Is phosphorylation the main physiological action of myosin light chain kinase?

1994 ◽  
Vol 72 (11) ◽  
pp. 1377-1379 ◽  
Author(s):  
Setsuro Ebashi ◽  
Hideto Kuwayama
Keyword(s):  
Amino Acid ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Actin Binding ◽  
In Vitro System ◽  
Beryllium Sulfate ◽  
Kinase Phosphorylation ◽  
Actin Binding Site

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.

scholarly journals Myosin light chain kinase binding to a unique site on F-actin revealed by three-dimensional image reconstruction

2001 ◽  
Vol 154 (3) ◽  
pp. 611-618 ◽  
Author(s):  
Victoria Hatch ◽  
Gang Zhi ◽  
Lula Smith ◽  
James T. Stull ◽  
Roger Craig ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Three Dimensional ◽  
Actin Binding ◽  
Structural Basis ◽  
Thin Filaments ◽  
Helical Reconstruction ◽  
Nonmuscle Cells

Ca2+–calmodulin-dependent phosphorylation of myosin regulatory light chains by the catalytic COOH-terminal half of myosin light chain kinase (MLCK) activates myosin II in smooth and nonmuscle cells. In addition, MLCK binds to thin filaments in situ and F-actin in vitro via a specific repeat motif in its NH2 terminus at a stoichiometry of one MLCK per three actin monomers. We have investigated the structural basis of MLCK–actin interactions by negative staining and helical reconstruction. F-actin was decorated with a peptide containing the NH2-terminal 147 residues of MLCK (MLCK-147) that binds to F-actin with high affinity. MLCK-147 caused formation of F-actin rafts, and single filaments within rafts were used for structural analysis. Three-dimensional reconstructions showed MLCK density on the extreme periphery of subdomain-1 of each actin monomer forming a bridge to the periphery of subdomain-4 of the azimuthally adjacent actin. Fitting the reconstruction to the atomic model of F-actin revealed interaction of MLCK-147 close to the COOH terminus of the first actin and near residues 228–232 of the second. This unique location enables MLCK to bind to actin without interfering with the binding of any other key actin-binding proteins, including myosin, tropomyosin, caldesmon, and calponin.

scholarly journals Properties of caldesmon isolated from chicken gizzard

1985 ◽  
Vol 230 (3) ◽  
pp. 695-707 ◽  
Author(s):  
P K Ngai ◽  
M P Walsh
Keyword(s):  
Amino Acid ◽  
Light Chain ◽  
Cyclic Nucleotide ◽  
Acid Content ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Amino Acid Content ◽  
Cyclic Nucleotide Phosphodiesterase ◽  
Chicken Gizzard

Chicken gizzard smooth muscle contains two major calmodulin-binding proteins: caldesmon (11.1 microM; Mr 141 000) and myosin light-chain kinase (4.6 microM; Mr 136 000), both of which are associated with the contractile apparatus. The amino acid composition of caldesmon is distinct from that of myosin light-chain kinase and is characterized by a very high glutamic acid content (25.5%), high contents of lysine (13.6%) and arginine (10.3%), and a low aromatic amino acid content (2.4%). Caldesmon lacked myosin light-chain kinase and phosphatase activities and did not compete with either myosin light-chain kinase or cyclic nucleotide phosphodiesterase (both calmodulin-dependent enzymes) for available calmodulin, suggesting that calmodulin may have distinct binding sites for caldesmon on the one hand and myosin light-chain kinase and cyclic nucleotide phosphodiesterase on the other. Consistent with the lack of effect of caldesmon on myosin phosphorylation, caldesmon did not affect the assembly or disassembly of myosin filaments in vitro. As previously shown [Ngai & Walsh (1984) J. Biol. Chem. 259, 13656-13659], caldesmon can be reversibly phosphorylated. The phosphorylation and dephosphorylation of caldesmon were further characterized and the Ca2+/calmodulin-dependent caldesmon kinase was purified; kinase activity correlated with a protein of subunit Mr 93 000. Caldesmon was not a substrate of myosin light-chain kinase or phosphorylase kinase, both calmodulin-activated protein kinases.

The Regulation of Myosin Light Chain Kinase by Ca++ and Calmodulin in Vitro and in Vivo

1982 ◽  
pp. 219-238 ◽  
Author(s):  
J. T. Stull ◽  
D. K. Blumenthal ◽  
B. R. Botterman ◽  
G. A. Klug ◽  
D. R. Manning ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain

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.

scholarly journals Phosphorylation by casein kinase II alters the biological activity of calmodulin

1992 ◽  
Vol 283 (1) ◽  
pp. 21-24 ◽  
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.

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

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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