研究v-HRas 泛素化後的作用

Abstract

Ras family proteins, a class of small GTP-binding proteins, contain effector domain for its signal transduction and the C-terminal hypervariable region that regulate their plasma membrane localization. Ras regulate many biological functions, including proliferation, differentiation, and survival. Ubiquitination of Ras has been implicated in its protein stability and cellular localization. Recent studies further indicate that mono─ubiquitination increases Ras protein activity by Ras 117 and 147 lysine (K). However, the function of other lysine residues on Ras protein is still unclear. In this study, we searched for potential ubiquitination residues on vH-Ras and studied their biological role. We first generated K-R (arginine) mutation in the C-terminal hypervariable region on H-Ras, i.e. 167K, 170K and 185K (designated as C3KR). Because 42K is located between Switch I and II domain, we also generated mutation in the N-terminal region, i.e.5K, 42K and 147K (designated as N3KR). Unexpectedly, instead of reducing protein ubiquitination, our data indicated that C3KR mutation exhibited more poly-ubiquitinated H-Ras than in wild types, and resulted in v-HRAS degradation. Also, we found that v-HRas degradation in C3KR and N3KR mutant types was higher than in wild types with cycloheximide (CHX) treatment. Interestingly, EPS8 and ERK activity was downregulation in H-Ras knockdown T-24 cells. To clarify how H-Ras affects Eps8 and ERK activity, we generated C3KR in HeLa cells to confirm this question. The data show that C3KR can promote Eps8 degradation and reduce ERK activity. Moreover, we found that C3KR mutant form also reduced cell colony numbers in HeLa cells and stable H-Ras knockdown T-24 cells growing in soft agar. Taken together, our results point out C-terminal lysine residues regulated the protein stability of H-Ras and Eps8 and in turn affected colony formation.