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 Ca(2+) 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.
Xie
Ce
C
Department of Molecular and Cellular Pharmacology, Gunma University Graduate School of Medicine, Maebashi, Japan.
Zhang
Yue
Y
Wang
Hong Hui
HH
Matsumoto
Atsushi
A
Nakamura
Akio
A
Ishikawa
Ryoki
R
Yoshiyama
Shinji
S
Hayakawa
Kohichi
K
Kohama
Kazuhiro
K
Gao
Ying
Y
D000199:Actins / Q000502:physiology; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology; D009154:Mutation; D018994:Myosin Light Chains / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism
D000199:Actins; D002135:Calcium-Binding Proteins; D018994:Myosin Light Chains; D011994:Recombinant Proteins; D009219:Myosin-Light-Chain Kinase; D009218:Myosins; D002118:Calcium