Characterization of RNF5 - a novel RING finger protein

描述(申请人提供)：我们搜索与c-Cb1、BRCA1和MDM2的环域同源的环指蛋白，从而在线虫中鉴定出RNF-5。RNF5的过度表达和抑制表明RNF5在雅致毛虫的生长发育和对胁迫的反应中起着重要的作用。RNF-5在体外表现出泛素连接酶活性，在体内可以被广泛的泛素化，这表明RNF-5参与了蛋白质稳定性和/或运输的调节。酵母菌双杂交鉴定为Paxlin，为RNF-5相关蛋白。巴西林作为一种支架，用于将分子重新招募到信号转导复合体中，该复合体在处理调节细胞黏附、运动和生长控制的外部刺激方面非常重要。我们证实了RNF5‘S与巴西林的结合和泛素化，表明RNF5在巴西林的调控中起着重要的作用。此外，人原发肿瘤的Northern研究表明，RNF-5在结肠癌和肾癌中的表达降低；RNF-5与RAS的共表达阻断了RAS介导的NIH3T3细胞的体外转化，提示RNF-5参与了肿瘤的发生。这些观察结果有力地支持了RNF-5在细胞生长发育以及转化过程中发挥重要作用的假设。本研究旨在对重组人神经营养因子-5‘S进行全面的表征如下：(1)确定RNF-5在暴露于各种形式的应激前后的表达模式和调节。(2)。研究重组核因子-5‘S稳定性的调节以及蛋白激酶在核因子-5的表达、定位和E3连接酶活性中的作用(3)。评估RN F-S在调节巴西林的定位、稳定性和功能中的作用。这些变化在正常和应激条件下对局灶性黏附和生长因子信号的影响将被确定。(4)在转化培养物和原发肿瘤中(在DNA、RNA和蛋白质水平)确定RNF-5基因组序列和表达的变化(5)。利用线虫鉴定RNF-5在调节Dauer形成和生长控制中的上游和下游成分。RNF-5-paxlin相互作用的功能意义也将被评估。总体而言，成功完成拟议的研究将确定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.