Kawano Y, Fukata Y, Oshiro N, Amano M, Nakamura T, Ito M, Matsumura F, Inagaki M, Kaibuchi K
Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, Japan.
J Cell Biol. 1999 Nov 29;147(5):1023-38. doi: 10.1083/jcb.147.5.1023.
Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, phosphorylates myosin-binding subunit (MBS) of myosin phosphatase and thereby inactivates the phosphatase activity in vitro. Rho-kinase is thought to regulate the phosphorylation state of the substrates including myosin light chain (MLC), ERM (ezrin/radixin/moesin) family proteins and adducin by their direct phosphorylation and by the inactivation of myosin phosphatase. Here we identified the sites of phosphorylation of MBS by Rho-kinase as Thr-697, Ser-854 and several residues, and prepared antibody that specifically recognized MBS phosphorylated at Ser-854. We found by use of this antibody that the stimulation of MDCK epithelial cells with tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF) induced the phosphorylation of MBS at Ser-854 under the conditions in which membrane ruffling and cell migration were induced. Pretreatment of the cells with Botulinum C3 ADP-ribosyltransferase (C3), which is thought to interfere with Rho functions, or Rho-kinase inhibitors inhibited the TPA- or HGF-induced MBS phosphorylation. The TPA stimulation enhanced the immunoreactivity of phosphorylated MBS in the cytoplasm and membrane ruffling area of MDCK cells. In migrating MDCK cells, phosphorylated MBS as well as phosphorylated MLC at Ser-19 were localized in the leading edge and posterior region. Phosphorylated MBS was localized on actin stress fibers in REF52 fibroblasts. The microinjection of C3 or dominant negative Rho-kinase disrupted stress fibers and weakened the accumulation of phosphorylated MBS in REF52 cells. During cytokinesis, phosphorylated MBS, MLC and ERM family proteins accumulated at the cleavage furrow, and the phosphorylation level of MBS at Ser-854 was increased. Taken together, these results indicate that MBS is phosphorylated by Rho-kinase downstream of Rho in vivo, and suggest that myosin phosphatase and Rho-kinase spatiotemporally regulate the phosphorylation state of Rho-kinase substrates including MLC and ERM family proteins in vivo in a cooperative manner.
Rho相关激酶（Rho激酶）由小GTP酶Rho激活，使肌球蛋白磷酸酶的肌球蛋白结合亚基（MBS）磷酸化，从而在体外使磷酸酶活性失活。Rho激酶被认为通过直接磷酸化以及使肌球蛋白磷酸酶失活来调节包括肌球蛋白轻链（MLC）、ERM（埃兹蛋白/根蛋白/膜突蛋白）家族蛋白和内收蛋白在内的底物的磷酸化状态。在此，我们确定了Rho激酶使MBS磷酸化的位点为苏氨酸-697、丝氨酸-854以及其他几个残基，并制备了特异性识别在丝氨酸-854处磷酸化的MBS的抗体。我们利用该抗体发现，在诱导膜皱褶和细胞迁移的条件下，用十四酰佛波醇-13-乙酸酯（TPA）或肝细胞生长因子（HGF）刺激MDCK上皮细胞会诱导MBS在丝氨酸-854处磷酸化。用据认为会干扰Rho功能的肉毒杆菌C3 ADP核糖基转移酶（C3）或Rho激酶抑制剂对细胞进行预处理可抑制TPA或HGF诱导的MBS磷酸化。TPA刺激增强了MDCK细胞细胞质和膜皱褶区域中磷酸化MBS的免疫反应性。在迁移的MDCK细胞中，磷酸化的MBS以及丝氨酸-19处磷酸化的MLC定位于前沿和后部区域。磷酸化的MBS定位于REF52成纤维细胞的肌动蛋白应力纤维上。显微注射C3或显性负性Rho激酶会破坏应力纤维，并削弱REF52细胞中磷酸化MBS的积累。在胞质分裂期间，磷酸化的MBS、MLC和ERM家族蛋白在分裂沟处积累，并且丝氨酸-854处MBS的磷酸化水平升高。综上所述，这些结果表明MBS在体内是由Rho下游的Rho激酶磷酸化的，并提示肌球蛋白磷酸酶和Rho激酶在体内以协同方式在时空上调节包括MLC和ERM家族蛋白在内的Rho激酶底物的磷酸化状态。
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