Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A severalfold activation of calcium transport and (Ca2+ + Mg2+)-activated ATPase activity by micromolar concentrations of calmodulin was observed in sarcoplasmic reticulum vesicles obtained from canine ventricles. This activation was seen in the presence of 120 mM KCl. The ratio of moles of calcium transported per mol of ATP hydrolyzed remained at about 0.75 when calcium transport and (Ca2+ + Mg2+)-activated ATPase activity were measured in the presence and absence of calmodulin. Thus, the efficiency of the calcium transport process did not change. Stimulation of calcium transport by calmodulin involves the phosphorylation of one or more proteins. The major 32P-labeled protein, as determined by sodium dodecyl sulfate slab gel electrophoresis, was the 22,000-dalton protein called phospholamban. The Ca2+ concentration dependency of calmodulin-stimulated microsomal phosphorylation corresponded to that of calmodulin-stimulated (Ca2+ + Mg2+)-activated ATPase activity. Proteins of 11,000 and 6,000 daltons and other proteins were labeled to a lesser extent. A similar phosphorylation pattern was obtained when microsomes were incubated with cAMP-dependent protein kinase and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Phosphorylation produced by added cAMP-dependent protein kinase and calmodulin was additive. These studies provided further evidence for Ca2+-dependent regulation of calcium transport by calmodulin in sarcoplasmic reticulum that could play a role in the beat-to-beat regulation of cardiac relaxation in the intact heart.
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PMID:Calmodulin-mediated regulation of calcium transport and (Ca2+ + Mg2+)-activated ATPase activity in isolated cardiac sarcoplasmic reticulum. 612 98

Cardiac sarcoplasmic reticulum plays a critical role in the excitation-contraction cycle and hormonal regulation of heart cells. Catecholamines exert their ionotropic action through the regulation of calcium transport into the sarcoplasmic reticulum. Cyclic 3'-5'-adenosine monophosphate (cAMP) causes the cAMP-dependent protein kinase to phosphorylate the regulatory protein phospholamban, which results in the stimulation of calcium transport. Calmodulin also phosphorylates phospholamban by a calcium-dependent mechanism. We have reported the isolation and purification of phospholamban with low deoxycholate (DOC) concentrations (5 X 10(-6) M). We have also reported the isolation and purification of Ca2+ + Mg2+-ATPase with a similar procedure. Both phospholamban and Ca2+ + Mg2+-ATPase retained their native properties associated with sarcoplasmic reticulum vesicles. Further, we have shown that the removal of phospholamban from membranes of sarcoplasmic reticulum vesicles uncouples Ca2+-uptake from ATPase without any effect on Ca2+ + Mg2+-ATPase activity or Ca2+ efflux. Phospholamban appears to be the substrate for both the Ca2+-calmodulin system and the cAMP-dependent protein kinase system. It is found that the phosphorylation of phospholamban by the Ca2+-calmodulin system is required for the normal basal level of Ca2+ transport, and that the phosphorylation of phospholamban at another site by the cAMP-dependent protein kinase system causes the stimulation of Ca2+-transport above the basal level. The functional effects of the phosphorylation of phospholamban by cAMP-dependent protein kinase system are expressed only after the phosphorylation of phospholamban with Ca2+-calmodulin system. We propose a model for the cardiac Ca2+ + Mg2+-ATPase, whereby the enzyme is normally uncoupled from Ca2+ uptake. The enzyme becomes coupled to Ca2+ transport after the first site of phospholamban is phosphorylated with the Ca2+-calmodulin system. When the second site of phospholamban is phosphorylated with cAMP-dependent protein kinase both Ca2+ transport and ATPase are stimulated and phospholamban becomes inaccessible to DOC solubilization and trypsin.
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PMID:Role of phospholamban in regulating cardiac sarcoplasmic reticulum calcium pump. 614 39

We recently reported that phospholamban, the activator of the cardiac sarcoplasmic reticulum calcium pump, is phosphorylated by both cAMP-dependent protein kinase and a membrane-bound, Ca2+/calmodulin-dependent phospholamban kinase. Phospholamban kinase and glycogen phosphorylase b kinase share the same substrate specificity. They differ however in that phospholamban kinase exhibits an absolute requirement for exogenous calmodulin. In line with the latter observation, phospholamban kinase is shown in this report to be inhibited by fluphenazine. Lower concentrations of the drug induced an activation of the kinase, presumably by hydrophobic interaction with either membrane phospholipids or integral proteins. Also, phospholamban kinase was found to be totally insensitive to antibodies elicited against phosphorylase kinase. Since antipsychotic drugs fail to inhibit the delta-subunit-dependent activity of phosphorylase kinase, the above findings confirm that the two kinases are distinct molecular entities. After detergent solubilization of the sarcoplasmic reticulum, the phospholamban-ATPase complex remains a substrate for phospholamban kinase activity, which retains the ability to catalyze the phosphorylation of exogenous phosphorylase b. However, the Ca2+ dependence is entirely lost upon solubilization and no kinase activity is retained on calmodulin-Sepharose in the presence of Ca2+ ions. Phospholamban and phosphorylase kinase activities copurify with the pump-phospholamban complex upon fractionation of the solubilized proteins by density gradient ultracentrifugation, suggesting a tight interaction between the ATPase, its activator, and the phospholamban kinase. A tentative schematic representation of this supramolecular assembly is based upon the results described in this and preceding papers.
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PMID:Ca2+/calmodulin-dependent phospholamban kinase from cardiac sarcoplasmic reticulum is distinct from phosphorylase kinase and forms a regulatory complex with phospholamban and the Ca2+-ATPase. 622 Jun 53

Phosphorylation of purified phospholamban isolated from canine cardiac sarcoplasmic reticulum vesicles decreased the electrophoretic mobility of the protein in sodium dodecyl sulfate (SDS)-polyacrylamide gels. Different mobility forms of phospholamban in SDS gels were visualized both by direct protein staining and by autoradiography. Unphosphorylated phospholamban migrated with an apparent Mr = 25,000 in SDS gels; maximal phosphorylation of phospholamban by cAMP- or Ca2+-calmodulin-dependent protein kinase increased the apparent Mr to 27,000. Partial phosphorylation of phospholamban by either protein kinase gave intermediate mobility forms of molecular weights between 25,000 and 27,000, suggesting that more than one phosphorylation site was present on the holoprotein for each activity. Boiling of phospholamban in SDS dissociated the holoprotein into an apparently homogeneous class of low molecular weight "monomers." Only two mobility forms of monomeric phospholamban were observed in SDS gels after phosphorylation by cAMP-dependent protein kinase, corresponding to 9-kDa dephospho- and 11-kDa phosphoproteins. All of the 9-kDa protein could be phosphorylated and converted into the 11-kDa mobility form, suggesting the presence of only one site of phosphorylation on a single type of monomer for cAMP-dependent protein kinase. Simultaneous phosphorylation of monomeric phospholamban by cAMP-dependent protein kinase and Ca2+-calmodulin-dependent protein kinase gave an additional mobility form of the protein, suggesting that different sites of phosphorylation were present for each activity on each monomer. Incomplete dissociation of the holoprotein by boiling it in a relatively low concentration of SDS facilitated the detection of five major mobility forms of the protein in SDS gels, and the mobilities of all of these forms were decreased by phosphorylation. We propose that the high molecular weight form of phospholamban is a multimer of electrophoretically indistinguishable monomers, each of which contains a different phosphorylation site for cAMP-dependent protein kinase activity and Ca2+-calmodulin-dependent protein kinase activity. Phosphorylation of phospholamban at multiple sites is responsible for the various mobility forms of the holoprotein detected in SDS-polyacrylamide gels.
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PMID:Phosphorylation-induced mobility shift in phospholamban in sodium dodecyl sulfate-polyacrylamide gels. Evidence for a protein structure consisting of multiple identical phosphorylatable subunits. 622 39

Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) is able to catalyze the phosphorylation of phospholamban in a canine cardiac sarcoplasmic reticulum preparation. This phosphorylation is associated with a 2-fold stimulation of Ca2+ uptake by cardiac sarcoplasmic reticulum similar to that seen following phosphorylation of phospholamban by an endogenous calmodulin-dependent protein kinase or by the catalytic subunit of cAMP-dependent protein kinase. Two-dimensional peptide maps of the tryptic fragments of phospholamban indicate that the three protein kinases differ in their selectivity for sites of phosphorylation. However, one common peptide appears to be phosphorylated by all three protein kinases. These findings suggest that protein kinase C may play a role similar to those played by cAMP- and calmodulin-dependent protein kinases in the regulation of Ca2+ uptake by cardiac sarcoplasmic reticulum, and raise the possibility that the effects of all three protein kinases are mediated through phosphorylation of a common peptide in phospholamban.
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PMID:Phosphorylation of phospholamban by calcium-activated, phospholipid-dependent protein kinase. Stimulation of cardiac sarcoplasmic reticulum calcium uptake. 623 8

Two endogenous protein kinase activities, cAMP-dependent and calmodulin-Ca2+-dependent, are associated with isolated cardiac sarcoplasmic reticulum (SR) vesicles. Both kinases phosphorylate an endogenous substrate of approximately 22,000 daltons (phospholamban). The phosphorylation of phospholamban by either the intrinsic or by exogenous cAMP-dependent protein kinase is found to be Ca2+-independent between 0.05 and 100 microM free Ca2+. Calmodulin-dependent phosphorylation, on the other hand, does not require cAMP and is absolutely dependent on the presence of free Ca2+ over a concentration range that corresponds to physiological levels (10(-7) to 10(-5) M). Phosphorylation of SR vesicles by both kinases is additive and the extent of saturation of the cAMP-specific sites has no effect on the degree of stimulation by calmodulin or its Ca2+-dependence. Trifluoperazine, an inhibitor of calmodulin, inhibits calmodulin-dependent phosphorylation without affecting cAMP-dependent phosphorylation, indicating the presence of two types of kinases. This is made further evident by the selectivity of each kinase for exogenous substrates. Whereas cAMP-dependent protein kinase appears to phosphorylate histone ILA (a basic protein) preferentially, calmodulin-dependent protein kinase prefers phosvitin (an acidic protein).
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PMID:Studies on phosphorylation of canine cardiac sarcoplasmic reticulum by calmodulin-dependent protein kinase. 627 7

Canine cardiac sarcoplasmic reticulum vesicles contain intrinsic cAMP-dependent and Ca2+ -calmodulin-dependent protein kinase (EC 2.7.1.37) activities and a common substrate, phospholamban, for these enzymes. Cyclic AMP-dependent protein kinase associated with sarcoplasmic reticulum membranes was solubilized with Triton X-100. Solubilization of the sarcoplasmic reticulum protein kinase did not affect its dependency on cAMP or its substrate specificity. The solubilized cAMP-dependent protein kinase was purified by DEAE-cellulose chromatography and was characterized as a type II enzyme on the basis of its elution at high ionic strength. DEAE-purified cAMP-dependent protein kinase exhibited no Ca2+ -calmodulin-dependent protein kinase activity. Cytosol from canine cardiac muscle cells, chromatographed on DEAE-cellulose under conditions identical to those used with sarcoplasmic reticulum, exhibited the presence of both type I and type II cAMP-dependent protein kinase isozymes. The properties of the DEAE-cellulose purified type II protein kinases from sarcoplasmic reticulum and cytosol were similar. We conclude that cardiac sarcoplasmic reticulum contains primarily type II cAMP-dependent protein kinase and this is probably the enzyme which phosphorylates sarcoplasmic reticulum in vivo and regulates Ca2+ transport.
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PMID:Characterization of cyclic 3':5'-amp-dependent protein kinase in sarcoplasmic reticulum and cytosol of canine myocardium. 629 89

Two substrate proteins for cAMP-dependent protein kinase detected in a rat heart sarcolemma preparation displayed molecular weights of 24,000 and 9000 in sodium dodecyl sulfate gels and were shown to be interconvertible. The 9000-dalton protein could readily be separated from other low molecular weight phosphoproteins (mol. wt. 14,000 and 7000) by the use of 15% polyacrylamide gels. In addition to an endogenous cAMP-dependent protein kinase the membrane preparation also contained a protein-phosphorylation system that required Ca2+ and calmodulin. It appeared that both 24,000- and 55,000-dalton proteins were substrates for the endogenous Ca2+- and calmodulin-dependent protein kinase. Contaminating sarcoplasmic reticulum vesicles, first loaded with calcium oxalate, could be separated from the enriched sarcolemma preparation by sucrose gradient centrifugation. The separation was confirmed by comparative analysis of 5'-nucleotidase, Na+ -Ca2+ antiporter, and (Ca2+ + Mg2+)-dependent ATPase activities and by determination of gel electrophoretic (phospho)protein composition, sialic acid, cholesterol, and phospholipid contents. The 24,000-dalton phosphoprotein complex was equally distributed between sarcolemmal and sarcoplasmic reticulum fractions, whereas the 55,000- and 7000-dalton proteins were predominantly found in the sarcolemmal fraction. The 24,000-dalton protein was most likely phospholamban, because no other phosphoprotein was found in the 20,000 molecular weight range.
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PMID:Phosphorylation of low-molecular-weight proteins in preparations of rat heart sarcolemma and sarcoplasmic reticulum. 630 73

The rate of calcium transport by sarcoplasmic reticulum vesicles from dog heart assayed at 25 degrees C, pH 7.0, in the presence of oxalate and a low free Ca2+ concentration (approx. 0.5 microM) was increased from 0.091 to 0.162 mumol . mg-1 . min-1 with 100 nM calmodulin, when the calcium-, calmodulin-dependent phosphorylation was carried out prior to the determination of calcium uptake in the presence of a higher concentration of free Ca2+ (preincubation with magnesium, ATP and 100 microM CaCl2; approx. 75 microM free Ca2+). Half-maximal activation of calcium uptake occurs under these conditions at 10-20 nM calmodulin. The rate of calcium-activated ATP hydrolysis by the Ca2+-, Mg2+-dependent transport ATPase of sarcoplasmic reticulum was increased by 100 nM calmodulin in parallel with the increase in calcium transport; calcium-independent ATP splitting was unaffected. The calcium-, calmodulin-dependent phosphorylation of sarcoplasmic reticulum, preincubated with approx. 75 microM Ca2+ and assayed at approx. 10 microM Ca2+ approaches maximally 3 nmol/mg protein, with a half-maximal activation at about 8 nM calmodulin; it is abolished by 0.5 mM trifluperazine. More than 90% of the incorporated [32P]phosphate is confined to a 9-11 kDa protein, which is also phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and most probably represents a subunit of phospholamban. The stimulatory effect of 100 nM calmodulin on the rate of calcium uptake assayed at 0.5 microM Ca2+ was smaller following preincubation of sarcoplasmic reticulum vesicles with calmodulin in the presence of approx. 75 microM Ca2+, but in the absence of ATP, and was associated with a significant degree of calmodulin-dependent phosphorylation. However, the stimulatory effect on calcium uptake and that on calmodulin-dependent phosphorylation were both absent after preincubation with calmodulin, without calcium and ATP, suggestive of a causal relationship between these processes.
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PMID:Calmodulin-dependent elevation of calcium transport associated with calmodulin-dependent phosphorylation in cardiac sarcoplasmic reticulum. 630 68

Stimulation of secretion in exocrine cells by agonists involving cAMP as second messenger is associated with the phosphorylation of a specific membrane-associated 22.4-kDa protein (protein III) (Jahn et al.). Here it is shown by subcellular fractionation of rat parotid gland lobules that protein III is associated with the endoplasmic reticulum. The submicrosomal fractions containing protein III, also contain the ATP-dependent microsomal calcium pump activity. Protein III in microsomal subfractions can be phosphorylated in vitro with catalytic subunit from cAMP-dependent protein kinase. Phosphorylated protein III contains exclusively P-serine. Protein III can be removed from ER-membranes with acid chloroform-methanol or Triton X-114, but not by high salt wash indicating that it is tightly associated with the membranes. Protein III is smaller than phospholamban and, in contrast to phospholamban, resistant to heating in SDS. A relationship between phosphorylation of protein III and microsomal calcium sequestration is discussed.
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PMID:Specific phosphorylation of a protein in calcium accumulating endoplasmic reticulum from rat parotid glands following stimulation by agonists involving cAMP as second messenger. 631 93


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