Phosphorylation of myosin-binding subunit (MBS) of myosin phosphatase by Rho-kinase in vivo.
Phosphorylation of myosin-binding subunit (MBS) of myosin phosphatase by Rho-kinase in vivo.
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Rho-kinase in Vivo的肌球蛋白磷酸酶的肌球蛋白结合亚基（MB）的磷酸化。
DOI：
10.1083/jcb.147.5.1023
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发表时间：
1999-11-29
期刊：
影响因子：
--
通讯作者：
Kaibuchi K
中科院分区：
文献类型：
--
作者：
Kawano Y;Fukata Y;Oshiro N;Amano M;Nakamura T;Ito M;Matsumura F;Inagaki M;Kaibuchi K
Rho相关激酶(Rho-kinaseRho)被小分子GTP酶Rho激活，使肌球蛋白磷酸酶的肌球蛋白结合亚单位(MBS)磷酸化，从而在体外使磷酸酶失活。Rho-Kinase被认为通过直接磷酸化和肌球蛋白磷酸酶失活来调节底物包括肌球蛋白轻链(MLC)、ERM(ezrin/Radioxin/moesin)家族蛋白和内收蛋白的磷酸化状态。在此，我们确定了Rho-Kinase磷酸化MBS的位点为Thr-697、Ser-854和几个残基，并制备了特异性识别Ser-854处的MBS的抗体。用该抗体刺激MDCK上皮细胞，发现TPA或肝细胞生长因子(HGF)刺激MDCK上皮细胞可诱导Ser-854上MBS的磷酸化，从而诱导细胞膜皱缩和细胞迁移。用干扰Rho功能的肉毒杆菌C3ADP核糖基转移酶(C3)或Rho激酶抑制剂预处理细胞，可抑制TPA或HGF诱导的MBS磷酸化。TPA刺激可增强MDCK细胞胞浆和膜皱褶区内磷酸化MBS的免疫反应性。在迁移的MDCK细胞中，磷酸化的MBS和Ser-19的磷酸化的MLC位于细胞的前缘和后部。在REF52成纤维细胞中，磷酸化的MBS定位于肌动蛋白应激纤维上。微量注射C3或显性阴性的Rho-Kinase可破坏应激纤维，并减弱磷酸化MBS在REF52细胞中的积聚。在胞质分裂过程中，MBS、MLC和ERM家族蛋白在卵裂沟处积累，MBS在Ser-854处的磷酸化水平增加。综上所述，这些结果表明MBS在体内被Rho下游的Rho-Kinase磷酸化，并提示肌球蛋白磷酸酶和Rho-Kinase在体内以协同的方式调节Rho-Kinase底物包括MLC和ERM家族蛋白的磷酸化状态。
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.
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影响因子：
4.8
作者：
Chihara, K;Amano, M;Kaibuchi, K
通讯作者：
Kaibuchi, K
影响因子：
4.8
作者：
Amano, M;Ito, M;Kaibuchi, K
通讯作者：
Kaibuchi, K
影响因子：
3.5
作者：
Ishizaki, T;Naito, M;Narumiya, S
通讯作者：
Narumiya, S
影响因子：
2.1
作者：
Amano, M;Chihara, K;Kaibuchi, K
通讯作者：
Kaibuchi, K
影响因子：
64.5
作者：
Cachero, TG;Morielli, AD;Peralta, EG
通讯作者：
Peralta, EG