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)

The contribution of lysine and arginine residues to the substrate specificity of the myosin light-chain kinase has been studied using chemically modified myosin light chains. Succinylation or maleylation of the myosin light chains caused complete inhibition of their phosphorylation. Modification of 50% of the lysine residues resulted in 90% inhibition of phosphorylation and this was accompanied by a 25-fold increase in the apparent Km. In contrast, phosphorylation of the myosin light chains by the cAMP-dependent protein kinase was relatively insensitive to lysine modification, with only a 15% reduction in phosphorylation following succinylation of 50% of the lysine residues. Treatment with either cyclohexane-1,2-dione or camphorquinone-10-sulfonic acid resulted in between 90 and 98% inhibition of myosin light-chain phosphorylation. These reagents caused modification of both lysine and arginine residues, and accordingly only part of the inhibition can be attributed to arginine modification. Modification of all of the cysteine and methionine residues caused only a 40% inhibition of phosphorylation. The results of this study support the concept that lysine and arginine residues act as essential specificity determinants for the myosin light-chain kinase in protein substrates.
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PMID:Chemical modification of lysine and arginine residues in the myosin regulatory light chain inhibits phosphorylation. 308 64

Microinjection of the catalytic subunit of cAMP-dependent protein kinase (A-kinase) into living fibroblasts or the treatment of these cells with agents that elevate the intracellular cAMP level caused marked alterations in cell morphology including a rounded phenotype and a complete loss of actin microfilament bundles. These effects were transient and fully reversible. Two-dimensional gel electrophoresis was used to analyze the changes in phosphoproteins from cells injected with A-kinase. These experiments showed that accompanying the disassembly of actin microfilaments, phosphorylation of myosin light chain kinase (MLCK) increased and concomitantly, the phosphorylation of myosin P-light chain decreased. Moreover, inhibiting MLCK activity via microinjection of affinity-purified antibodies specific to native MLCK caused a complete loss of microfilament bundle integrity and a decrease in myosin P-light chain phosphorylation, similar to that seen after injection of A-kinase. These data support the idea that A-kinase may regulate microfilament integrity through the phosphorylation and inhibition of MLCK activity in nonmuscle cells.
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PMID:Regulation of actin microfilament integrity in living nonmuscle cells by the cAMP-dependent protein kinase and the myosin light chain kinase. 329 Feb 22

Catecholamines are known to influence the contractility of cardiac and skeletal muscles, presumably via cAMP-dependent phosphorylation of specific proteins. We have investigated the in vitro phosphorylation of myofibrillar proteins by the catalytic subunit of cAMP-dependent protein kinase of fast- and slow-twitch skeletal muscles and cardiac muscle with a view to gaining a better understanding of the biochemical basis of catecholamine effects on striated muscles. Incubation of canine red skeletal myofibrils with the isolated catalytic subunit of cAMP-dependent protein kinase and Mg-[gamma-32P]ATP led to the rapid incorporation of [32P]phosphate into five major protein substrates of subunit molecular weights (MWs) 143,000, 60,000, 42,000, 33,000, and 11,000. The 143,000 MW substrate was identified as C-protein; the 42,000 MW substrate is probably actin; the 33,000 MW substrate was shown not to be a subunit of tropomyosin and, like the 60,000 and 11,000 MW substrates, is an unidentified myofibrillar protein. Isolated canine red skeletal muscle C-protein as phosphorylated to the extent of approximately 0.5 mol Pi/mol C-protein. Rabbit white skeletal muscle and bovine cardiac muscle C-proteins were also phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, both in myofibrils and in the isolated state. Cardiac C-protein was phosphorylated to the extent of 5-6 mol Pi/mol C-protein, whereas rabbit white skeletal muscle C-protein was phosphorylated at the level of approximately 0.5 mol Pi/mol C-protein. As demonstrated earlier by others, C-protein of skeletal and cardiac muscles inhibited the actin-activated myosin Mg2+-ATPase activity at low ionic strength in a system reconstituted from the purified skeletal muscle contractile proteins (actin and myosin).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phosphorylation of skeletal and cardiac muscle C-proteins by the catalytic subunit of cAMP-dependent protein kinase. 375 98

The level of phosphorylation of myosin regulatory light chain in BALB/c 3T3 and certain other cultured substrate-attached fibroblasts has been shown to be altered by several agents which influence cell shape, attachment and/or surface receptors. This was investigated by metabolic labelling with [32P]orthophosphate, followed by exposure of the cells to the chosen conditions, rapid freezing to 'fix' phosphorylation levels, extraction and concentration in the presence of kinase and phosphatase inhibitors, and final analysis by two-dimensional gel electrophoresis. Gel patterns were interpreted by comparison with immunoprecipitates with antiserum to mouse nonmuscle myosin. Treatment of cells either with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or dibutyryl-cAMP suppressed light chain phosphorylation as predicted from the control mechanisms proposed previously from in vitro studies for Ca++ calmodulin and cAMP-dependent protein kinase respectively. Other effects were less easily explained: in BALB/c 3T3 cells, contrasting with previously reported behaviour of CHO cells, the cAMP-induced decline was small and transitory; and in at least one cell line (16C) the EGTA-induced decline was preceded by a strong pulse of enhanced phosphorylation. A striking and unexpected result was that azide, almost certainly acting on mitochondrial function, caused myosin light chain phosphorylation to be maintained over a long period even in the presence of EGTA which would otherwise bring about an immediate drop. The cleavage (by trypsin) or binding (by con A) of surface receptors was also shown to trigger the biochemical modulation of cellular myosin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Myosin light chain phosphorylation in fibroblast shape change, detachment and patching. 379 39

One of the two regulatory light chains, RLC-a, of scallop smooth muscle myosin was fully phosphorylated by myosin light chain kinase of chicken gizzard muscle. The residue phosphorylated was Ser. It may be the Ser at number 11 from the N-terminal. The sequence of 9 residues around the Ser-11, QRATSNVFA, is identical with that around the phosphorylatable Ser of LC20 of chicken gizzard myosin. RLC-a was also phosphorylated slowly by cAMP-dependent protein kinase. The phosphorylation of RLC-a may be involved in the regulatory system for the catch contraction of scallop muscle.
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PMID:Phosphorylation of regulatory light chain a (RLC-a) in smooth muscle myosin of scallop, Patinopecten yessoensis. 384 Aug 2

The 20,000-dalton light chain of chicken gizzard myosin was phosphorylated in vitro by the cAMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) from bovine heart. The enzyme catalyzed incorporation of 1 mol of Pi/mol of light chain in a reaction that was completely dependent upon cAMP and independent of Ca2+. Two-dimensional peptide mapping of alpha-chymotryptic digests, as well as phosphoamino acid analysis of acid hydrolysates, were used to compare the site phosphorylated by cAMP-dependent protein kinase to that phosphorylated by turkey gizzard myosin light chain kinase. The results indicate that both enzymes phosphorylate the same serine residue. However, the light chains were a better in vitro substrate for myosin light chain kinase than for cAMP-dependent protein kinase. The amino acid sequence around the phosphorylated serine is characteristic of substrates of cAMP-dependent protein kinases.
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PMID:Phosphorylation of smooth muscle myosin light chains by cAMP-dependent protein kinase. 625 55

In vitro experiments support the ideal that the actin-activated MgATPase activity of smooth muscle myosin and myosin from nonmuscle cells is regulated by the phosphorylation of the 20,000 dalton light chain of myosin. Experiments with intact smooth muscles support this mechanism but also raise the possibility that tension may be maintained in the presence of partial dephosphorylation (12). The possibility that smooth muscle contraction may also be modulated by additional regulatory systems (13,29) is to be expected based on experience with other types of muscle. The enzyme myosin light chain kinase catalyzes the phosphorylation of the 20,000 dalton light chain of myosin. This enzyme requires Ca2+-calmodulin for activity. The activity of myosin kinases that have been isolated from avian smooth muscle cells (8) or human platelets (16) can be decreased by phosphorylation. This phosphorylation is catalyzed by cAMP-dependent protein kinase and decreases myosin kinase activity by interfering with the binding of Ca2+-calmodulin. A number of different phosphatases have been purified from smooth muscle (22). These phosphatases play an important role in determining the state of phosphorylation of myosin and myosin kinase. Two areas of particular interest at present are the regulation of phosphatase activity and the physiological significance of myosin kinase phosphorylation.
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PMID:Regulation of contractile proteins by reversible phosphorylation of myosin and myosin kinase. 629 99

A phosphatase that is active in dephosphorylating the isolated 20,000-Da light chain of myosin, as well as the enzyme myosin light chain kinase, has been purified to apparent homogeneity from turkey gizzards. The enzyme has a molecular weight of 165,000 by sedimentation-equilibrium centrifugation under nondenaturing conditions and is composed of three subunits (Mr = 60,000, 55,000, and 38,000) in a 1:1:1 molar ratio. The properties of the holoenzyme, as well as the purified catalytic subunit (Mr = 38,000) were compared using myosin light chains, intact myosin, and myosin light chain kinase as substrates. Although the holoenzyme is active in dephosphorylating the isolated myosin light chains and the enzyme myosin light chain kinase, the holoenzyme does not dephosphorylate myosin. On the other hand, the catalytic subunit of the holoenzyme dephosphorylates all three substrates. When myosin light chain kinase, which has been phosphorylated at two sites is used as substrate, both sites are rapidly dephosphorylated by the phosphatase in the absence of bound calmodulin. If calmodulin is bound to the diphosphorylated kinase, only one site is dephosphorylated. Interestingly, the single site dephosphorylated when calmodulin is bound to myosin light chain kinase is the site that is not phosphorylated when the calmodulin-myosin kinase complex is phosphorylated by cAMP-dependent protein kinase.
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PMID:Purification and characterization of a multisubunit phosphatase from turkey gizzard smooth muscle. The effect of calmodulin binding to myosin light chain kinase on dephosphorylation. 630 72

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

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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