The degradation of p53 and its major E3 ligase Mdm2 is differentially dependent on the proteasomal ubiquitin receptor S5a.

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

The lysine48-based polyubiquitin chain proteasomal signal: not a single child anymore

The conjugation of ubiquitin (Ub) to proteins is involved in the regulation of many processes. The modification serves as a recognition element in trans, in which downstream effectors bind to the modified protein and determine its fate and/or function. A polyUb chain that is linked through internal lysine (Lys)-48 of Ub and anchored to an internal Lys residue of the substrate has become the accepted "canonical" signal for proteasomal targeting and degradation. However, recent studies show that the signal is far more diverse and that chains based on other internal linkages, as well as linear or heterologous chains made of Ub and Ub-like proteins and even monoUb, are recognized by the proteasome. In addition, chains linked to residues other than internal Lys were described, all challenging the current paradigm

KPC1-mediated ubiquitination and proteasomal processing of NF-κB1 p105 to p50 restricts tumor growth

NF-{kappa}B is a key transcriptional regulator involved in inflammation and cell proliferation, survival, and transformation. Several key steps in its activation are mediated by the ubiquitin (Ub) system. One uncharacterized step is limited proteasomal processing of the NF-{kappa}B1 precursor p105 to the p50 active subunit. Here, we identify KPC1 as the Ub ligase (E3) that binds to the ankyrin repeats domain of p105, ubiquitinates it, and mediates its processing both under basal conditions and following signaling. Overexpression of KPC1 inhibits tumor growth likely mediated via excessive generation of p50. Also, overabundance of p50 downregulates p65, suggesting that a p50-p50 homodimer may modulate transcription in place of the tumorigenic p50-p65. Transcript analysis reveals increased expression of genes associated with tumor-suppressive signals. Overall, KPC1 regulation of NF-{kappa}B1 processing appears to constitute an important balancing step among the stimulatory and inhibitory activities of the transcription factor in cell growth control

The N-terminal domain of MyoD is necessary and sufficient for its nuclear localization-dependent degradation by the ubiquitin system

A growing number of proteins, including the myogenic transcription factor MyoD, are targeted for proteasomal degradation after N-terminal ubiquitination (NTU) where the first ubiquitin moiety is conjugated to the N-terminal residue rather than to an internal lysine. NTU might be essential in targeting both lysine-containing and naturally occurring lysine-less proteins such as p16INK4a and p14ARF; however, the mechanisms that underlie this process are largely unknown. Specifically, the recognition motif(s) in the target substrates and the ubiquitin ligase(s) that catalyze NTU are still obscure. Here we show that the N-terminal domain of MyoD is critical for its degradation and that its destabilizing effect depends on nuclear localization of the protein. Deletion of the first 15 aa of MyoD blocked completely its lysine-independent degradation. Importantly, transfer of the first 30 N-terminal residues of MyoD to GFP destabilized this otherwise stable protein, and, here too, targeting for degradation depended on localization of the protein to the nucleus. Deletion of the N-terminal domain of lysine-less MyoD did not abolish completely ubiquitination of the protein, suggesting that this domain may be required for targeting the protein also in a postubiquitination step. Interestingly, NTU is evolutionarily conserved: in the yeast Saccharomyces cerevisiae lysine-less (LL) MyoD is degraded in a ubiquitin-, N-terminal domain-, and nuclear localization-dependent manner. Taken together, our data suggest that a short N-terminal segment of MyoD is necessary and sufficient to render MyoD susceptible for ubiquitin- and nuclear-dependent degradation

Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis

Ubiquitin modification is mediated by a large family of specificity determining ubiquitin E3 ligases. To facilitate ubiquitin transfer, RING E3 ligases bind both substrate and a ubiquitin E2 conjugating enzyme linked to ubiquitin via a thioester bond, but the mechanism of transfer has remained elusive. Here we report the crystal structure of the dimeric RING of RNF4 in complex with E2 (UbcH5a) linked by an isopeptide bond to ubiquitin. While the E2 contacts a single protomer of the RING, ubiquitin is folded back onto the E2 by contacts from both RING protomers. The C-terminal tail of ubiquitin is locked into an active site groove on the E2 by an intricate network of interactions, resulting in changes at the E2 active site. This arrangement is primed for catalysis as it can deprotonate the incoming substrate lysine residue and stabilise the consequent tetrahedral transition state intermediate