Boundary of the autoinhibitory region of smooth muscle myosin light-chain kinase.
It has been proposed that myosin light-chain kinase (MLCK) activity is inhibited in the absence of Ca2+/calmodulin by a pseudosubstrate sequence [Kemp, B. E., Pearson, R. B., Guerriero, V. J., Bagchi, I., & Means, A. R. (1987) J. Biol. Chem. 262, 2542-2548]. To evaluate this hypothesis, the role of a cluster of basic residues, Arg797-Arg798-Lys799, which are essential for the pseudosubstrate sequence, in the inhibition of MLCK was studied. A full-length cDNA of chicken gizzard MLCK was obtained, and the recombinant MLCK which contains the entire amino acid sequence was expressed in Escherichia coli. The Ca2+/calmodulin-dependent activity of the recombinant MLCK was comparable to that of the naturally isolated MLCK. Two truncation mutants, MT799 and MT796, were produced, of which MT799 but not MT796 contained a cluster of basic residues. Neither MT799 nor MT796 bound calmodulin, and kinase activity was inhibited (similar to MLCK activity in the absence of Ca2+/calmodulin). However, the kinase activity of the mutants was increased markedly by subsequent tryptic proteolysis. The tryptic digestion of the mutants initially produced a 64-kDa fragment then, subsequently, the 61-kDa fragment, and the increase in activity coincided with the appearance of the 61-kDa fragment. This was similar to the digestion profile of native MLCK, and it is known that the 61-kDa fragment is the constitutively active kinase [Ikebe, M., Stepinska, M., Kemp, B. E., Means, A. R., & Hartshorne, D. J. (1987) J. Biol. Chem. 262, 13828-13834].(ABSTRACT TRUNCATED AT 250 WORDS)
Yano
K
K
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970.
Araki
Y
Y
Hales
S J
SJ
Tanaka
M
M
Ikebe
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014422:Turkeys
D017931:DNA Primers; D009219:Myosin-Light-Chain Kinase