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)