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.     

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.     

Mdmx enhances p53 ubiquitination by altering the substrate preference of the Mdm2 ubiquitin ligase

mdm2 and mdmx oncogenes play essential yet non-redundant roles in synergistic inactivation of the tumor suppressor, p53. While Mdm2 inhibits p53 activity mainly by augmenting its ubiquitination, the functional role of Mdmx on p53 ubiquitination remains obscure. In transfected H1299 cells, Mdmx augmented Mdm2-mediated ubiquitination of p53. In in vitro ubiquitination assays, the Mdmx/Mdm2 heteromeric complex, in comparison to the Mdm2 homomer, showed enhanced ubiquitinase activity toward p53 and the reduced auto-ubiquitination of Mdm2. Alteration of the substrate specificity via binding to Mdmx may contribute to efficient ubiquitination and inactivation of p53 by Mdm2.

Structured summary

MINT-7219995: P53 (uniprotkb:P04637) physically interacts (MI:0914) with Ubiquitin (uniprotkb:P62988) by anti bait coimmunoprecipitation (MI:0006)MINT-7220023: Ubiquitin (uniprotkb:P62988) physically interacts (MI:0914) with P53 (uniprotkb:P04637) by pull down (MI:0096)     

HAUSP-regulated switch from auto- to p53 ubiquitination by Mdm2 (in silico discovery)

Stability of the 'guardian of the genome' tumor suppressor protein p53 is regulated predominantly through its ubiquitination. The ubiquitin-specific protease HAUSP plays an important role in this process. Recent experiments showed that p53 demonstrates a differential response to changes in HAUSP which nature and significance are not understood yet. Here a data-driven mathematical model of the Mdm2-mediated p53 ubiquitination network is presented which offers an explanation for the cause of such a response. The model predicts existence of the HAUSP-regulated switch from auto- to p53 ubiquitination by Mdm2. This switch suggests a potential role of HAUSP as a downstream target of stress signals in cells. The model accounts for a significant amount of experimental data, makes predictions for some rate constants, and can serve as a building block for the larger model describing a complex dynamic response of p53 to cellular stresses.     

Tissue-specific differences of p53 inhibition by Mdm2 and Mdm4

The function of the p53 tumor suppressor to inhibit proliferation or initiate apoptosis is often abrogated in tumor cells. Mdm2 and its homolog, Mdm4, are critical inhibitors of p53 that are often overexpressed in human tumors. In mice, loss of Mdm2 or Mdm4 leads to embryonic lethal phenotypes that are completely rescued by concomitant loss of p53. To examine the role of Mdm2 and Mdm4 in a temporal and tissue-specific manner and to determine the relationships of these inhibitors to each other, we generated conditional alleles. We deleted Mdm2 and Mdm4 in cardiomyocytes, since proliferation and apoptosis are important processes in heart development. Mice lacking Mdm2 in the heart were embryonic lethal and showed defects at the time recombination occurred. A critical number of cardiomyocytes were lost by embryonic day 13.5, resulting in heart failure. This phenotype was completely rescued by deletion of p53. Mice lacking Mdm4 in the heart were born at the correct ratio and appeared to be normal. Our studies provide the first direct evidence that Mdm2 can function in the absence of Mdm4 to regulate p53 activity in a tissue-specific manner. Moreover, Mdm4 cannot compensate for the loss of Mdm2 in heart development.     

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.     

Cocompartmentalization of p53 and Mdm2 is a major determinant for Mdm2-mediated degradation of p53

The product of the Mdm2 oncogene directly interacts with p53 and promotes its ubiquitination and proteasomal degradation. Initial biological studies identified nuclear export sequences (NES), similar to that of the Rev protein from the human immunodeficiency virus, both in Mdm2 and p53. The reported phenotypes resulting from mutation of these NESs, together with results obtained using the nuclear export inhibitor leptomycin B (LMB), have led to a model according to which nuclear export of p53 (via either the NES of Mdm2 or its own NES) is required for efficient p53 degradation. In this study we demonstrate that Mdm2 can promote degradation of p53 in the nucleus or in the cytoplasm, provided both proteins are colocalized. We also investigated if nuclear export is an obligate step on the p53 degradation pathway. We find that (1) when proteasome activity is inhibited, ubiquitinated p53 accumulates in the nucleus and not in the cytoplasm; (2) Mdm2 with a mutated NES can efficiently mediate degradation of wild type p53 or p53 with a mutated NES; (3) the nuclear export inhibitor LMB can increase the steady-state level of p53 by inhibiting Mdm2-mediated ubiquitination of p53; and (4) LMB fails to inhibit Mdm2-mediated degradation of the p53NES mutant, demonstrating that Mdm2-dependent proteolysis of p53 is feasible in the nucleus in the absence of any nuclear export. Therefore, given cocompartmentalization, Mdm2 can promote ubiquitination and proteasomal degradation of p53 with no absolute requirement for nuclear to cytoplasmic transport.     

Mdm2 and Mdm4 loss regulates distinct p53 activities

Mutational inactivation of p53 is a hallmark of most human tumors. Loss of p53 function also occurs by overexpression of negative regulators such as MDM2 and MDM4. Deletion of Mdm2 or Mdm4 in mice results in p53-dependent embryo lethality due to constitutive p53 activity. However, Mdm2(-/-) and Mdm4(-/-) embryos display divergent phenotypes, suggesting that Mdm2 and Mdm4 exert distinct control over p53. To explore the interaction between Mdm2 and Mdm4 in p53 regulation, we first generated mice and cells that are triple null for p53, Mdm2, and Mdm4. These mice had identical survival curves and tumor spectrum as p53(-/-) mice, substantiating the principal role of Mdm2 and Mdm4 as negative p53 regulators. We next generated mouse embryo fibroblasts null for p53 with deletions of Mdm2, Mdm4, or both; introduced a retrovirus expressing a temperature-sensitive p53 mutant, p53A135V; and examined p53 stability and activity. In this system, p53 activated distinct target genes, leading to apoptosis in cells lacking Mdm2 and a cell cycle arrest in cells lacking Mdm4. Cells lacking both Mdm2 and Mdm4 had a stable p53 that initiated apoptosis similar to Mdm2-null cells. Additionally, stabilization of p53 in cells lacking Mdm4 with the Mdm2 antagonist nutlin-3 was sufficient to induce a cell death response. These data further differentiate the roles of Mdm2 and Mdm4 in the regulation of p53 activities.     

MdmX protein is essential for Mdm2 protein-mediated p53 polyubiquitination

Genetic evidence has implicated both Mdm2 and MdmX as essential in negative regulation of p53. However, the exact role of MdmX in this Mdm2-dependent protein degradation is not well understood. Most, if not all, previous Mdm2 studies used GST-Mdm2 fusion proteins in the in vitro assays. Here, we show that the p53 polyubiquitination activity of GST-Mdm2 is conferred by the GST tag and non-GST-tagged Mdm2 only catalyzes monoubiquitination of p53 even at extremely high concentrations. We further demonstrate that MdmX is a potent activator of Mdm2, facilitating dose-dependent p53 polyubiquitination. This activation process requires the RING domains of both MdmX and Mdm2 proteins. The polyubiquitination activity of Mdm2/MdmX is Mdm2-dependent. Unlike Mdm2 or MdmX overexpression alone, co-overexpression of MdmX and Mdm2 consistently triggered p53 degradation in cells. Moreover, cellular polyubiquitination of p53 was only observable in the cytoplasm where both Mdm2 and MdmX are readily detectable. Importantly, RNAi knockdown of MdmX increased levels of endogenous p53 accompanied by reduced p53 polyubiquitination. In conclusion, our work has resolved a major confusion in the field derived from using GST-Mdm2 and demonstrated that MdmX is the cellular activator that converts Mdm2 from a monoubiquitination E3 ligase to a polyubiquitination E3 ligase toward p53. Together, our findings provide a biochemical basis for the requirement of both Mdm2 and MdmX in the dynamic regulation of p53 stability.     

Cutaneous HPV23 E6 prevents p53 phosphorylation through interaction with HIPK2

Ultraviolet irradiation (UV) is the major risk factor for the development of skin cancer. Moreover, increasing evidence suggests cutaneotropic human papillomaviruses (HPV) from the beta genus to play a causal role as a co-factor in the development of cutaneous squamous cell carcinoma. Homeodomain-interacting protein kinase 2 (HIPK2) operates as a potential suppressor in skin tumorigenesis and is stabilized by UV-damage. HIPK2 is an important regulator of apoptosis, which forms a complex with the tumor suppressor p53, mediating p53 phosphorylation at Ser 46 and thus promoting pro-apoptotic gene expression. In our study, we demonstrate that cutaneous HPV23 E6 protein directly targets HIPK2 function. Accordingly, HPV23 E6 interacts with HIPK2 both in vitro and in vivo. Furthermore, upon massive UVB-damage HPV23 E6 co-localizes with endogenous HIPK2 at nuclear bodies. Functionally, we demonstrate that HPV23 E6 inhibits HIPK2-mediated p53 Ser 46 phosphorylation through enforcing dissociation of the HIPK2/p53 complex. In addition, HPV23 E6 co-accumulates with endogenous HIPK2 upon UV damage suggesting a mechanism by which HPV23 E6 keeps HIPK2 in check after UV damage. Thus, cutaneous HPV23 E6 prevents HIPK2-mediated p53 Ser 46 phosphorylation, which may favour survival of UV-damaged keratinocytes and skin carcinogenesis by apoptosis evasion.     

Deciphering the mechanisms of HPV E6 mutations in the destabilization of E6/E6AP/p53 complex

In epithelial tumors, oncoprotein E6 binds with the ubiquitin ligase E6AP to form E6/E6AP heterodimer; then this heterodimer recruits p53 to form E6/E6AP/p53 heterotrimer and induces p53 degradation. Recent experiments demonstrated that three E6 single-site mutants (F47R, R102A, and L50E) can inhibit the E6/E6AP/p53 heterotrimer formation and rescue p53 from the degradation pathway. However, the molecular mechanism underlying mutation-induced heterotrimer inhibition remains largely elusive. Herein, we performed extensive molecular dynamics simulations (totally ～13 μs) on both heterodimer and heterotrimer to elucidate at an atomic level how each p53-degradation-defective HPV16 E6 mutant reduces the structural stabilities of the two complexes. Our simulations reveal that the three E6 mutations destabilize the structure of E6/E6AP/p53 complex through distinct mechanisms. Although F47R^E6 mutation has no effect on the structure of E6/E6AP heterodimer, it results in an electrostatic repulsion between R47^E6 and R290^p53, which is unfavorable for E6-p53 binding. R102A^E6 mutation destabilizes the structure of E6/E6AP heterodimer and significantly disrupts hydrophobic and cation-π interactions between F47^E6 and E286^p53/L298^p53/R290^p53. L50E^E6 mutation impairs both E6 interdomain interactions (especially F47-K108 cation-π interaction) and E6-E6AP intermolecular interactions important for the stabilization of E6/E6AP heterodimer. This study identifies the intra- and intermolecular interactions crucial for the complex stability, which may provide mechanistic insights into the inhibition of complex formation by the three HPV16 E6 mutations.