Myosin light chain kinase mediates sarcomere organization during cardiac hypertrophy in vitro

The 155-kDa component of bovine stomach, which exhibits a strong actomyosin (AM) activating activity and a relatively weak myosin light chain kinase (MLCK) activity, has a strong affinity for the actin filament and the actin-binding site is confined to an 80 amino acid residue on its N-terminal side. This affinity may play a crucial role in AM activation. Some reagents preferentially abolish either the AM-activating effect or MLCK activity. In conclusion, MLCK of the 155-kDa component does not play a fundamental role in activating the AM system as far as the in vitro system is concerned. The possible mechanism of AM activation by the component is discussed.Key words: myosin light chain kinase, phosphorylation of myosin light chain, leiotonin, wortmannin, beryllium sulfate.

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

Circulation ◽

10.1161/circ.116.suppl_16.ii_221-a ◽

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.104.5.1309 ◽

1987 ◽

Vol 104 (5) ◽

pp. 1309-1323 ◽

Cited By ~ 76

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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Phosphorylation by casein kinase II alters the biological activity of calmodulin

Biochemical Journal ◽

10.1042/bj2830021 ◽

1992 ◽

Vol 283 (1) ◽

pp. 21-24 ◽

Cited By ~ 32

Author(s):

D B Sacks ◽

H W Davis ◽

J P Williams ◽

E L Sheehan ◽

J G N Garcia ◽

...

Keyword(s):

Light Chain ◽

Cyclic Nucleotide ◽

Myosin Light Chain Kinase ◽

Myosin Light Chain ◽

Casein Kinase Ii ◽

Casein Kinase ◽

Cyclic Nucleotide Phosphodiesterase ◽

Maximal Velocity ◽

Intact Cells

Calmodulin is the major intracellular Ca(2+)-binding protein, providing Ca(2+)-dependent regulation of numerous intracellular enzymes. The phosphorylation of calmodulin may provide an additional mechanism for modulating its function as a signal transducer. Phosphocalmodulin has been identified in tissues and cells, and calmodulin is phosphorylated both in vitro and in intact cells by various enzymes. Phosphorylation of calmodulin on serine/threonine residues by casein kinase II decreases its ability to activate both myosin-light-chain kinase and cyclic nucleotide phosphodiesterase. For myosin-light-chain kinase the primary effect is an inhibition of the Vmax. of the reaction, with no apparent change in the concentration at which half-maximal velocity is attained (K0.5) for either Ca2+ or calmodulin. In contrast, for phosphodiesterase, phosphorylation of calmodulin significantly increases the K0.5 for calmodulin without noticeably altering the Vmax. or the K0.5 for Ca2+. The higher the stoichiometry of phosphorylation of calmodulin, the greater the inhibition of calmodulin-stimulated activity for both enzymes. Therefore the phosphorylation of calmodulin by casein kinase II appears to provide a Ca(2+)-independent mechanism whereby calmodulin regulates at least two important target enzymes, myosin-light-chain kinase and cyclic nucleotide phosphodiesterase.

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CHD1L prevents lipopolysaccharide-induced hepatocellular carcinomar cell death by activating hnRNP A2/B1-nmMYLK axis

Cell Death and Disease ◽

10.1038/s41419-021-04167-9 ◽

2021 ◽

Vol 12 (10) ◽

Author(s):

Guangliang Wang ◽

Xiaofeng Zhang ◽

Wei Cheng ◽

Yanxuan Mo ◽

Juan Chen ◽

...

Keyword(s):

Cell Death ◽

Tumor Cell ◽

Light Chain ◽

Myosin Light Chain Kinase ◽

Myosin Light Chain ◽

Driver Gene ◽

Caspase 8 ◽

Mechanism Analysis ◽

Tumor Cell Death

AbstractChromodomain helicase/ATPase DNA-binding protein 1-like gene (CHD1L) has been characterized to be a driver gene in hepatocellular carcinoma (HCC). However, the intrinsic connections between CHD1L and intestinal dysbacteriosis-related inflammation reaction in HCC progression remain incompletely understood. In this study, a specific correlation between CHD1L and nonmuscle isoform of myosin light chain kinase (nmMLCK/nmMYLK), a newly identified molecule associated NF-κB signaling transduction, was disclosed in HCC. CHD1L promotes nmMYLK expression and prevents lipopolysaccharide (LPS) induced tumor cell death. In vitro experiment demonstrated that overexpressed nmMYLK is essential for CHD1L to maintain HCC cell alive, while knocking down nmMYLK significantly attenuate the oncogenic roles of CHD1L. Mechanism analysis revealed that nmMYLK can prevent Caspase-8 from combining with MyD88, an important linker of TLRs signaling pathway, while, knocking down nmMYLK facilitate the MyD88 combines with Caspase-8 and lead to the proteolytic cascade of Caspase as well as the consequent cell apoptosis. Mechanism analysis showed that CHD1L promotes the nmMYLK expression potentially through upregulating the heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP A2/B1) expression, which can bind to myosin light chain kinase (MYLK) pre-mRNA and lead to the regnant translation of nmMYLK. In summary, this work characterizes a previously unknown role of CHD1L in preventing LPS-induced tumor cell death through activating hnRNP A2/B1-nmMYLK axis. Further inhibition of CHD1L and its downstream signaling could be a novel promising strategy in HCC treatment.

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Tumor necrosis factor (TNF) increases myosin light chain kinase isoform 1 (MLCK1) transcription and targets MLCK1 to the perijunctional actomyosin ring in vitro and in vivo .

The FASEB Journal ◽

10.1096/fasebj.21.5.a587 ◽

2007 ◽

Vol 21 (5) ◽

Author(s):

W. Vallen Graham ◽

Yingmin Wang ◽

Brad T. Schwarz ◽

Amanda M. Marchiando ◽

Jerrold R. Turner

Keyword(s):

Tumor Necrosis Factor ◽

Light Chain ◽

Myosin Light Chain Kinase ◽

Tumor Necrosis ◽

Myosin Light Chain ◽

Necrosis Factor

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Substance P contracts bovine tracheal smooth muscle via activation of myosin light chain kinase

AJP Cell Physiology ◽

10.1152/ajpcell.1990.259.2.c258 ◽

1990 ◽

Vol 259 (2) ◽

pp. C258-C265 ◽

Cited By ~ 42

Author(s):

M. A. Corson ◽

J. R. Sellers ◽

R. S. Adelstein ◽

M. Schoenberg

Keyword(s):

Substance P ◽

Light Chain ◽

Myosin Light Chain Kinase ◽

Time Course ◽

Myosin Light Chain ◽

Immunoblot Analysis ◽

Isometric Tension ◽

Peak Tension ◽

Tension Response

At near-threshold substance P concentrations, the isometric tension response of bovine tracheal strips is almost completely abolished by atropine, indicating mediation of contraction via substance P-stimulated release of acetylcholine from prejunctional nerve terminals. At near-maximal concentrations, the atropine-inhibited component of the tension response is less than 25%, indicating mainly direct activation. Under conditions in which activation by substance P is direct, peak tension is reached in approximately 11 min. Immunoblot analysis of the time course of phosphorylation of the 20-kDa myosin light chain (LC20) reveals incorporation of approximately 0.5 mol phosphate/mol light chain at 10 min. Two-dimensional tryptic phosphopeptide analysis of phosphorylated light chain reveals a single major phosphopeptide. The peptide migrates identically with that produced by myosin light chain kinase phosphorylation of purified tracheal myosin in vitro. Contraction stimulated by acetylcholine is more rapid, with attainment of peak tension in 2.5 min and a peak LC20 phosphorylation of 0.65 mol/mol. These results indicate that 1) substance P mediates contraction of bovine trachea both directly and indirectly, and 2) under conditions in which activation is direct, the tension and phosphorylation responses qualitatively resemble those observed with acetylcholine.

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Abstract 747: A Novel Cardiac Myosin Light Chain Kinase Regulates Sarcomere Assembly in the Vertebrate Heart

Circulation ◽

10.1161/circ.116.suppl_16.ii_141-c ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Osamu Seguchi ◽

Seiji Takashima ◽

Satoru Yamazaki ◽

Masanori Asakura ◽

Yoshihiro Asano ◽

...

Keyword(s):

Light Chain ◽

Myosin Light Chain Kinase ◽

Myosin Light Chain ◽

Neonatal Rat ◽

Specific Gene ◽

Rat Cardiomyocytes ◽

Failing Heart ◽

Cardiac Ventricle ◽

Sarcomere Assembly

Background Cardiomyocytes mainly consist of sarcomere structure and marked sarcomere disorganization is a well-documented characteristic of cardiomyocytes in the failing myocardium. Among several constituents of sarcomeres, myosin regulatory light chain 2 (MLC2v) affects physiological cardiac sarcomere formation and heart development through its phosphorylation. Exact upstream kinase of MLC2v in cardiomyocytes however, have not been elucidated so far. Mathods and Results We identified a novel cardiac-specific gene whose expression was upregulated in failing myocardium and was also correlated with the values of pulmonary arterial pressure of each patient. This gene has kinase domain and MLC2v was identified as a putative substrate by in vitro kinase reaction using mouse heart extracts and recombinant protein of this gene. Therefore, we termed this novel gene as a cardiac-specific myosin light chain kinase (cardiac-MLCK, gene symbol: MYLK3 ). To further investigate the function of cardiac-MLCK, we targeted cardiac-MLCK using specific siRNAs in cultured neonatal rat cardiomyocytes. Reduced expression of cardiac-MLCK caused the impairment of sarcomere reassembly on epinephrine treatment. Also knockdown of cardiac-MLCK expression in zebrafish embryos using specific morpholino antisense oligonucleotides led to dilation of the cardiac ventricle with incomplete sarcomere formation. We also analyzed the nature of cardiac-MLCK and MLC2v in mouse models of heart failure. Expression levels of cardiac-MLCK proteins and phosphorylation levels of MLC2v were severely reduced in the failing heart of trans-aortic constriction mice. Conclusions We identified a novel cardiac-MLCK which contributed mainly to cardiac MLC2v phosphorylation. Attenuation of cardiac-MLCK expression caused severe sarcomere disorganization both in vitro and in vivo implies its important roles in cardiac functions. Our results suggest that cardiac-MLCK mRNA expression may be upregulated to compensate for the lower expression and reduced phosphorylation of MLC2v in failing myocardium. As a possible therapeutic modality in patients with CHF, upregulation of cardiac-MLCK may promote sarcomere assembly and enhanced contractility of the failing heart

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