Mdm2

Discovery and expression in tumor cells

The murine double minute (mdm2) oncogene, which codes for the Mdm2 protein, was originally cloned, along with two other genes (mdm1 and mdm3) from the transformed mouse cell line 3T3-DM. Mdm2 overexpression, in cooperation with oncogenic Ras, promotes transformation of primary rodent fibroblasts, and mdm2 expression led to tumor formation in nude mice. The human homologue of this protein was later identified and is sometimes called Hdm2. Further supporting the role of mdm2 as an oncogene, several human tumor types have been shown to have increased levels of Mdm2, including soft tissue sarcomas and osteosarcomas as well as breast tumors.

An additional Mdm2 family member, Mdm4 (also called MdmX), has been discovered and is also an important negative regulator of p53.

Ubiquitination target: p53

The key target of Mdm2 is the p53 tumor suppressor. Mdm2 has been identified as a p53 interacting protein that represses p53 transcriptional activity. Mdm2 achieves this repression by binding to and blocking the N-terminal trans-activation domain of p53. Mdm2 is a p53 responsive gene—that is, its transcription can be activated by p53. Thus when p53 is stabilized, the transcription of Mdm2 is also induced, resulting in higher Mdm2 protein levels.

E3 ligase activity

The E3 ubiquitin ligase MDM2 is a negative regulator of the p53 tumor suppressor protein. MDM2 binds and ubiquitinates p53, facilitating it for degradation. p53 can induce transcription of MDM2, generating a negative feedback loop. Mdm2 also acts as an E3 ubiquitin ligase, targeting both itself and p53 for degradation by the proteasome (see also ubiquitin). Several lysine residues in p53 C-terminus have been identified as the sites of ubiquitination, and it has been shown that p53 protein levels are downregulated by Mdm2 in a proteasome-dependent manner. Mdm2 is capable of auto-polyubiquitination, and in complex with p300, a cooperating E3 ubiquitin ligase, is capable of polyubiquitinating p53. In this manner, Mdm2 and p53 are the members of a negative feedback control loop that keeps the level of p53 low in the absence of p53-stabilizing signals. This loop can be interfered with by kinases and genes like p14arf when p53 activation signals, including DNA damage, are high.

Structure and function

The full-length transcript of the mdm2 gene encodes a protein of 491 amino acids with a predicted molecular weight of 56kDa. This protein contains several conserved structural domains including an N-terminal p53 interaction domain, the structure of which has been solved using x-ray crystallography. The Mdm2 protein also contains a central acidic domain (residues 230–300). The phosphorylation of residues within this domain appears to be important for regulation of Mdm2 function. In addition, this region contains nuclear export and import signals that are essential for proper nuclear-cytoplasmic trafficking of Mdm2. Another conserved domain within the Mdm2 protein is a zinc finger domain, the function of which is poorly understood.

Mdm2 also contains a C-terminal RING domain (amino acid residues 430–480), which contains a Cis3-His2-Cis3 consensus that coordinates two ions of zinc. These residues are required for zinc binding, which is essential for proper folding of the RING domain. The RING domain of Mdm2 confers E3 ubiquitin ligase activity and is sufficient for E3 ligase activity in Mdm2 RING autoubiquitination. The RING domain of Mdm2 is unique in that it incorporates a conserved Walker A or P-loop motif characteristic of nucleotide binding proteins, as well as a nucleolar localization sequence. The RING domain also binds specifically to RNA, although the function of this is poorly understood.

Regulation

There are several known mechanisms for regulation of Mdm2. One of these mechanisms is phosphorylation of the Mdm2 protein. Mdm2 is phosphorylated at multiple sites in cells. Following DNA damage, phosphorylation of Mdm2 leads to changes in protein function and stabilization of p53. Additionally, phosphorylation at certain residues within the central acidic domain of Mdm2 may stimulate its ability to target p53 for degradation. HIPK2 is a protein that regulates Mdm2 in this way. The induction of the p14arf protein, the alternate reading frame product of the p16INK4a locus, is also a mechanism of negatively regulating the p53-Mdm2 interaction. p14arf directly interacts with Mdm2 and leads to up-regulation of p53 transcriptional response. ARF sequesters Mdm2 in the nucleolus, resulting in inhibition of nuclear export and activation of p53, since nuclear export is essential for proper p53 degradation.

Inhibitors of the MDM2-p53 interaction include the cis-imidazoline analog nutlin.

Levels and stability of Mdm2 are also modulated by ubiquitylation. Mdm2 auto ubiquitylates itself, which allows for its degradation by the proteasome. Mdm2 also interacts with a ubiquitin specific protease, USP7, which can reverse Mdm2-ubiquitylation and prevent it from being degraded by the proteasome. USP7 also protects from degradation the p53 protein, which is a major target of Mdm2. Thus Mdm2 and USP7 form an intricate circuit to finely regulate the stability and activity of p53, whose levels are critical for its function.

Interactions

Mdm2 has been shown to interact with:

• ABL1,
• ARRB1,
• ARRB2,
• CCNG1,
• CTBP1,
• CTBP2,
• DAXX,
• DHFR,
• EP300,
• ERICH3,
• FKBP3,
• FOXO4,
• GNL3,
• HDAC1,
• HIF1A,
• HTATIP,
• IGF1R,
• MDM4,
• NUMB,
• P16,
• P53,
• P73,
• PCAF,
• PSMD10,
• PSME3,
• RPL5,
• RPL11,
• PML,
• RPL26,
• RRM2B,
• RYBP,
• TBP, and
• UBC.

Mdm2 p53-independent role

Mdm2 overexpression was shown to inhibit DNA double-strand break repair mediated through a novel, direct interaction between Mdm2 and Nbs1 and independent of p53. Regardless of p53 status, increased levels of Mdm2, but not Mdm2 lacking its Nbs1-binding domain, caused delays in DNA break repair, chromosomal abnormalities, and genome instability. These data demonstrated Mdm2-induced genome instability can be mediated through Mdm2:Nbs1 interactions and independent from its association with p53.

References

References

1. (July 1992). "Amplification of a gene encoding a p53-associated protein in human sarcomas". Nature.
2. (November 2006). "Hdmx modulates the outcome of p53 activation in human tumor cells". The Journal of Biological Chemistry.
3. (October 2017). "The Functional Roles of the MDM2 Splice Variants P2-MDM2-10 and MDM2-∆5 in Breast Cancer Cells". Translational Oncology.
4. (February 2004). "In vivo activation of the p53 pathway by small-molecule antagonists of MDM2". Science.
5. (July 2002). "Tyrosine phosphorylation of Mdm2 by c-Abl: implications for p53 regulation". The EMBO Journal.
6. (March 2003). "Subcellular localization of beta-arrestins is determined by their intact N domain and the nuclear export signal at the C terminus". The Journal of Biological Chemistry.
7. (August 2008). "Nedd4 mediates agonist-dependent ubiquitination, lysosomal targeting, and degradation of the beta2-adrenergic receptor". The Journal of Biological Chemistry.
8. (February 2003). "Beta-arrestin 2 functions as a G-protein-coupled receptor-activated regulator of oncoprotein Mdm2". The Journal of Biological Chemistry.
9. (January 2003). "Cyclin G1 has growth inhibitory activity linked to the ARF-Mdm2-p53 and pRb tumor suppressor pathways". Molecular Cancer Research.
10. (July 2003). "Hdm2 recruits a hypoxia-sensitive corepressor to negatively regulate p53-dependent transcription". Current Biology.
11. (June 2008). "p14ARF interacts with DAXX: effects on HDM2 and p53". Cell Cycle.
12. (May 2008). "MDM2 regulates dihydrofolate reductase activity through monoubiquitination". Cancer Research.
13. (October 1998). "p300/MDM2 complexes participate in MDM2-mediated p53 degradation". Molecular Cell.
14. (Feb 2010). "A comprehensive resource of interacting protein regions for refining human transcription factor networks". PLOS ONE.
15. (February 2009). "FKBP25, a novel regulator of the p53 pathway, induces the degradation of MDM2 and activation of p53". FEBS Letters.
16. (2008). "Mdm2 induces mono-ubiquitination of FOXO4". PLOS ONE.
17. (July 2008). "Aberrant expression of nucleostemin activates p53 and induces cell cycle arrest via inhibition of MDM2". Molecular and Cellular Biology.
18. (November 2002). "MDM2-HDAC1-mediated deacetylation of p53 is required for its degradation". The EMBO Journal.
19. (April 2003). "Direct interactions between HIF-1 alpha and Mdm2 modulate p53 function". The Journal of Biological Chemistry.
20. (January 2000). "Regulation of tumor angiogenesis by p53-induced degradation of hypoxia-inducible factor 1alpha". Genes & Development.
21. (April 2002). "Tip60 is targeted to proteasome-mediated degradation by Mdm2 and accumulates after UV irradiation". The EMBO Journal.
22. (July 2008). "Identification of c-Cbl as a new ligase for insulin-like growth factor-I receptor with distinct roles from Mdm2 in receptor ubiquitination and endocytosis". Cancer Research.
23. (December 2002). "MdmX inhibits Smad transactivation". Oncogene.
24. (March 1999). "MDM2 interacts with MDMX through their RING finger domains". FEBS Letters.
25. (December 2002). "MdmX is a RING finger ubiquitin ligase capable of synergistically enhancing Mdm2 ubiquitination". The Journal of Biological Chemistry.
26. (May 2008). "Structure of the MDM2/MDMX RING domain heterodimer reveals dimerization is required for their ubiquitylation in trans". Cell Death and Differentiation.
27. (March 2003). "Mammalian Numb is a target protein of Mdm2, ubiquitin ligase". Biochemical and Biophysical Research Communications.
28. (January 2008). "NUMB controls p53 tumour suppressor activity". Nature.
29. (March 1998). "ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways". Cell.
30. (July 2002). "Multiple interacting domains contribute to p14ARF mediated inhibition of MDM2". Oncogene.
31. (March 1998). "The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2's inhibition of p53". Cell.
32. (May 1997). "Mdm2 promotes the rapid degradation of p53". Nature.
33. (December 1997). "Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53". FEBS Letters.
34. (July 1999). "Mdm2 binds p73 alpha without targeting degradation". Oncogene.
35. (May 1999). "MDM2 suppresses p73 function without promoting p73 degradation". Molecular and Cellular Biology.
36. (August 2002). "MDM2 inhibits PCAF (p300/CREB-binding protein-associated factor)-mediated p53 acetylation". The Journal of Biological Chemistry.
37. (July 2008). "Retinoblastoma protein modulates gankyrin-MDM2 in regulation of p53 stability and chemosensitivity in cancer cells". Oncogene.
38. (March 2008). "Proteasome activator PA28 gamma regulates p53 by enhancing its MDM2-mediated degradation". The EMBO Journal.
39. (December 2003). "Ribosomal protein L11 negatively regulates oncoprotein MDM2 and mediates a p53-dependent ribosomal-stress checkpoint pathway". Molecular and Cellular Biology.
40. (November 1994). "The ribosomal L5 protein is associated with mdm-2 and mdm-2-p53 complexes". Molecular and Cellular Biology.
41. (July 2004). "PML regulates p53 stability by sequestering Mdm2 to the nucleolus". Nature Cell Biology.
42. (December 2003). "MDM2 and promyelocytic leukemia antagonize each other through their direct interaction with p53". The Journal of Biological Chemistry.
43. (October 2003). "Cellular stress and DNA damage invoke temporally distinct Mdm2, p53 and PML complexes and damage-specific nuclear relocalization". Journal of Cell Science.
44. (August 2003). "Physical and functional interactions between PML and MDM2". The Journal of Biological Chemistry.
45. (October 2008). "Mdm2 regulates p53 mRNA translation through inhibitory interactions with ribosomal protein L26". Molecular Cell.
46. (November 2008). "ATM-mediated serine 72 phosphorylation stabilizes ribonucleotide reductase small subunit p53R2 protein against MDM2 to DNA damage". Proceedings of the National Academy of Sciences of the United States of America.
47. (February 2009). "RYBP stabilizes p53 by modulating MDM2". EMBO Reports.
48. (December 1997). "The MDM2 C-terminal region binds to TAFII250 and is required for MDM2 regulation of the cyclin A promoter". The Journal of Biological Chemistry.
49. (August 1997). "Repression of p53-mediated transcription by MDM2: a dual mechanism". Genes & Development.
50. (March 2008). "CARPs enhance p53 turnover by degrading 14-3-3sigma and stabilizing MDM2". Cell Cycle.
51. (2005). "Mdm2 Binds to Nbs1 at Sites of DNA Damage and Regulates Double Strand Break Repair". Journal of Biological Chemistry.