Abstract
Both p53 and its repressor Mdm2 are subject to ubiquitination and proteasomal degradation. We show that knockdown of the deubiquitinating enzyme USP5 (isopeptidase T) results in an increase in the level and transcriptional activity of p53. Suppression of USP5 stabilizes p53, whereas it has little or no effect on the stability of Mdm2. This provides a mechanism for transcriptional activation of p53. USP5 knockdown interferes with the degradation of ubiquitinated p53 rather than attenuating p53 ubiquitination. In vitro studies have shown that a preferred substrate for USP5 is unanchored polyubiquitin. Consistent with this, we observed for the first time in a mammalian system that USP5 makes a major contribution to Lys-48-linked polyubiquitin disassembly and that suppression of USP5 results in the accumulation of unanchored polyubiquitin chains. Ectopic expression of a C-terminal mutant of ubiquitin (G75A/G76A), which also causes the accumulation of free polyubiquitin, recapitulates the effects of USP5 knockdown on the p53 pathway. We propose a model in which p53 is selectively stabilized because the unanchored polyubiquitin that accumulates after USP5 knockdown is able to compete with ubiquitinated p53 but not with Mdm2 for proteasomal recognition. This raises the possibility that there are significant differences in proteasomal recognition of p53 and Mdm2. These differences could be exploited therapeutically. Our study reveals a novel mechanism for regulation of p53 and identifies USP5 as a potential target for p53 activating therapeutic agents for the treatment of cancer.
Highlights
5030 JOURNAL OF BIOLOGICAL CHEMISTRY protease activity of the proteasome, is used in cancer therapy
USP5 Knockdown Activates p53—To identify deubiquitinating enzymes (DUBs) whose suppression activates p53, we carried out a screen using a library consisting of pools of 4 shRNA targeting 50 members of the DUB family [39]
We looked at the effect of USP5 knockdown on the level of p53 and Mdm2
Summary
Cell Culture—ARN8 cells were derived from wild-type p53expressing A375 human melanoma cells by stable transfection with a p53-responsive reporter construct (RGC⌬Fos-LacZ) [40]. After transfection with synthetic siRNA duplexes, cells were fixed with ice-cold methanol-acetone and incubated with primary antibodies followed by Alexa Fluor 488 dye-conjugated anti-mouse and Alexa Fluor 594 dye-conjugated anti-rabbit secondary antibodies (Invitrogen) as described previously [45]. After transfection with siRNA duplexes, cells were lysed in SDS immunoprecipitation lysis buffer: phosphate-buffered saline containing 1% Nonidet P-40, 1% SDS, 5 mM EDTA, 10 mM iodoacetamide, 1 mM DTT, and protease inhibitor mixture (Roche Applied Science). The samples normalized for protein were diluted 10-fold with immunoprecipitation wash buffer: phosphate-buffered saline containing 1% Nonidet P-40, 5 mM EDTA, 1 mM DTT, and protease inhibitor mixture (Roche Applied Science). In Vitro Deubiquitination Assay—After transfection with siRNA, cells were lysed in Nonidet P-40 buffer: 0.1% Nonidet P-40, 50 mM Tris, pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM DTT protease inhibitor mixture (Roche Applied Science).
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