Markey Cancer Center, University of Kentucky, Lexington, KY, USA.
Oncogene. 2013 Aug 8;32(32):3754-64. doi: 10.1038/onc.2012.383. Epub 2012 Sep 10.
Rho signaling is increasingly recognized to contribute to invasion and metastasis. In this study, we discovered that metastasis-associated protein S100A4 interacts with the Rho-binding domain (RBD) of Rhotekin, thus connecting S100A4 to the Rho pathway. Glutathione S-transferase pull-down and immunoprecipitation assays demonstrated that S100A4 specifically and directly binds to Rhotekin RBD, but not the other Rho effector RBDs. S100A4 binding to Rhotekin is calcium-dependent and uses residues distinct from those bound by active Rho. Interestingly, we found that S100A4 and Rhotekin can form a complex with active RhoA. Using RNA interference, we determined that suppression of both S100A4 and Rhotekin leads to loss of Rho-dependent membrane ruffling in response to epidermal growth factor, an increase in contractile F-actin 'stress' fibers and blocks invasive growth in three-dimensional culture. Accordingly, our data suggest that interaction of S100A4 and Rhotekin permits S100A4 to complex with RhoA and switch Rho function from stress fiber formation to membrane ruffling to confer an invasive phenotype.
Rho 信号通路被越来越多地认为与侵袭和转移有关。在这项研究中，我们发现转移相关蛋白 S100A4 与 Rhotekin 的 Rho 结合域 (RBD) 相互作用，从而将 S100A4 与 Rho 通路联系起来。谷胱甘肽 S-转移酶下拉和免疫沉淀实验表明，S100A4 特异性且直接结合 Rhotekin RBD，但不与其他 Rho 效应物 RBD 结合。S100A4 与 Rhotekin 的结合依赖于钙，并且使用与活性 Rho 结合的不同残基。有趣的是，我们发现 S100A4 和 Rhotekin 可以与活性 RhoA 形成复合物。通过 RNA 干扰，我们确定抑制 S100A4 和 Rhotekin 均可导致表皮生长因子诱导的 Rho 依赖性细胞膜皱襞减少，增加收缩性 F-肌动蛋白“应激”纤维，并阻止三维培养中的侵袭性生长。因此，我们的数据表明，S100A4 和 Rhotekin 的相互作用允许 S100A4 与 RhoA 形成复合物，并将 Rho 功能从应力纤维形成切换到细胞膜皱襞，从而赋予侵袭表型。
