title: The degradation of p53 and its major E3 ligase Mdm2 is differentially dependent on the proteasomal ubiquitin receptor S5a.
abstract: p53 and its major E3 ligase Mdm2 are both ubiquitinated and targeted to the proteasome for degradation. Despite the importance of this in regulating the p53 pathway, little is known about the mechanisms of proteasomal recognition of ubiquitinated p53 and Mdm2. In this study, we show that knockdown of the proteasomal ubiquitin receptor S5a/PSMD4/Rpn10 inhibits p53 protein degradation and results in the accumulation of ubiquitinated p53. Overexpression of a dominant-negative deletion of S5a lacking its ubiquitin-interacting motifs (UIM)s, but which can be incorporated into the proteasome, also causes the stabilization of p53. Furthermore, small-interferring RNA (siRNA) rescue experiments confirm that the UIMs of S5a are required for the maintenance of low p53 levels. These observations indicate that S5a participates in the recognition of ubiquitinated p53 by the proteasome. In contrast, targeting S5a has no effect on the rate of degradation of Mdm2, indicating that proteasomal recognition of Mdm2 can be mediated by an S5a-independent pathway. S5a knockdown results in an increase in the transcriptional activity of p53. The selective stabilization of p53 and not Mdm2 provides a mechanism for p53 activation. Depletion of S5a causes a p53-dependent decrease in cell proliferation, demonstrating that p53 can have a dominant role in the response to targeting S5a. This study provides evidence for alternative pathways of proteasomal recognition of p53 and Mdm2. Differences in recognition by the proteasome could provide a means to modulate the relative stability of p53 and Mdm2 in response to cellular signals. In addition, they could be exploited for p53-activating therapies. This work shows that the degradation of proteins by the proteasome can be selectively dependent on S5a in human cells, and that this selectivity can extend to an E3 ubiquitin ligase and its substrate.
authors.affiliation: Division of Cancer Research, Medical Research Institute, Jacqui Wood Cancer Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK.
authors.forename: A
authors.initials: A
authors.lastname: Sparks
authors.affiliation: Division of Cancer Research, Medical Research Institute, Jacqui Wood Cancer Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK.
authors.forename: S
authors.initials: S
authors.lastname: Dayal
authors.affiliation: Division of Cancer Research, Medical Research Institute, Jacqui Wood Cancer Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK.
authors.forename: J
authors.initials: J
authors.lastname: Das
authors.affiliation: Division of Molecular Medicine, College of Life Sciences, University of Dundee, Dundee, UK.
authors.forename: P
authors.initials: P
authors.lastname: Robertson
authors.affiliation: MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK.
authors.forename: S
authors.initials: S
authors.lastname: Menendez
authors.affiliation: Division of Cancer Research, Medical Research Institute, Jacqui Wood Cancer Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK.
authors.forename: M K
authors.initials: MK
authors.lastname: Saville
mesh_terms: D045325:HCT116 Cells; D006801:Humans; D061986:MCF-7 Cells; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism / Q000502:physiology*; D059748:Proteolysis; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism*; D016601:RNA-Binding Proteins; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: C498506:PSMD4 protein, human; D016601:RNA-Binding Proteins; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D046988:Proteasome Endopeptidase Complex