Yano K, Araki Y, Hales S J, Tanaka M, Ikebe M
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970.
Biochemistry. 1993 Nov 16;32(45):12054-61. doi: 10.1021/bi00096a016.
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)
有人提出，在没有Ca2+/钙调蛋白的情况下，肌球蛋白轻链激酶（MLCK）的活性会被一个假底物序列所抑制[肯普，B.E.，皮尔逊，R.B.，圭列罗，V.J.，巴吉，I.，& 米恩斯，A.R.（1987年）《生物化学杂志》262，2542 - 2548]。为了评估这一假设，研究了假底物序列所必需的一组碱性残基精氨酸797 - 精氨酸798 - 赖氨酸799在MLCK抑制中的作用。获得了鸡砂囊MLCK的全长cDNA，并在大肠杆菌中表达了包含完整氨基酸序列的重组MLCK。重组MLCK的Ca2+/钙调蛋白依赖性活性与天然分离的MLCK相当。产生了两个截短突变体，MT799和MT796，其中MT799而非MT796包含一组碱性残基。MT799和MT796都不结合钙调蛋白，激酶活性受到抑制（类似于在没有Ca2+/钙调蛋白时的MLCK活性）。然而，随后的胰蛋白酶消化显著提高了突变体的激酶活性。突变体的胰蛋白酶消化最初产生一个64 kDa的片段，随后产生61 kDa的片段，活性的增加与61 kDa片段的出现一致。这与天然MLCK的消化模式相似，并且已知61 kDa片段是组成型活性激酶[池部，M.，斯特平斯卡，M.，肯普，B.E.，米恩斯，A.R.，& 哈茨霍恩，D.J.（1987年）《生物化学杂志》262，13828 - 13834]。（摘要截短于250字）
