TRIAD1 inhibits MDM2-mediated p53 ubiquitination and degradation

Murine double minute (MDM2) is an E3 ligase that promotes ubiquitination and degradation of tumor suppressor protein 53 (p53). MDM2-mediated regulation of p53 has been investigated as a classical tumorigenesis pathway. Here, we describe TRIAD1 as a novel modulator of the p53-MDM2 axis that induces p53 activation by inhibiting its regulation by MDM2. Ablation of TRIAD1 attenuates p53 levels activity upon DNA damage, whereas ectopic expression of TRIAD1 promotes p53 stability by inhibiting MDM2-mediated ubiquitination/degradation. Moreover, TRIAD1 binds to the C-terminus of p53 to promote its dissociation from MDM2. These results implicate TRIAD1 as a novel regulatory factor of p53-MDM2.Structured summary of protein interactions:p53 physically interacts with Mdm2 and Triad1 by anti tag coimmunoprecipitation (View Interaction: 1, 2, 3)Mdm2physically interacts with Triad1 by anti tag coimmunoprecipitation (View interaction)p53physically interacts with Mdm2 by anti tag coimmunoprecipitation (View interaction)Triad1binds to p53 by pull down (View interaction)Mdm2physically interacts with p53 by anti tag coimmunoprecipitation (View interaction)p53physically interacts with Triad1 by anti tag coimmunoprecipitation (View interaction)     

Mechanistic studies of MDM2-mediated ubiquitination in p53 regulation

As a central regulator for cell cycle arrest, apoptosis, and cellular senescence, p53 requires multiple layers of regulatory control to ensure correct temporal and spatial functions. It is well accepted that Mdm2-mediated ubiquitination plays a crucial role in p53 regulation. In addition to proteasome-mediated degradation, ubiquitination of p53 by Mdm2 acts a key signal for its nuclear export. Nuclear export has previously been thought to require the disassociation of the p53 tetramer and exposure of the intrinsic nuclear export signal. To elucidate the molecular mechanism of degradation-independent repression on p53 by Mdm2, we have developed a two-step approach to purify ubiquitinated forms of p53 induced by Mdm2 from human cells. Surprisingly, however, we found that ubiquitination has no effect on the tetramerization/oligomerization of p53, arguing against this seemingly well accepted model. Moreover, nuclear export of p53 alone is not sufficient to completely abolish p53 activity. Ubiquitination-mediated repression of p53 by Mdm2 acts at least, in part, through inhibiting the sequence-specific DNA binding activity. Thus, our results have important implications regarding the mechanisms by which Mdm2 acts on p53.     

UBE4B promotes Hdm2-mediated degradation of the tumor suppressor p53

The TP53 gene (encoding the p53 tumor suppressor) is rarely mutated, although frequently inactivated, in medulloblastoma and ependymoma. Recent work in mouse models showed that the loss of p53 accelerated the development of medulloblastoma. The mechanism underlying p53 inactivation in human brain tumors is not completely understood. We show that ubiquitination factor E4B (UBE4B), an E3 and E4 ubiquitin ligase, physically interacts with p53 and Hdm2 (also known as Mdm2 in mice). UBE4B promotes p53 polyubiquitination and degradation and inhibits p53-dependent transactivation and apoptosis. Notably, silencing UBE4B expression impairs xenotransplanted tumor growth in a p53-dependent manner and overexpression of UBE4B correlates with decreased expression of p53 in these tumors. We also show that UBE4B overexpression is often associated with amplification of its gene in human brain tumors. Our data indicate that amplification and overexpression of UBE4B represent previously undescribed molecular mechanisms of inactivation of p53 in brain tumors.     

CSA and CSB proteins interact with p53 and regulate its Mdm2-dependent ubiquitination

Mutations in Cockayne syndrome (CS) A and B genes (CSA and CSB) result in a rare genetic disease that affects the development and homeostasis of a wide range of tissues and organs. We previously correlated the degenerative phenotype of patients to the enhanced apoptotic response, exhibited by CS cells, which is associated with the exceptional induction of p53 protein, upon a variety of stress stimuli. Here we showed that the elevated and persistent levels of p53 displayed by CS cells are due to the insufficient ubiquitination of the p53 protein. We further demonstrated that CSA and CSB proteins associate in a unique complex with p53 and Mdm2; this interaction greatly stimulates the ubiquitination of p53 in an Mdm2-dependent manner. Tandem affinity purification and immunoprecipitations combined with mass spectrometry studies indicate that CSA and CSB associate within a Cullin Ring Ubiquitin Ligase complex responsible, under certain circumstances, for p53 ubiquitination. This study identifies CSA and CSB as the key elements of a regulatory mechanism that equilibrate beneficial and detrimental effects of p53 activity upon cellular stress. The deregulation of p53, in absence of either of the CS proteins, can potentially explain the early onset degeneration of tissues and organs observed in CS patients.     

p53 ubiquitination: Mdm2 and beyond

Although early studies have suggested that the oncoprotein Mdm2 is the primary E3 ubiquitin ligase for the p53 tumor suppressor, an increasing amount of data suggests that p53 ubiquitination and degradation are more complex than once thought. The discoveries of MdmX, HAUSP, ARF, COP1, Pirh2, and ARF-BP1 continue to uncover the multiple facets of this pathway. There is no question that Mdm2 plays a pivotal role in downregulating p53 activities in numerous cellular settings. Nevertheless, growing evidence challenges the conventional view that Mdm2 is essential for p53 turnover.     

Nucleocytoplasmic shuttling of p53 is essential for MDM2-mediated cytoplasmic degradation but not ubiquitination

As a shuttling protein, p53 is constantly transported through the nuclear pore complex. p53 nucleocytoplasmic transport is carried out by a bipartite nuclear localization signal (NLS) located at its C-terminal domain and two nuclear export signals (NES) located in its N- and C-terminal regions, respectively. The role of nucleocytoplasmic shuttling in p53 ubiquitination and degradation has been a subject of debate. Here we show that the two basic amino acid groups in the p53 bipartite NLS function collaboratively to import p53. Mutations disrupting individual amino acids in the NLS, although causing accumulation of p53 in the cytoplasm to various degrees, reduce but do not eliminate the NLS activity, and these mutants remain sensitive to MDM2 degradation. However, disrupting both parts of the bipartite NLS completely blocks p53 from entering the nucleus and causes p53 to become resistant to MDM2-mediated degradation. Similarly, mutations disrupting four conserved hydrophobic amino acids in the p53 C-terminal NES block p53 export and prohibit it from MDM2 degradation. We also show that colocalization of a nonshuttling p53 with MDM2 either in the nucleus or in the cytoplasm is sufficient for MDM2-induced p53 polyubiquitination but not degradation. Our data provide new insight into the mechanism and regulation of p53 nucleocytoplasmic shuttling and degradation.     

Yes-associated protein and p53-binding protein-2 interact through their WW and SH3 domains

To understand the role of the Yes-associated protein (YAP), binding partners of its WW1 domain were isolated by a yeast two-hybrid screen. One of the interacting proteins was identified as p53-binding protein-2 (p53BP-2). YAP and p53BP-2 interacted in vitro and in vivo using their WW1 and SH3 domains, respectively. The YAP WW1 domain bound to the YPPPPY motif of p53BP-2, whereas the p53BP-2 SH3 domain interacted with the VPMRLR sequence of YAP, which is different from other known SH3 domain-binding motifs. By mutagenesis, we showed that this unusual SH3 domain interaction was due to the presence of three consecutive tryptophans located within the betaC strand of the SH3 domain. A point mutation within this triplet, W976R, restored the binding selectivity to the general consensus sequence for SH3 domains, the PXXP motif. A constitutively active form of c-Yes was observed to decrease the binding affinity between YAP and p53BP-2 using chloramphenicol acetyltransferase/enzyme-linked immunosorbent assay, whereas the overexpression of c-Yes did not modify this interaction. Since overexpression of an activated form of c-Yes resulted in tyrosine phosphorylation of p53BP-2, we propose that the p53BP-2 phosphorylation, possibly in the WW1 domain-binding motif, might negatively regulate the YAP.p53BP-2 complex.     

Jab1 induces the cytoplasmic localization and degradation of p53 in coordination with Hdm2

The biological mechanisms for maintaining the basal level of p53 in normal cells require nuclear exclusion and cytoplasmic degradation. Here, we showed that Jab1 facilitates p53 nuclear exclusion and its subsequent degradation in coordination with Hdm2. p53 was excluded from the nucleus in the presence of Jab1; this exclusion was prevented by leptomycin B treatment. Nuclear export of p53 was accompanied by a decrease in the levels of p53, as well as of its target proteins, which include p21 and Bax. Domain analyses of Jab1 showed that the N-terminal domain, 1-110, was capable of inducing cytoplasmic translocation of p53. Furthermore, 110-191 was required to facilitate the degradation of p53. Neither of these mutants incorporated into the CSN complex, indicating that Jab1 could affect the levels of p53 independent of intact CSN complex. Conversely, Jab1 was incapable of translocating and degrading two p53 mutants, W23S and 6KR, neither of which could be modified by Hdm2. Moreover, Jab1 did not affect the cellular localization or protein levels of p53 in p53 and Hdm2 double-null mouse embryo fibroblasts. We further observed that the ablation of endogenous Jab1 by small interfering RNA prevented Hdm2-mediated p53 nuclear exclusion. Under stressed conditions, which could sequester Hdm2 in its inactive state, Jab1 did not affect p53. Our studies implicate that Jab1 is required to remove post-translationally modified p53 and provide a novel target for p53-related cancer therapies.     

Acetylation of p53 inhibits its ubiquitination by Mdm2

In response to DNA damage, the activity of the p53 tumor suppressor is modulated by protein stabilization and post-translational modifications including acetylation. Interestingly, both acetylation and ubiquitination can modify the same lysine residues at the C terminus of p53, implicating a role of acetylation in the regulation of p53 stability. However, the direct effect of acetylation on Mdm2-mediated ubiquitination of p53 is still lacking because of technical difficulties. Here, we have developed a method to obtain pure acetylated p53 proteins from cells, and by using an in vitro purified system, we provide the direct evidence that acetylation of the C-terminal domain is sufficient to abrogate its ubiquitination by Mdm2. Importantly, even in the absence of DNA damage, acetylation of the p53 protein is capable of reducing the ubiquitination levels and extending its half-life in vivo. Moreover, we also show that acetylation of p53 can affect its ubiquitination through other mechanisms in addition to the site competition. This study has significant implications regarding a general mechanism by which protein acetylation modulates ubiquitination-dependent proteasome proteolysis.     

RNAi knock of HdmX or Hdm2 leads to new insights into p53 signaling

Comment on: Heminger et.al. Aging 2009; 1:89-108.     

MDM2 and promyelocytic leukemia antagonize each other through their direct interaction with p53

p53 can be regulated through post-translational modifications and through interactions with positive and negative regulatory factors. MDM2 binding inhibits p53 and promotes its degradation by the proteasome, whereas promyelocytic leukemia (PML) activates p53 by recruiting it to multiprotein complexes termed PML-nuclear bodies. We reported previously an in vivo and in vitro interaction between PML and MDM2 that is independent of p53. In the current study, we investigated whether interaction between MDM2 and PML can indirectly affect p53 activity. Increasing amounts of MDM2 inhibited p53 activation by PML but could not inhibit PML-mediated activation of a p53 fusion protein that lacked the MDM2-binding domain. Conversely, increasing amounts of PML could overcome p53 inhibition by MDM2 but could not overcome MDM2-mediated inhibition of a p53 fusion protein that lacked the PML-binding domain. These results demonstrate that MDM2 and PML can antagonize each other through their direct interaction with p53 and suggest the combined effects of MDM2 and PML on p53 function are determined by the relative level of each protein. Furthermore, these results imply that interactions between MDM2 and PML by themselves have little or no effect on p53 activity.