Inhibitory effect of the catalytic domain of myosin light chain kinase on actin-myosin interaction: Insight into the mode of inhibition

The catalytic domain of myosin light chain kinase (MLCK) not only exerts ki nase activity to phosphorylate the 20 kDa light chain but also inhibits the actin-myosin interaction. The site of action of this novel role of the dom ain has been suggested to be myosin [Okagaki et al, (1999) J, Biochem, 125, 619-626], Tn this study, we have analyzed the amino acid sequences of MLCK and myosin that are involved in the inhibition. The ATP-binding peptide of Gly(526)-Lys(548) Of chicken gizzard MLCK exerted the inhibitory effect on the movement of actin filaments on a myosin-coated glass surface. However, the peptide that neighbors the sequence failed to inhibit the movement. Th e inhibition of the ATP-binding peptide was confirmed by measuring ATPase a ctivities of the myosin, The inhibition by parent MLCK of the movement was relieved by the 20 kDa light chain, but not by the 17 kDa myosin light chai n. The peptide of the 20 kDa light chain sequence of Ser(1)-Glu(29) also re lieved the inhibition. Thus, the interaction of the ATP-binding sequence wi th the 20 kDa light chain sequence should cause the inhibition of the actin -myosin interaction. Concerning the regulation of the inhibition, calmoduli n relieved the inhibitory effect of MLCK on the movement of actin filaments . The calmodulin-binding peptide (Ala(796) Ser(815)) prevented the relief, suggesting the involvement of this sequence. Thus, the mode of regulation b y Ca2+ and calmodulin of the novel role of the catalytic domain is similar, but not identical, to the mode of regulation of the kinase activity of the domain.