstrated recently that in cardiac sarcoplasmic reticulum (SR), a membrane-associated Ca2+/calmodulin-dependent protein kinase (CaM kinase) phosphorylates and activates the Ca(2+)-pumping ATPase (Ca(2+)-ATPase) in addition to phosphorylating the previously characterized substrates, phospholamban, and Ca2+ release channel (ryanodine receptor) (Xu, A., Hawkins, C., and Narayanan, N. (1993) J. Biol. Chem. 268, 8394-8397). The present study shows that a CaM kinase regulatory system capable of modulating SR Ca2+ pump activity through direct phosphorylation of the Ca(2+)-ATPase is functional in slow twitch but not fast twitch skeletal muscle. Incubation of SR vesicles isolated from rabbit slow twitch (soleus) and fast twitch (adductor magnus) skeletal muscles in the presence of Ca2+ and calmodulin resulted in phosphorylation of the Ca(2+)-ATPase in slow twitch muscle SR but not in fast twitch muscle SR. Exogenous CaM kinase II, which stimulated phosphorylation of the cardiac and slow twitch muscle SR Ca(2+)-ATPase, failed to phosphorylate fast twitch muscle SR Ca(2+)-ATPase. These observations demonstrate that CaM kinase-catalyzed phosphorylation of the Ca2+ pump is isoform-specific since heart and slow twitch muscle express the same Ca(2+)-ATPase isoform (SERCA2a), which is distinct from that of fast twitch muscle (SERCA1). As in the case of cardiac SR Ca(2+)-ATPase, phosphorylation of the slow twitch muscle SR Ca(2+)-ATPase (occurring at a serine residue) resulted in a 2-fold increase in catalytic activity of the enzyme without alteration in its Ca2+ sensitivity. In addition, Ca2+/calmodulin-dependent prephosphorylation of slow twitch muscle SR resulted in a greater than 2-fold increase in its Ca2+ transport activity. In both cardiac and slow twitch muscle SR, phosphorylation of the Ca(2+)-ATPase by the endogenous CaM kinase occurred rapidly (maximum within 2 min at 37 degrees C), had similar pH optimum (8.5-9.0), temperature optimum (30 degrees C), and calmodulin concentration-dependence (k0.5 50-60 nM). cAMP-dependent protein kinase did not phosphorylate the Ca(2+)-ATPase appreciably in either cardiac or slow twitch muscle SR. These findings suggest a muscle-specific role for the membrane-associated CaM kinase in the modulation of Ca2+ uptake and release functions of the SR. In cardiac and slow twitch muscle, phosphorylation of the SR Ca(2+)-ATPase by CaM kinase might provide a novel mechanism for the modulation of the enzymatic and Ca2+ transport functions of this enzyme.
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PMID:Sarcoplasmic reticulum calcium pump in cardiac and slow twitch skeletal muscle but not fast twitch skeletal muscle undergoes phosphorylation by endogenous and exogenous Ca2+/calmodulin-dependent protein kinase. Characterization of optimal conditions for calcium pump phosphorylation. 798 62

It is well known that phosphorylation of the membrane protein phospholamban by cAMP-dependent or Ca2+/calmodulin-dependent protein kinase results in the activation of the Ca(2+)-pumping ATPase of cardiac sarcoplasmic reticulum (SR); such enzyme activation is thought to be due to the disruption of an inhibitory interaction of non-phosphorylated phospholamban with the ATPase. We describe here a novel mechanism for the regulation of the ATPase through direct phosphorylation of this enzyme by a Ca2+/calmodulin-dependent protein kinase (CaM kinase) associated with the SR membrane. It is shown that incubation of cardiac SR in the presence of Ca2+ and calmodulin results in the phosphorylation of the ATPase in addition to the previously recognized substrates of CaM kinase, viz. phospholamban and Ca2+ channel. The phosphorylated amino acid in the ATPase has been identified as serine. Phosphorylation of the membrane-bound ATPase is stimulated by exogenous CaM kinase. Furthermore, ATPase purified from cardiac SR is phosphorylated by exogenous CaM kinase and the phosphorylated enzyme displays 2-fold increase in catalytic activity without any appreciable change in its Ca2+ sensitivity. Thus, direct phosphorylation of the Ca(2+)-pumping ATPase by CaM kinase can stimulate its enzymatic activity and, therefore, Ca2+ transport function.
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PMID:Phosphorylation and activation of the Ca(2+)-pumping ATPase of cardiac sarcoplasmic reticulum by Ca2+/calmodulin-dependent protein kinase. 838 59

The preproendothelin-1 (preproET-1) gene is induced by thrombin after phosphorylation of nonreceptor protein tyrosine kinase pathways. This study investigated the contribution of Ca2+/calmodulin-dependent intracellular signaling cascades to this pathway and measured ET-1 mRNA levels by Northern blot analysis in human endothelial cells. Increased intracellular Ca2+ levels in response to Ca2+ ionophore or Ca2+ ATPase inhibitors tert-butylhydroquinone and thapsigargin mimicked thrombin actions on ET-1 mRNA induction. Thrombin-mediated activation of ET-1 mRNA was reduced by specific calmodulin antagonists W7 or calmidazolium and after inhibition of CaM kinase II by KN-62. Inhibition of calcium/calmodulin-dependent phosphatase calcineurin by cyclosporin A, however, stimulated ET-1 mRNA in human endothelial cells. Phosphotyrosine immunoblot assays show that calcium/calmodulin-dependent signaling pathways precede thrombin-induced tyrosine phosphorylation, and that the calcium/calmodulin-dependent phosphatase calcineurin also exerts its effects via activation of protein tyrosine kinases. These observations demonstrate that thrombin stimulates the preproET-1 gene in human endothelial cells through calcium-dependent activation of CaM kinase and protein tyrosine kinases, and that calcineurin may also participate in regulation of the prepro ET-1 gene.
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PMID:Thrombin-mediated ET-1 gene regulation involves CaM kinases and calcineurin in human endothelial cells. 858 30

In cardiac muscle, a Ca2+/calmodulin-dependent protein kinase (CaM kinase) associated with the sarcoplasmic reticulum (SR) is known to phosphorylate the membrane proteins phospholamban, Ca(2+)-ATPase, and Ca(2+)-release channel (ryanodine receptor). Phosphorylation of phospholamban and Ca(2+)-ATPase is recognized to stimulate Ca2+ sequestration by the SR but the functional consequence of Ca2+ channel phosphorylation has not been clearly established. In this study, we investigated the effects of the SR Ca(2+)-release inhibitor, ruthenium red (RR), and the SR Ca(2+)-release activator, ryanodine (at submicromolar concentrations), on CaM kinase-mediated phosphorylation of the Ca(2+)-cycling proteins in rabbit cardiac SR. Incubation of SR with RR (5-30 microM) for 3 min at 37 degrees C resulted in marked (up to 85%) inhibition of Ca2+ channel phosphorylation (50% inhibition with 15 +/- 2 microM RR) by the endogenous membrane-associated CaM kinase. Phosphorylation of the Ca2+ channel by exogenously added multifunctional alpha CaM kinase II was also inhibited similarly by RR. Phosphorylation of the Ca(2+)-ATPase by endogenous and exogenous CaM kinase was inhibited only modestly (25-30%) by RR, and phospholamban phosphorylation was unaffected by RR. The magnitude of RR-induced inhibition of Ca2+ channel phosphorylation did not differ appreciably at saturating or subsaturating concentrations of Ca2+ or calmodulin, and in the absence or presence of protein phosphatase inhibitors. In contrast to the effects of RR, low concentrations of ryanodine (0.25-1 microM) caused significant stimulation (up to approximately 50%) of Ca2+ channel phosphorylation but had no effect on Ca(2+)-ATPase and phospholamban phosphorylation. These findings suggest that interaction of RR with the ryanodine receptor induces a "nonphosphorylatable state" of the Ca(2+)-release channel, likely through a conformational change involving occlusion of the CaM kinase phosphorylation site. On the other hand, ryanodine binding to the receptor may serve to maintain an open, "phosphorylatable state" of the channel.
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PMID:Divergent effects of ruthenium red and ryanodine on Ca2+/calmodulin-dependent phosphorylation of the Ca2+ release channel (ryanodine receptor) in cardiac sarcoplasmic reticulum. 880 75

In cardiac muscle, a membrane-associated Ca2+/calmodulin-dependent protein kinase (CaM kinase) phosphorylates the Ca(2+)-pumping ATPase in addition to its previously characterized substrates, phospholamban and Ca(2+)-release channel (ryanodine receptor). The phosphorylated amino acid in the Ca(2+)-ATPase has been identified as serine. Posphorylation of the Ca(2+)-ATPase is rapid and is reversible by a membrane-associated protein phosphatase, Ca(2+)-ATPase purified from cardiac SR underwent phosphorylation by exogenous CaM kinase, and the phosphorylated enzyme displayed twofold greater catalytic activity without alteration in its Ca(2+)-sensitivity. The phosphorylation of the Ca(2+)-ATPase was found to be isoform-specific in that the cardiac and slow-twitch skeletal muscle isoform (SERCA 2), but not the fast-twitch skeletal muscle isoform (SERCA 1), underwent phosphorylation by CaM kinase. Studies using SERCA 1 and SERCA 2 isoforms and their mutants expressed in a heterelogous cell system have resulted in i) confirmation of the isoform specificity of Ca(2+)-ATPase phosphorylation by CaM kinase, ii) identification of Ser38 as the site in SERCA 2 phosphorylated by CaM kinase, and iii) demonstration of phosphorylation-induced increase in Vmax of Ca2+ transport by the SERCA 2 enzyme. These observations suggest that in cardiac and slow-twitch skeletal muscle direct phosphorylation of the SR Ca(2+)-ATPase by the membrane-bound CaM kinase may serve to stimulate Ca2+ sequestration and therefore, the speed of muscle relaxation.
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PMID:Phosphorylation and regulation of the Ca(2+)-pumping ATPase in cardiac sarcoplasmic reticulum by calcium/calmodulin-dependent protein kinase. 920 41

Na+-K+ ATPase is known to be involved in the transport of sodium and potassium across the cell membrane. We describe here a novel mechanism for the regulation of cardiac Na+-K+ ATPase through phosphorylation by a Ca2+/calmodulin-dependent protein kinase (CaM kinase) present in the sarcolemmal membrane. Incubation of cardiac sarcolemma in the presence of Ca2+ and calmodulin resulted in phosphorylation of a 110 kDa protein, identified as the alpha-subunit of Na+-K+ ATPase. The compound W-7, a potent inhibitor of calmodulin, caused significant inhibition of the CaM kinase-mediated phosphorylation while ouabain, a potent inhibitor of Na+-K+ ATPase, had no effect. Furthermore, phosphorylation of the sarcolemmal membrane with Ca2+/calmodulin caused significant reduction in the activity of Na+-K+ ATPase. These results suggest that phosphorylation of the alpha-subunit of Na+-K+ ATPase by an endogenous CaM kinase may lead to an inhibition of its catalytic activity.
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PMID:Phosphorylation of cardiac Na+-K+ ATPase by Ca2+/calmodulin dependent protein kinase. 929 48


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