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首页> 外文期刊>Molecular and Cellular Biology >The p160 RhoA-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton.
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The p160 RhoA-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton.

机译：p160 RhoA结合激酶ROKα是激酶家族的成员，参与细胞骨架的重组。

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The GTPase RhoA has been implicated in various cellular activities, including the formation of stress fibers, motility, and cytokinesis. We recently reported on a p150 serine/threonine kinase (termed ROK alpha) binding RhoA only in its active GTP-bound state and on its cDNA; introduction of RhoA into HeLa cells resulted in translocation of the cytoplasmic kinase to plasma membranes, consistent with ROK alpha being a target for RhoA (T. Leung, E. Manser, L. Tan, and L. Lim, J. Biol. Chem. 256:29051-29054, 1995). Reanalysis of the cDNA revealed that ROK alpha contains an additional N-terminal region. We also isolated another cDNA which encoded a protein (ROK beta) with 90% identity to ROK alpha in the kinase domain. Both ROK alpha and ROK beta, which had a molecular mass of 160 kDa, contained a highly conserved cysteine/histidine-rich domain located within a putative pleckstrin homology domain. The kinases bound RhoA, RhoB, and RhoC but not Rac1 and Cdc42. The Rho-binding domain comprises about 30 amino acids. Mutations within this domain caused partial or complete loss of Rho binding. The morphological effects of ROK alpha were investigated by microinjecting HeLa cells with DNA constructs encoding various forms of ROK alpha. Full-length ROK alpha promoted formation of stress fibers and focal adhesion complexes, consistent with its being an effector of RhoA. ROK alpha truncated at the C terminus promoted this formation and also extensive condensation of actin microfilaments and nuclear disruption. The proteins exhibited protein kinase activity which was required for stress fiber formation; the kinase-dead ROK alpha K112A and N-terminally truncated mutants showed no such promotion. The latter mutant instead induced disassembly of stress fibers and focal adhesion complexes, accompanied by cell spreading. These effects were mediated by the C-terminal region containing Rho-binding, cysteine/histidine-rich, and pleckstrin homology domains. Thus, the multidomained ROK alpha appears to be involved in reorganization of the cytoskeleton, with the N and C termini acting as positive and negative regulators, respectively, of the kinase domain whose activity is crucial for formation of stress fibers and focal adhesion complexes.
机译：GTPase RhoA与多种细胞活动有关，包括应激纤维的形成，运动性和胞质分裂。我们最近报道了一个p150丝氨酸/苏氨酸激酶（称为ROKα）仅在其活性GTP结合状态及其cDNA上结合RhoA。将RhoA引入HeLa细胞后，胞质激酶易位至质膜，这与ROKα是RhoA的靶标一致（T.Leung，E.Manser，L.Tan和L.Lim，J.Biol。 256：29051-29054，1995）。 cDNA的重新分析表明，ROK alpha包含一个额外的N端区域。我们还分离了另一种cDNA，它编码一种蛋白（ROK beta），与激酶域中的ROK alpha具有90％的同一性。分子量为160 kDa的ROK alpha和ROK beta都包含一个高度保守的富含半胱氨酸/组氨酸的域，位于一个假定的pleckstrin同源域内。激酶结合RhoA，RhoB和RhoC，但不结合Rac1和Cdc42。 Rho结合结构域包含约30个氨基酸。该结构域内的突变导致Rho结合的部分或全部丧失。通过将HeLa细胞与编码各种形式的ROKα的DNA构建体一起显微注射来研究ROKα的形态学效应。全长ROKα促进了应力纤维和粘着斑复合物的形成，这与其作为RhoA的效应子一致。在C末端截短的ROKα促进了这种形成，并且还导致肌动蛋白微丝的广泛凝结和核破坏。蛋白质显示出应力纤维形成所必需的蛋白激酶活性。激酶死亡的ROK alpha K112A和N端截短的突变体均未显示出这种促进作用。后者突变体反而引起应力纤维和粘着斑复合物的分解，并伴随细胞扩散。这些作用是由包含Rho结合，富含半胱氨酸/组氨酸和pleckstrin同源结构域的C末端区域介导的。因此，多域ROKα似乎参与了细胞骨架的重组，其中N和C末端分别充当了激酶结构域的正向和负向调节剂，其活性对于形成应激纤维和粘着斑复合物至关重要。

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