Mouse double minute 2 homolog (MDM2) also known as E3 ubiquitin-protein ligase Mdm2 is a protein that in humans is encoded by the MDM2gene. Mdm2 is an important negative regulator of the p53 tumor suppressor. Mdm2 protein functions both as an E3 ubiquitin ligase that recognizes the N-terminal trans-activation domain (TAD) of the p53 tumor suppressor and an inhibitor of p53 transcriptional activation.
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.
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.
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.
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 resdiues 430-480), which contains a Cis3-His2-Cis3 consensus that coordinates two molecules 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.
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. 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. It is interesting to note that 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.
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.
^Uhrinova S, Uhrin D, Powers H, et al (2005). "Structure of free MDM2 N-terminal domain reveals conformational adjustments that accompany p53-binding". J. Mol. Biol.350 (3): 587–98. doi:10.1016/j.jmb.2005.05.010. PMID15953616.
^Oliner JD, Kinzler KW, Meltzer PS, George DL, Vogelstein B (July 1992). "Amplification of a gene encoding a p53-associated protein in human sarcomas". Nature358 (6381): 80–3. doi:10.1038/358080a0. PMID1614537.
^Wade M, Wong ET, Tang M, Stommel JM, Wahl GM (November 2006). "Hdmx modulates the outcome of p53 activation in human tumor cells". J. Biol. Chem.281 (44): 33036–44. doi:10.1074/jbc.M605405200. PMID16905769.
^Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, Kong N, Kammlott U, Lukacs C, Klein C, Fotouhi N, Liu EA (2004). "In vivo activation of the p53 pathway by small-molecule antagonists of MDM2". Science303 (5659): 844–848. doi:10.1126/science.1092472. PMID14704432.
^ abWang P, Wu Y, Ge X, Ma L, Pei G (March 2003). "Subcellular localization of beta-arrestins is determined by their intact N domain and the nuclear export signal at the C terminus". J. Biol. Chem.278 (13): 11648–53. doi:10.1074/jbc.M208109200. PMID12538596.
^Wang P, Gao H, Ni Y, Wang B, Wu Y, Ji L, Qin L, Ma L, Pei G (February 2003). "Beta-arrestin 2 functions as a G-protein-coupled receptor-activated regulator of oncoprotein Mdm2". J. Biol. Chem.278 (8): 6363–70. doi:10.1074/jbc.M210350200. PMID12488444.
^Zhao L, Samuels T, Winckler S, Korgaonkar C, Tompkins V, Horne MC, Quelle DE (January 2003). "Cyclin G1 has growth inhibitory activity linked to the ARF-Mdm2-p53 and pRb tumor suppressor pathways". Mol. Cancer Res.1 (3): 195–206. PMID12556559.
^ abMirnezami AH, Campbell SJ, Darley M, Primrose JN, Johnson PW, Blaydes JP (July 2003). "Hdm2 recruits a hypoxia-sensitive corepressor to negatively regulate p53-dependent transcription". Curr. Biol.13 (14): 1234–9. doi:10.1016/S0960-9822(03)00454-8. PMID12867035.
^Grossman SR, Perez M, Kung AL, Joseph M, Mansur C, Xiao ZX, Kumar S, Howley PM, Livingston DM (October 1998). "p300/MDM2 complexes participate in MDM2-mediated p53 degradation". Mol. Cell2 (4): 405–15. doi:10.1016/S1097-2765(00)80140-9. PMID9809062.
^Ochocka AM, Kampanis P, Nicol S, Allende-Vega N, Cox M, Marcar L, Milne D, Fuller-Pace F, Meek D (February 2009). "FKBP25, a novel regulator of the p53 pathway, induces the degradation of MDM2 and activation of p53". FEBS Lett.583 (4): 621–6. doi:10.1016/j.febslet.2009.01.009. PMID19166840.
^Sehat B, Andersson S, Girnita L, Larsson O (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 Res.68 (14): 5669–77. doi:10.1158/0008-5472.CAN-07-6364. PMID18632619.
^Badciong JC, Haas AL (December 2002). "MdmX is a RING finger ubiquitin ligase capable of synergistically enhancing Mdm2 ubiquitination". J. Biol. Chem.277 (51): 49668–75. doi:10.1074/jbc.M208593200. PMID12393902.
^Linke K, Mace PD, Smith CA, Vaux DL, Silke J, Day CL (May 2008). "Structure of the MDM2/MDMX RING domain heterodimer reveals dimerization is required for their ubiquitylation in trans". Cell Death Differ.15 (5): 841–8. doi:10.1038/sj.cdd.4402309. PMID18219319.
^Yogosawa S, Miyauchi Y, Honda R, Tanaka H, Yasuda H (March 2003). "Mammalian Numb is a target protein of Mdm2, ubiquitin ligase". Biochem. Biophys. Res. Commun.302 (4): 869–72. doi:10.1016/S0006-291X(03)00282-1. PMID12646252.
^Zhang Y, Xiong Y, Yarbrough WG (March 1998). "ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways". Cell92 (6): 725–34. doi:10.1016/S0092-8674(00)81401-4. PMID9529249.
^Clark PA, Llanos S, Peters G (July 2002). "Multiple interacting domains contribute to p14ARF mediated inhibition of MDM2". Oncogene21 (29): 4498–507. doi:10.1038/sj.onc.1205558. PMID12085228.
^Pomerantz J, Schreiber-Agus N, Liégeois NJ, Silverman A, Alland L, Chin L, Potes J, Chen K, Orlow I, Lee HW, Cordon-Cardo C, DePinho RA (March 1998). "The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2's inhibition of p53". Cell92 (6): 713–23. doi:10.1016/S0092-8674(00)81400-2. PMID9529248.
^Jin Y, Zeng SX, Dai MS, Yang XJ, Lu H (August 2002). "MDM2 inhibits PCAF (p300/CREB-binding protein-associated factor)-mediated p53 acetylation". J. Biol. Chem.277 (34): 30838–43. doi:10.1074/jbc.M204078200. PMID12068014.
^Qiu W, Wu J, Walsh EM, Zhang Y, Chen CY, Fujita J, Xiao ZX (July 2008). "Retinoblastoma protein modulates gankyrin-MDM2 in regulation of p53 stability and chemosensitivity in cancer cells". Oncogene27 (29): 4034–43. doi:10.1038/onc.2008.43. PMID18332869.
^Zhu H, Wu L, Maki CG (December 2003). "MDM2 and promyelocytic leukemia antagonize each other through their direct interaction with p53". J. Biol. Chem.278 (49): 49286–92. doi:10.1074/jbc.M308302200. PMID14507915.
^Kurki S, Latonen L, Laiho M (October 2003). "Cellular stress and DNA damage invoke temporally distinct Mdm2, p53 and PML complexes and damage-specific nuclear relocalization". J. Cell. Sci.116 (Pt 19): 3917–25. doi:10.1242/jcs.00714. PMID12915590.
^Wei X, Yu ZK, Ramalingam A, Grossman SR, Yu JH, Bloch DB, Maki CG (August 2003). "Physical and functional interactions between PML and MDM2". J. Biol. Chem.278 (31): 29288–97. doi:10.1074/jbc.M212215200. PMID12759344.
^Léveillard T, Wasylyk B (December 1997). "The MDM2 C-terminal region binds to TAFII250 and is required for MDM2 regulation of the cyclin A promoter". J. Biol. Chem.272 (49): 30651–61. doi:10.1074/jbc.272.49.30651. PMID9388200.
Fang S, Jensen JP, Ludwig RL, Vousden KH, Weissman AM (March 2000). "Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53". J. Biol. Chem.275 (12): 8945–51. doi:10.1074/jbc.275.12.8945. PMID10722742.
Hay TJ, Meek DW (July 2000). "Multiple sites of in vivo phosphorylation in the MDM2 oncoprotein cluster within two important functional domains". FEBS Lett.478 (1-2): 183–6. doi:10.1016/S0014-5793(00)01850-0. PMID10922493.
Honda R, Yasuda H (March 2000). "Activity of MDM2, a ubiquitin ligase, toward p53 or itself is dependent on the RING finger domain of the ligase". Oncogene19 (11): 1473–6. doi:10.1038/sj.onc.1203464. PMID10723139.
Kussie PH, Gorina S, Marechal V, Elenbaas B, Moreau J, Levine AJ, Pavletich NP (November 1996). "Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain". Science274 (5289): 948–53. doi:10.1126/science.274.5289.948. PMID8875929.
Meek DW, Knippschild U (December 2003). "Posttranslational modification of MDM2". Mol. Cancer Res.1 (14): 1017–26. PMID14707285.
Midgley CA, Desterro JM, Saville MK, Howard S, Sparks A, Hay RT, Lane DP (May 2000). "An N-terminal p14ARF peptide blocks Mdm2-dependent ubiquitination in vitro and can activate p53 in vivo". Oncogene19 (19): 2312–23. doi:10.1038/sj.onc.1203593. PMID10822382.
Momand J, Zambetti GP, Olson DC, George D, Levine AJ (June 1992). "The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation". Cell69 (7): 1237–45. doi:10.1016/0092-8674(92)90644-R. PMID1535557.