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)

Muscarinic receptor, from porcine synaptic membrane, was purified by affinity chromatography. Molecular weight analysis by SDS-gel electrophoresis revealed one major peptide with an apparent Mr of 68 +/- 2 Kda. The purified receptor was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase resulting in a concomitant loss in specific binding, and this loss was reversed by calcineurin.
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PMID:Phosphorylation of brain muscarinic receptor: evidence of receptor regulation. 303 Mar 6

Bovine brain contains two major calmodulin (CaM) dependent phosphodiesterase isozymes which are homodimeric proteins with subunit molecular masses of 60 and 63 kilodaltons (kDa), respectively. The 60-kDa subunit isozyme can be phosphorylated by cAMP-dependent protein kinase, resulting in a decrease in the enzyme affinity towards CaM. The phosphorylation is blocked by Ca2+ and CaM and reversed by the CaM-stimulated phosphatase (calcineurin). The 63-kDa subunit isozymes can also be phosphorylated, but in this case by a CaM-dependent protein kinase(s). This phosphorylation is also accompanied by a decrease in the isozyme affinity towards CaM and can be reversed by the CaM-dependent phosphatase. Analysis of the complex regulatory properties of the phosphodiesterase isozymes has led to the suggestion that fluxes of cAMP and Ca2+ during cell activations are closely coupled and that the CaM-dependent phosphodiesterase isozymes play key roles in this signal coupling phenomenon.
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PMID:Regulation of cAMP concentration by calmodulin-dependent cyclic nucleotide phosphodiesterase. 303 Mar 66

A calmodulin-stimulated protein phosphatase has been purified from bovine myocardium. The purification procedure involves sequential DEAE-Sephacel ion exchange chromatography, calmodulin-Sepharose affinity chromatography, and high performance liquid chromatography using a Spherogel TSK DEAE 5PW column. By SDS polyacrylamide gel electrophoresis, the purified cardiac phosphatase consists of two subunits of Mr 61,000 and 19,000, similar to the brain enzyme, calcineurin. Protein phosphatase activity of the cardiac enzyme is stimulated by Ca2+-calmodulin and inhibited by the calmodulin antagonist drug, calmidazolium. Effects of a series of divalent cations on catalytic activity of the cardiac calmodulin-stimulated protein phosphatase are similar to those observed with calcineurin, when the two enzymes are assayed under identical conditions. Highly enriched preparations of bovine cardiac sarcolemma contain substrates of cAMP-dependent protein kinase of Mr 166 K, 133 K, 108 K, 79 K, 39 K, and 14 K, which are specifically dephosphorylated by the calmodulin-stimulated phosphatase with pseudofirst-order rate constants of 0.23, 0.46, 0.69, 0.35, 0.69, and 0.115 min-1, respectively. These substrates are not present in purified preparations of cardiac sarcoplasmic reticulum. These results support a role of the calmodulin-stimulated phosphatase in the Ca2+-regulation of specific sarcolemmal processes by protein dephosphorylation.
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PMID:Cardiac calmodulin-stimulated protein phosphatase: purification and identification of specific sarcolemmal substrates. 303 93

Calcineurin, a Ca2+ and calmodulin (CM)-dependent phosphatase, has been shown to be present in high concentrations in the striatum. Using inhibitor-1(phosphorylated by cAMP-dependent protein kinase) as a substrate, we found Ca2+/CM-dependent phosphatase (calcineurin) to be more than 2-fold higher than non-Ca2+/CM-dependent phosphatase in the rat striatum. In order to determine the cellular localization of calcineurin, striatal kainic acid injections were used to destroy neurons whose cell bodies are present at the site of injection. Glutamic acid decarboxylase (GAD) activity was measured as an indicator of destruction of striatal GABA-ergic neurons. After intrastriatal injection of 1 and 2 ug of kainic acid, there was a significant decrease of both calcineurin and GAD. However, injection of 0.5 ug kainic acid into the striatum reduced GAD activity by 81%, but had no effect on calcineurin phosphatase activity. Thus calcineurin does not appear to be equally distributed among all types of striatal neurons, but rather may be concentrated in neurons that are less sensitive to kainic acid than the GABA-ergic neuron.
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PMID:Ca2+/calmodulin-dependent phosphoprotein phosphatase activity of calcineurin in rat striatum: effect of kainic acid lesions. 303 26

A novel Mr 17,000 Ca2+-binding protein isolated from bovine brain was found to be a potent inhibitor of the Ca2+- and phospholipid-dependent protein kinase (protein kinase C), also isolated from bovine brain. Half-maximal inhibition by this calciprotein of the initial rate of phosphorylation of histone III-S by protein kinase C occurred at a calciprotein concentration of 2.2 microM under standard conditions. Comparison of the effects of a number of Ca2+-binding proteins on protein kinase C activity indicated that the Mr 17,000 Ca2+-binding protein was the most potent inhibitor, followed by the intestinal Ca2+-binding protein and calcineurin. Calmodulin, troponin C, S-100 protein and a Mr 21,000 Ca2+-binding protein of bovine brain were relatively weak inhibitors of protein kinase C. The inhibitory effect of the Mr 17,000 Ca2+-binding protein was apparently not due to its interaction with phospholipid or the basic protein substrate and therefore appears to be due to a direct effect on the protein kinase C. These observations suggest that the novel Mr 17,000 Ca2+-binding protein, and possibly other Ca2+-binding proteins, may play a physiological role in regulating the activity of protein kinase C.
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PMID:Inhibition of the Ca2+- and phospholipid-dependent protein kinase by a novel Mr 17,000 Ca2+-binding protein. 316 Mar 47

cAMP-dependent protein kinase, protein kinase C, cGMP-dependent protein kinase, smooth muscle myosin light-chain kinase, and phosphorylase kinase were examined with respect to their ability to phosphorylate porcine atrial muscarinic receptors (mAcChRs). Experiments were performed both in detergent solution and in a reconstituted system containing the mAcChR alone or in the presence of the purified porcine atrial inhibitor guanine nucleotide binding protein (Gi). Only cAMP-dependent protein kinase was capable of phosphorylating the receptor under any of the experimental conditions examined. Phosphorylation of the mAcChR in the detergent-solubilized state resulted in a loss of ligand binding sites that was reversible upon treatment with calcineurin in the presence of calcium and calmodulin. Upon reconstitution, the apparent stoichiometry of phosphorylation was increased by about 15-fold. Carbachol-stimulated covalent incorporation of phosphate was found only in the reconstituted system in the presence of Gi, suggesting that the large agonist-stimulated increase in phosphorylation observed in vivo [Kwatra, M. M., & Hosey, M. M. (1986) J. Biol. Chem. 261, 12429-12432] may in part result from a unique receptor conformation that occurs upon association with this protein. Ligand binding studies indicated that phosphorylation of the mAcChR in the detergent-solubilized or reconstituted state did not affect its interaction with carbachol or L-quinuclidinyl benzilate in vitro. Carbachol-induced stimulation of the GTPase activity of Gi in the reconstituted system was also unaffected by phosphorylation.
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PMID:Phosphorylation of the porcine atrial muscarinic acetylcholine receptor by cyclic AMP dependent protein kinase. 344 51

The Ca2+-dependent binding of [125I]calmodulin (CaM) to hepatic proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was utilized to identify CaM binding or "acceptor" proteins or CAPs. Two proteins of apparent molecular weight of 60,000 (CAP-60) and 45,000 (CAP-45) comprised greater than 80% of the Ca2+-dependent CaM binding in rat liver cytosol. CAP-60 and CAP-45 were partially purified by a variety of chromatographic steps, including affinity chromatography on CaM Sepharose. CAP-60 possessed a native molecular size of 400,000, indicating it to be the CaM-binding "subunit" of a larger oligomeric complex. In contrast, CAP-45 was monomeric as judged by gel filtration. Neither CAP-60 nor CAP-45 possessed chromatographic properties consistent with known CaM-dependent enzymes reported in the literature. Two-dimensional peptide mapping provided convincing evidence that CAP-60 and CAP-45 were unrelated to other well-characterized CAPs, namely Ca2+ (CaM)-dependent protein kinase II, calcineurin, or the CaM-dependent cyclic nucleotide phosphodiesterase. The relative abundance and high affinity for CaM could suggest that these novel target proteins, CAP-60 and CAP-45, represent a dominant pathway for CaM action in the mammalian liver.
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PMID:Identification of high-affinity calmodulin-binding proteins in rat liver. 346 18

A 20-residue peptide analogue (IASGRTGRRNAIHDILVSSA) of the 8000-dalton heat-stable cAMP-dependent protein kinase inhibitor undergoes efficient calcium-dependent binding by calmodulin, with Kd approximately 70 nM when calcium is present. It is a potent inhibitor of smooth muscle myosin light chain kinase and of the calmodulin-dependent phosphatase activity of calcineurin. At concentrations above 3 microM, the peptide stimulates the basal activity of calcineurin. The native protein kinase inhibitor has no effect on the catalytic activity of myosin light chain kinase and is moderately inhibitory to both the calmodulin-dependent and -independent phosphatase activity of calcineurin. Competition experiments using excess concentrations of calcineurin and calmodulin suggest that the primary interaction of the native heat-stable inhibitor is with the catalytic subunit of protein kinase. Dansylcalmodulin exhibits only a weak interaction with the inhibitor. Observations on deletion peptides of the 20-residue analogue help to delineate the overlapping peptide binding specificities of the cAMP-dependent protein kinase [Scott, J. D., Glaccum, M. B., Fischer, E. H., & Krebs, E. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1613-1616] and calmodulin. In both cases, the most effectively bound peptides contain the RTGRR sequence.
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PMID:Association of calmodulin with peptide analogues of the inhibitory region of the heat-stable protein inhibitor of adenosine cyclic 3',5'-phosphate dependent protein kinase. 375 57

Purified bovine brain calmodulin was biotinylated with biotinyl-epsilon-aminocaproic acid N-hydroxysuccinimide. Biotinylated calmodulin was used to detect and quantify calmodulin-binding proteins following both protein blotting and slot-blot procedures by using alkaline phosphatase or peroxidase coupled to avidin. When purified bovine brain calcineurin, a calmodulin-dependent protein phosphatase, was immobilized on nitrocellulose slot blots, biotinylated calmodulin bound in a calcium-dependent saturable manner; these blots were then quantified by densitometry. Biotinylated calmodulin was able to detect as little as 10 ng of calcineurin, and the binding was competitively inhibited by addition of either native calmodulin or trifluoperazine. When biotinylated calmodulin was used to probe protein blots of crude brain cytosol and membrane preparations after gel electrophoresis, only protein bands characteristic of known calmodulin-binding proteins (i.e., calmodulin-dependent protein kinase, calcineurin, spectrin) were detected with avidin-peroxidase or avidin-alkaline phosphatase procedures. Purified calcineurin was subjected to one- and two-dimensional gel electrophoresis and protein blotting; as expected, only the 61-kDa calmodulin-binding subunit was detected. When the two-dimensional protein blot was incubated with biotinylated calmodulin and detected with avidin-alkaline phosphatase, several apparent forms of the 61-kDa catalytic subunit were detected, consistent with isozymic species of the enzyme. The results of these studies suggest that biotinylated calmodulin can be used as a simple, sensitive, and quantifiable probe for the study of calmodulin-binding proteins.
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PMID:A rapid and sensitive method for detection and quantification of calcineurin and calmodulin-binding proteins using biotinylated calmodulin. 386 79

Bovine brain contains calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes which are composed of two distinct subunits: Mr 60,000 and 63,000. The 60-kDa but not the 63-kDa subunit-containing isozyme can be phosphorylated by cAMP-dependent protein kinase resulting in decreased affinity of this subunit toward calmodulin (Sharma, R. K., and Wang, J. H. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 2603-2607). In contrast, purified 63-kDa subunit-containing isozyme has been found to be phosphorylated by a preparation of bovine brain calmodulin-binding proteins in the presence of Ca2+ and calmodulin. The phosphorylation resulted in the maximal incorporation of 2 mol of phosphate/mol of the phosphodiesterase subunit with a 50% decrease in the enzyme affinity toward calmodulin. At a constant calmodulin concentration of 6 nM, the phosphorylated isozyme required a higher concentration of Ca2+ for activation than the nonphosphorylated phosphodiesterase. The Ca2+ concentrations at 50% activation by calmodulin of the nonphosphorylated and phosphorylated isozymes were 1.1 and 1.9 microM, respectively. Phosphorylation can be reversed by the calmodulin-dependent phosphatase, calcineurin, but not by phosphoprotein phosphatase 1. The results suggest that the Ca2+ sensitivities of brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes can be modulated by protein phosphorylation and dephosphorylation mechanisms in response to different second messengers.
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PMID:Calmodulin and Ca2+-dependent phosphorylation and dephosphorylation of 63-kDa subunit-containing bovine brain calmodulin-stimulated cyclic nucleotide phosphodiesterase isozyme. 394 89


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