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

肌球蛋白轻链激酶(MLCK)在调节肌动蛋白-肌球蛋白相互作用中起核心作用。MLCK在钙离子和钙调蛋白(CaM)存在的情况下使肌球蛋白轻链磷酸化，从而激活肌球蛋白，使其与肌动蛋白相互作用。除该激酶活性外，MLCK还表现为：1)肌动蛋白结合活性，可将肌动蛋白细丝组装成肌球蛋白束；2)肌球蛋白结合活性，可形成肌球蛋白细丝。为了定位肌动蛋白和肌球蛋白在MLCK分子中的结合活性，并研究它们在肌动蛋白肌球蛋白相互作用中的可能作用，我们在大肠杆菌中表达了编码MLCK的各种片段作为重组蛋白。我们发现MLCK由一个N端的肌动蛋白结合域、一个中央激活区和一个C端的肌球蛋白结合域组成。Met^1-Pro^&lt；41&gt；序列负责与肌动蛋白的钙敏感结合。只有当肌球蛋白被磷酸化时，这个结合部位才会对肌动蛋白-肌球蛋白相互作用产生抑制作用。MLCK在C-末端与肌球蛋白结合，其序列与端粒蛋白完全相同，端粒蛋白是一种在平滑肌细胞中含量丰富的肌球蛋白结合蛋白。这个域本身在相互作用中没有调节作用。然而，当这个结构域扩展到包括已知的调节激酶域活性的序列时，相互作用就被刺激了。只有当肌球蛋白未磷酸化时，才能观察到这种刺激。

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.