Phosphorylation of the myosin-binding subunit of myosin phosphatase by Raf-1 and inhibition of phosphatase activity
Phosphorylation of the myosin-binding subunit of myosin phosphatase by Raf-1 and inhibition of phosphatase activity
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DOI：
10.1074/jbc.m106343200
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发表时间：
2002-01-25
影响因子：
4.8
通讯作者：
Kasid, U
中科院分区：
文献类型：
--
作者：
Broustas, CG;Grammatikakis, N;Kasid, U
Raf-1丝氨酸/苏氨酸蛋白激酶在细胞存活、增殖和迁移中发挥重要作用；然而，Raf-1 在不同细胞过程中的具体靶点尚未明确定义。肌球蛋白磷酸酶活性对于细胞骨架重组、胞质分裂和细胞运动的调节至关重要。在这里，我们描述了 Raf-1 与肌球蛋白磷酸酶的关联以及 Raf-1 对肌球蛋白磷酸酶调节肌球蛋白结合亚基 (MBS) 的磷酸化。用佛波醇 12-肉豆蔻酸酯 13-乙酸酯处理细胞已被证明可以刺激 Raf-1 蛋白激酶。为了确定 Raf-1 酶促激活对 MBS 磷酸化的影响，用 FLAG 标记的全长 Raf-1 瞬时转染 COS-1 细胞。与未处理的细胞相比，在从佛波醇 12-肉豆蔻酸酯 13-乙酸酯处理的细胞中免疫沉淀的 FLAG-Raf-1 存在的情况下，纯化的谷胱甘肽 S-转移酶标记的截短 MBS 蛋白（氨基酸 654-880）的磷酸化显着升高（类似于 3.0 倍）。使用连续激酶磷酸酶测定和磷酸化肌球蛋白轻链作为磷酸酶反应中的底物，我们发现 Raf-1 相关蛋白磷酸酶特异性活性受到抑制（没有和有腺苷 5'-O-(3-硫代三磷酸) 的相对磷酸酶活性：分别为 100 和相似于 30%）。此前，电离辐射已被证明可激活 Raf-1（Kasid, U.、Suy, S.、Dent, P.、Ray, S.、Whiteside, T. L. 和 Sturgill, T. W. (1996) Nature 382, 813-816）。细胞暴露于电离辐射导致 Raf-1 与 MBS 的关联增加（与未辐射对照相比增加 3-6 倍），并抑制 Raf-1 相关蛋白磷酸酶特异性活性（无电离辐射和有电离辐射的相对磷酸酶活性：分别为 100 和相似于 54%）。我们的研究确定 MBS 是 Raf-1 的新底物，并暗示 Raf-1 在调节涉及肌球蛋白磷酸酶活性的途径中的作用。
Raf-1 serine/threonine protein kinase plays an important role in cell survival, proliferation, and migration; however, the specific targets of Raf-1 in diverse cellular processes are not clearly defined. Myosin phosphatase activity is critical to the regulation of cytoskeletal reorganization, cytokinesis, and cell motility. Here, we describe the association of Raf-1 with myosin phosphatase and phosphorylation of the regulatory myosin-binding subunit (MBS) of myosin phosphatase by Raf-1. Treatment of cells with phorbol 12-myristate 13-acetate has been shown to stimulate Raf-1 protein kinase. To determine the effect of enzymatic activation of Raf-1 on MBS phosphorylation, COS-1 cells were transiently transfected with FLAG-tagged full-length Raf-1. A significantly higher phosphorylation of purified glutathione S-transferase-tagged truncated MBS protein (amino acids 654-880) occurred in the presence of FLAG-Raf-1 immunoprecipitated from phorbol 12-myristate 13-acetate-treated cells compared with untreated cells (similar to3.0-fold). Using a sequential kinase-phosphatase assay and phosphorylated myosin light chain as substrate in the phosphatase reaction, we showed that Raf-1-associated protein phosphatase-specific activity was inhibited (relative phosphatase activity without and with adenosine 5'-O-(3-thiotriphosphate): 100 and similar to30%, respectively). Previously, ionizing radiation has been shown to activate Raf-1 (Kasid, U., Suy, S., Dent, P., Ray, S., Whiteside, T. L., and Sturgill, T. W. (1996) Nature 382, 813-816). Exposure of cells to ionizing radiation resulted in the increased association of Raf-1 with MBS (3-6-fold versus unirradiated control) and inhibition of Raf-1-associated protein phosphatase-specific activity (relative phosphatase activity without and with ionizing radiation: 100 and similar to54%, respectively). Our studies identify MBS as a new substrate of Raf-1 and implicate a role for Raf-1 in the regulation of pathways involving myosin phosphatase activity.