MDM2 promotes proteasomal degradation of p21Waf1 via a conformation change

Abstract

MDM2 plays a major role in cancer development and progression via both p53-dependent and -independent functions. One of its p53-independent functions is the induction of the ubiquitin-independent proteasomal degradation of p21Waf1. The present study was designed to characterize the mechanism(s) by which MDM2 induces p21Waf1 degradation. We first determined the regions of MDM2 required for p21Waf1 degradation using pulldown assays and Western blotting and then examined the mechanisms using limited proteolysis and fluorescence resonance energy transfer assays. We found that the MDM2-p21Waf1 interaction depended on the central domain of MDM2 and that nuclear localization of both proteins was necessary for p21 Waf1 degradation. Specifically, amino acids 226-250 of MDM2were required for p21Waf1 binding and degradation, and amino acids 251-260 were necessary for p21Waf1 degradation. The latter region induced a conformation change in p21Waf1, increasing its interaction with the C8 subunit of the proteasome, leading to its degradation. When MDM2 lacked either segment (aa 226-250 or aa 251-260), its capacity to promote p21Waf1 degradation and cell cycle progression was significantly reduced. In summary, the present study elucidated a previously unknown mechanism by which MDM2 promotes the degradation of an intact protein (p21Waf1) through an ubiquitin-independent proteasomal degradation pathway. Because MDM2 also increases the degradation of other proteins in a ubiquitinindependent manner, this mechanism may underlie part of its tumorigenic properties. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.