Suppression of the deubiquitinating enzyme USP5 causes the accumulation of unanchored polyubiquitin and the activation of p53.
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.
Dayal
Saurabh
S
CR-UK Cell Transformation Research Group, Department of Surgery and Molecular Oncology, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland.
Sparks
Alison
A
Jacob
Jimmy
J
Allende-Vega
Nerea
N
Lane
David P
DP
Saville
Mark K
MK
D019943:Amino Acid Substitution; D045744:Cell Line, Tumor; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006801:Humans; D020125:Mutation, Missense; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism; D051736:Proto-Oncogene Proteins c-mdm2 / Q000235:genetics / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology*
C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D010450:Endopeptidases; D046988:Proteasome Endopeptidase Complex; C091939:ubiquitin isopeptidase