scholarly journals Myosin light-chain phosphatase

1976 ◽  
Vol 157 (3) ◽  
pp. 687-697 ◽  
Author(s):  
M Morgan ◽  
S V Perry ◽  
J Ottaway
Keyword(s):  
Skeletal Muscle ◽  
Light Chain ◽  
Sodium Dodecyl Sulphate ◽  
Myosin Light Chain Kinase ◽  
Adenosine Triphosphatase ◽  
Myosin Light Chain ◽  
Sodium Dodecyl ◽  
Affinity Column ◽  
Heavy Meromyosin ◽  
Myosin Light Chain Phosphatase

1. A method for the isolation of a new enzyme, myosin light-chain phosphatase, from rabbit white skeletal muscle by using a Sepharose-phosphorylated myosin light-chain affinity column is described. 2. The enzyme migrated as a single component on electrophoresis in sodium dodecyl sulphate/polyacrylamide gel at pH7.0, with apparent mol.wt. 70000. 3. The enzyme was highly specific for the phosphorylated P-light chain of myosin, had pH optima at 6.5 and 8.0 and was not inhibited by NaF. 4. A Ca2+-sensitive ‘ATPase’ (adenosine triphosphatase) system consisting of myosin light-chain kinase, myosin light-chain phosphatase and the P-light chain is described. 5. Evidence is presented for a phosphoryl exchange between Pi, phosphorylated P-light chain and myosin light-chain phosphatase. 6. Heavy meromyosin prepared by chymotryptic digestion can be phosphorylated by myosin light-chain kinase. 7. The ATPase activities of myosin and heavy meromyosin, in the presence and absence of F-actin, were not significantly changed (+/- 10%) by phosphorylation of the P-light chain.

scholarly journals Calmodulin and myosin light-chain kinase of rabbit fast skeletal muscle

1979 ◽  
Vol 179 (1) ◽  
pp. 89-97 ◽  
Author(s):  
A C Nairn ◽  
S V Perry
Keyword(s):  
Skeletal Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Troponin I ◽  
Maximal Activity ◽  
Bovine Brain ◽  
Affinity Column ◽  
Fast Skeletal Muscle ◽  
Skeletal Muscle Myosin

1. It is confirmed that myosin light-chain kinase is a protein of mol.wt. about 80,000 that is inactive in the absence of calmodulin. 2. In the presence of 1 mol of calmodulin/mol of kinase 80-90% of the maximal activity is obtained. 3. Crude preparations of the whole light-chain fraction of rabbit fast-skeletal-muscle myosin contain enough calmodulin to activate the enzyme. A method for the preparation of calmodulin-free P light chain is described. 4. A procedure is described for the isolation of calmodulin from rabbit fast skeletal muscle. 5. Rabbit fast-skeletal-muscle calmodulin is indistinguishable from bovine brain calmodulin in its ability to activate myosin light-chain kinase. The other properties of these two proteins are also very similar. 6. Rabbit fast-skeletal-muscle troponin C was about 10% as effective as calmodulin as activator for myosin light-chain kinase. 7. By chromatography on a Sepharose-calmodulin affinity column evidence was obtained for the formation of a Ca2+-dependent complex between calmodulin and myosin light-chain kinase. 8. Troponin I from rabbit fast skeletal muscle and histone IIAS were phosphorylated by fully activated myosin light-chain kinase at about 1% of the rate of the P light chain.

scholarly journals Smooth muscle myosin light chain kinase expression in cardiac and skeletal muscle

2000 ◽  
Vol 279 (5) ◽  
pp. C1656-C1664 ◽  
Author(s):  
B. Paul Herring ◽  
Shelley Dixon ◽  
Patricia J. Gallagher
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Cardiac Myocyte ◽  
Cardiac Tissue ◽  
Specific Protein ◽  
Myoblast Differentiation ◽  
Adult Skeletal Muscle

The purpose of this study was to characterize myosin light chain kinase (MLCK) expression in cardiac and skeletal muscle. The only classic MLCK detected in cardiac tissue, purified cardiac myocytes, and in a cardiac myocyte cell line (AT1) was identical to the 130-kDa smooth muscle MLCK (smMLCK). A complex pattern of MLCK expression was observed during differentiation of skeletal muscle in which the 220-kDa-long or “nonmuscle” form of MLCK is expressed in undifferentiated myoblasts. Subsequently, during myoblast differentiation, expression of the 220-kDa MLCK declines and expression of this form is replaced by the 130-kDa smMLCK and a skeletal muscle-specific isoform, skMLCK in adult skeletal muscle. These results demonstrate that the skMLCK is the only tissue-specific MLCK, being expressed in adult skeletal muscle but not in cardiac, smooth, or nonmuscle tissues. In contrast, the 130-kDa smMLCK is ubiquitous in all adult tissues, including skeletal and cardiac muscle, demonstrating that, although the 130-kDa smMLCK is expressed at highest levels in smooth muscle tissues, it is not a smooth muscle-specific protein.

Abstract 1102: Cardiac-Specific Myosin Light Chain Kinase (MLCK), a Third Member of MLCK Family, Potentiates Sarcomere Formation and Cardiac Contraction

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Jason Y Chan ◽  
Morihiko Takeda ◽  
Laura E Briggs ◽  
Jonathan T Lu ◽  
Nobuo Horikoshi ◽  
...  
Keyword(s):  
Heart Failure ◽  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Cardiac Hypertrophy ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Contractile Function ◽  
Severe Heart Failure ◽  
Cardiac Contraction

Background: Two myosin light chain kinase (MLCK) proteins, skeletal (encoded by mylk2 gene) and smooth muscle MLCK (encoded by mylk1 gene) have been shown to be expressed in mammals. Human mylk2 has been mapped as a disease locus for familial cardiac hypertrophy (OMIM 606566 ), suggesting that abnormal function of skeletal MLCK stimulates cardiac hypertrophy. While phosphorylation of the putative substrate of skeletal MLCK, myosin light chain 2 (MLC2), is recognized as a key regulator of cardiac contraction, the abundance of skeletal MLCK in the heart is controversial, suggesting the existence of an additional MLCK that is preferentially expressed in cardiac muscle. Methods and Results: We characterized a new kinase named cardiac MLCK that is encoded by a gene homologous to mylk1 and 2 and is specifically expressed in the heart in both atrium and ventricle. Expression of cardiac MLCK was highly regulated by the cardiac homeobox transcription factor, Nkx2.5, in neonatal cardiomyocytes. The overall structure of cardiac MLCK protein is conserved with skeletal and smooth muscle MLCK including putative catalytic and adjacent Ca2+/calmodulin binding domains at the carboxyl-terminus. The amino-terminus is unique without significant homology to other known proteins. Cardiac MLCK phosphorylated MLC2v with a catalytic value of Km=4.3 micro M (Lineweaver-Burk analysis) indicating high affinity of cardiac MLCK to MLC2v, similar to the affinity of skeletal muscle MLCK to skeletal muscle MLC2 and smooth muscle MLCK to smooth muscle MLC2. Adenoviral-mediated overexpression of cardiac MLCK and knockdown of cardiac MLCK using RNAi in cultured cardiomyocytes revealed that cardiac MLCK regulates MLC2v phosphorylation, sarcomere organization and cardiac myocyte contraction. Expression of cardiac MLCK protein was significantly decreased in severe heart failure in vivo (post-myocardial infarction heart failure mouse model). Conclusion: Cardiac MLCK is a new key regulator of cardiac contraction and sarcomere organization. Reduction of cardiac MLCK function leading to decreased phosphorylation of MLC2v may contribute to compromised contractile function in the failing heart.

Expression of the catalytic domain of myosin light chain kinase increases paracellular permeability

1996 ◽  
Vol 271 (5) ◽  
pp. C1678-C1684 ◽  
Author(s):  
G. Hecht ◽  
L. Pestic ◽  
G. Nikcevic ◽  
A. Koutsouris ◽  
J. Tripuraneni ◽  
...  
Keyword(s):  
Tight Junction ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Catalytic Domain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Epithelial Tight Junction ◽  
Tight Junction Permeability ◽  
Mlc20 Phosphorylation

Contractile events resulting from phosphorylation of the 20-kDa myosin light chain (MLC20) have been implicated in the regulation of epithelial tight junction permeability. To address this question, Madin-Darby canine kidney cells were transfected with a murine leukemia retroviral vector containing DNA encoding either the catalytic domain of myosin light chain kinase (tMK) or the beta-galactosidase gene (beta-gal). Autoradiograms of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of myosin immunoprecipitated from 32Pi-labeled transfected cells demonstrated that MLC20 phosphorylation was increased 3.1 +/- 0.9-fold in cells expressing tMK compared with cells expressing beta-gal. Phosphopeptide mapping confirmed that myosin light chain kinase was responsible for the increased MLC20 phosphorylation. Transepithelial electrical resistance, a measurement of barrier function, of tMK cell monolayers was consistently < 10% (123 +/- 20 omega.cm2) of that of monolayers comprised of wild-type cells (1,456 +/- 178 omega.cm2) or cells expressing beta-gal (1,452 +/- 174 omega.cm2). Dual 22Na+ and [3H]mannitol flux studies indicated that the decrease in resistance in tMK cells was attributable to increased paracellular flow. These data support the idea that MLC20 phosphorylation by myosin light chain kinase is involved in regulating epithelial tight junction permeability.

Modulator Binding Protein in Platelets

1979 ◽  
Author(s):  
L. Muszbek ◽  
J. Hàrsfalvi
Keyword(s):  
Skeletal Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Binding Protein ◽  
Troponin C ◽  
Detectable Amount ◽  
Virtual Absence ◽  
Platelet Protein ◽  
Regulator Protein

In a previous paper (Muszbek et al. FEBS Letters 1977, 80, 308) the existence of a troponin C-like protein has been revealed in platelets. This protein was isolated, characterized and identified as the multifunctional Cadependent regulator protein (modulator). Here we show that there is a platelet protein which in 6·3 M urea if Ca2+ present can form a complex with modulator protein and with skeletal muscle troponin C, too. It withstands aceton treatment and can be extracted from platelet aceton powder by 1 M KCl. Further purification could be achieved by affinity chromatography on an Affi-Gel 10-troponin C column. Modulator binding protein also copurified with platelet actomyosin though there was no detectable amount of modulator protein in this preparation. Since modulator protein appears to be responsible for the Ca2+ regulation of purified platelet myosin light chain kinase (Dabrowska and Harsthorne BBRC 1978, 85, 1352) the low Ca2+ sensitivity of platelet actomyosin may be due to the virtual absence of modulator and/or to the presence of modula tor binding prote in in thrombosthenin.

scholarly journals Myosin light chain kinase and myosin light chain phosphatase from Dictyostelium: effects of reversible phosphorylation on myosin structure and function.

1987 ◽  
Vol 104 (5) ◽  
pp. 1309-1323 ◽  
Author(s):  
L M Griffith ◽  
S M Downs ◽  
J A Spudich
Keyword(s):  
Enzyme Activity ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Tight Binding ◽  
Subsequent Treatment ◽  
Myosin Light Chain Phosphatase ◽  
In Vitro Motility Assay ◽  
Dictyostelium Myosin

We have partially purified myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) from Dictyostelium discoideum. MLCK was purified 4,700-fold with a yield of approximately 1 mg from 350 g of cells. The enzyme is very acidic as suggested by its tight binding to DEAE. Dictyostelium MLCK has an apparent native molecular mass on HPLC G3000SW of approximately 30,000 D. Mg2+ is required for enzyme activity. Ca2+ inhibits activity and this inhibition is not relieved by calmodulin. cAMP or cGMP have no effect on enzyme activity. Dictyostelium MLCK is very specific for the 18,000-D light chain of Dictyostelium myosin and does not phosphorylate the light chain of several other myosins tested. Myosin purified from log-phase amebas of Dictyostelium has approximately 0.3 mol Pi/mol 18,000-D light chain as assayed by glycerol-urea gel electrophoresis. Dictyostelium MLCK can phosphorylate this myosin to a stoichiometry approaching 1 mol Pi/mol 18,000-D light chain. MLCP, which was partially purified, selectively removes phosphate from the 18,000-D light chain but not from the heavy chain of Dictyostelium myosin. Phosphatase-treated Dictyostelium myosin has less than or equal to 0.01 mol Pi/mol 18,000-D light chain. Phosphatase-treated myosin could be rephosphorylated to greater than or equal to 0.96 mol Pi/mol 18,000-D light chain by incubation with MLCK and ATP. We found myosin thick filament assembly to be independent of the extent of 18,000-D light-chain phosphorylation when measured as a function of ionic strength. However, actin-activated Mg2+-ATPase activity of Dictyostelium myosin was found to be directly related to the extent of phosphorylation of the 18,000-D light chain. MLCK-treated myosin moved in an in vitro motility assay (Sheetz, M. P., and J. A. Spudich, 1983, Nature (Lond.), 305:31-35) at approximately 1.4 micron/s whereas phosphatase-treated myosin moved only slowly or not at all. The effects of phosphatase treatment on the movement were fully reversed by subsequent treatment with MLCK.

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