HDMX and its homologue HDM2 are two essential proteins for the cell; after genotoxic stress, both are phosphorylated near to their RING domain, specifically at serine 403 and 395, respectively. Once phosphorylated, both can bind the p53 mRNA and enhance its translation; however, both recognize p53 protein and provoke its degradation under normal conditions. HDM2 has been well-recognized as an E3 ubiquitin ligase, whereas it has been reported that even with the high similarity between the RING domains of the two homologs, HDMX does not have the E3 ligase activity. Despite this, HDMX is needed for the proper p53 poly-ubiquitination. Phosphorylation at serine 395 changes the conformation of HDM2, helping to explain the switch in its activity, but no information on HDMX has been published. Here, we study the conformation of HDMX and its phospho-mimetic mutant S403D, investigate its E3 ligase activity and dissect its binding with p53. We show that phospho-mutation does not change the conformation of the protein, but HDMX is indeed an E3 ubiquitin ligase in vitro; however, in vivo, no activity was found. We speculated that HDMX is regulated by induced fit, being able to switch activity accordingly to the specific partner as p53 protein, p53 mRNA or HDM2. Our results aim to contribute to the elucidation of the contribution of the HDMX to p53 regulation.

Catedra CONACyT Laboratorio de Biomarcadores Moleculares Instituto de Física Universidad Autónoma de San Luis Potosí México City México

Department of Medical Biosciences Umeå University SE 90185 Umeå Sweden

Équipe Labellisée Ligue Contre le Cancer Université Paris 7 INSERM UMR 1162 27 Rue Juliette Dodu 75010 Paris France

Laboratorio de Interacciones Biomoleculares y cáncer Instituto de Física Universidad Autónoma de San Luis Potosí Av Parque Chapultepec 1570 Privadas del pedregal 78210 SLP México

Regional Centre for Applied Molecular Oncology Masaryk Memorial Cancer Institute Zluty kopec 7 656 53 Brno Czech Republic

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Li C., Chen L. and Chen J. (2002) DNA damage induces MDMX nuclear translocation by p53-dependent and -independent mechanisms. Mol. Cell. Biol. 22, 7562–7571 10.1128/MCB.22.21.7562-7571.2002 PubMed DOI PMC

Chen L., Gilkes D.M., Pan Y., Lane W.S. and Chen J. (2005) ATM and Chk2-dependent phosphorylation of MDMX contribute to p53 activation after DNA damage. EMBO J. 24, 3411–3422 10.1038/sj.emboj.7600812 PubMed DOI PMC

Malbert-Colas L., Ponnuswamy A., Olivares-Illana V., Tournillon A.S., Naski N. and Fahraeus R. (2014) HDMX folds the nascent p53 mRNA following activation by the ATM kinase. Mol. Cell. 54, 500–511 10.1016/j.molcel.2014.02.035 PubMed DOI

Fahraeus R. and Olivares-Illana V. (2014) MDM2’s social network. Oncogene 33, 4365–4376 10.1038/onc.2013.410 PubMed DOI

Gajjar M., Candeias M.M., Malbert-Colas L., Mazars A., Fujita J., Olivares-Illana V.et al. . (2012) The p53 mRNA-Mdm2 interaction controls Mdm2 nuclear trafficking and is required for p53 activation following DNA damage. Cancer Cell. 21, 25–35 10.1016/j.ccr.2011.11.016 PubMed DOI

Gu L., Zhu N., Zhang H., Durden D.L., Feng Y. and Zhou M. (2009) Regulation of XIAP translation and induction by MDM2 following irradiation. Cancer Cell. 15, 363–375 10.1016/j.ccr.2009.03.002 PubMed DOI PMC

Jung C.H., Kim J., Park J.K., Hwang S.G., Moon S.K., Kim W.J.et al. . (2013) Mdm2 increases cellular invasiveness by binding to and stabilizing the Slug mRNA. Cancer Lett. 335, 270–277 10.1016/j.canlet.2013.02.035 PubMed DOI

Gu L., Zhang H., He J., Li J., Huang M. and Zhou M. (2012) MDM2 regulates MYCN mRNA stabilization and translation in human neuroblastoma cells. Oncogene 31, 1342–1353 10.1038/onc.2011.343 PubMed DOI PMC

Hernandez-Monge J., Martinez-Sanchez M., Rousset-Roman A., Medina-Medina I. and Olivares-Illana V. (2021) MDM2 regulates RB levels during genotoxic stress. EMBO Rep. 22, e50615 10.15252/embr.202050615 PubMed DOI PMC

Gnanasundram S.V., Malbert-Colas L., Chen S., Fusee L., Daskalogianni C., Muller P.et al. . (2020) MDM2’s dual mRNA binding domains co-ordinate its oncogenic and tumour suppressor activities. Nucleic Acids Res. 48, 6775–6787 10.1093/nar/gkaa431 PubMed DOI PMC

Medina-Medina I., Martinez-Sanchez M., Hernandez-Monge J., Fahraeus R., Muller P. and Olivares-Illana V. (2018) p53 promotes its own polyubiquitination by enhancing the HDM2 and HDMX interaction. Protein Sci. 27, 976–986 10.1002/pro.3405 PubMed DOI PMC

Maya R., Balass M., Kim S.T., Shkedy D., Leal J.F., Shifman O.et al. . (2001) ATM-dependent phosphorylation of Mdm2 on serine 395: role in p53 activation by DNA damage. Genes Dev. 15, 1067–1077 10.1101/gad.886901 PubMed DOI PMC

Medina-Medina I., Garcia-Beltran P., de la Mora-de la Mora I., Oria-Hernandez J., Millot G., Fahraeus R.et al. . (2016) Allosteric interactions by p53 mRNA Govern HDM2 E3 ubiquitin ligase specificity under different conditions. Mol. Cell. Biol. 36, 2195–2205 10.1128/MCB.00113-16 PubMed DOI PMC

Uhrik L., Wang L., Haronikova L., Medina-Medina I., Rebolloso-Gomez Y., Chen S.et al. . (2019) Allosteric changes in HDM2 by the ATM phosphomimetic S395D mutation: implications on HDM2 function. Biochem. J. 476, 3401–3411 10.1042/BCJ20190687 PubMed DOI PMC

Candeias M.M., Malbert-Colas L., Powell D.J., Daskalogianni C., Maslon M.M., Naski N.et al. . (2008) P53 mRNA controls p53 activity by managing Mdm2 functions. Nat. Cell Biol. 10, 1098–1105 10.1038/ncb1770 PubMed DOI

Naski N., Gajjar M., Bourougaa K., Malbert-Colas L., Fahraeus R. and Candeias M.M. (2009) The p53 mRNA-Mdm2 interaction. Cell Cycle 8, 31–34 10.4161/cc.8.1.7326 PubMed DOI

Haupt Y., Maya R., Kazaz A. and Oren M. (1997) Mdm2 promotes the rapid degradation of p53. Nature 387, 296–299 10.1038/387296a0 PubMed DOI

Honda R., Tanaka H. and Yasuda H. (1997) Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS Lett. 420, 25–27 10.1016/S0014-5793(97)01480-4 PubMed DOI

Sharp D.A., Kratowicz S.A., Sank M.J. and George D.L. (1999) Stabilization of the MDM2 oncoprotein by interaction with the structurally related MDMX protein. J. Biol. Chem. 274, 38189–38196 10.1074/jbc.274.53.38189 PubMed DOI

Tanimura S., Ohtsuka S., Mitsui K., Shirouzu K., Yoshimura A. and Ohtsubo M. (1999) MDM2 interacts with MDMX through their RING finger domains. FEBS Lett. 447, 5–9 10.1016/S0014-5793(99)00254-9 PubMed DOI

Iyappan S., Wollscheid H.P., Rojas-Fernandez A., Marquardt A., Tang H.C., Singh R.K.et al. . (2010) Turning the RING domain protein MdmX into an active ubiquitin-protein ligase. J. Biol. Chem. 285, 33065–33072 10.1074/jbc.M110.115113 PubMed DOI PMC

Yang J., Jin A., Han J., Chen X., Zheng J. and Zhang Y. (2021) MDMX Recruits UbcH5c to Facilitate MDM2 E3 Ligase Activity and Subsequent p53 Degradation In Vivo. Cancer Res. 81, 898–909 10.1158/0008-5472.CAN-20-0790 PubMed DOI PMC

Wang X., Wang J. and Jiang X. (2011) MdmX protein is essential for Mdm2 protein-mediated p53 polyubiquitination. J. Biol. Chem. 286, 23725–23734 10.1074/jbc.M110.213868 PubMed DOI PMC

Linares L.K., Hengstermann A., Ciechanover A., Muller S. and Scheffner M. (2003) HdmX stimulates Hdm2-mediated ubiquitination and degradation of p53. Proc. Natl. Acad. Sci. U. S. A. 100, 12009–12014 10.1073/pnas.2030930100 PubMed DOI PMC

Egorova O., Mis M. and Sheng Y. (2014) A site-directed mutagenesis study of the MdmX RING domain. Biochem. Biophys. Res. Commun. 447, 696–701 10.1016/j.bbrc.2014.04.065 PubMed DOI

Badciong J.C. and Haas A.L. (2002) MdmX is a RING finger ubiquitin ligase capable of synergistically enhancing Mdm2 ubiquitination. J. Biol. Chem. 277, 49668–49675 10.1074/jbc.M208593200 PubMed DOI

Midgley C.A., Fisher C.J., Bartek J., Vojtesek B., Lane D. and Barnes D.M. (1992) Analysis of p53 expression in human tumours: an antibody raised against human p53 expressed in Escherichia coli. J. Cell Sci. 101, 183–189 10.1242/jcs.101.1.183 PubMed DOI

Li X., Romero P., Rani M., Dunker A.K. and Obradovic Z. (1999) Predicting protein disorder for N-, C-, and internal regions. Genome Inform. Ser. Workshop Genome Inform. 10, 30–40 PubMed

Uhrik L., Wang L., Haronikova L., Medina-Medina I., Rebolloso-Gomez Y., Chen S.et al. . (2019) Allosteric changes in HDM2 by the ATM phospho-mimetic S395D mutation: Implications on HDM2 function. Biochem. J. 10.1042/BCJ20190687 PubMed DOI PMC

Jumper J., Evans R., Pritzel A., Green T., Figurnov M., Ronneberger O.et al. . (2021) Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 10.1038/s41586-021-03819-2 PubMed DOI PMC

Varadi M., Anyango S., Deshpande M., Nair S., Natassia C., Yordanova G.et al. . (2021) AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Res. 50, D439–D444 PubMed PMC

Shvarts A., Steegenga W.T., Riteco N., van Laar T., Dekker P., Bazuine M.et al. . (1996) MDMX: a novel p53-binding protein with some functional properties of MDM2. EMBO J. 15, 5349–5357 10.1002/j.1460-2075.1996.tb00919.x PubMed DOI PMC

Danovi D., Meulmeester E., Pasini D., Migliorini D., Capra M., Frenk R.et al. . (2004) Amplification of Mdmx (or Mdm4) directly contributes to tumor formation by inhibiting p53 tumor suppressor activity. Mol. Cell. Biol. 24, 5835–5843 10.1128/MCB.24.13.5835-5843.2004 PubMed DOI PMC

Shvarts A., Bazuine M., Dekker P., Ramos Y.F., Steegenga W.T., Merckx G.et al. . (1997) Isolation and identification of the human homolog of a new p53-binding protein, Mdmx. Genomics 43, 34–42 10.1006/geno.1997.4775 PubMed DOI

Wei X., Wu S., Song T., Chen L., Gao M., Borcherds W.et al. . (2016) Secondary interaction between MDMX and p53 core domain inhibits p53 DNA binding. Proc. Natl. Acad. Sci. U. S. A. 113, E2558–E2563 10.1073/pnas.1603838113 PubMed DOI PMC

Tournillon A.S., Lopez I., Malbert-Colas L., Naski N., Olivares-Illana V. and Fahraeus R. (2015) The alternative translated MDMX(p60) isoform regulates MDM2 activity. Cell Cycle 14, 449–458 10.4161/15384101.2014.977081 PubMed DOI PMC

Tackmann N.R. and Zhang Y. (2017) Mouse modelling of the MDM2/MDMX-p53 signalling axis. J. Mol. Cell Biol. 9, 34–44 10.1093/jmcb/mjx006 PubMed DOI PMC

Montes de Oca Luna R., Wagner D.S. and Lozano G. (1995) Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53. Nature 378, 203–206 10.1038/378203a0 PubMed DOI

Jones S.N., Roe A.E., Donehower L.A. and Bradley A. (1995) Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53. Nature 378, 206–208 10.1038/378206a0 PubMed DOI

Parant J., Chavez-Reyes A., Little N.A., Yan W., Reinke V., Jochemsen A.G.et al. . (2001) Rescue of embryonic lethality in Mdm4-null mice by loss of Trp53 suggests a nonoverlapping pathway with MDM2 to regulate p53. Nat. Genet. 29, 92–95 10.1038/ng714 PubMed DOI