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.
Highlights
The covalent conjugation of ubiquitin to proteins can target them for degradation by the 26S proteasome.[1]
Our investigation of the regulation of p53 by the deubiquitinating enzyme USP5 raised the possibility that there are differences in proteasomal recognition of p53 and Mdm2.49 As a first step to explore this, we have looked at the role of the proteasomal ubiquitin receptor S5a
To investigate the pathway of proteasomal recognition of p53 and Mdm[2], the ubiquitin receptor S5a was knocked down using synthetic small-interferring RNA (siRNA)
Summary
The covalent conjugation of ubiquitin to proteins can target them for degradation by the 26S proteasome.[1] This is composed of two multi-protein complexes:[2,3,4] the 20S core and the 19S regulatory particle (RP). The 19S RP participates in the recruitment of ubiquitinated proteins. It mediates protein deubiquitination, unfolding and translocation, and opens the gated entrance to the 20S core. Ubiquitin-dependent recognition of substrates by the proteasome involves ubiquitin-binding receptors. Studies in lower organisms show that particular ubiquitin receptors participate in the recognition of specific subsets of ubiquitinated proteins.[5,6] Much remains to be learned regarding the identity and precise roles of proteasome-targeting ubiquitin receptors in man. Bortezomib, an inhibitor of the proteolytic activity of the proteasome, is used in the treatment of some cancers.[7,8] Blocking proteasomal recruitment is an alternative mechanism for interfering with protein degradation by the proteasome
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