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Molecular approach to the structure and function of myosin light chain kinase

肌球蛋白轻链激酶结构和功能的分子方法

基本信息

批准号：
08457633
负责人：
KOHAMA Kazuhiro
金额：
$ 4.61万
依托单位：
Gunma University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

Myosin light chain kinase (MLCK) plays a central role in regulating the actin-myosin interaction of smooth muscle. MLCK phosphorylates the light chain of myosin in the presence of Ca^<2+> and calmodulin (CaM) thereby activating myosin so that it can interact with actin. Besides this kinase activity, MLCK shows i) actin-binding activity that can assemble actin filaments into their bundles and ii) myosin-binding activity that can form myosin filaments. To localize the actin- and myosin-binding activities in the MLCK molecule and to examine their possible role in regulating the actin myosin interaction, we expressed various fragments of cDNA encoding MLCK in Escherichia coli as recombinant proteins. We found that MLCK consists of an N-terminal actin-binging domain, a central kinase domain, and a C-terminal myosin-binding domain. The Met^1-Pro^<41> sequence is responsible for Ca^<2+>/CaM-sensitive binding to actin. This binding site exerts an inhibitory effect on the actin-myosin interaction only when myosin is phosphorylated. MLCK binds to myosin at the C-terminal domain, the sequence of which is identical to telokin, an abundant myosin-binding protein in smooth muscle cells. This domain itself has no regulatory role in the interaction. However, the interaction was stimulated when this domain was extended to include the sequence known to regulate the activity of the kinase domain. The stimulation was observed only when myosin was unphosphorylated.

肌球蛋白轻链激酶（MLCK）在调节平滑肌肌动蛋白-肌球蛋白相互作用中起重要作用。MLCK在Ca^2+和钙调蛋白（CaM）存在下磷酸化肌球蛋白的轻链，从而激活肌球蛋白，使其能够与肌动蛋白相互作用。除了这种激酶活性外，MLCK还表现出i）肌动蛋白结合活性，可以将肌动蛋白丝组装成它们的束，ii）肌球蛋白结合活性，可以形成肌球蛋白丝。本地化的肌动蛋白和肌球蛋白的MLCK分子的结合活性，并检查其可能的作用，在调节肌动蛋白肌球蛋白的相互作用，我们表达了各种片段的cDNA编码MLCK在大肠杆菌中的重组蛋白。我们发现MLCK由一个N端肌动蛋白结合结构域，一个中央激酶结构域和一个C端肌球蛋白结合结构域组成。Met^1-Pro^<41>序列负责Ca^<2+>/CaM敏感性地与肌动蛋白结合。只有当肌球蛋白被磷酸化时，这个结合位点才对肌动蛋白-肌球蛋白相互作用产生抑制作用。MLCK在C-末端结构域与肌球蛋白结合，其序列与telokin相同，telokin是平滑肌细胞中丰富的肌球蛋白结合蛋白。该结构域本身在相互作用中没有调节作用。然而，当该结构域被扩展到包括已知调节激酶结构域活性的序列时，相互作用被刺激。只有当肌球蛋白未磷酸化时才观察到刺激。