Nonkinase activity of MLCK in elongated filopodia formation and chemotaxis of vascular smooth muscle cells toward sphingosylphosphorylcholine.
Nonkinase activity of MLCK in elongated filopodia formation and chemotaxis of vascular smooth muscle cells toward sphingosylphosphorylcholine.
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DOI：
10.1152/ajpheart.00965.2008
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发表时间：
2009-02
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影响因子：
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通讯作者：
Hong Hui Wang;A. Nakamura;A. Matsumoto;S. Yoshiyama;Xiao-ran Qin;L. Ye;Ce Xie;Yue Zhang;Ying Gao;R. Ishikawa;K. Kohama
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作者：
Hong Hui Wang;A. Nakamura;A. Matsumoto;S. Yoshiyama;Xiao-ran Qin;L. Ye;Ce Xie;Yue Zhang;Ying Gao;R. Ishikawa;K. Kohama
肌动蛋白轻链激酶（myosin light chain kinase， MLCK）是血管平滑肌细胞（VSMCs）肌动蛋白-肌球蛋白相互作用的调控蛋白，肌动蛋白轻链激酶是一种具有n端肌动蛋白结合域和c端肌动蛋白结合域的中心催化结构域的融合蛋白。除了由催化结构域介导的激酶活性的调节作用外，来自两个末端的非激酶活性也能够发挥调节作用，Nakamura等人（32）综述了这一点。我们之前的研究表明，非激酶活性在鞘甲酰基磷酸胆碱（SPC）刺激下介导丝状足（25）。为了探索非激酶活性在趋化性中的调节作用，我们利用慢病毒介导的RNAi系统构建了完全消除MLCK表达的VSMCs。我们假设mlck下调的VSMCs在SPC刺激下不能形成丝状足和迁移，并证实了这一假设。我们进一步从编码野生型（WT） MLCK的牛cDNA构建了一个激酶失活突变体，通过突变位于催化结构域的atp结合位点，随后确认了WT（激酶失活突变体）的激酶活性的存在（缺失）。我们将WT和突变体转染到mlck下调的VSMCs中。我们期望转染后的VSMCs能够恢复诱导丝状足的能力和对SPC的趋化性，结果发现这两种结构都恢复了这种能力。由于它们共享肌动蛋白和肌球蛋白结合结构域，我们得出结论，非激酶活性在spc诱导的迁移中起主要作用。
The actin-myosin interaction of vascular smooth muscle cells (VSMCs) is regulated by myosin light chain kinase (MLCK), which is a fusion protein of the central catalytic domain with the N-terminal actin-binding and C-terminal myosin-binding domains. In addition to the regulatory role of kinase activity mediated by the catalytic domain, nonkinase activity that derives from both terminals is able to exert a regulatory role as reviewed by Nakamura et al. (32). We previously showed that nonkinase activity mediated the filopodia upon the stimulation by sphingosylphosphorylcholine (SPC) (25). To explore the regulatory role of nonkinase activity in chemotaxis, we constructed VSMCs where the expression of MLCK was totally abolished by using a lentivirus-mediated RNAi system. We hypothesized that the MLCK-downregulated VSMCs were unable to form filopodia and to migrate upon SPC stimulation and confirmed the hypothesis. We further constructed a kinase-inactive mutant from bovine cDNA coding wild-type (WT) MLCK by mutating the ATP-binding sites located in the catalytic domain, followed by confirming the presence (absence) of the kinase activity of WT (kinase-inactive mutant). We transfected WT and the mutant into MLCK-downregulated VSMCs. We expected that the transfected VSMCs will recover the ability to induce filopodia and chemotaxis toward SPC and found both constructs rescued the ability. Because they share the actin- and myosin-binding domains, we concluded nonkinase activity plays a major role for SPC-induced migration.