p53 is an intrinsically disordered protein with a large number of post-translational modifications and interacting partners. The hierarchical order and subcellular location of these events are still poorly understood. The activation of p53 during the DNA damage response (DDR) requires a switch in the activity of the E3 ubiquitin ligase MDM2 from a negative to a positive regulator of p53. This is mediated by the ATM kinase that regulates the binding of MDM2 to the p53 mRNA facilitating an increase in p53 synthesis. Here we show that the binding of MDM2 to the p53 mRNA brings ATM to the p53 polysome where it phosphorylates the nascent p53 at serine 15 and prevents MDM2-mediated degradation of p53. A single synonymous mutation in p53 codon 22 (L22L) prevents the phosphorylation of the nascent p53 protein and the stabilization of p53 following genotoxic stress. The ATM trafficking from the nucleus to the p53 polysome is mediated by MDM2, which requires its interaction with the ribosomal proteins RPL5 and RPL11. These results show how the ATM kinase phosphorylates the p53 protein while it is being synthesized and offer a novel mechanism whereby a single synonymous mutation controls the stability and activity of the encoded protein.

Department of Medical Biosciences Umeå University Umeå Sweden

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

Instituto de Física Universidad Autónoma de San Luis Potosí SLP México

RECAMO Masaryk Memorial Cancer Institute Zluty kopec 7 Brno Czech Republic

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Banin S., Moyal L., Shieh S., et al. . (1998). Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science 281, 1674–1677. PubMed

Bursac S., Brdovcak M.C., Donati G., et al. . (2014). Activation of the tumor suppressor p53 upon impairment of ribosome biogenesis. Biochim. Biophys. Acta 1842, 817–830. PubMed

Candeias M.M., Malbert-Colas L., Powell D.J., et al. . (2008). P53 mRNA controls p53 activity by managing Mdm2 functions. Nat. Cell Biol. 10, 1098–1105. PubMed

Canman C.E., Lim D.S., Cimprich K.A., et al. . (1998). Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science 281, 1677–1679. PubMed

Chen L., Gilkes D.M., Pan Y., et al. . (2005). ATM and Chk2-dependent phosphorylation of MDMX contribute to p53 activation after DNA damage. EMBO J. 24, 3411–3422. PubMed PMC

Chene P. (2003). Inhibiting the p53-MDM2 interaction: an important target for cancer therapy. Nat. Rev. Cancer 3, 102–109. PubMed

Cheng Q., and Chen J. (2010). Mechanism of p53 stabilization by ATM after DNA damage. Cell Cycle 9, 472–478. PubMed PMC

Cheok C.F., Verma C.S., Baselga J., et al. . (2011). Translating p53 into the clinic. Nat. Rev. Clin. Oncol. 8, 25–37. PubMed

Coffill C.R., Lee A.P., Siau J.W., et al. . (2016). The p53–Mdm2 interaction and the E3 ligase activity of Mdm2/Mdm4 are conserved from lampreys to humans. Genes Dev. 30, 281–292. PubMed PMC

Donati G., Peddigari S., Mercer C.A., et al. . (2013). 5S ribosomal RNA is an essential component of a nascent ribosomal precursor complex that regulates the Hdm2-p53 checkpoint. Cell Rep. 4, 87–98. PubMed PMC

Fahraeus R., Marin M., and Olivares-Illana V. (2016). Whisper mutations: cryptic messages within the genetic code. Oncogene 35, 3753–3759. PubMed

Gajjar M., Candeias M.M., Malbert-Colas L., 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. PubMed

Gandin V., Sikstrom K., Alain T., et al. . (2014). Polysome fractionation and analysis of mammalian translatomes on a genome-wide scale. J. Vis. Exp. 87, e51455. PubMed PMC

Gannon H.S., Woda B.A., and Jones S.N. (2012). ATM phosphorylation of Mdm2 Ser394 regulates the amplitude and duration of the DNA damage response in mice. Cancer Cell 21, 668–679. PubMed PMC

Gartner J.J., Parker S.C., Prickett T.D., et al. . (2013). Whole-genome sequencing identifies a recurrent functional synonymous mutation in melanoma. Proc. Natl Acad. Sci. USA 110, 13481–13486. PubMed PMC

Grover R., Candeias M.M., Fahraeus R., et al. . (2009). p53 and little brother p53/47: linking IRES activities with protein functions. Oncogene 28, 2766–2772. PubMed

Gullberg M., Goransson C., and Fredriksson S. (2011). Duolink-‘In-cell Co-IP’ for visualization of protein interactions in situ. Nat. Methods. 8, 982.

Guo H., Ingolia N.T., Weissman J.S., et al. . (2010). Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466, 835–840. PubMed PMC

Haupt Y., Maya R., Kazaz A., et al. . (1997). Mdm2 promotes the rapid degradation of p53. Nature 387, 296–299. PubMed

Hickson I., Zhao Y., Richardson C.J., et al. . (2004). Identification and characterization of a novel and specific inhibitor of the ataxia-telangiectasia mutated kinase ATM. Cancer Res. 64, 9152–9159. PubMed

Hosp F., Vossfeldt H., Heinig M., et al. . (2015). Quantitative interaction proteomics of neurodegenerative disease proteins. Cell Rep. 11, 1134–1146. PubMed PMC

Joerger A.C., and Fersht A.R. (2010). The tumor suppressor p53: from structures to drug discovery. Cold Spring Harb. Perspect. Biol. 2, a000919. PubMed PMC

Kannan S., Lane D.P., and Verma C.S. (2016). Long range recognition and selection in IDPs: the interactions of the C-terminus of p53. Sci. Rep. 6, 23750. PubMed PMC

Karakostis K., Ponnuswamy A., Fusee L.T., et al. . (2016). p53 mRNA and p53 protein structures have evolved independently to interact with MDM2. Mol. Biol. Evol. 33, 1280–1292. PubMed

Kimchi-Sarfaty C., Oh J.M., Kim I.W., et al. . (2007). A ‘silent’ polymorphism in the MDR1 gene changes substrate specificity. Science 315, 525–528. PubMed

Komatsu K., Matsuura S., Tauchi H., et al. . (1996). The gene for Nijmegen breakage syndrome (V2) is not located on chromosome 11. Am. J. Hum. Genet. 58, 885–888. PubMed PMC

Koos B., Andersson L., Clausson C.M., et al. . (2014). Analysis of protein interactions in situ by proximity ligation assays. Curr. Top. Microbiol. Immunol. 377, 111–126. PubMed

Kubbutat M.H., Jones S.N., and Vousden K.H. (1997). Regulation of p53 stability by Mdm2. Nature 387, 299–303. PubMed

Kubbutat M.H., Ludwig R.L., Levine A.J., et al. . (1999). Analysis of the degradation function of Mdm2. Cell Growth Differ. 10, 87–92. PubMed

Lambert P.F., Kashanchi F., Radonovich M.F., et al. . (1998). Phosphorylation of p53 serine 15 increases interaction with CBP. J. Biol. Chem. 273, 33048–33053. PubMed

Lee H.J., Lan L., Peng G., et al. . (2015). Tyrosine 370 phosphorylation of ATM positively regulates DNA damage response. Cell Res. 25, 225–236. PubMed PMC

Lindstrom M.S., Deisenroth C., and Zhang Y. (2007. a). Putting a finger on growth surveillance: insight into MDM2 zinc finger-ribosomal protein interactions. Cell Cycle 6, 434–437. PubMed

Lindstrom M.S., Jin A., Deisenroth C., et al. . (2007. b). Cancer-associated mutations in the MDM2 zinc finger domain disrupt ribosomal protein interaction and attenuate MDM2-induced p53 degradation. Mol. Cell. Biol. 27, 1056–1068. PubMed PMC

Lopez I., Tournillon A.S., Prado Martins R., et al. . (2017). p53-mediated suppression of BiP triggers BIK-induced apoptosis during prolonged endoplasmic reticulum stress. Cell Death Differ. 24, 1717–1729. PubMed PMC

Loughery J., Cox M., Smith L.M., et al. . (2014). Critical role for p53-serine 15 phosphorylation in stimulating transactivation at p53-responsive promoters. Nucleic Acids Res. 42, 7666–7680. PubMed PMC

MacLaine N.J., and Hupp T.R. (2011). How phosphorylation controls p53. Cell Cycle 10, 916–921. PubMed

Malbert-Colas L., Ponnuswamy A., Olivares-Illana V., et al. . (2014). HDMX folds the nascent p53 mRNA following activation by the ATM kinase. Mol. Cell 54, 500–511. PubMed

Marechal A., and Zou L. (2013). DNA damage sensing by the ATM and ATR kinases. Cold Spring Harbor Perspect. Biol. 5, a012716. PubMed PMC

Marine J.C., Dyer M.A., and Jochemsen A.G. (2007). MDMX: from bench to bedside. J. Cell Sci. 120, 371–378. PubMed

Marine J.C., and Lozano G. (2010). Mdm2-mediated ubiquitylation: p53 and beyond. Cell Death Differ. 17, 93–102. PubMed

Matsuura S., Weemaes C., Smeets D., et al. . (1997). Genetic mapping using microcell-mediated chromosome transfer suggests a locus for Nijmegen breakage syndrome at chromosome 8q21-24. Am. J. Hum. Genet. 60, 1487–1494. PubMed PMC

Maya R., Balass M., Kim S.T., et al. . (2001). ATM-dependent phosphorylation of Mdm2 on serine 395: role in p53 activation by DNA damage. Genes Dev. 15, 1067–1077. PubMed PMC

Medina-Medina I., Garcia-Beltran P., de la Mora-de la Mora I., et al. . (2016). Allosteric interactions by p53 mRNA govern HDM2 E3 ubiquitin ligase specificity under different conditions. Mol. Cell. Biol. 36, 2195–2205. PubMed PMC

Meek D.W. (2009). Tumour suppression by p53: a role for the DNA damage response? Nat. Rev. Cancer 9, 714–723. PubMed

Meek D.W., and Anderson C.W. (2009). Posttranslational modification of p53: cooperative integrators of function. Cold Spring Harbor Perspect. Biol. 1, a000950. PubMed PMC

Morgan S.E., and Kastan M.B. (1997). p53 and ATM: cell cycle, cell death, and cancer. Adv. Cancer Res. 71, 1–25. PubMed

Naski N., Gajjar M., Bourougaa K., et al. . (2009). The p53 mRNA–Mdm2 interaction. Cell Cycle 8, 31–34. PubMed

Ofir-Rosenfeld Y., Boggs K., Michael D., et al. . (2008). Mdm2 regulates p53 mRNA translation through inhibitory interactions with ribosomal protein L26. Mol. Cell 32, 180–189. PubMed PMC

Pant V., Xiong S., Jackson J.G., et al. . (2013). The p53-Mdm2 feedback loop protects against DNA damage by inhibiting p53 activity but is dispensable for p53 stability, development, and longevity. Genes Dev. 27, 1857–1867. PubMed PMC

Pereg Y., Shkedy D., de Graaf P., et al. . (2005). Phosphorylation of Hdmx mediates its Hdm2- and ATM-dependent degradation in response to DNA damage. Proc. Natl Acad. Sci. USA 102, 5056–5061. PubMed PMC

Ren J., Wen L., Gao X., et al. . (2009). DOG 1.0: illustrator of protein domain structures. Cell Res. 19, 271–273. PubMed

Saito S., Yamaguchi H., Higashimoto Y., et al. . (2003). Phosphorylation site interdependence of human p53 post-translational modifications in response to stress. J. Biol. Chem. 278, 37536–37544. PubMed

Sauna Z.E., and Kimchi-Sarfaty C. (2011). Understanding the contribution of synonymous mutations to human disease. Nat. Rev. Genet. 12, 683–691. PubMed

Schwanhausser B., Busse D., Li N., et al. . (2011). Global quantification of mammalian gene expression control. Nature 473, 337–342. PubMed

Scott S.P., Bendix R., Chen P., et al. . (2002). Missense mutations but not allelic variants alter the function of ATM by dominant interference in patients with breast cancer. Proc. Natl Acad. Sci. USA 99, 925–930. PubMed PMC

Soderberg O., Gullberg M., Jarvius M., et al. . (2006). Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat. Methods 3, 995–1000. PubMed

Stracker T.H., Roig I., Knobel P.A., et al. . (2013). The ATM signaling network in development and disease. Front. Genet. 4, 37. PubMed PMC

Supek F., Minana B., Valcarcel J., et al. . (2014). Synonymous mutations frequently act as driver mutations in human cancers. Cell 156, 1324–1335. PubMed

Takagi M., Tsuchida R., Oguchi K., et al. . (2004). Identification and characterization of polymorphic variations of the ataxia telangiectasia mutated (ATM) gene in childhood Hodgkin disease. Blood 103, 283–290. PubMed

Teufel D.P., Bycroft M., and Fersht A.R. (2009). Regulation by phosphorylation of the relative affinities of the N-terminal transactivation domains of p53 for p300 domains and Mdm2. Oncogene 28, 2112–2118. PubMed PMC

Tompa P. (2002). Intrinsically unstructured proteins. Trends Biochem. Sci. 27, 527–533. PubMed

Tompa P. (2005). The interplay between structure and function in intrinsically unstructured proteins. FEBS Lett. 579, 3346–3354. PubMed

Tournillon A.S., Lopez I., Malbert-Colas L., et al. . (2016). p53 binds the mdmx mRNA and controls its translation. Oncogene 36, 723–730. PubMed

Uversky V.N. (2016). p53 proteoforms and intrinsic disorder: an illustration of the protein structure-function continuum concept. Int. J. Mol. Sci. 17, 1874. PubMed PMC

Weibrecht I., Lundin E., Kiflemariam S., et al. . (2013). In situ detection of individual mRNA molecules and protein complexes or post-translational modifications using padlock probes combined with the in situ proximity ligation assay. Nat. Protoc. 8, 355–372. PubMed

Wright P.E., and Dyson H.J. (2009). Linking folding and binding. Curr. Opin. Struct. Biol. 19, 31–38. PubMed PMC

Yang D.Q., Halaby M.J., and Zhang Y. (2006). The identification of an internal ribosomal entry site in the 5′-untranslated region of p53 mRNA provides a novel mechanism for the regulation of its translation following DNA damage. Oncogene 25, 4613–4619. PubMed

Zhang Q., Xiao H., Chai S.C., et al. . (2011). Hydrophilic residues are crucial for ribosomal protein L11 (RPL11) interaction with zinc finger domain of MDM2 and p53 protein activation. J. Biol. Chem. 286, 38264–38274. PubMed PMC

Zhou X., Liao J.M., Liao W.J., et al. . (2012). Scission of the p53-MDM2 loop by ribosomal proteins. Genes Cancer 3, 298–310. PubMed PMC

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