Characterization of a novel nuclear Rab protein

描述(申请人提供)：RAB GTP结合蛋白是RAS超基因家族的成员。RAB蛋白通常参与蛋白质的转运，并决定细胞内膜转运步骤的特异性。我们已经发现了Rab蛋白家族中的一个新成员，我们将其命名为Nu-Rab，因为它具有独特的核定位。NU-Rab mRNA在所研究的大多数癌细胞系中都高度表达，并且与其匹配的正常组织相比，在60%以上的人类原发乳腺癌中过表达。GFP和Myc标记的Nu-Rab主要定位于细胞核，也有一些分布在细胞质的亚细胞室。使用本实验室产生的Nu-Rab抗体进行免疫组化也检测到内源性Nu-Rab主要定位于细胞核。NU-Rab的表达被紫外线(UV)、thapsigarin和舒林酸硫化物等凋亡诱导剂下调。PI3-激酶抑制剂LY294002、PI3-K磷酸酶PTEN和显性负性突变体-Akt也下调Nu-Rab的mRNA表达，提示Nu-Rab是PI3-K/Akt信号通路的潜在下游靶点。我们现在建议进一步研究Nu-Rab在细胞生长和/或存活中的作用，并阐明其在人类乳腺癌中过表达的分子基础。在具体目标1中，我们将分析更大的新鲜冷冻和石蜡包埋的组织标本池，以评估Nu-Rab在mRNA和蛋白质水平的表达。在特定的目标2中，我们将启动功能研究，以探索野生型和GTP结合的Nu-Rab蛋白突变体在致癌转化中的作用。我们还将探讨Nu-Rab过表达或缺乏是否会影响遗传毒性和非遗传毒性药物诱导的细胞凋亡。将启动特定目标3的研究，以确定潜在的Nu-Rab效应器/调节蛋白，以探索Nu-Rab是否在核质蛋白运输中发挥作用，并研究Nu-Rab调节的潜在信号事件。这些是探索性/发育性研究，一旦完成，将产生足够的新数据和试剂，为未来深入研究这种新型三磷酸腺苷结合蛋白在乳腺癌发生和/或进展中的分子机制(S)奠定基础。

DESCRIPTION (provided by applicant): Rab GTP-binding proteins are members of the Ras supergene family. Rab proteins are generally involved in protein transport/trafficking and determine the specificity of membrane transport steps within cell. We have identified a novel member of the Rab protein family, which we have named Nu-Rab, for its unique nuclear localization. Nu-Rab mRNA is highly expressed in most of cancer cell lines examined, and it is overexpressed in more than 60% of human primary breast cancers when compared to their matching normal tissues. The GFP- and Myc-tagged Nu-Rab mainly localizes to nucleus with some distribution noted in the cytoplasmic subcellular compartment. Immnunochemistry using Nu-Rab antibodies generated in our laboratory also detects that the endogenous Nu-Rab predominantly localizes to nucleus. Nu-Rab expression is down-regulated by apoptosis inducing agents such as ultraviolet radiation (UV), thapsigargin and sulindac sulfide. The PI3-kinase inhibitor LY294002, PI3-K phosphotase PTEN and dominant-negative mutant-Akt also down-regulate Nu-Rab mRNA expression, suggesting that Nu-Rab is a potential downstream target of the PI3-K/Akt signaling pathway. We are now proposing to further investigate the role of Nu-Rab in cell growth and/or survival and to elucidate the molecular basis of its overexpression in human breast cancer. In specific aim 1, we will analyze a larger pool of fresh-frozen and paraffin-embedded tissue specimens to evaluate the expression of Nu-Rab at the mRNA and protein levels. In specific aim 2, we will initiate functional studies to explore the effect of wild type-and GTP-binding mutants of Nu-Rab protein in oncogenic transformation. We will also explore whether Nu-Rab overexpression or deficiency effects apoptosis induced by genotoxic and non-genotoxic agents. Studies in specific aim 3 will be initiated to identify potential Nu-Rab effectors/regulator proteins in order to explore whether Nu-Rab plays a role in nuclear-cytoplasmic protein trafficking and study potential signaling events modulated by Nu-Rab. These are exploratory/developmental studies that, upon completion, will generate sufficient new data and reagents that will form the basis of future in-depth studies investigating the molecular mechanisms (s) of action of this novel GTP-binding protein in breast cancer development and/or progression.