RhoA-mediated Phospholipase D1 signaling is not required for the formation of stress fibers and focal adhesions.
RhoA-mediated Phospholipase D1 signaling is not required for the formation of stress fibers and focal adhesions.
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DOI：
10.1016/j.cellsig.2005.05.027
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发表时间：
2006-04
影响因子：
4.8
通讯作者：
W. Su;P. Chardin;Masakazu Yamazaki;Y. Kanaho;G. Du
中科院分区：
文献类型：
--
作者：
W. Su;P. Chardin;Masakazu Yamazaki;Y. Kanaho;G. Du
小的GTPase RhoA调节广泛的细胞功能，包括转化和细胞骨架重组。大量蛋白质已被确定为RhoA的靶标，但它们在这些过程中的具体作用尚不清楚。磷脂酶D （PLD）几年前被证明是这样一个靶标；我们实验室和其他人最近的研究表明，在两种哺乳动物PLD同工酶中，PLD1而不是PLD2被RhoA激活，并且这种激活是通过体外和体内的直接结合进行的。在这项研究中，我们利用一系列RhoA突变体，利用酵母双杂交系统、共免疫沉淀和PLD体内实验，在RhoA上定义了一个由Asn41、trp58和Asp76残基组成的pld1特异性相互作用位点。结果进一步证实了我们之前的发现，RhoA通过直接相互作用激活PLD1。这些突变体随后被用来研究PLD1在RhoA信号刺激的细胞骨架重组中的作用。我们的研究结果表明，PLD1不是rhoa介导的应力纤维和黏附灶形成所必需的。PLD1信号在rhoa介导的细胞骨架重组中缺乏重要性，这进一步得到了观察结果的支持，使用shRNA方法耗尽PLD1和四环素诱导的PLD1野生型和催化失活突变体在稳定细胞系中的过表达不会改变应激纤维和局灶黏着形成。
The small GTPase RhoA regulates a wide spectrum of cellular functions including transformation and cytoskeletal reorganization. A large number of proteins have been identified as targets of RhoA, but their specific roles in these processes are not clear. Phospholipase D (PLD) was shown to be one such target several years ago; more recent work from our laboratory and others has demonstrated that of the two mammalian PLD isozymes, PLD1 but not PLD2 is activated by RhoA and this activation proceeds through direct binding both in vitro and in vivo. In this study, using a series of RhoA mutants, we have defined a PLD1-specific interacting site on RhoA composed of the residues Asn41, Trp58and Asp76, using the yeast two-hybrid system, co-immunoprecipitation, and a PLD in vivo assay. The results further substantiate our previous finding that RhoA activates PLD1 through direct interaction. These mutants were then used to investigate the role of PLD1 in the cytoskeletal reorganization stimulated by RhoA signaling. Our results show that PLD1 is not required for the RhoA-mediated stress fiber and focal adhesion formation. The lack of importance of PLD1 signaling in RhoA-mediated cytoskeletal reorganization is further supported by the observation that PLD1 depletion using an shRNA approach and tetracycline-induced overexpression of the wild-type and the catalytically inactive mutant of PLD1 in stable cell lines do not alter stress fiber and focal adhesion formation.