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)

Phosphorylation of purified Na+,K(+)-ATPase by cAMP-dependent protein kinase (protein kinase A) decreases the activity of this enzyme. We have now shown, using several experimental approaches, that a highly conserved seryl residue on the catalytic (alpha) subunit of Na+,K(+)-ATPase, corresponding to Ser943 of the rat alpha 1 isoform, is the phosphorylation site for protein kinase A. cDNAs corresponding to wild-type Na+,K(+)-ATPase and Na+,K(+)-ATPase in which Ser943 was mutated to Ala were transfected into COS cells. Treatment of the transfected cells with forskolin plus 3-isobutyl-1-methylxanthine resulted in a decrease in the activity of the wild-type enzyme but not in that of the mutated enzyme. The results suggest that, in intact cells, the activity of the Na+,K(+)-ATPase is regulated in part by signal transduction pathways that use protein kinase A-dependent phosphorylation of the Na+,K(+)-ATPase alpha subunit.
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PMID:Identification of the phosphorylation site for cAMP-dependent protein kinase on Na+,K(+)-ATPase and effects of site-directed mutagenesis. 751 Jul 9

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

Phospholamban is a putative suppressor of the Ca2+ ATPase of the cardiac sarcoplasmic reticulum. The level of mRNA encoding the Ca2+ ATPase has been shown to be increased, whereas the phospholamban mRNA level to be decreased in the ventricles obtained from hyperthyroid rabbits [Nagai R, Zarain-Herzberg A. Brandl CJ, Fujii J. Tada M. MacLennan DH, Alpert NR, Periasamy M. (1989) Proc Natl Acad Sci USA 86: 2966-2970]. The present study was designed to examine whether these effects of thyroid hormone on the expression of the Ca2+ ATPase and phospholamban are exerted directly on cardiac myocytes and whether the resultant incoordinate expression of these proteins alters Ca2+ pumping activity. We studied the levels of phospholamban and Ca2+ ATPase mRNA in primary isolated neonatal rat myocardial cells incubated with triiodothyronine (T3) for 3-48 h and the Ca2+ uptake activity of the microsomes prepared from the cells. Northern blot analysis showed that T3 decreased phospholamban mRNA levels to about a half of control in 24 h. On the other hand, Ca2+ ATPase mRNA gradually increased with time. EC50 for phospholamban mRNA expression was 2.5 x 10(-10) M which was approximately 10 times higher than that for the Ca2+ ATPase. T3 increased Vmax of Ca2+ uptake with the significant reduction of K0.5 for Ca2+ (0.40 +/- 0.02 microM for control v 0.31 +/- 0.02 microM for T3-treated vesicles), indicating that thyroid hormone stimulates Ca2+ pumping activity not only by increasing the Ca2+ ATPase but also decreasing phospholamban. These results suggested that phospholamban regulates the Ca2+ ATPase in dual modes; in short time range, by decreasing the affinity of the Ca2+ ATPase for Ca2+ by phosphorylation of phospholamban with cAMP-dependent protein kinase, and in long time range, by changing the molecular ratio between the two proteins through the regulation of gene expression.
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PMID:Thyroid hormone enhances Ca2+ pumping activity of the cardiac sarcoplasmic reticulum by increasing Ca2+ ATPase and decreasing phospholamban expression. 781 58

Phospholamban is a negative regulator of the sarcoplasmic reticulum Ca(2+)-pumping ATPase. Phosphorylation of phospholamban activates the ATPase and decreases the level of cytosolic calcium. Phospholamban is phosphorylated in heart by cAMP-dependent protein kinase, cGMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase II (CM-kinase-II) and in smooth muscle cells by cGMP-dependent protein kinase. In contrast to heart muscle, phospholamban is poorly phosphorylated by CM-kinase-II in extracts of rat aortic smooth muscle cells. Rat aorta phospholamban amino acid sequence was identical to dog heart. The peptide substrate specificity of CM-kinase-II from rat aorta was the same as that from rat heart. The lack of phosphorylation of rat aorta phospholamban by the CM-kinase-II appears to result from the relatively low abundance of phospholamban in smooth muscle.
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PMID:Phosphorylation of phospholamban in aortic smooth muscle cells and heart by calcium/calmodulin-dependent protein kinase II. 785 66

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

Second messenger regulation of IRK1 (Kir2.1) inward rectifier K+ channels was investigated in giant inside-out patches from Xenopus oocytes. Kir2.1-mediated currents that run down completely within minutes upon excision of the patches could be partly restored by application of Mg-ATP together with > 10 microM free Mg2+ to the cytoplasmic side of the patch. As restoration could not be induced by the ATP analogs AMP-PNP or ATP gamma S, this suggests an ATPase-like mechanism. In addition to ATP, the catalytic subunit of cAMP-dependent protein kinase (PKA) induced an increase in current amplitude, which could, however, only be observed if channels were previously or subsequently stimulated by Mg-ATP and free Mg2+. This indicates that functional activity of Kir2.1 channels requires both phosphorylation by PKA and ATP hydrolysis. Moreover, currents could be down-regulated by N-heptyl-5-chloro-1-naphthalenesulfonamide, a specific stimulator of protein kinase C (PKC), suggesting that PKA and PKC mediate inverse effects on Kir2.1 channels. Regulation of Kir2.1 channels described here may be an important mechanism for regulation of excitability.
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PMID:Kir2.1 inward rectifier K+ channels are regulated independently by protein kinases and ATP hydrolysis. 799 32

To elucidate the mechanism of Na+ retention by insulin in vivo, the direct tubular effect of insulin on NaCl transport in the in vitro microperfused medullary thick ascending limb of Henle (MTAL) was examined. Insulin at 10(-6) mol/l in the bath increased transepithelial voltage (Vte) from 3.1 +/- 0.3 to 5.7 +/- 0.3 mV (n = 12, P < 0.0001). The effect of insulin on Vte was dependent on its concentration, and the half-maximal effect of insulin was observed at 5 x 10(-9) mol/l. Insulin at 10(-6) mol/l also caused a significant decrease of luminal Cl- concentration from 85.4 +/- 5.0 to 62.8 +/- 3.0 mmol/l (n = 5, P < 0.002) when the lumen was microperfused constantly at less than 1 nl/min. Insulin at 10(-6) mol/l also increased net lumen-to-bath Cl- flux (JCl) from 143 +/- 15 to 292 +/- 37 pmol.mm-1.min-1 (n = 5, P < 0.004). When the Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) in the basolateral membrane was blocked by 10(-4) mol/l ouabain, the insulin-mediated increase in Vte was completely suppressed. When the Na(+)-K(+)-2Cl- cotransporter in the luminal membrane of the MTAL was blocked by 10(-4) mol/l furosemide, the insulin-mediated increase in Vte was also abolished. To test whether adenosine 3',5'-cyclic monophosphate (cAMP) contributes to the action of insulin, we examined the effect of cAMP analogue and cAMP-dependent protein kinase inhibitor on the action of insulin. A maximal concentration (5 x 10(-4) mol/l) of dibutyryl-cAMP (DBcAMP) increased Vte and JCl.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Insulin stimulates NaCl transport in isolated perfused MTAL of Henle's loop of rabbit kidney. 806 87

Paramecium dyneins were tested as substrates for phosphorylation by cAMP-dependent protein kinase, cGMP-dependent protein kinase, and two Ca(2+)-dependent protein kinases that were partially purified from Paramecium extracts. Only cAMP-dependent protein kinase caused significant phosphorylation. The major phosphorylated species was a 29 kDa protein that was present in both 22 S and 12 S dyneins; its phosphate-accepting activity peaked with 22 S dynein. In vitro phosphorylation was maximal at five minutes, then decreased. This decrease in phosphorylation was inhibited by the addition of vanadate or NaF. The 29 kDa protein was not phosphorylated by a heterologous cAMP-dependent protein kinase, the bovine catalytic subunit. Phosphorylation of dynein did not change its ATPase activity. In sucrose gradient fractions from the last step of dynein purification, phosphorylation by an endogenous kinase occurred. This phosphorylation could not be attributed to the small amounts of cAMP- and cGMP-dependent protein kinases known to be present, nor was it Ca(2+)-dependent. This previously uncharacterized ciliary protein kinase used casein as an in vitro substrate.
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PMID:In vitro phosphorylation of ciliary dyneins by protein kinases from Paramecium. 812 14

Renal basolateral membranes contain protein kinase A (PKA) and Ca-dependent protein kinases. We studied the effect of cyclic adenosine monophosphate (cAMP), the active phorbol ester phorbol 12-myristate 13-acetate (PMA) and calmodulin on Na-phosphate cotransporter. Rabbit renal basolateral membranes, enriched 15-fold in Na-K-ATPase activity, were phosphorylated with 50 microM ATP, and 32P uptake was measured in the presence of Na or K. 32P uptake was greater in the presence of Na than in the presence of K, indicating the existence of Na-dependent phosphate uptake, i.e., Na-phosphate cotransporter. cAMP, 1 microM, and the catalytic subunit of cAMP-dependent protein kinase (PKA-CSU, 15 mU/ml) inhibited Na-phosphate cotransporter activity by 30-50%, respectively. The effect of CSU was prevented by the PKA inhibitor (1 microgram/ml). Calmodulin, 1 microM, also inhibited Na-phosphate cotransporter by 48% (p < 0.05), and this effect of calmodulin was prevented by the inhibitor naphthalene sulfonamide W-13 (100 microM). In contrast, the active phorbol ester PMA, 1 microM, increased the Na-phosphate cotransporter by 62%, while the inactive analog 4-alpha phorbol failed to elicit such a stimulation. The results demonstrate the presence of Na-dependent phosphate transport in rabbit renal basolateral membranes which is modulated by PKA and by Ca-dependent protein kinases.
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PMID:Modulation of renal basolateral Na-phosphate cotransporter by protein kinase A and Ca-dependent protein kinases. 816 19

The catalytic (C) subunit of cAMP-dependent protein kinase interacts with two classes of inhibitors. The regulatory (R) subunits, types I and II, associate to form an inactive holoenzyme complex that is activated in response to cAMP. The C-subunit is also inhibited by small heat-stable protein kinase inhibitors (PKI's). Inhibition by both PKI and RI-subunit requires the synergistic high-affinity binding of MgATP. The stabilizing effect of ATP was quantitated by using analytical gel chromatography. Both the type I holoenzyme and the C.PKI complex in the presence of MgATP show apparent Kd's for subunit association that are below 0.1 nM, while in the absence of MgATP the apparent Kd's are 125 nM and 2.3 microM, respectively, for the two complexes. In the absence of MgATP both complexes also can be dissociated readily and, hence, activated by salt-induced dissociation. Under physiological salt concentrations, salt-induced dissociation would be substantial in the absence of the high-affinity binding of MgATP. In both complexes, the ATPase activity of the free C-subunit is abolished. The off rates for MgATP also indicate that the type I holoenzyme is more stable than the C.PKI complex. The off rate (t1/2) for MgATP from the C.PKI complex is 17 min, while the off rate for the type I holoenzyme is 11.7 h. When the C.PKI complex is incubated with RI-subunit in the presence or absence of MgATP, the C-subunit preferentially reassociates with the RI-subunit, forming holoenzyme. In contrast, free PKI cannot compete for the C-subunit when it is part of a holoenzyme complex.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Physiological inhibitors of the catalytic subunit of cAMP-dependent protein kinase: effect of MgATP on protein-protein interactions. 826 80


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