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蛋白的激活受一系列被称为鸟嘌呤核苷酸交换因子(Rho GEF)的酶的控制，因此了解调控Rho蛋白活性的机制对于设计阻止Rho蛋白介导的转化的策略至关重要。Net1是一种针对RhoA亚家族小G蛋白的核Rho全环蛋白。Net1的过表达导致其在细胞质中的错误定位，并刺激结构性的RhoA激活和细胞转化。我们观察到Net1亚型在原发乳腺肿瘤中过度表达，并且Net1蛋白在乳腺癌细胞系中的过度表达显著刺激了它们的增殖。我们还观察到，Net1需要一个C-末端的PDZ结合位点来刺激细胞的增殖和转化，并且这个结合位点介导了与由肿瘤抑制因子Dlg1、相关蛋白Cask1和DNA修饰酶Topo I组成的蛋白质复合体的相互作用。由于Dlg1是致癌病毒(如人乳头瘤病毒和人T细胞白血病病毒1)癌蛋白的必需靶点，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。支持这项研究的假设是，Net1的过度表达导致其在细胞质中的错误定位，并导致RhoA的结构性激活和Dlg1的肿瘤抑制功能。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研究Net1亚型与Dlg1、CaskI和Topo I的相互作用。在目标2中，我们将阐明调节激酶对Net1异构体的磷酸化如何控制Net1与Dlg1的相互作用。在目标3中，我们将检测Net1过表达、与Dlg1、CaskI和Topo I形成复合体，以及Net1磷酸化对乳腺上皮细胞增殖和转化的影响。这些目标的完成将勾勒出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的作用，并确定干预这种疾病的潜在新靶点。

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.