Izawa T, Fukata Y, Kimura T, Iwamatsu A, Dohi K, Kaibuchi K
Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan.
Biochem Biophys Res Commun. 2000 Nov 11;278(1):72-8. doi: 10.1006/bbrc.2000.3772.
Rho-associated kinase (Rho-kinase), which is activated by the Rho small GTPase, phosphorylates the myosin-binding subunit (MBS) of myosin phosphatase, myosin light chain (MLC), the ERM family proteins, and adducin, thereby regulating the formation of stress fibers, focal adhesions, microvillus formation, and cell motility. Here, to further understand the role of Rho-kinase in the regulation of the numerous cellular processes by Rho, we purified a novel substrate of Rho-kinase having a molecular mass of 48 kDa (p48) from a rat liver cytosol extract. Mass spectral analysis revealed p48 to be elongation factor-1 alpha (EF-1 alpha), which is known as an actin-binding protein besides a cofactor of polypeptide elongation. Rho-kinase directly phosphorylated recombinant EF-1alpha in vitro. A high- speed cosedimentation assay revealed that phosphorylation of EF-1 alpha by Rho-kinase decreased the binding activity of EF-1 alpha to filamentous actin (F-actin). A low-speed sedimentation assay revealed that phosphorylation of EF-1 alpha by Rho-kinase decreased the F-actin-bundling activity. In addition, EF-1 alpha bound to MBS of myosin phosphatase, suggesting that both Rho-kinase and myosin phosphatase regulate the phosphorylation state of EF-1 alpha downstream of Rho as other substrates of Rho-kinase, i.e., MLC, adducin, and the ERM family. These results suggest that the Rho/Rho-kinase pathway regulates the organization of actin cytoskeleton via the phosphorylation of EF-1 alpha.
Rho相关激酶（Rho激酶）由Rho小GTP酶激活，可使肌球蛋白磷酸酶的肌球蛋白结合亚基（MBS）、肌球蛋白轻链（MLC）、ERM家族蛋白和内收蛋白磷酸化，从而调节应力纤维的形成、粘着斑、微绒毛形成和细胞运动。在此，为了进一步了解Rho激酶在Rho调节众多细胞过程中的作用，我们从大鼠肝脏胞质溶胶提取物中纯化了一种分子量为48 kDa的新型Rho激酶底物（p48）。质谱分析显示p48是延伸因子-1α（EF-1α），它除了作为多肽延伸的辅因子外，还是一种肌动蛋白结合蛋白。Rho激酶在体外直接使重组EF-1α磷酸化。高速共沉降分析表明，Rho激酶使EF-1α磷酸化会降低EF-1α与丝状肌动蛋白（F-肌动蛋白）的结合活性。低速沉降分析表明，Rho激酶使EF-1α磷酸化会降低F-肌动蛋白的成束活性。此外，EF-1α与肌球蛋白磷酸酶的MBS结合，这表明Rho激酶和肌球蛋白磷酸酶都像Rho激酶的其他底物（即MLC、内收蛋白和ERM家族）一样，在Rho下游调节EF-1α的磷酸化状态。这些结果表明，Rho/Rho激酶途径通过EF-1α的磷酸化调节肌动蛋白细胞骨架的组织。
