Mark K. Saville

Mdm2-Mediated NEDD8 Conjugation of p53 Inhibits Its Transcriptional Activity

The only reported role for the conjugation of the NEDD8 ubiquitin-like molecule is control of the activity of SCF ubiquitin ligase complexes. Here, we show that the Mdm2 RING finger E3 ubiquitin ligase can also promote NEDD8 modification of the p53 tumor suppressor protein. Mdm2 is itself modified with NEDD8 with very similar characteristics to the autoubiquitination activity of Mdm2. By using a cell line (TS-41) with a temperature-sensitive mutation in the NEDD8 conjugation pathway and a p53 mutant that cannot be NEDDylated (3NKR), we demonstrate that Mdm2-dependent NEDD8 modification of p53 inhibits its transcriptional activity. These findings expand the role for Mdm2 as an E3 ligase, providing evidence that Mdm2 is a common component of the ubiquitin and NEDD8 conjugation pathway and indicating the diverse mechanisms by which E3 ligases can control the function of substrate proteins.

An N-terminal p14ARF peptide blocks Mdm2-dependent ubiquitination in vitro and can activate p53 in vivo.

The p53 tumour suppressor protein is down-regulated by the action of Mdm2, which targets p53 for rapid degradation by the ubiquitin-proteasome pathway. The p14ARF protein is also a potent tumour suppressor that acts by binding to Mdm2 and blocking Mdm2-dependent p53 degradation and transcriptional silencing. We have screened a series of overlapping synthetic peptides derived from the p14ARF protein sequence and found that a peptide corresponding to the first 20 amino acids of ARF (Peptide 3) could bind human Mdm2. The binding site for Peptide 3 on Mdm2 was determined by deletion mapping and lies adjacent to the binding site of the anti-Mdm2 antibody 2A10, which on microinjection into cells can activate p53-dependent transactivation of a reporter plasmid. To determine whether Peptide 3 could similarly activate p53, we expressed a fusion of green fluorescent protein and Peptide 3 in MCF7 and U-2 OS cells and were able to demonstrate induction of p53 protein and p53-dependent transcription. Peptide 3 was able to block in vitro ubiquitination of p53 mediated by Mdm2. Small peptides which are sufficient to block degradation of p53 could provide therapeutic agents able to restore p53-dependent cell death pathways in tumours that retain wild-type p53 expression.

Lysine‐63‐linked ubiquitination is required for endolysosomal degradation of class I molecules

MHC class I molecules display peptides from endogenous and viral proteins for immunosurveillance by cytotoxic T lymphocytes (CTL). The importance of the class I pathway is emphasised by the remarkable strategies employed by different viruses to downregulate surface class I and avoid CTL recognition. The K3 gene product from Kaposis sarcoma‐associated herpesvirus (KSHV) is a viral ubiquitin E3 ligase which ubiquitinates and degrades cell surface MHC class I molecules. We now show that modification of K3‐associated class I by lysine‐63‐linked polyubiquitin chains is necessary for their efficient endocytosis and endolysosomal degradation and present three lines of evidence that monoubiquitination of class I molecules provides an inefficient internalisation signal. This lysine‐63‐linked polyubiquitination requires both UbcH5b/c and Ubc13‐conjugating enzymes for initiating mono‐ and subsequent polyubiquitination of class I, and the clathrin‐dependent internalisation is mediated by the epsin endocytic adaptor. Our results explain how lysine‐63‐linked polyubiquitination leads to degradation by an endolysosomal pathway and demonstrate a novel mechanism for endocytosis and endolysosomal degradation of class I, which may be applicable to other receptors.

Mdmx stabilizes p53 and Mdm2 via two distinct mechanisms

The p53 protein maintains genomic integrity through its ability to induce cell cycle arrest or apoptosis in response to various forms of stress. Substantial regulation of p53 activity occurs at the level of protein stability, largely determined by the activity of the Mdm2 protein. Mdm2 targets both p53 and itself for ubiquitylation and subsequent proteasomal degradation by acting as an ubiquitin ligase, a function that needs an intact Mdm2 RING finger. For efficient degradation of p53 nuclear export appears to be required. The Mdmx protein, structurally homologous to Mdm2, does not target p53 for degradation, but even stabilizes both p53 and Mdm2, an activity most likely mediated by heterodimerization of the RING fingers of Mdm2 and Mdmx. Here we show that Mdmx expression leads to accumulation of ubiquitylated, nuclear p53 but does not significantly affect the Mdm2‐mediated ubiquitylation of p53. In contrast, Mdmx stabilizes Mdm2 by inhibiting its self‐ubiquitylation.

The deubiquitinating enzyme USP2a regulates the p53 pathway by targeting Mdm2

Mdm2 is an E3 ubiquitin ligase that promotes its own ubiquitination and also ubiquitination of the p53 tumour suppressor. In a bacterial two‐hybrid screen, using Mdm2 as bait, we identified an Mdm2‐interacting peptide that bears sequence similarity to the deubiquitinating enzyme USP2a. We have established that full‐length USP2a associates with Mdm2 in cells where it can deubiquitinate Mdm2 while demonstrating no deubiquitinating activity towards p53. Ectopic expression of USP2a causes accumulation of Mdm2 in a dose‐dependent manner and consequently promotes Mdm2‐mediated p53 degradation. This differs from the behaviour of HAUSP, which deubiquitinates p53 in addition to Mdm2 and thus protects p53 from Mdm2‐mediated degradation. We further demonstrate that suppression of endogenous USP2a destabilises Mdm2 and causes accumulation of p53 protein and activation of p53. Our data identify the deubiquitinating enzyme USP2a as a novel regulator of the p53 pathway that acts through its ability to selectively target Mdm2.

Different effects of p14ARF on the levels of ubiquitinated p53 and Mdm2 in vivo.

Mdm2 has been shown to promote its own ubiquitination and the ubiquitination of the p53 tumour suppressor by virtue of its E3 ubiquitin ligase activity. This modification targets Mdm2 and p53 for degradation by the proteasome. The p14ARF tumour suppressor has been shown to inhibit degradation of p53 mediated by Mdm2. Several models have been proposed to explain this effect of p14ARF. Here we have compared the effects of p14ARF overexpression on the in vivo ubiquitination of p53 and Mdm2. We report that the inhibition of the Mdm2-mediated degradation of p53 by p14ARF is associated with a decrease in the proportion of ubiquitinated p53. The levels of polyubiquitinated p53 decreased preferentially compared to monoubiquitinated species. p14ARF overexpression increased the levels of Mdm2 but it did not reduce the overall levels of ubiquitinated Mdm2 in vivo. This is unexpected because p14ARF has been reported to inhibit the ubiquitination of Mdm2 in vitro. In addition we show that like p14ARF, the proteasome inhibitor MG132 can promote the accumulation of Mdm2 in the nucleolus and that this can occur in the absence of p14ARF expression. We also show that the mutation of the nucleolar localization signal of Mdm2 does not impair the overall ubiquitination of Mdm2 but is necessary for the effective polyubiquitination of p53. These studies reveal important differences in the regulation of the stability of p53 and of Mdm2.

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.

The cell-cycle regulated transcription factor B-Myb is phosphorylated by cyclin A/Cdk2 at sites that enhance its transactivation properties

Expression of the B-Myb transcription factor is upregulated during late G1 phase of the cell cycle by an E2F-dependent transcriptional mechanism. B-Myb is specifically phosphorylated during S phase, suggesting that a cyclin-dependent kinase (Cdk) regulates its activity. Consistent with this notion, the S phase-specific cyclin A/Cdk2 was found previously to enhance B-Myb transactivation activity in cotransfected cells. In this study we provide evidence that B-Myb is a direct physiological target for cyclin A/Cdk2. We demonstrate that B-Myb is an in vitro substrate for cyclin A/Cdk2, but not for cyclin D1/Cdk4 or cyclin E/Cdk2. By mutating candidate Cdk2 phosphorylation sites, we show that B-Myb is phosphorylated at Thr447, Thr490, Thr497 and Ser581 by cyclin A/Cdk2 in vitro and that these sites are also phosphorylated in cycling U-2 OS cells. Inhibition of endogenous Cdk2 by dominant negative Cdk2 attenuated phosphorylation of Thr447, Thr490 and Thr497, but had no effect upon Ser581 modification. B-Myb transactivation activity was significantly reduced in a mutant containing amino acid substitutions at all four identified cyclin A/Cdk2 sites and was constitutively low in Saos-2 cells where endogenous cyclin A/Cdk2 activity was unable to phosphorylate ectopically expressed B-Myb. These data indicate that phosphorylation by cyclin A/Cdk2 is directly involved in enhancing B-Myb transactivation activity and that levels of endogenous cyclin A/Cdk2 activity may contribute to cell line-specific B-Myb function.

MdmX is a substrate for the deubiquitinating enzyme USP2a

It has previously been shown that ubiquitin-specific protease 2a (USP2a) is a regulator of the Mdm2/p53 pathway. USP2a binds to Mdm2 and can deubiquitinate Mdm2 without reversing Mdm2-mediated p53 ubiquitination. Overexpression of USP2a causes accumulation of Mdm2 and promotes p53 degradation. We now show that MdmX is also a substrate for USP2a. MdmX associates with USP2a independently of Mdm2. Ectopic expression of wild-type USP2a but not a catalytic mutant prevents Mdm2-mediated degradation of MdmX. This correlates with the ability of wild-type USP2a to deubiquitinate MdmX. siRNA-mediated knockdown of USP2a in NTERA-2 testicular embryonal carcinoma cells and MCF7 breast cancer cells causes destabilization of MdmX and results in a decrease in MdmX protein levels, showing that endogenous USP2a participates in the regulation of MdmX stability. The therapeutic drug, cisplatin decreases MdmX protein expression. USP2a mRNA and protein levels were also reduced after cisplatin exposure. The magnitude and time course of USP2a downregulation suggests that the reduction in USP2a levels could contribute to the decrease in MdmX expression following treatment with cisplatin. Knockdown of USP2a increases the sensitivity of NTERA-2 cells to cisplatin, raising the possibility that suppression of USP2a in combination with cisplatin may be an approach for cancer therapy.

Elevated Levels of Oncogenic Protein Kinase Pim-1 Induce the p53 Pathway in Cultured Cells and Correlate with Increased Mdm2 in Mantle Cell Lymphoma

Mutation of the p53 gene is a common event during tumor pathogenesis. Other mechanisms, such as mdm2 amplification, provide alternative routes through which dysfunction of the p53 pathway is promoted. Here, we address the hypothesis that elevated expression of pim oncogenes might suppress p53 by regulating Mdm2. At a physiological level, we show that endogenous Pim-1 and Pim-2 interact with endogenous Mdm2. Additionally, the Pim kinases phosphorylate Mdm2 in vitro and in cultured cells at Ser166 and Ser186, two previously identified targets of other signaling pathways, including Akt. Surprisingly, at high levels of Pim expression, as would occur in tumors, active, but not inactive, Pim-1 or Pim-2 blocks the degradation of both p53 and Mdm2 in a manner that is independent of Mdm2 phosphorylation, leading to increased p53 levels and, proportionately, p53-dependent transactivation. Additionally, Pim-1 induces endogenous ARF, p53, Mdm2, and p21 in primary murine embryo fibroblasts and stimulates senescence-associated β-galactosidase levels, consistent with the induction of senescence. Immunohistochemical analysis of a cohort of 33 human mantle cell lymphomas shows that elevated expression of Pim-1 occurs in 42% of cases, with elevated Pim-2 occurring in 9% of cases, all of which also express Pim-1. Notably, elevated Pim-1 correlates with elevated Mdm2 in MCL with a p value of 0.003. Taken together, our data are consistent with the idea that Pim normally interacts with the p53 pathway but, when expressed at pathological levels, behaves as a classic dominant oncogene that stimulates a protective response through induction of the p53 pathway.