Definition of the inhibitory domain of smooth muscle myosin light chain kinase by site-directed mutagenesis.
Definition of the inhibitory domain of smooth muscle myosin light chain kinase by site-directed mutagenesis.
复制标题
通过定点诱变定义平滑肌肌球蛋白轻链激酶的抑制域。
DOI：
10.1021/bi00228a021
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发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Hartshorne,DJ
中科院分区：
文献类型：
--
作者：
Ito,M;GuerrieroJr,V;Chen,XM;Hartshorne,DJ
摘要：平滑肌肌球蛋白轻链激酶的定点突变被应用于确定其自身抑制结构域。突变体均起始于Leu-447，但含有不同长度的C-末端序列。那些含有Glu-972的完整C-末端序列的具有钙调素依赖的激酶活性。通过截短至Thr-778去除推定的抑制结构域导致产生组成型活性（钙调素非依赖性）物种。因此，抑制结构域位于Thr-778的C-末端侧。截断赖氨酸-793和色氨酸-800也导致组成型活性突变体，虽然后者的比活性小于其他突变体。没有截短的突变体结合钙调蛋白。对于每种突变体，相对于ATP和20 000-道尔顿轻链的Km值与用天然酶获得的值相似。通过用胰蛋白酶有限蛋白水解后激活激酶活性来检测抑制结构域的存在。使用该程序，确定抑制结构域仅在截短至Trp-800的突变体中表现，而在终止于Lys-793的突变体中不存在。这些结果表明，抑制结构域的关键区域包含在序列Tyr-794至Trp-800内。该区域与钙调素结合位点重叠5个残基。我们对抑制序列的分配与通过假底物结构域的自抑制一致。IV^肌球蛋白轻链激酶（MLCK）1是调节平滑肌收缩活动的关键酶。MLCK对两条20 000-道尔顿轻链上的Ser-19进行磷酸化被认为是收缩起始所必需的（哈茨霍恩，1987）。此事件
Revised Manuscript Received January 2, 1991 abstract: Site-directed mutagenesis of smooth muscle myosin light chain kinase was applied to define its autoinhibitory domain. Mutants were all initiated at Leu-447 butcontained varying lengths of C-terminal sequence. Those containing the complete C-terminal sequence to Glu-972 possessed kinase activitiesthat were calmodulin-dependent. Removal of the putative inhibitory domain by truncation to Thr-778 resulted in generation of a constitutively active (calmodulin-independent) species. Thus, the inhibitory domain lies to the C-terminal side of Thr-778. Truncation to Lys-793 and to Trp-800 also resulted in constitutively active mutants, although the specific activity of the latter was less than the other mutants. None of the truncated mutants bound calmodulin. For each mutant, the Km values with respect to ATP and to the 20 000-dalton light chain were similar to values obtained with the native enzyme. The presence of the inhibitory domain was detected by activation of kinase activity following limited proteolysis with trypsin. Using this procedure, it was determined that the inhibitory domain was manifest only in the mutant truncated to Trp-800 and was absent from that endingat Lys-793. These results indicatethat a critical region of the inhibitory domain is contained within the sequence Tyr-794 to Trp-800. This region overlaps with the calmodulin-binding site for five residues. Our assignment of the inhibitory sequence is consistent with autoinhibition via a pseudosubstrate domain.IV^ yosin light chain kinase (MLCK) 1 is a key enzyme in the regulation of contractile activity in smooth muscle. Phosphorylation of Ser-19 on each of the two 20 000-dalton light chains by MLCK is thought to be essential for the initiation of contraction (Hartshorne, 1987). This event is
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DOI：
10.1073/pnas.87.6.2284
发表时间：
1990-03-01
影响因子：
11.1
作者：
OLSON, NJ;PEARSON, RB;MEANS, AR
通讯作者：
MEANS, AR
兔骨骼肌肌球蛋白轻链激酶的氨基酸序列。
影响因子：
2.9
作者：
K. Takio;D. Blumenthal;K. Walsh;K. Titani;E. Krebs
通讯作者：
E. Krebs
[30] 平滑肌肌球蛋白轻链激酶
DOI：
--
发表时间：
1983
期刊：
影响因子：
--
作者：
M. Walsh;S. Hinkins;R. Da̧browska;D. Hartshorne
通讯作者：
D. Hartshorne
骨骼肌肌球蛋白轻链激酶中钙调蛋白结合和催化结构域的表征。
影响因子：
4.8
作者：
A. Edelman;Koji Takio;Donald K. Blumenthal;R. Hansen;Kenneth A. Walsh;K. Titani;E. Krebs
通讯作者：
E. Krebs
单克隆抗体对平滑肌肌球蛋白特性的影响。
影响因子：
2.9
作者：
Ito,M;Pierce,PR;Allen,RE;Hartshorne,DJ
通讯作者：
Hartshorne,DJ