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 kinase 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 domain has been suggested to be myosin [Okagaki et al. (1999) J. Biochem. 125, 619-626]. In this study, we have analyzed the amino acid sequences of MLCK and myosin that are involved in the inhibition. The ATP-binding peptide of Gly526-Lys548 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. The inhibition of the ATP-binding peptide was confirmed by measuring ATPase activities 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 chain. The peptide of the 20 kDa light chain sequence of Ser1-Glu29 also relieved the inhibition. Thus, the interaction of the ATP-binding sequence with the 20 kDa light chain sequence should cause the inhibition of the actin-myosin interaction. Concerning the regulation of the inhibition, calmodulin relieved the inhibitory effect of MLCK on the movement of actin filaments. The calmodulin-binding peptide (Ala796 Ser815) prevented the relief, suggesting the involvement of this sequence. Thus, the mode of regulation by 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.
Okagaki
T
T
Department of Pharmacology, Gunma University School of Medicine, Maebashi, Gunma, 371-8511, Japan. okagaki@sb.gunma-u.ac.jp
Ye
L H
LH
Samizo
K
K
Tanaka
T
T
Kohama
K
K
D000199:Actins / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D002417:Cattle; D002645:Chickens; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D018994:Myosin Light Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009218:Myosins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000199:Actins; D002147:Calmodulin; D018994:Myosin Light Chains; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D009219:Myosin-Light-Chain Kinase; D009218:Myosins