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.
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