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...

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Published in:The Journal of biological chemistry 1997-12, Vol.272 (51), p.32182-32189
Main Authors: Ye, Li-Hong, Hayakawa, Kohichi, Kishi, Hiroko, Imamura, Michihiro, Nakamura, Akio, Okagaki, Tsuyoshi, Takagi, Takashi, Iwata, Akiko, Tanaka, Takeshi, Kohama, Kazuhiro
Format: Article
Language:English
Subjects:
Actins - metabolism
Amino Acid Sequence
Animals
Calmodulin - metabolism
Chickens
Molecular Sequence Data
Muscle, Smooth - enzymology
Myosin-Light-Chain Kinase - chemistry
Myosin-Light-Chain Kinase - metabolism
Myosins - metabolism
Peptide Fragments - metabolism
Protein Binding
Recombinant Proteins - metabolism
Sequence Homology, Amino Acid
Structure-Activity Relationship
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.272.51.32182