Deng J T, Van Lierop J E, Sutherland C, Walsh M P
Smooth Muscle Research Group and Canadian Institutes of Health Research Group in Regulation of Vascular Contractility, Department of Biochemistry, University of Calgary Faculty of Medicine, Calgary, Alberta T2N 4N1, Canada.
J Biol Chem. 2001 May 11;276(19):16365-73. doi: 10.1074/jbc.M011634200. Epub 2001 Feb 8.
Smooth muscle contraction follows an increase in cytosolic Ca(2+) concentration, activation of myosin light chain kinase, and phosphorylation of the 20-kDa light chain of myosin at Ser(19). Several agonists acting via G protein-coupled receptors elicit a contraction without a change in Ca(2+) via inhibition of myosin light chain phosphatase and increased myosin phosphorylation. We showed that microcystin (phosphatase inhibitor)-induced contraction of skinned smooth muscle occurred in the absence of Ca(2+) and correlated with phosphorylation of myosin light chain at Ser(19) and Thr(18) by a kinase distinct from myosin light chain kinase. In this study, we identify this kinase as integrin-linked kinase. Chicken gizzard integrin-linked kinase cDNA was cloned, sequenced, expressed in E. coli, and shown to phosphorylate myosin light chain in the absence of Ca(2+) at Ser(19) and Thr(18). Subcellular fractionation revealed two distinct populations of integrin-linked kinase, including a Triton X-100-insoluble component that phosphorylates myosin in a Ca(2+)-independent manner. These results suggest a novel function for integrin-linked kinase in the regulation of smooth muscle contraction via Ca(2+)-independent phosphorylation of myosin, raise the possibility that integrin-linked kinase may also play a role in regulation of nonmuscle motility, and confirm that integrin-linked kinase is indeed a functional protein-serine/threonine kinase.
平滑肌收缩伴随着胞质Ca(2+)浓度升高、肌球蛋白轻链激酶激活以及肌球蛋白20-kDa轻链在Ser(19)位点的磷酸化。几种通过G蛋白偶联受体起作用的激动剂，通过抑制肌球蛋白轻链磷酸酶并增加肌球蛋白磷酸化，引发收缩而不改变Ca(2+)。我们发现，微囊藻毒素（磷酸酶抑制剂）诱导的去皮平滑肌收缩在无Ca(2+)的情况下发生，并且与一种不同于肌球蛋白轻链激酶的激酶使肌球蛋白轻链在Ser(19)和Thr(18)位点磷酸化相关。在本研究中，我们确定这种激酶为整合素连接激酶。鸡胗整合素连接激酶cDNA被克隆、测序，在大肠杆菌中表达，并显示在无Ca(2+)的情况下使肌球蛋白轻链在Ser(19)和Thr(18)位点磷酸化。亚细胞分级分离揭示了整合素连接激酶的两个不同群体，包括一种Triton X-100不溶性成分，其以不依赖Ca(2+)的方式使肌球蛋白磷酸化。这些结果提示整合素连接激酶在通过肌球蛋白不依赖Ca(2+)的磷酸化调节平滑肌收缩中具有新功能，增加了整合素连接激酶也可能在非肌肉运动调节中起作用的可能性，并证实整合素连接激酶确实是一种功能性蛋白丝氨酸/苏氨酸激酶。
