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 assaying in 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.
Ye
L H
LH
Department of Pharmacology, Gunma University School of Medicine, Maebashi, Gunma 371 Japan.
Hayakawa
K
K
Kishi
H
H
Imamura
M
M
Nakamura
A
A
Okagaki
T
T
Takagi
T
T
Iwata
A
A
Tanaka
T
T
Kohama
K
K
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000378:metabolism; D002645:Chickens; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D000199:Actins; D002147:Calmodulin; D010446:Peptide Fragments; D011994:Recombinant Proteins; D009219:Myosin-Light-Chain Kinase; D009218:Myosins