Kohichi Hayakawa

Myosin light chain kinase: an actin-binding protein that regulates an ATP-dependent interaction with myosin

Myosin light chain kinase (MLCK) is a key regulator of smooth muscle contraction. The most conspicuous form of regulation is achieved by phosphorylation of the myosin light chain, allowing myosin to interact with actin. This interaction is regulated by actin-binding proteins that modulate actin filaments. In this review Kazuhiro Kohama and colleagues consider MLCK as an actin-binding protein and attempt to shed light on the cross-talk between the different kinds of regulation of the actin-myosin interaction in smooth muscle. An understanding of these mechanisms will assist the development of compounds with therapeutic importance in muscular disorders.

The Structure and Function of the Actin-binding Domain of Myosin Light Chain Kinase of Smooth Muscle

In addition to its kinase activity, the myosin light chain kinase (MLCK) of smooth muscle has an actin binding activity through which it can regulate the actin-myosin interaction of smooth muscle (Kohama, K., Okagaki, T., Hayakawa, K., Lin, Y., Ishikawa, R., Shimmen, T., and Inoue, A. (1992) Biochem. Biophys. Res. Commun. 184, 1204–1211). In this study, we have analyzed the actin binding activity of MLCK and related it to its amino acid sequence by producing native and recombinant fragments of MLCK. Parent MLCK exhibited both calcium ion (Ca2+) and calmodulin (Ca2+/CaM)-sensitive and Ca2+/CaM-insensitive binding to actin filaments. The native fragment, which consists of the Met1–Lys114 sequence (Kanoh, S., Ito, M., Niwa, E., Kawano, Y., and Hartshorne, D. J. (1993)Biochemistry 32, 8902–8907), and the recombinant NN fragment, which contains this 1–114 sequence, showed only Ca2+/CaM-sensitive binding. An inhibitory effect of the NN fragment on the actin-myosin interaction was observed by assayingin vitro motility and by measuring the actin-activated ATPase activity of myosin. The recombinant NN/41 fragment, which is constructed without the Met1–Pro41 sequence of the NN fragment, lost both the actin binding activity and the inhibitory effect. We confirmed the importance of the 1–41 sequence by using a few synthetic peptides to compete against the NN fragment in binding to actin filaments. The experiments using recombinant fragments and synthetic peptides also revealed that the site for CaM-binding is the Pro26–Pro41 sequence. The site for the Ca2+/CaM-insensitive binding, which is shown to be localized between the Ca2+/CaM-sensitive site and the central kinase domain of MLCK, exerted no regulatory effects on the actin-myosin interaction.

A novel regulatory effect of myosin light chain kinase from smooth muscle on the ATP-dependent interaction between actin and myosin.

The actin-binding activity of myosin light chain kinase (MLCK) from smooth muscle was studied with special reference to the ATP-dependent interaction between actin and myosin. MLCK in the presence of calmodulin endowed sensitivity to Ca2+ on the movement of actin filaments on phosphorylated myosin from smooth muscle that was fixed on a coverslip. This regulatory effect was not attributable to the kinase activity of MLCK but could be explained by its actin-binding activity. The importance of the actin-binding activity was further substantiated by results of an experiment with Nitellopsis actin-cables in which MLCK regulated the interaction under conditions where MLCK was exclusively associated with the actin-cables.

Characterization of the myosin light chain kinase from smooth muscle as an actin-binding protein that assembles actin filaments in vitro

In addition to its kinase activity, myosin light chain kinase has an actin-binding activity, which results in bundling of actin filaments [Hayakawa et al., Biochem. Biophys. Res. Commun. 199, 786-791, 1994]. There are two actin-binding sites on the kinase: calcium- and calmodulin-sensitive and insensitive sites [Ye et al., J. Biol. Chem. 272, 32182-32189, 1997]. The calcium/calmodulin-sensitive, actin-binding site is located at Asp2-Pro41 and the insensitive site is at Ser138-Met213. The cyanogen bromide fragment, consisting of Asp2-Met213, is furnished with both sites and is the actin-binding core of myosin light chain kinase. Cross-linking between the two sites assembles actin filaments into bundles. Breaking of actin-binding at the calcium/calmodulin-sensitive site by calcium/calmodulin disassembles the bundles.

Okadaic acid stimulates the ATP-dependent interaction between actin and myosin of smooth muscle via a direct effect on myosin

The direct effect of okadaic acid (OA) on the ATP-dependent interaction between actin and myosin of smooth muscle was examined not only by the conventional measurement of ATPase activity but also by application of in vitro motility assay developed recently. The motility was effectively enhanced by microM levels of OA. Measurements of the activities of myosin confirmed that the myosin mediated this effect. The result of this study, which was carried out in the absence of protein phosphatase, are not compatible with the recent reports that the stimulatory effect of OA on smooth muscle contraction is attributable to its inhibitory effect on the activity of the protein phosphatase.

Calcium regulation of non‐kinase and kinase activities of recombinant myosin light‐chain kinase and its mutants

Myosin light‐chain kinase (MLCK) comprised of N‐terminal actin‐binding domain, central catalytic domain, and C‐terminal myosin‐binding domain. It exerted not only kinase activity to phosphorylate 20 kDa regulatory light chain of smooth muscle but also exerted non‐kinase activity on myosin motor and myosin ATPase activities (Nakamura et al., Biochem. Biophys. Res. Commun. 2008, 369, 135). The previous studies on the multiple MLCK functions were done using MLCK fragments. The present study reported the expression of whole MLCK molecules in Escherichia coli in a large amount. The construct in which the calmodulin (CaM) binding domain for regulating kinase activity was mutated lost the kinase activity. However, the mutant exerted non‐kinase activity and inhibited both myosin motor and ATPase activities. The domain that regulated kinase activity was also shown to be involved in the Ca2+ regulation of non‐kinase activity. The deletion mutants of actin‐binding domain which located at N‐terminal 1–41 amino acids demonstrated that non‐kinase activity was mediated through actin filaments.

Actin-Binding Property of Myosin Light Chain Kinase and Its Role in Regulating Actin-Myosin Interaction of Smooth Muscle

Myosin light chain kinase (MLCK), a key regulator of smooth muscle contraction, has a property to bind to actin filaments that is obviously unrelated to the kinase activity innate in MLCK. We have found that this property exerts an inhibitory effect on the ATP-dependent interaction between actin and myosin. The N-terminal, actin-binding fragment was responsible for the inhibition. Calmodulin in the presence of Ca2+ (Ca2+/CaM) efficiently relieved the inhibition imposed by MLCK. The inhibitory effect was potent enough to explain physiological involvement in terms of the amount of MLCK that was present as a cytoskeletal protein in smooth muscle.