Balance between activities of Rho kinase and type 1 protein phosphatase modulates turnover of phosphorylation and dynamics of desmin/vimentin filaments
Balance between activities of Rho kinase and type 1 protein phosphatase modulates turnover of phosphorylation and dynamics of desmin/vimentin filaments
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DOI：
10.1074/jbc.274.49.34932
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发表时间：
1999-12-03
影响因子：
4.8
通讯作者：
Inagaki, M
中科院分区：
文献类型：
--
作者：
Inada, H;Togashi, H;Inagaki, M
为了分析 Rho 激酶引起的细胞周期依赖性结蛋白磷酸化，我们开发了特异性识别结蛋白 Thr-16、Thr-75 和 Thr-76 激酶依赖性磷酸化的抗体。使用这些抗体，在有丝分裂晚期 Saos-2 细胞的卵裂沟处特异地观察到结蛋白的磷酸化。然后我们发现用花萼蛋白 A 处理间期细胞显示结蛋白的所有三个位点均发生磷酸化。我们还发现，一种特异性识别由 Rho 激酶在 Ser-71 位点磷酸化的波形蛋白的抗体，在花萼蛋白 A 处理后变得具有免疫反应性。这种花萼蛋白 A 诱导的波形蛋白 Ser-71 位点的间期磷酸化可被 Rho 激酶抑制剂或显性失活 Rho 激酶的表达所阻断。综上所述，我们的结果表明，Rho 激酶不仅在有丝分裂细胞中被激活，而且在间期细胞中也被激活，并且磷酸化中间丝蛋白，尽管由于 1 型蛋白磷酸酶的组成作用，表观磷酸化水平降低至不可检测的水平。 Rho 激酶引起的中间丝蛋白磷酸化与 I 型蛋白磷酸酶引起的去磷酸化之间的平衡可能会影响可溶性池和聚合中间丝之间的中间丝亚基的连续交换。
To analyze the cell cycle-dependent desmin phosphorylation by Rho kinase, we developed antibodies specifically recognizing the kinase-dependent phosphorylation of desmin at Thr-16, Thr-75, and Thr-76. With these antibodies, phosphorylation of desmin was observed specifically at the cleavage furrow in late mitotic Saos-2 cells. We then found that treatment of the interphase cells with calyculin A revealed phosphorylation at all the three sites of desmin. We also found that an antibody, which specifically recognizes vimentin phosphorylated at Ser-71 by Rho kinase, became immunoreactive after calyculin A treatment. This calyculin A-induced interphase phosphorylation of vimentin at Ser-71 was blocked by Rho kinase inhibitor or by expression of the dominant-negative Rho kinase. Taken together, our results indicate that Rho kinase is activated not only in mitotic cells but also interphase ones, and phosphorylates intermediate filament proteins, although the apparent phosphorylation level is diminished to an undetectable level due to the constitutive action of type 1 protein phosphatase. The balance between intermediate filament protein phosphorylation by Rho kinase and dephosphorylation by type I protein phosphatase may affect the continuous exchange of intermediate filament subunits between a soluble pool and polymerized intermediate filaments.