Characterization of RNF5 - a novel RING finger protein

描述（由申请人提供）：我们寻找与c-Cbl， BRCAI和Mdm2的RING结构域具有同源性的RING指蛋白，从而鉴定出秀丽隐杆线虫中的RNF-5。RNF5的过表达和抑制表明了RNF5在秀丽隐杆线虫生长发育和应激反应中的作用。RNF-5在体外表现出泛素酶活性，并在体内受到广泛的泛素化，这表明RNF-5参与了蛋白质稳定性和/或运输的调节。酵母2杂交筛选证实paxillin为RNF-5相关蛋白。帕罗西林的功能是作为一种支架，将分子募集到信号转导复合体中，在处理调节细胞粘附、运动和生长控制的外部刺激中发挥重要作用。我们已经证实了RNF-5与paxillin的关联和泛素化，表明RNF-5在paxillin的调控中起重要作用。此外，人类原发肿瘤的北部显示结肠癌和肾癌中RNF-5表达降低；RNF-5与Ras共表达可阻断Ras介导的NIH3T3细胞体外转化，提示RNF-5参与肿瘤发生。这些观察结果有力地支持了RNF-5在细胞生长发育和转化过程中发挥重要作用的假设。拟开展的研究旨在对RNF-5进行如下综合表征：(1)。确定各种应激前后RNF-5表达的模式和调控。(2)。表征RNF-5稳定性的调控以及蛋白激酶在RNF-5表达、定位和E3连接酶活性中的作用(3)。评估rsf在帕罗西林定位、稳定性和功能调控中的作用。在正常和应激条件下，这种变化对局灶黏附和生长因子信号的影响将被确定。(4)确定转化培养物和原发肿瘤中RNF-5基因组序列和表达的变化（在DNA、RNA和蛋白质水平上）(5)。利用线虫鉴定RNF-5在水形成调控和生长控制中的上下游组分。RNF-5- paxillin相互作用的功能意义也将被评估。总之，这些研究的成功完成将确定RNF-5在正常细胞维持和应激反应中的作用。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.