Localization and activity of myosin light chain kinase isoforms during the cell cycle

Phosphorylation on Ser 19 of the myosin II regulatory light chain by myosin light chain kinase (MLCK) regulates actomyosin contractility in smooth muscle and vertebrate nonmuscle cells. The smooth/nonmuscle MLCK gene locus produces two kinases, a high molecular weight isoform (long MLCK) and a low molecular weight isoform (short MLCK), that are differentially expressed in smooth and nonmuscle tissues. To study the relative localization of the MLCK isoforms in cultured nonmuscle cells and to determine the spatial and temporal dynamics of MLCK localization during mitosis, we constructed green fluorescent protein fusions of the long and short MLCKs. In interphase cells, localization of the long MLCK to stress fibers is mediated by five DXRXXL motifs, which span the junction of the NH(2)-terminal extension and the short MLCK. In contrast, localization of the long MLCK to the cleavage furrow in dividing cells requires the five DXRXXL motifs as well as additional amino acid sequences present in the NH(2)-terminal extension. Thus, it appears that nonmuscle cells utilize different mechanisms for targeting the long MLCK to actomyosin structures during interphase and mitosis. Further studies have shown that the long MLCK has twofold lower kinase activity in early mitosis than in interphase or in the early stages of postmitotic spreading. These findings suggest a model in which MLCK and the myosin II phosphatase (Totsukawa, G., Y. Yamakita, S. Yamashiro, H. Hosoya, D.J. Hartshorne, and F. Matsumura. 1999. J. Cell Biol. 144:735-744) act cooperatively to regulate the level of Ser 19-phosphorylated myosin II during mitosis and initiate cytokinesis through the activation of myosin II motor activity.     

Localization of an actin binding domain in smooth muscle myosin light chain kinase

Phosphorylation of the regulatory light chain of myosin II by myosinlight chain kinase is important for regulating many contractile processes.Smooth muscle myosin light chain kinase has been shown to be associated withboth actin and myosin filaments in vitro and in vivo. In this report wedefine an actin binding region by using molecular deletions to generaterecombinant mutant proteins that were analyzed by co-sedimentation withF-actin. An actin binding region restricted to residues 2-42 in the animoterminus of the rabbit smooth muscle myosin light chain kinase wasidentified.     

Phosphorylation of myosin light chain kinase: a cellular mechanism for Ca2+ desensitization

Phosphorylation of the regulatory light chain of myosin by the Ca²⁺/calmodulin-dependent myosin light chain kinase plays an important role in smooth muscle contraction, nonmuscle cell shape changes, platelet contraction, secretion, and other cellular processes. Smooth muscle myosin light chain kinase is also phosphorylated, and recent results from experiments designed to satisfy the criteria of Krebs and Beavo for establishing the physiological significance of enzyme phosphorylation have provided insights into the cellular regulation and function of this phosphorylation in smooth muscle. The multifunctional Ca²⁺/calmodulin-dependent protein kinase II phosphorylates myosin light chain kinase at a regulatory site near the calmodulin-binding domain. This phosphorylation increases the concentration of Ca²⁺/calmodulin required for activation and hence increases the Ca²⁺ concentrations required for myosin light chain kinase activity in cells. However, the concentration of cytosolic Ca²⁺ required to effect myosin light chain kinase phosphorylation is greater than that required for myosin light chain phosphorylation. Phosphorylation of myosin light chain kinase is only one of a number of mechanisms used by the cell to down regulate the Ca²⁺ signal in smooth muscle. Since both smooth and nonmuscle cells express the same form of myosin light chain kinase, this phosphorylation may play a regulatory role in cellular processes that are dependent on myosin light chain phosphorylation.     

Effects of purified myosin light chain kinase on myosin light chain phosphorylation and catecholamine secretion in digitonin-permeabilized chromaffin cells

Many non-muscle cells including chromaffin cells contain actin and myosin. The 20,000 dalton light chain subunits of myosin can be phosphorylated by a Ca²⁺/calmodulin-dependent enzyme, myosin light chain kinase. In tissues other than striated muscle, light chain phosphorylation is required for actin-induced myosin ATPase activity. The possibility that actin and myosin are involved in catecholamine secretion was investigated by determining whether increased phosphorylation in the presence of [-³²P]ATP of myosin light chain by myosin light chain kinase enhances secretion from digitonin-treated chromaffin cells. In the absence of exogenous myosin light chain kinase, 1 M Ca²⁺ caused a 30–40% enhancement of the phosphorylation of a 20 kDa protein. This protein was identified on 2-dimensional gels as myosin light chain by its comigration with purified myosin light chain. Purified myosin light chain kinase (400 g/ml) in the presence of calmodulin (10 M) caused little or no enhancement of myosin light chain phosphorylation in the absence of Ca²⁺ in digitonin-treated cells. In the presence of 1 M Ca²⁺, myosin light chain kinase (400 g/ml) caused an approximately two-fold increase in myosin light chain phosphorylation in digitonin-treated cells in 5 min. The phosphorylation required permeabilization of the cells by digitonin and occurred within the cells rather than in the medium. Myosin light chain kinase-induced phosphorylation of myosin light chain was maximal at 1 M. Ca²⁺. Under identical conditions to those of the phosphorylation experiments, secretion was unaltered by myosin light chain kinase. The experiments indicate that the phosphorylation of myosin light chain by myosin light chain kinase is not a limiting factor in secretion in digitonin-treated chromaffin cells and suggest that the activation of myosin is not directly involved in secretion from the cells. The experiments also demonstrate the feasibility of investigation of effects of exogenously added proteins on secretion in digitonin-treated cells.Abbreviations EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - KGEPM solution containing potassium glutamate, EGTA, PIPES and MgCl₂ - NE norepinephrine - PIPES piperazine-N,-N-bis-(2-ethanesulfonic acid) - PSS physiological salt solution     

Phosphorylation and partial sequence of pregnant sheep myometrium myosin light chain kinase

The function of the uterine smooth muscle in gestation and parturition is affected by a variety of hormones and biomolecules, some of which alter the intracellular levels of cAMP and Ca²⁺. Since the activity of smooth muscle MLCK has been shown to be modulated by phosphorylation, the effect of this modification of pregnant sheep myometrium (psm) MLCK by the catalytic subunit of cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) was studied. In contrast to other smooth muscle MLCK reported, PKA incorporates 2.0–2.2 moles phosphate into a mole of psm MLCK both in the presence and absence of Ca²⁺-calmodulin. Modification of serine residues inhibited the activity of the enzyme. PKC also incorporated 2.0–2.1 moles of phosphate per mole psmMLCK under both conditions but had no effect on the MLCK activity. Sequential phosphorylation by PKC and PKA incorporated 3.8–4.1 moles phosphate suggesting that the amino acid residues modified by the two kinases are different. Phosphoamino acid analysis of the MLCK revealed that PKC phosphorylated serine and threonine residues. The double reciprocal plots of the enzyme activity and calmodulin concentrations showed that the V_max of the reaction is not altered by phosphorylation by PKA but the calmodulin concentration require for half-maximal activation is increased about 4-fold. Only 10 out of 17 monoclonal antibodies to various regions of the turkey gizzard MLCK cross-reacted with psmMLCK suggesting structural differences between these enzymes. Comparison of the deduced amino acid sequence of the cDNA encoding the C-terminal half of the psmMLCK molecule showed that while cgMLCK and psmMLCK are highly homologous, a number of nonconservative substitutions are present, particularly near the PKA phosphrylation site B (S⁸²⁸).     

Distinct roles of ROCK (Rho-kinase) and MLCK in spatial regulation of MLC phosphorylation for assembly of stress fibers and focal adhesions in 3T3 fibroblasts

ROCK (Rho-kinase), an effector molecule of RhoA, phosphorylates the myosin binding subunit (MBS) of myosin phosphatase and inhibits the phosphatase activity. This inhibition increases phosphorylation of myosin light chain (MLC) of myosin II, which is suggested to induce RhoA-mediated assembly of stress fibers and focal adhesions. ROCK is also known to directly phosphorylate MLC in vitro; however, the physiological significance of this MLC kinase activity is unknown. It is also not clear whether MLC phosphorylation alone is sufficient for the assembly of stress fibers and focal adhesions.We have developed two reagents with opposing effects on myosin phosphatase. One is an antibody against MBS that is able to inhibit myosin phosphatase activity. The other is a truncation mutant of MBS that constitutively activates myosin phosphatase. Through microinjection of these two reagents followed by immunofluorescence with a specific antibody against phosphorylated MLC, we have found that MLC phosphorylation is both necessary and sufficient for the assembly of stress fibers and focal adhesions in 3T3 fibroblasts. The assembly of stress fibers in the center of cells requires ROCK activity in addition to the inhibition of myosin phosphatase, suggesting that ROCK not only inhibits myosin phosphatase but also phosphorylates MLC directly in the center of cells. At the cell periphery, on the other hand, MLCK but not ROCK appears to be the kinase responsible for phosphorylating MLC. These results suggest that ROCK and MLCK play distinct roles in spatial regulation of MLC phosphorylation.     

Structure and function of the essential light chain of myosin

The review summarizes the recent data on the structure and function of the essential light chain of myosin. It is known that the essential light chain of myosin stabilizes the lever arm. Consistent with the model of the shift of the dynamic population of conformations, the conformational flexibility of the essential light chain is emphasized, which opens the way to determining its new functions. It is proposed that the interaction between the C-terminal domain of the essential light chain and the N-terminal subdomain of the heavy chain of myosin may be involved in the coupling of ATP hydrolysis and rotation of the lever arm. The recent data indicate that the isoforms of the essential light chain with the additional N-terminal peptide are capable of interacting with actin and src-homologous domain 3 of myosin. The structural aspects of these interactions and the modulatory role of the isoforms of the essential light chain of myosin are discussed.     

Two-site phosphorylation of the phosphorylatable light chain (20-kDa light chain) of chicken gizzard myosin

When prepared under specified conditions chicken gizzard myosin was obtained which when incubated with ATP gave rise to a diphosphorylated as well as the monophosphorylated form of P light chain. Formation of the diphosphorylated light chain occurred more readily with these myosin preparations, but could also be obtained by prolonged incubation of the isolated whole light chain fraction with kinase preparations from rabbit skeletal and chicken gizzard muscles. Using isolated light chains as substrate the more readily formed monophosphorylated light chain contained serine phosphate while the diphosphorylated form contained serine and threonine phosphates.     

Protein kinase network in the regulation of phosphorylation and dephosphorylation of smooth muscle myosin light chain

The contraction of smooth muscle is regulated primarily by intracellular Ca²⁺ signal. It is well established that the elevation of the cytosolic Ca²⁺ level activates myosin light chain kinase, which phosphorylates 20 kDa regulatory myosin light chain and activates myosin ATPase. The simultaneous measurement of cytosolic Ca²⁺ concentration and force development revealed that the alteration of the Ca²⁺-sensitivity of the contractile apparatus as well as the Ca²⁺ signal plays a critical role in the regulation of smooth muscle contraction. The fluctuation of an extent of myosin phosphorylation for a given change in Ca²⁺ concentration is considered to contribute to the major mechanisms regulating the Ca²⁺-sensitivity. The level of myosin phosphorylation is determined by the balance between phosphorylation and dephosphorylation. The phosphorylation level for a given Ca²⁺ elevation is increased either by Ca²⁺-independent activation of phosphorylation process or inhibition of dephosphorylation. In the last decade, the isolation and cloning of myosin phosphatase facilitated the understanding of regulatory mechanism of dephosphorylation process at the molecular level. The inhibition of myosin phosphatase can be achieved by (1) alteration of hetrotrimeric structure, (2) phosphorylation of 110 kDa regulatory subunit MYPT1 at the specific site and (3) inhibitory protein CPI-17 upon its phosphorylation. Rho-kinase was first identified to phosphorylate MYPT1, and later many kinases were found to phosphorylate MYPT1 and inhibit dephosphorylation of myosin. Similarly, the phosphorylation of CPI-17 can be catalysed by multiple kinases. Moreover, the myosin light chain can be phosphorylated by not only authentic myosin light chain kinase in a Ca²⁺-dependent manner but also by multiple kinases in a Ca²⁺-independent manner, thus adding a novel mechanism to the regulation of the Ca²⁺-sensitivity by regulating the phosphorylation process. It is now clarified that the protein kinase network is involved in the regulation of myosin phosphorylation and dephosphorylation. However, the physiological role of each component remains to be determined. One approach to accomplish this purpose is to investigate the effects of the dominant negative mutants of the signalling molecule on the smooth muscle contraction. In this regards, a protein transduction technique utilizing the cell-penetrating peptides would provide a useful tool. In the preliminary study, we succeeded in introducing a fragment of MYPT1 into the arterial strips, and found enhancement of contraction.     

Theoretical model of the interactions between Ca2+, calmodulin and myosin light chain kinase

Active Ca²⁺/calmodulin (CaM)-dependent myosin light chain kinase (MLCK) plays an important role in the process of MLC phosphorylation and consecutive smooth muscle contraction. Here, we propose a mathematical model of a detailed kinetic scheme describing interactions among Ca²⁺, CaM and MLCK and taking into account eight different aggregates. The main model result is the prediction of the Ca²⁺ dependent active form of MLCK, which is in the model taken as proportional to the concentration of Ca₄CaM · MLCK complex. Wegscheider’s condition is additionally applied as a constraint enabling the prediction of some parameter values that have not yet been obtained by experiments.     

Intracellular interaction of myosin light chain kinase with macrophage migration inhibition factor (MIF) in endothelium

The endothelial cell Ca2+/calmodulin (CaM)-dependent myosin light chain kinase isoform (EC MLCK) is a multifunctional contractile effector involved in vascular barrier regulation, leukocyte diapedesis, apoptosis, and angiogenesis. The EC MLCK isoform and its splice variants contain a unique N-terminal sequence not present in the smooth muscle MLCK isoform (SM MLCK), which allows novel upregulation of MLCK activation by signaling cascades including p60src. The yeast two-hybrid assay system using the entire EC MLCK1 N-terminus (922 aa) as bait, identified additional stable MLCK binding partners including the 12 KDa macrophage migration inhibitory factor (MIF). This finding was confirmed by cross immunoprecipitation assays under non-denaturing conditions and by GST pull down experiments using GST-N-terminal MLCK (#1-923) and MLCK N-terminal deletion mutants in TNFalpha- and thrombin-stimulated endothelium. This EC MLCK-MIF interaction was shown biochemically and by immunofluorescent microscopy to be enhanced in TNFalpha- and thrombin-stimulated endothelium, both of which induce increased MLCK activity. Thrombin induced the colocalization of an epitope-tagged, full-length MIF fusion protein with phosphorylated MLC along peripheral actin stress fibers. Together these studies suggest that the novel interaction between MIF and MLCK may have important implications for the regulation of both non-muscle cytoskeletal dynamics as well as pathobiologic vascular events that involve MLCK.     

Novel role for non-muscle myosin light chain kinase (MLCK) in hyperoxia-induced recruitment of cytoskeletal proteins, NADPH oxidase activation, and reactive oxygen species generation in lung endothelium

We recently demonstrated that hyperoxia (HO) activates lung endothelial cell NADPH oxidase and generates reactive oxygen species (ROS)/superoxide via Src-dependent tyrosine phosphorylation of p47(phox) and cortactin. Here, we demonstrate that the non-muscle ~214-kDa myosin light chain (MLC) kinase (nmMLCK) modulates the interaction between cortactin and p47(phox) that plays a role in the assembly and activation of endothelial NADPH oxidase. Overexpression of FLAG-tagged wild type MLCK in human pulmonary artery endothelial cells enhanced interaction and co-localization between cortactin and p47(phox) at the cell periphery and ROS production, whereas abrogation of MLCK using specific siRNA significantly inhibited the above. Furthermore, HO stimulated phosphorylation of MLC and recruitment of phosphorylated and non-phosphorylated cortactin, MLC, Src, and p47(phox) to caveolin-enriched microdomains (CEM), whereas silencing nmMLCK with siRNA blocked recruitment of these components to CEM and ROS generation. Exposure of nmMLCK(-/-) null mice to HO (72 h) reduced ROS production, lung inflammation, and pulmonary leak compared with control mice. These results suggest a novel role for nmMLCK in hyperoxia-induced recruitment of cytoskeletal proteins and NADPH oxidase components to CEM, ROS production, and lung injury.     

Myosin light chain kinase and acto-myosin contractility modulate activation of the ERK cascade downstream of oncogenic Ras

The actin cytoskeleton is recognized as an important component of both adhesion- and growth factor-dependent signaling, but its role in oncogene-dependent signaling has received much less attention. In this study, we investigated the role played by the acto-myosin cytoskeleton and its main regulators, i.e., myosin light chain kinase and Rho kinase, in oncogenic Ki-Ras-induced signaling. We found that activation of the ERK cascade by Ras is dependent on acto-myosin contractility, under the regulation of myosin light chain kinase but not Rho kinase. Inhibition of myosin II or myosin light chain kinase caused a complete loss of ERK phosphorylation in a time- and dose-dependent manner, but proved dispensable for activation of the PI3K pathway. We also provide evidence that the target of myosin light chain kinase lays at the level of Raf activation. Since myosin light chain kinase is a target of ERK, these results suggest a previously uncharacterized signaling pathway involving Ras-mediated alterations of the actin cytoskeleton, which might play a critical role in ERK activation by the Ras oncogene and contribute to aberrant signaling and enhanced cell motility. In addition, restoration of stress fibers following ectopic expression of tropomyosin 2 resulted in reduced levels of ERK phosphorylation. Finally, these studies suggest that myosin light chain kinase but not Rho kinase plays an essential role in the generation of ERK signaling in transformed cells and indicate distinct cellular roles for Rho-kinase and myosin light chain kinase-dependent functions involving the regulation of acto-myosin contractility.     

Identification of an allelic variant of isoform MLC1-V/sB (human myosin light chain)

A study of 250 specimens of human myocardium by two-dimensional gel electrophoresis revealed an allelic variant of isoform MLC1-V/sB, which was identified by immunoblotting with monoclonal antibody against MLC1-V/sB and peptide mapping afterin situ tryptic digestion of electroblotted proteins. The substitution Asn-144 for His-144 was found in this new allelic variant of MLC1-V/sB.This work was supported by a grant from Russian Human Genome Project.     

Preparation of Monoclonal Antibodies against Human Ventricular Myosin Light Chain 1 （HVMLC1） for Functional Studies

Using purified recombinant human ventricular myosin light chain 1 (HVMLC 1) as the antigen,three monoclonal antibodies,designated C8,C9 and B 12,were prepared.Immunoblot experiments demonstratedthat all monoclonal antibodies could react with the ventricular myosin light chain 1 isolated from differentsources,such as human,rat or pig.It was also demonstrated that C8 was directed against the NN part of theN-fragment (amino acid 1-40) of HVMLC1,and both C9 and B12 against the C-fragment (amino acid 99-195).The affinity constants of C8,C9 and B12 were 3.20×10~8,8.60×10~7 and 1.77×10~8 M~(-1),respectively,determined by non-competitive ELISA.The isotype of B12 was determined as lgG2a,whereas the isotype ofboth C8 and C9 were IgG1.In the presence of C9 or B12,the actin-activated Mg~(2 )ATPase activity of myosinwas greatly inhibited,but there was almost no effect on the Mg~(2 )ATPase activity for C8.B12 and C9 alsoinhibited the superprecipitation of porcine cardiac native actomyosin (myosin B) and reconstituted actomyosin,but C8 did not.The results indicate that all three monoclonal antibodies could bind the intact myosin molecule,but B12 and C9 might more easily react with epitopes located in the C-fragment of HVMLC1.The inhibitoryeffects of B 12 and C9 on ATPase activity and superprecipitation assays show that light chain 1,particularlythe C-fragment domain,is involved in the modulation of the actin-activated Mg~(2 )ATPase activity of myosinand,as a consequence,plays an essential role in the interaction of actin and myosin.     

Identification of SNPs,mapping and analysis of allele frequencies in two candidate genes for meat production traits: the porcine myosin heavy chain 2B (MYH4) and the skeletal muscle myosin regulatory light chain 2 (HUMMLC2B)

Myosin is one of the most important skeletal muscle proteins. It is composed of myosin heavy chains and myosin light chains that exist with different isoforms coded by different genes. We studied the porcine myosin heavy chain 2B (MYH4) and the porcine skeletal muscle myosin regulatory light chain 2 (HUMMLC2B) genes. A single nucleotide polymorphism (SNP), identified for each gene, was used for linkage mapping of MYH4 and HUMMLC2B to porcine chromosome (Sscr) 12 and Sscr 3, respectively. The mapping of these two genes was confirmed by using a porcine-rodent radiation hybrid panel, even if for MYH4 the LOD score and the retention fraction were low. Allele frequencies at the two loci were studied in a sample of 307 unrelated pigs belonging to seven different pig breeds. Moreover the distribution of the alleles at these two loci was analysed in groups of pigs with extreme divergent (positive and negative) estimated breeding values (EBV) for four meat production traits that have undergone selection in Italian heavy pigs.     

Interaction of pollen F-actin with rabbit muscle myosin and its subfragments (HMM,S1)

Summary Actin purified from maize pollen grains can be polymerized into F-actin which increased the ATPase activities of proteolytic fragments (HMM, S₁) of rabbit muscle myosin. The values of K_app is 232 M for HMM and 290 M for S₁, which are six- and seven-fold higher than those of rabbit muscle F-actin under the same conditions. Pollen actin and rabbit muscle myosin form hybrid actomyosin showing increase in viscosity and turbidity of solution. Viscosity and turbidity of the actomyosin dropped and then increased again with addition of ATP. Polymerized pollen actin can be decorated in vitro with both rabbit muscle HMM and S₁ to form an arrowhead-shaped structure like that observed in living plant cells. The results show that pollen actin is similar to muscle actin at a qualitative level. But there are differences between them at a quantitative level.Abbreviations HMM heavy meromyosin - S₁ myosin subfragment 1 - ATP adenosine-5-triphosphate