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 purpose of this review is to describe the importance of calmodulin as a mediator of the effects of calcium ions in living systems, particularly in the process of skeletal muscle contraction. Calmodulin is a low molecular weight, acidic, calcium binding protein which mediates the Ca2+ regulation of a wide range of physiological processes throughout eukaryotic organisms. At low free Ca2+ concentrations, such as exist in resting muscle sarcoplasm, calmodulin exists in the Ca2+-free form in which state it does not generally interact with a target protein. Following an appropriate stimulus, the free Ca2+ concentration rises whereupon Ca2+ binds to calmodulin which undergoes a conformational change enabling it to interact with a target protein(s). The overall result of this protein-protein interaction is a physiological effect, e.g., Ca2+ binding to calmodulin in smooth muscle allows it to interact with and activate myosin light chain kinase which catalyzes the phosphorylation of myosin. This reaction results in contraction of the smooth muscle. Recent studies have implicated calmodulin in the Ca2+ control of three enzymes in skeletal muscle: phosphorylase kinase, myosin light chain kinase and a protein kinase of the sarcoplasmic reticulum. Various classes of drugs, including certain local anaesthetics, have been shown to affect calmodulin-dependent processes. It is likely that the effects of such drugs result from their interaction with calmodulin.
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PMID:Calmodulin and its roles in skeletal muscle function. 630 99

In previous work from this laboratory, a partially purified protein kinase from the soil amoeba Acanthamoeba castellanii was shown to phosphorylate the heavy chain of the two single-headed Acanthamoeba myosin isoenzymes, myosin IA and IB, resulting in a 10- to 20-fold increase in their actin-activated Mg2+-ATPase activities (Maruta, H., and Korn, E.D. (1977) J. Biol. Chem. 252, 8329-8332). A myosin I heavy chain kinase has now been purified to near homogeneity from Acanthamoeba by chromatography on DE-52 cellulose, phosphocellulose, and Procion red dye, followed by chromatography on histone-Sepharose. Myosin I heavy chain kinase contains a single polypeptide of 107,000 Da by electrophoretic analysis. Molecular sieve chromatography yields a Stokes radius of 4.1 nm, consistent with a molecular weight of 107,000 for a native protein with a frictional ratio of approximately 1.3:1. The kinase catalyzes the incorporation of 0.9 to 1.0 mol of phosphate into the heavy chain of both myosins IA and IB. Phosphoserine has been shown to be the phosphorylated amino acid in myosin IB. The kinase has highest specific activity toward myosin IA and IB, about 3-4 mumol of phosphate incorporated/min/mg (30 degrees C) at concentrations of myosin I that are well below saturating levels. The kinase also phosphorylates histone 2A, isolated smooth muscle light chains, and, to a very small extent, casein, but has no activity toward phosvitin or myosin II, a third Acanthamoeba myosin isoenzyme with a very different structure from myosin IA and IB. Myosin I heavy chain kinase requires Mg2+ but is not dependent on Ca2+, Ca2+/calmodulin, or cAMP for activity. The kinase undergoes an apparent autophosphorylation.
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PMID:Purification and characterization of a myosin I heavy chain kinase from Acanthamoeba castellanii. 630 72

Phosphorylation of myosin increases rapidly upon stimulation of an arterial smooth muscle. However, peak values are not maintained and phosphorylation declines, while active stress increases monotonically to a sustained steady state. The aim of this study was to determine the reason(s) for the transient change in myosin phosphorylation. Four hypotheses were considered: 1) reduced substrate, i.e., ATP depletion, 2) altered access of either the myosin kinase or phosphatase to the cross bridge, 3) reduced myosin kinase activity secondary to its phosphorylation by adenosine 3',5'-cyclic monophosphate-dependent protein kinase, and 4) reduced myoplasmic [Ca2+] during the contraction. Our results suggest that the most likely explanation is that there are two Ca2+-dependent regulatory processes: 1) myosin phosphorylation and 2) a second, unidentified site allowing stress maintenance with reduced cross-bridge cycling rates. A higher cell Ca2+ concentration appears to be necessary to activate myosin kinase and produce myosin phosphorylation than is needed for force maintenance. We suggest that agonist-induced Ca2+ transients, coupled with the differential Ca2+ sensitivity of the two regulatory systems, may explain the observed transient in myosin phosphorylation during a maintained contraction in smooth muscle.
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PMID:Ca2+, cAMP, and changes in myosin phosphorylation during contraction of smooth muscle. 631 Oct 24

A calmodulin-dependent glycogen synthase kinase distinct from phosphorylase kinase has been purified approximately equal to 5000-fold from rabbit skeletal muscle by a procedure involving fractionation with ammonium sulphate (0-33%), and chromatographies on phosphocellulose, calmodulin-Sepharose and DEAE-Sepharose. 0.75 mg of protein was obtained from 5000 g of muscle within 4 days, corresponding to a yield of approximately equal to 3%. The Km for glycogen synthase was 3.0 microM and the V 1.6-2.0 mumol min-1 mg-1. The purified enzyme showed a major protein staining band (Mr 58 000) and a minor component (Mr 54 000) when examined by dodecyl sulphate polyacrylamide gel electrophoresis. The molecular weight of the native enzyme was determined to be 696 000 by sedimentation equilibrium centrifugation, indicating a dodecameric structure. Electron microscopy suggested that the 12 subunits were arranged as two hexameric rings stacked one upon the other. Following incubation with Mg-ATP and Ca2+-calmodulin, the purified protein kinase underwent an 'autophosphorylation reaction'. The reaction reached a plateau when approximately equal to 5 mol of phosphate had been incorporated per 58 000-Mr subunit. Both the 58 000-Mr and 54 000-Mr species were phosphorylated to a similar extent. Autophosphorylation did not affect the catalytic activity. The calmodulin-dependent protein kinase initially phosphorylated glycogen synthase at site-2, followed by a slower phosphorylation of site-1 b. The protein kinase also phosphorylated smooth muscle myosin light chains, histone H1, acetyl-CoA carboxylase and ATP-citrate lyase. These findings suggest that the calmodulin-dependent glycogen synthase kinase may be a enzyme of broad specificity in vivo. Glycogen synthase kinase-4 is an enzyme that resembles the calmodulin-dependent glycogen synthase kinase in phosphorylating glycogen synthase (at site-2), but not glycogen phosphorylase. Glycogen synthase kinase-4 was unable to phosphorylate any of the other proteins phosphorylated by the calmodulin-dependent glycogen synthase kinase, nor could it phosphorylate site 1 b of glycogen synthase. The results demonstrate that glycogen synthase kinase-4 is not a proteolytic fragment of the calmodulin-dependent glycogen synthase kinase, that has lost its ability to be regulated by Ca2+-calmodulin.
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PMID:The calmodulin-dependent glycogen synthase kinase from rabbit skeletal muscle. Purification, subunit structure and substrate specificity. 631 30

Calmodulin-dependent glycogen synthase kinase from rabbit skeletal muscle and calmodulin-dependent protein kinase-II from rat brain were found to have remarkably similar substrate specificities. Both protein kinases phosphorylated synapsin -I, glycogen synthase, smooth muscle myosin light chains, histone H1 and acetyl-CoA carboxylase at the same relative rates. Site-2 of glycogen synthase was preferentially phosphorylated by both enzymes, followed by a slower phosphorylation of site-1b. Each protein kinase catalysed a 2-fold activation of tryptophan 5-monooxygenase. Calmodulin-dependent protein kinase-II and glycogen synthase kinase exhibited similar immunological cross-reactivity in the presence of Ca2+ and calmodulin, using monoclonal antibody raised against the rat brain enzyme. In the absence of Ca2+ and calmodulin, cross-reactivity of glycogen synthase kinase was decreased, whereas that of calmodulin-dependent protein kinase-II was not. The two enzymes appear to represent different isoenzymes of a multifunctional calmodulin-dependent protein kinase that may mediate many of the actions of Ca2+ in mammalian tissues. The results demonstrate that calmodulin-dependent protein kinase-II is identical to calmodulin-dependent synapsin -I kinase-II, previously shown to be very similar to calmodulin-dependent glycogen synthase kinase [(1983) FEBS Lett. 163, 329-334].
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PMID:Comparison of calmodulin-dependent glycogen synthase kinase from skeletal muscle and calmodulin-dependent protein kinase-II from brain. 632 77

A high salt extract of bovine brain was found to contain a protein kinase which catalyzed the phosphorylation of heavy chain of brain myosin. The protein kinase, designated as myosin heavy chain kinase, has been purified by column chromatography on phosphocellulose, Sephacryl S-300, and hydroxylapatite. During the purification, the myosin heavy chain kinase was found to co-purify with casein kinase II. Furthermore, upon polyacrylamide gel electrophoresis of the purified enzyme under non-denaturing conditions, both the heavy chain kinase and casein kinase activities were found to comigrate. The purified enzyme phosphorylated casein, phosvitin, troponin T, and isolated 20,000-dalton light chain of gizzard myosin, but not histone or protamine. The kinase did not require Ca2+-calmodulin, or cyclic AMP for activity. Heparin, which is known to be a specific inhibitor of casein kinase II, inhibited the heavy chain kinase activity. These results indicate that the myosin heavy chain kinase is identical to casein kinase II. The myosin heavy chain kinase catalyzed the phosphorylation of the heavy chains in intact brain myosin. The heavy chains in intact gizzard myosin were also phosphorylated, but to a much lesser extent. The heavy chains of skeletal muscle and cardiac muscle myosins were not phosphorylated to an appreciable extent. Although the light chains isolated from brain and gizzard myosins were efficiently phosphorylated by the same enzyme, the rates of phosphorylation of these light chains in the intact myosins were very small. From these results it is suggested that casein kinase II plays a role as a myosin heavy chain kinase for brain myosin rather than as a myosin light chain kinase.
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PMID:Purification and identification of myosin heavy chain kinase from bovine brain. 632 58

The src gene product of Rous sarcoma virus (pp60(src)) was highly purified from a rat tumor cell line and shown to have physiological actin transformation activity in a cellular microinjection assay that measures the dissolution of actin microfilament bundles in vivo. The purified pp60(src) fraction consisted of two major proteins, seen on silver-stained sodium dodecyl sulfate-polyacrylamide gels: a 60,000-dalton (60K) protein, identified as pp60(src) by immunoprecipitation with tumor-bearing rabbit immunoglobulin G (IgG) and peptide mapping, and an unrelated 65K protein. There was no evidence for proteolytic cleavage of pp60(src). A 7,000-fold purification of the tyrosine-specific protein kinase activity of pp60(src) was achieved by this procedure. Purified pp60(src) phosphorylated tumor-bearing rabbit IgG heavy chains, casein, histones H1 and H2B, tubulin, and microtubule-associated proteins when assayed in vitro. When incubated with [gamma-(32)P]ATP in the absence of exogenous phosphoacceptor substrates, purified pp60(src) became labeled with (32)P at the tyrosine residues exclusively. Phosphatase and cyclic AMP-dependent protein kinase activities were undetectable in the purified fraction. Microinjection of highly purified pp60(src) into the cytoplasm of normal Swiss 3T3 mouse fibroblasts caused rapid and reversible dissolution of actin stress fibers, as visualized by indirect immunofluorescence with actin antibodies. The actin-disrupting activity was thermolabile and sensitive to inhibition by coinjection of tumor-bearing rabbit IgG, and purified to about the same extent (8,000-fold) as did the IgG kinase activity of pp60(src), thus implicating pp60(src) as the active agent. Examination of actin-associated proteins as substrates for the pp60(src) kinase in vitro showed that vinculin was phosphorylated directly by pp60(src), although to a small extent. Actin, myosin, and tropomyosin were not phosphorylated. Thus, pp60(src) purified by this procedure retains native functional properties and provides a useful probe for analyzing transformation-dependent changes in actin cytoarchitecture.
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PMID:Highly purified pp60src induces the actin transformation in microinjected cells and phosphorylates selected cytoskeletal proteins in vitro. 640 62

It has previously been shown that the regulatory light chains of myosin from Limulus, the horseshoe crab, can be phosphorylated either by purified turkey gizzard smooth muscle myosin light chain (MLC) kinase or by a crude kinase fraction prepared from Limulus muscle [Sellers, J. R. (1981) J. Biol. Chem. 256, 9274-9278]. This phosphorylation was shown to be associated with a 20-fold increase in the actin-activated MgATPase activity of the myosin. We have now purified the Ca2+-calmodulin-dependent MLC kinase from Limulus muscle to near homogeneity by using a combination of low ionic strength extraction, ammonium sulfate fractionation, and chromatography on Sephacryl S-300 and DEAE-Sephacel. The final purification was achieved by affinity chromatography on a calmodulin-Sepharose 4B column. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed 95% of the protein to be comprised of a doublet with Mr = 39000 and 37000. Electrophoresis of the kinase fraction under nondenaturing conditions resulted in a partial separation of the two major bands and demonstrated that each had catalytic activity. An SDS-polyacrylamide gel overlayed with 125I-calmodulin demonstrated that both the Mr 39K and the Mr 37K proteins bind calmodulin. Neither of the bands could be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. With Limulus myosin light chains as a substrate, the Vmax was 15.4 mumol min-1 mg-1, and the Km was 15.6 microM. The KD for calmodulin was determined to be 6 nM. The enzyme did not phosphorylate histones, casein, actin, or tropomyosin.
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PMID:Purification of myosin light chain kinase from Limulus muscle. 654 61

A crude myosin fraction from bovine brain has been found to contain a Ca2+-independent myosin kinase that catalyzes the phosphorylation of 20,000-Da light chain of gizzard myosin. The myosin kinase has been separated from the myosin by Sepharose CL-4B gel filtration and purified further by chromatography on phosphocellulose, Sephacryl S-300, and hydroxylapatite. The myosin kinase was found to copurify with casein kinase II and show the same substrate specificity with the casein kinase. These results indicate that the myosin kinase is identical to casein kinase II. The purified myosin kinase catalyzed the preferential phosphorylation of the threonyl residues of 20,000-Da light chains of gizzard and brain myosins. The 17,000-Da light chains of these myosins and the mixed light chains of skeletal and cardiac muscle myosins were not phosphorylated by the enzyme to an appreciable extent.
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PMID:Identification of calcium-independent myosin kinase with casein kinase II. 657 82

Several characteristics of receptor capping in lymphocyte membranes suggest similarities with mechanisms underlying control of contraction in smooth muscle fibers. Both capping and contraction are Ca2+ dependent and require metabolic energy. Contractile proteins such as actin and myosin are associated with the cap, as is calmodulin, which mediates the Ca2+ dependence of smooth muscle contraction. Recent studies have shown that myosin light chain kinase (MLCK), which plays a central role in regulation of smooth muscle contraction, is also present in isolated lymphocyte membrane-cytoskeleton complexes. We have explored this analogy further, using mouse lymphoma T cells whose membranes were rendered permeable to small proteins by using a low-Ca2+ EGTA solution similar to that used to chemically skin smooth muscle cells. Permeabilized lymphocytes were then exposed to solutions containing various combinations of high or low Ca2+, ATP, or other nucleotides (5'-adenylyl imidodiphosphate, adenosine 5'-[gamma-thio]triphosphate, guanosine 5'-[gamma-thio]triphosphate, CTP, ITP, UTP, and GTP), calmodulin, Ca2+-insensitive MLCK (MLCK subunit that has been stripped of the Ca2+ binding site), and the catalytic subunit of cAMP-dependent protein kinase that phosphorylates (and thereby inactivates) MLCK. Capping of concanavalin A-labeled receptors in these various test solutions was scored. In all solutions the capping observed in permeable lymphoma cells correlated well with contraction previously observed in similarly treated skinned smooth muscle fibers, providing strong evidence for the involvement of myosin light chain phosphorylation in the regulation of receptor capping.
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PMID:Regulation of receptor capping in mouse lymphoma T cells by Ca2+-activated myosin light chain kinase. 658 74


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