Characterization of RNF5 - a novel RING finger protein

描述（由申请人提供）：我们对与c-Cbl、BRCAI和Mdm 2的RING结构域共享同源性的RING指蛋白的搜索使我们鉴定了C.优雅RNF-5的过度表达和抑制表明RNF-5在C. elegan的生长发育和对压力的反应。RNF-5在体外表现出泛素连接酶活性，并且在体内经历广泛的泛素化，表明RNF-5参与蛋白质稳定性和/或运输的调节。酵母双杂交筛选鉴定桩蛋白为RNF-5相关蛋白。桩蛋白作为一种支架，用于将分子募集到信号转导复合物中，该复合物在调节细胞粘附、运动和生长控制的外部刺激的处理中是重要的。我们已经证实了RNF-5与桩蛋白的关联和泛素化，表明RNF-5在桩蛋白的调节中起重要作用。此外，原发性人类肿瘤的北方研究揭示了结肠癌和肾癌中RNF-5表达的降低; RNF-5与Ras的共表达阻断了Ras介导的NIH 3 T3细胞的体外转化，这表明RNF-5与肿瘤发生有关。这些观察结果为RNF-5在细胞生长和发育以及转化过程中起重要作用的假设提供了强有力的支持。本研究的主要目的是对RNF-5进行全面的表征，具体如下：（1）。确定暴露于各种形式的压力之前和之后RNF-5表达的模式和调节。（二）.表征RNF-5稳定性的调节以及蛋白激酶在RNF-5表达、定位和E3连接酶活性中的作用（3）。评估RNF-S在桩蛋白定位、稳定性和功能调节中的作用。将确定在正常和应激条件下这种变化对粘着斑和生长因子信号传导的影响。(4)确定转化培养物和原发性肿瘤中RNF-5基因组序列和表达的变化（在DNA、RNA和蛋白质水平）（5）。使用C。线虫的研究，以确定RNF-5的上游和下游组分在dauer形成和生长控制的调节。还将评估RNF-5-桩蛋白相互作用的功能意义。总的来说，成功完成拟议的研究将建立RNF-5在正常细胞维持，应激反应中的作用。人类癌症的发展和进展。

DESCRIPTION (provided by applicant): Our search for RING finger proteins that share homology with RING domains of c-Cbl, BRCAI and Mdm2 led us to identify RNF-5 in C. elegans. Over expression and inhibition of RNF5 pointed to the role of RNF-5 in C. elegan's growth, development and response to stress. RNF-5, exhibit ubiquitin Iigase activity in vitro and are subject to extensive ubiquitination in vivo, suggesting that RNF-5 is involved in regulation of protein stability and/or trafficking. A yeast 2-hybrid screen identified paxillin, as RNF-5 associated protein. Paxillin functions as a scaffold for the recruitment of molecules into a signal transduction complex important in processing of external stimuli that modulate cell adhesion, motility and growth control. We have confirmed RNF-5's association with, and ubiquitination of, paxillin, suggesting that RNF-5 is playing important role in the regulation of paxillin. Furthermore, Northern of primary human tumors revealed decreased RNF-5 expression in colon and renal cancer; co-expression of RNF-5 with Ras blocked Ras-mediated in vitro transformation of NIH3T3 cells -implicating RNF-5 in tumorigenesis. These observations provide strong support for the hypothesis that RNF-5 plays an important role in cell growth and development as well as in transformation processes. The proposed studies aim at comprehensive characterization of RNF-5's as follows: (1). Determine the pattern and regulation of RNF-5 expression before and after exposure to various forms of stress. (2). Characterize the regulation of RNF-5's stability and the role of protein kinases in RNF-5 expression, localization and E3 ligase activities (3). Assess the role of RN F-S in the regulation of paxillin localization, stability and function. The implications of such changes for focal adhesion and growth factor signaling under normal and stressed conditions will be determined.(4) Determine changes in RNF-5 genomic sequence and expression in transformed cultures and in primary tumors (at the level of DNA, RNA and protein) (5). Use C. elegans to identify the upstream and downstream components of RNF-5 in the regulation of dauer formation and growth control. The functional significance of the RNF-5- paxillin interaction will also be assessed. Overall, successful completion of the proposed studies will establish the role of RNF-5 in normal cell maintenance, the stress response. development and Progression of human cancer.