MDM2 Binding Induces a Conformational Change in p53 That Is Opposed by Heat-shock Protein 90 and Precedes p53 Proteasomal Degradation

Mark Sasaki,Linghu Nie,Carl G Maki

doi:10.1074/jbc.m610514200

Abstract

p53 protein conformation is an important determinant of its localization and activity. Changes in p53 conformation can be monitored by reactivity with wild-type conformation-specific (pAb-1620) or mutant conformation-specific (pAb-240) p53 antibodies. Wild-type p53 accumulated in a mutant (pAb-240 reactive) form when its proteasome-dependent degradation was blocked during recovery from stress treatment and in cells co-expressing p53 and MDM2. This suggests that conformational change precedes wild-type p53 degradation by the proteasome. MDM2 binding to the p53 N terminus could induce a conformational change in wild-type p53. Interestingly, this conformational change was opposed by heat-shock protein 90 and did not require the MDM2 RING-finger domain and p53 ubiquitination. Finally, ubiquitinated p53 accumulated in a pAb-240 reactive form when p53 degradation was blocked by proteasome inhibition, and a p53-ubiquitin fusion protein displayed a mutant-only conformation in MDM2-null cells. These results support a model in which MDM2 binding induces a conformational change that is opposed by heat-shock protein 90 and precedes p53 ubiquitination. The covalent attachment of ubiquitin may "lock" p53 in a mutant conformation in the absence of MDM2-binding and prior to its degradation by the proteasome.

Highlights

Wild-type p53 was markedly shifted toward a mutant conformation when cells co-expressing p53 and MDM2 were treated with Heat-shock protein 90 (Hsp90) inhibitors (17-AAG, GA, radicicol)
This effect required the MDM2 binding domain in the p53 N terminus and the p53-binding domain in the MDM2 N terminus but did not require the MDM2 RING-finger domain that is necessary for its ubiquitination activity
Overexpression of Hsp90 partially blocked the change in p53 conformation seen when cells expressing p53 and MDM2 were treated with GA

Summary

EXPERIMENTAL PROCEDURES

Plasmid DNAs—DNA encoding p53 ⌬42N has been described [23] and was from Peter Howley (Harvard Medical School). FLAG-tagged wild-type p53 has been described [24] and was from Zhimin Yuan (Harvard School of Public Health). This DNA contains wild-type p53 sequences cloned into BamHI and XbaI sites downstream of the FLAG epitope. DNA encoding wild-type MDM2 and MDM2 ⌬p53BD were from Steve Grossman (University of Massachusetts Medical School). MDM2 ⌬RING DNA encodes residues 6 –339 [27]. DNA encoding FLAG-tagged Hsp was provided by Len Neckers (NCI, National Institutes of Health). The proteasome inhibitor MG132 (Boston Biochem) was added to a final concentration of 30 ␮M 18 h after transfection, and the cells were incubated for an additional 5–7 h before harvesting.

RESULTS

Findings

DISCUSSION