Rho GEFs in Cellular Proliferation and Transformation

描述（由申请人提供）：Rho家族小G蛋白激活与癌症的发展和转移进展有关。Rho蛋白的激活是由鸟嘌呤核苷酸交换因子（Rho GEF）家族控制的，因此了解Rho GEF活性的调节机制对于设计阻断Rho蛋白介导转化的策略至关重要。NET1是一个核Rho GEF，对小G蛋白的RhoA亚家族具有特异性。NET1过表达导致其在细胞质中的错误定位，刺激组成型RhoA激活和细胞转化。我们已经观察到NET1亚型在原发性乳腺肿瘤中过表达，并且在乳腺癌细胞系中过表达NET1蛋白会显著刺激其增殖。我们还观察到，NET1需要c端PDZ结合位点来刺激细胞增殖和转化，并且该结合位点介导与由肿瘤抑制因子Dlg1、相关蛋白Cask和DNA修饰酶Topo i组成的蛋白质复合物的相互作用。由于Dlg1是来自致癌病毒（如人乳头瘤病毒和人T细胞白血病病毒1）的癌蛋白的必要靶标，NET1和Dlg1之间的相互作用可能对NET1控制细胞增殖的机制特别重要。本研究的假设是，NET1的过表达导致其在细胞质中的错误定位，并导致RhoA的组成性激活和Dlg1抑瘤功能的抑制。我们将在以下具体目标中解决这一假设。在Aim1中，我们将在体外和乳腺上皮细胞中表征NET1亚型与Dlg1、Cask和Topo I的相互作用。在目标2中，我们将阐明调节激酶对NET1亚型的磷酸化如何控制NET1与Dlg1的相互作用。在Aim 3中，我们将研究NET1过表达、与Dlg1、Cask和Topo I形成复合物以及NET1磷酸化对乳腺上皮细胞增殖和转化的影响。这些目标的完成将描述NET1和Dlg1在控制乳腺上皮细胞转化中的独特和以前未被认识到的作用，并确定干预该疾病的潜在新靶点。

DESCRIPTION (provided by applicant): Rho family small G protein activation has been implicated in the development of cancer and in metastatic progression. Rho protein activation is controlled by a family of enzymes known as guanine nucleotide exchange factors (Rho GEFs), so understanding the mechanisms regulating Rho GEF activity is critical to devising strategies to block Rho protein-mediated transformation. NET1 is a nuclear Rho GEF that is specific for the RhoA subfamily of small G proteins. Overexpression of NET1 results in its mislocalization in the cytoplasm, and stimulates constitutive RhoA activation and cell transformation. We have observed that NET1 isoforms are overexpressed in primary breast tumors, and that overexpression of NET1 proteins in breast cancer cell lines dramatically stimulates their proliferation. We have also observed that NET1 requires a C-terminal PDZ binding site to stimulate cell proliferation and transformation, and that this binding site mediates interaction with a protein complex consisting of the tumor suppressor Dlg1, the related protein Cask, and the DNA modifying enzyme Topo I. Since Dlg1 is a requisite target for oncoproteins from cancer causing viruses such as the human papilloma virus and the human T cell leukemia virus 1, the interaction between NET1 and Dlg1 is likely to be especially important to the mechanism by which NET1 controls cell proliferation. The hypothesis driving the proposed research is that overexpression of NET1 causes its mislocalization in the cytoplasm, and results in the constitutive activation of RhoA and an inhibition of the tumor suppressor function of Dlg1. We will address this hypothesis in the following specific aims. In Aim1 we will characterize the interaction of NET1 isoforms with Dlg1, Cask and Topo I in vitro and in breast epithelial cells. In Aim 2 we will elucidate how the phosphorylation of NET1 isoforms by regulatory kinases controls the interaction of NET1 with Dlg1. In Aim 3 we will examine the effects of NET1 overexpression, complex formation with Dlg1, Cask and Topo I, and NET1 phosphorylation on breast epithelial cell proliferation and transformation. Completion of these aims will delineate a unique and previously unrecognized role for NET1 and Dlg1 in controlling breast epithelial cell transformation, and identify potential new targets for intervention in this disease.