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A p53–phosphoinositide signalosome regulates nuclear AKT activation

Nature Cell Biology volume 24, pages 1099–1113 (2022)Cite this article

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Abstract

The tumour suppressor p53 and PI3K–AKT pathways have fundamental roles in the regulation of cell growth and apoptosis, and are frequently mutated in cancer. Here, we show that genotoxic stress induces nuclear AKT activation through a p53-dependent mechanism that is distinct from the canonical membrane-localized PI3K–AKT pathway. Following genotoxic stress, a nuclear PI3K binds p53 in the non-membranous nucleoplasm to generate a complex of p53 and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), which recruits AKT, PDK1 and mTORC2 to activate AKT and phosphorylate FOXO proteins, thereby inhibiting DNA damage-induced apoptosis. Wild-type p53 activates nuclear AKT in an on/off fashion following stress, whereas mutant p53 dose-dependently stimulates high basal AKT activity. The p53–PtdIns(3,4,5)P3 complex is dephosphorylated to p53–phosphatidylinositol 4,5-bisphosphate by PTEN to inhibit AKT activation. The nuclear p53–phosphoinositide signalosome is distinct from the canonical membrane-localized pathway and insensitive to PI3K inhibitors currently in the clinic, which underscores its therapeutic relevance.

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Fig. 1: p53 interacts with nuclear AKT and regulates its activation.
Fig. 2: p53 recruits upstream AKT-activating kinases in the nucleus.
Fig. 3: The nuclear p53–AKT complex targets FOXO proteins.
Fig. 4: p53 binds PtdIns(3,4,5)P3, and IPMK and PTEN regulate the interconversion of nuclear p53–PIPn complexes.
Fig. 5: p53–PtdIns(3,4,5)P3 recruits and activates the nuclear AKT pathway.
Fig. 6: The p53–PIPn signalosome links the nuclear PI3K–AKT pathway to DNA repair and cell survival.
Fig. 7: The interaction of p53 with AKT depends on PIPn and is mediated by the C terminus of p53.
Fig. 8: Regulation of the nuclear AKT pathway by p53 depends on the PIPn-binding motif of p53.

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Data availability

All data supporting the findings of this study are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

References

  1. Choi, S. et al. Agonist-stimulated phosphatidylinositol-3,4,5-trisphosphate generation by scaffolded phosphoinositide kinases. Nat. Cell Biol. 18, 1324–1335 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Thapa, N., Horn, H. T. & Anderson, R. A. Phosphoinositide spatially free AKT/PKB activation to all membrane compartments. Adv. Biol. Regul. 72, 1–6 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Fruman, D. A. et al. The PI3K pathway in human disease. Cell 170, 605–635 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Hoxhaj, G. & Manning, B. D. The PI3K–AKT network at the interface of oncogenic signalling and cancer metabolism. Nat. Rev. Cancer 20, 74–88 (2020).

    Article  CAS  PubMed  Google Scholar 

  5. Manning, B. D. & Toker, A. AKT/PKB signaling: navigating the network. Cell 169, 381–405 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Stokoe, D. et al. Dual role of phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B. Science 277, 567–570 (1997).

    Article  CAS  PubMed  Google Scholar 

  7. Sarbassov, D. D., Guertin, D. A., Ali, S. M. & Sabatini, D. M. Phosphorylation and regulation of Akt/PKB by the rictor–mTOR complex. Science 307, 1098–1101 (2005).

    Article  CAS  PubMed  Google Scholar 

  8. Ebner, M., Lucic, I., Leonard, T. A. & Yudushkin, I. PI(3,4,5)P3 engagement restricts Akt activity to cellular membranes. Mol. Cell 65, 416–431.e6 (2017).

    Article  CAS  PubMed  Google Scholar 

  9. Alessi, D. R. et al. Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Bα. Curr. Biol. 7, 261–269 (1997).

    Article  CAS  PubMed  Google Scholar 

  10. Martelli, A. M. et al. The emerging multiple roles of nuclear Akt. Biochim. Biophys. Acta 1823, 2168–2178 (2012).

    Article  CAS  PubMed  Google Scholar 

  11. Meier, R., Alessi, D. R., Cron, P., Andjelkovic, M. & Hemmings, B. A. Mitogenic activation, phosphorylation, and nuclear translocation of protein kinase Bβ. J. Biol. Chem. 272, 30491–30497 (1997).

    Article  CAS  PubMed  Google Scholar 

  12. Andjelkovic, M. et al. Role of translocation in the activation and function of protein kinase B. J. Biol. Chem. 272, 31515–31524 (1997).

    Article  CAS  PubMed  Google Scholar 

  13. Borgatti, P. et al. Threonine 308 phosphorylated form of Akt translocates to the nucleus of PC12 cells under nerve growth factor stimulation and associates with the nuclear matrix protein nucleolin. J. Cell. Physiol. 196, 79–88 (2003).

    Article  CAS  PubMed  Google Scholar 

  14. Wang, R. & Brattain, M. G. AKT can be activated in the nucleus. Cell Signal. 18, 1722–1731 (2006).

    Article  CAS  PubMed  Google Scholar 

  15. Bozulic, L., Surucu, B., Hynx, D. & Hemmings, B. A. PKBα/Akt1 acts downstream of DNA-PK in the DNA double-strand break response and promotes survival. Mol. Cell 30, 203–213 (2008).

    Article  CAS  PubMed  Google Scholar 

  16. Boehme, K. A., Kulikov, R. & Blattner, C. p53 stabilization in response to DNA damage requires Akt/PKB and DNA-PK. Proc. Natl Acad. Sci. USA 105, 7785–7790 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Muller, P. A. & Vousden, K. H. p53 mutations in cancer. Nat. Cell Biol. 15, 2–8 (2013).

    Article  CAS  PubMed  Google Scholar 

  18. Kastenhuber, E. R. & Lowe, S. W. Putting p53 in context. Cell 170, 1062–1078 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Choi, S., Chen, M., Cryns, V. L. & Anderson, R. A. A nuclear phosphoinositide kinase complex regulates p53. Nat. Cell Biol. 21, 462–475 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Xu, R. et al. Inositol polyphosphate multikinase is a coactivator of p53-mediated transcription and cell death. Sci. Signal. 6, ra22 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Freeman, D. J. et al. PTEN tumor suppressor regulates p53 protein levels and activity through phosphatase-dependent and -independent mechanisms. Cancer Cell 3, 117–130 (2003).

    Article  CAS  PubMed  Google Scholar 

  22. Blind, R. D., Suzawa, M. & Ingraham, H. A. Direct modification and activation of a nuclear receptor-PIP2 complex by the inositol lipid kinase IPMK. Sci. Signal. 5, ra44 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  23. Blind, R. D. et al. The signaling phospholipid PIP3 creates a new interaction surface on the nuclear receptor SF-1. Proc. Natl Acad. Sci. USA 111, 15054–15059 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Huang, Y. et al. Global tumor protein p53/p63 interactome: making a case for cisplatin chemoresistance. Cell Cycle 11, 2367–2379 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Liu, Q., Turner, K. M., Alfred Yung, W. K., Chen, K. & Zhang, W. Role of AKT signaling in DNA repair and clinical response to cancer therapy. Neuro Oncol. 16, 1313–1323 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Mayer, I. A. & Arteaga, C. L. The PI3K/AKT pathway as a target for cancer treatment. Annu. Rev. Med. 67, 11–28 (2016).

    Article  CAS  PubMed  Google Scholar 

  27. Wang, Y. H. et al. DNA damage causes rapid accumulation of phosphoinositides for ATR signaling. Nat. Commun. 8, 2118 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Casamayor, A., Morrice, N. A. & Alessi, D. R. Phosphorylation of Ser-241 is essential for the activity of 3-phosphoinositide-dependent protein kinase-1: identification of five sites of phosphorylation in vivo. Biochem. J. 342, 287–292 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Moon, Z., Wang, Y., Aryan, N., Mousseau, D. D. & Scheid, M. P. Serine 396 of PDK1 is required for maximal PKB activation. Cell Signal. 20, 2038–2049 (2008).

    Article  CAS  PubMed  Google Scholar 

  30. Liu, P. et al. PtdIns(3,4,5)P3-dependent activation of the mTORC2 kinase complex. Cancer Discov. 5, 1194–1209 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Webb, A. E. & Brunet, A. FOXO transcription factors: key regulators of cellular quality control. Trends Biochem. Sci. 39, 159–169 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Chung, Y. M. et al. FOXO3 signalling links ATM to the p53 apoptotic pathway following DNA damage. Nat. Commun. 3, 1000 (2012).

    Article  PubMed  CAS  Google Scholar 

  33. Wang, F. et al. Biochemical and structural characterization of an intramolecular interaction in FOXO3a and its binding with p53. J. Mol. Biol. 384, 590–603 (2008).

    Article  CAS  PubMed  Google Scholar 

  34. Fu, W. et al. MDM2 acts downstream of p53 as an E3 ligase to promote FOXO ubiquitination and degradation. J. Biol. Chem. 284, 13987–14000 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Huang, H. & Tindall, D. J. Regulation of FOXO protein stability via ubiquitination and proteasome degradation. Biochim. Biophys. Acta 1813, 1961–1964 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Zhang, X., Tang, N., Hadden, T. J. & Rishi, A. K. Akt, FoxO and regulation of apoptosis. Biochim. Biophys. Acta 1813, 1978–1986 (2011).

    Article  CAS  PubMed  Google Scholar 

  37. Semenas, J. et al. The role of PI3K/AKT-related PIP5K1α and the discovery of its selective inhibitor for treatment of advanced prostate cancer. Proc. Natl Acad. Sci. USA 111, E3689–E3698 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Currie, R. A. et al. Role of phosphatidylinositol 3,4,5-trisphosphate in regulating the activity and localization of 3-phosphoinositide-dependent protein kinase-1. Biochem. J. 337, 575–583 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Calleja, V. et al. Intramolecular and intermolecular interactions of protein kinase B define its activation in vivo. PLoS Biol. 5, e95 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Chen, M. et al. The nuclear phosphoinositide response to stress. Cell Cycle 19, 268–289 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Barlow, C. A., Laishram, R. S. & Anderson, R. A. Nuclear phosphoinositides: a signaling enigma wrapped in a compartmental conundrum. Trends Cell Biol. 20, 25–35 (2010).

    Article  CAS  PubMed  Google Scholar 

  42. Boronenkov, I. V., Loijens, J. C., Umeda, M. & Anderson, R. A. Phosphoinositide signaling pathways in nuclei are associated with nuclear speckles containing pre-mRNA processing factors. Mol. Biol. Cell 9, 3547–3560 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Sharma, A., Singh, K. & Almasan, A. Histone H2AX phosphorylation: a marker for DNA damage. Methods Mol. Biol. 920, 613–626 (2012).

    Article  CAS  PubMed  Google Scholar 

  44. Liu, L. et al. DNA-PK promotes activation of the survival kinase AKT in response to DNA damage through an mTORC2–ECT2 pathway. Sci. Signal. 15, eabh2290 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Plo, I. et al. AKT1 inhibits homologous recombination by inducing cytoplasmic retention of BRCA1 and RAD51. Cancer Res. 68, 9404–9412 (2008).

    Article  CAS  PubMed  Google Scholar 

  46. Liu, P. et al. Akt-mediated phosphorylation of XLF impairs non-homologous end-joining DNA repair. Mol. Cell 57, 648–661 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Xu, N., Lao, Y., Zhang, Y. & Gillespie, D. A. Akt: a double-edged sword in cell proliferation and genome stability. J. Oncol. 2012, 951724 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  48. Toulany, M. et al. Akt promotes post-irradiation survival of human tumor cells through initiation, progression, and termination of DNA-PKcs-dependent DNA double-strand break repair. Mol. Cancer Res. 10, 945–957 (2012).

    Article  CAS  PubMed  Google Scholar 

  49. Huang, W. C. & Chen, C. C. Akt phosphorylation of p300 at Ser-1834 is essential for its histone acetyltransferase and transcriptional activity. Mol. Cell. Biol. 25, 6592–6602 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Liu, S. L. et al. Quantitative lipid imaging reveals a new signaling function of phosphatidylinositol-3,4-bisphophate: isoform- and site-specific activation of Akt. Mol. Cell 71, 1092–1104.e5 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Schober, P., Boer, C. & Schwarte, L. A. Correlation coefficients: appropriate use and interpretation. Anesth. Analg. 126, 1763–1768 (2018).

    Article  PubMed  Google Scholar 

  52. Miyaguchi, Y., Tsuchiya, K. & Sakamoto, K. P53 negatively regulates the transcriptional activity of FOXO3a under oxidative stress. Cell Biol. Int. 33, 853–860 (2009).

    Article  CAS  PubMed  Google Scholar 

  53. Cocco, L. et al. Synthesis of polyphosphoinositides in nuclei of Friend cells. Evidence for polyphosphoinositide metabolism inside the nucleus which changes with cell differentiation. Biochem. J. 248, 765–770 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Kikani, C. K. et al. Proliferative and antiapoptotic signaling stimulated by nuclear-localized PDK1 results in oncogenesis. Sci. Signal. 5, ra80 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  55. Shiraishi, I. et al. Nuclear targeting of Akt enhances kinase activity and survival of cardiomyocytes. Circ. Res. 94, 884–891 (2004).

    Article  CAS  PubMed  Google Scholar 

  56. Fu, W. & Hall, M. N. Regulation of mTORC2 signaling. Genes (Basel) 11, 1045 (2020).

    Article  CAS  Google Scholar 

  57. Thapa, N. et al. Phosphatidylinositol-3-OH kinase signalling is spatially organized at endosomal compartments by microtubule-associated protein 4. Nat. Cell Biol. 22, 1357–1370 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Chen, M. et al. The specificity of EGF-stimulated IQGAP1 scaffold towards the PI3K–Akt pathway is defined by the IQ3 motif. Sci. Rep. 9, 9126 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  59. Tzivion, G., Dobson, M. & Ramakrishnan, G. FoxO transcription factors; regulation by AKT and 14-3-3 proteins. Biochim. Biophys. Acta 1813, 1938–1945 (2011).

    Article  CAS  PubMed  Google Scholar 

  60. Olive, K. P. et al. Mutant p53 gain of function in two mouse models of Li–Fraumeni syndrome. Cell 119, 847–860 (2004).

    Article  CAS  PubMed  Google Scholar 

  61. Muller, P. A. & Vousden, K. H. Mutant p53 in cancer: new functions and therapeutic opportunities. Cancer Cell 25, 304–317 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Schulz-Heddergott, R. et al. Therapeutic ablation of gain-of-function mutant p53 in colorectal cancer inhibits Stat3-mediated tumor growth and invasion. Cancer Cell 34, 298–314.e7 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Hanel, W. et al. Two hot spot mutant p53 mouse models display differential gain of function in tumorigenesis. Cell Death Differ. 20, 898–909 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Iggo, R., Gatter, K., Bartek, J., Lane, D. & Harris, A. L. Increased expression of mutant forms of p53 oncogene in primary lung cancer. Lancet 335, 675–679 (1990).

    Article  CAS  PubMed  Google Scholar 

  65. Yemelyanova, A. et al. Immunohistochemical staining patterns of p53 can serve as a surrogate marker for TP53 mutations in ovarian carcinoma: an immunohistochemical and nucleotide sequencing analysis. Mod. Pathol. 24, 1248–1253 (2011).

    Article  CAS  PubMed  Google Scholar 

  66. Muller, P. A. et al. Mutant p53 drives invasion by promoting integrin recycling. Cell 139, 1327–1341 (2009).

    Article  PubMed  Google Scholar 

  67. Andre, F. et al. Alpelisib for PIK3CA-mutated, hormone receptor-positive advanced breast cancer. N. Engl. J. Med. 380, 1929–1940 (2019).

    Article  CAS  PubMed  Google Scholar 

  68. Mellman, D. L. et al. A PtdIns4,5P2-regulated nuclear poly(A) polymerase controls expression of select mRNAs. Nature 451, 1013–1017 (2008).

    Article  CAS  PubMed  Google Scholar 

  69. Malin, D., Chen, F., Schiller, C., Koblinski, J. & Cryns, V. L. Enhanced metastasis suppression by targeting TRAIL receptor 2 in a murine model of triple-negative breast cancer. Clin. Cancer Res. 17, 5005–5015 (2011).

    Article  CAS  PubMed  Google Scholar 

  70. Asmari, M., Ratih, R., Alhazmi, H. A. & El Deeb, S. Thermophoresis for characterizing biomolecular interaction. Methods 146, 107–119 (2018).

    Article  CAS  PubMed  Google Scholar 

  71. Costes, S. V. et al. Automatic and quantitative measurement of protein–protein colocalization in live cells. Biophys. J. 86, 3993–4003 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Chen, M., Horn, H. T., Wen, T., Cryns, V. L. & Anderson, R. A. Assessing in situ phosphoinositide–protein interactions through fluorescence proximity ligation assay in cultured cells. Methods Mol. Biol. 2251, 133–142 (2021).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank A. Audhya, M. Sussman, A. Gasch and W. Peti for discussions and comments, and L. Rodenkirch for technical support. This work was supported in part by a National Institutes of Health grant (R35GM134955 to R.A.A.), Department of Defense Breast Cancer Research Programme grants (W81XWH-17-1-0258 to R.A.A.; W81XWH-17-1-0259 and W81XWH-21-1-0129 to V.L.C.) and a grant from the Breast Cancer Research Foundation (V.L.C.).

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  1. These authors contributed equally: Vincent L. Cryns, Richard A. Anderson.

Authors and Affiliations

  1. University of Wisconsin-Madison, School of Medicine and Public Health, Madison, WI, USA

    Mo Chen, Suyong Choi, Tianmu Wen, Changliang Chen, Narendra Thapa, Jeong Hyo Lee & Richard A. Anderson

  2. Department of Medicine, University of Wisconsin Carbone Cancer Center, University of Wisconsin-Madison, School of Medicine and Public Health, Madison, WI, USA

    Changliang Chen, Jeong Hyo Lee & Vincent L. Cryns

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Contributions

M.C., S.C., T.W., C.C., N.T., V.L.C. and R.A.A. designed the experiments. M.C., S.C., T.W., C.C., N.T. and J.H.L. performed the experiments. M.C., V.L.C. and R.A.A. wrote the manuscript.

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Correspondence to Vincent L. Cryns or Richard A. Anderson.

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Extended data

Extended Data Fig. 1 p53 associates with active Akt in the nucleus and their interaction is enhanced by stress.

a,b, IF staining against AKT and p53 in MDA-MB-231 cells treated with 30 µM cisplatin for 24 h. The nuclear levels of Akt (b) were quantified. N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. c, WB analysis of pAktT308/S473 in MDA-MB-231 cells after 24 h starvation and treatment with 10 µM mTOR inhibitor Torin 1/KU-0063794/Palomid 529 or Akt inhibitor Miransertib/MK-2206/Tricirbine/Capivasertib/Ipatasertib, followed by EGF stimulation. Data shown represent 3 independent experiments. d–g, IF staining of pAktT308/S473 in MDA-MB-231 cells treated with 30 µM cisplatin and the mTOR inhibitor Torin 1 (10 µM) or the Akt inhibitor Miransertib (10 µM) for 24 h. The nuclear pAktT308/S473 levels were quantified (e,g). N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. h, WB analysis of pAktS473 in MDA-MB-231 cells after 24 h starvation and treatment with 10 µM PI3Kα inhibitor alpelisib, 2 µM pan-PI3K inhibitor buparlisib, or vehicle, followed by EGF stimulation. N = 3 independent experiments. i,j, IF staining of pAktS473 in MDA-MB-231 cells treated with 30 µM cisplatin and the PI3Kα inhibitor alpelisib (10 µM) or the pan-PI3K inhibitor buparlisib (2 µM) for 24 h. Nuclear pAktS473 levels were quantified (j). N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. k–n, IF staining of p53 and pAktS473 in MDA-MB-231 cells treated with 100 µM tBHQ, 100 µM hydroxyurea, 100 µM etoposide, 30 µM cisplatin, or vehicle for 24 h. The nuclear levels of pAktS473 (l) and p53 (m) and their colocalization (n) were quantified. N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. o–r, IF staining of p53 and pAktS473 in A549 and MDA-MB-231 cells treated with 30 µM cisplatin for 24 h. The nuclear levels of p53 and pAktS473 (q) and their colocalization (r) were quantified. N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. s–u, Two-panel section (s) of confocal IF staining against p53 and pAktS473 in A549 cells treated with 30 µM cisplatin for 24 h. The nuclear and cytosolic colocalization of p53 and pAktS473 was quantified (u). N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. For all graph panels, data are represented as mean ± SD, p value denotes two-sided paired t-test. Scale bar: 5 µm.

Source data

Extended Data Fig. 2 p53 binds nuclear Akt and regulates its activation.

a, PLA of p53-Akt/pAktT308/pAktS473 in A549 and MDA-MB-231 cells treated with vehicle or 30 µM cisplatin for 24 h. See quantification in Fig. 1g. b,c PLA of p53-pAktS473 in MDA-MB-231 cells treated with 100 µM tBHQ, 100 µM hydroxyurea, 100 µM etoposide, 30 µM cisplatin, or vehicle for 24 h. The nuclear PLA foci were quantified (c). N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. d, 3D sections of PLA between p53 and pAktS473 overlaid with Lamin A/C in MDA-MB-231 cells treated with vehicle or 30 µM cisplatin for 24 h. Each frame of the 3D sections was over a 0.2 μm thickness. Data shown represent 3 independent experiments. e, Co-IP of p53 and Akt pathway components from MDA-MB-231 cells treated with 30 µM cisplatin or vehicle for 24 h. N = 3 independent experiments. f, MDA-MB-231 cells were transiently transfected with control siRNAs or siRNAs against p53 for 48 h. Cell lysates were then analyzed by WB. Representative data of three independent experiments are shown. g,h Confocal IF staining of p53 and pAktT308 in MDA-MB-231 cells 48 h after transient transfection with control siRNAs or siRNAs against p53. The nuclear p53 and pAktT308 levels normalized to siCon transfected cells were quantified (h). N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. For all graph panels, data are represented as mean ± SD, p value denotes two-sided paired t-test. Scale bar: 5 µm.

Source data

Extended Data Fig. 3 p53 associates with PDK1, FOXOs and IPMK.

a, PLA of p53-pPDK1S241/pPDK1S396 in A549 and MDA-MB-231 cells treated with vehicle or 30 µM cisplatin for 24 h. See quantification in Fig. 2e. b–e, Confocal images of IF staining against PDK1/Sin1/FOXO3 and p53 in MDA-MB-231 cells treated with vehicle or 30 µM cisplatin for 24 h. The nuclear levels of PDK1 (c), Sin1 (d), FOXO3 (e) normalized to vehicle treated cells were quantified. N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. f, Quantification of nuclear pFOXO3S253 in A549 and MDA-MB-231 cells treated with vehicle or 30 µM cisplatin for 24 h (levels normalized to vehicle treated cells). N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. Representative images are shown in Fig. 3e. g, PLA of p53-FOXOs in A549 and MDA-MB-231 cells treated with vehicle or 30 µM cisplatin for 24 h. See quantification in Fig. 3g. h, MDA-MB-231 cells were transiently transfected with control siRNAs or siRNAs against p53. 48 h later, cell lysates were analyzed by WB. Representative data of three independent experiments are shown. i, Co-IP of IPMK with wild-type p53 and mutant p53 from HCT116 and Cal33 cells, respectively, treated with 30 µM cisplatin or vehicle for 24 h. Representative data of three independent experiments are shown. For all graph panels, data are represented as mean ± SD, p value denotes two-sided paired t-test. Scale bar: 5 µm.

Source data

Extended Data Fig. 4 p53 associates with PI4,5P2 and PI3,4,5P3 in the nucleus.

a–c, Confocal IF staining for p53 and PI3,4,5P3 in MDA-MB-231 cells treated with 30 µM cisplatin or vehicle for 24 h. The nuclear level of PI3,4,5P3 normalized to vehicle treated cells (b) and its colocalization with p53 (c) were quantified. N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. d,e, PLA of p53-PI4,5P2/PI3,4,5P3 in A549, MCF-10A, MDA-MB-231, and SUM1315 cells treated with 30 µM cisplatin or vehicle for 24 h. The nuclear PLA foci were quantified (e). N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. f–h, PLA of p53-PI4,5P2/PI3,4,5P3 in MDA-MB-231 cells treated with 100 µM tBHQ, 100 µM hydroxyurea, 100 µM etoposide, 30 µM cisplatin, or vehicle for 24 h. The nuclear PLA foci were quantified (g,h). N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. i,j PLA of p53-PI3,4,5P3 in MDA-MB-231 cells treated with vehicle or 30 µM cisplatin with or without the presence of the PI3Kα inhibitor alpelisib (10 µM), the pan-PI3K inhibitor buparlisib (2 µM), or the PIPKIα inhibitor ISA-2011B (50 µM) for 24 h. The nuclear PLA foci were quantified (j). N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. k, 3D sections of PLA between p53 and PI3,4,5P3 overlaid with Lamin A/C in MDA-MB-231 cells treated with vehicle or 30 µM cisplatin treatment for 24 h. Each frame of the 3D sections was over a 0.2 μm thickness. Data shown represent 3 independent experiments. For all graph panels, data are represented as mean ± SD, p value denotes two-sided paired t-test. Scale bar: 5 µm.

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Extended Data Fig. 5 IPMK and PTEN associate with and regulate p53-PIPn complexes and FOXO phosphorylation.

a–c, Confocal IF staining against p53 and IPMK in MDA-MB-231 cells treated with 30 µM cisplatin or vehicle for 24 h. The nuclear level of IPMK normalized to vehicle treated cells (b) and its colocalization with p53 (c) were quantified. N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. d–g, Confocal IF staining against p53 and PTEN in A549 or MDA-MB-231 cells treated with 30 µM cisplatin or vehicle for 24 h. The nuclear level of PTEN normalized to vehicle treated cells (f) and its colocalization with p53 (g) were quantified. N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. h–j, PLA of p53-IPMK/PTEN in A549 and MDA-MB-231 cells treated with vehicle or 30 µM cisplatin for 24 h. The nuclear PLA foci were quantified (i,j). N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. k-l, PLA of p53-PI4,5P2/PI3,4,5P3 in MDA-MB-231 cells 48 h after transient transfection with control siRNAs or siRNAs against PIPKIα, IPMK, or PTEN (k). See quantification in Fig. 4h, i. The knockdown efficiency was validated by WB (l). m,n, Confocal IF staining of p53 and pFOXO3S253 in MDA-MB-231 cells 48 h after transient transfection with control siRNAs or siRNAs against p53, PIPKIα, IPMK, or PTEN. The nuclear pFOXO3S253 levels normalized to siCon transfected cells were quantified (n). N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. o,p, MDA-MB-231 cells were transfected with control siRNAs or siRNAs against p53, PIPKIα, IPMK, or PTEN. 24 h later, cells were treated with 30 µM cisplatin or vehicle for 24 h before being processed for PLA between p53 and pFOXO3S253. The nuclear PLA foci were quantified (p). N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. For all graph panels, data are represented as mean ± SD, p value denotes two-sided paired t-test. Scale bar: 5 µm.

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Extended Data Fig. 6 The nuclear p53-PIPn signalosome regulates DNA damage repair and cell survival.

a, Confocal IF staining against PI4,5P2 and pPDK1S396/pFOXO3S253 in MDA-MB-231 cells treated with 30 µM cisplatin or vehicle for 24 h. See Fig. 6a, b for the nuclear region and quantification of the nuclear colocalization of PI4,5P2 and pPDK1S396/pFOXO3S253. b, Confocal and STED super-resolution images of IF staining against p53, pAktS473, and the DNA DSB marker γ-H2A.X in MDA-MB-231 and A549 cells treated with 30 µM cisplatin or vehicle for 24 h. See quantification in Fig. 6d. c–f, MDA-MB-231 cells were transfected with control siRNAs or siRNAs against p53, PIPKIα, or IPMK. 24 h later, cells were treated with 30 µM cisplatin, 10 µM mTOR inhibitor Torin1, 10 µM Akt inhibitor Miransertib or vehicle for 24 h before being processed for IF staining of p53 and pDNA-PKS2056/phosphor-p300S1834. The nuclear levels of p53, pDNA-PKS2056 and phospho-p300S1834 normalized to vehicle treated cells were quantified (d,f). N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. g–i, MDA-MB-231, HS578T, and MDA-MB-468 cells were transfected with control siRNAs or siRNAs against p53, PIPKIα, IPMK, or PTEN. 24 h later, cells were treated with 30 µM cisplatin or vehicle for 24 h before being processed for MTT cell viability assay. Cell viability was normalized to siCon transfected cells. N = 3 independent experiments. j, MDA-MB-231 cells were transfected with control siRNAs or siRNAs against p53, PIPKIα, IPMK, or PTEN. 24 h later, cells were treated with 30 µM cisplatin or vehicle for 24 h before being processed for Caspase-3 activity assay. Activity was normalized to siCon transfected cells. N = 3 independent experiments. For all graph panels, data are represented as mean ± SD, p value denotes two-sided paired t-test. Scale bar: 5 µm.

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Extended Data Fig. 7 p53 interaction with Akt requires the p53 C-terminal PIPn-binding motif and correlates with nuclear Akt activation.

a, Schematic representation of p53 domains (top). 1 or 6 basic residues in the CTD were mutated to glutamine (Q) to generate the PIPn-binding defective 6Q or 379Q mutants, respectively. b,c, The interaction of Akt with mutant p53 is regulated by the PIPn-binding motif of p53. HEK293FT cells transfected with HA-tagged Akt1 and FLAG-tagged mutant p53R175H, p53R248Q, or their corresponding 6Q or 379Q mutants were processed for IP with HA antibody, and then analyzed by WB. N = 3 independent experiments. d–g, A549 cells treated with 30 µM cisplatin for 24 h were processed for IF staining of p53 and pAktS473. The nuclear levels of p53 and pAktS473 were quantified. The low (L) and high (H) levels of nuclear p53 were determined based on the average p53 levels in the corresponding groups (e). The correlation between nuclear p53 and pAktS473 was determined by Pearson’s r (f,g). N = 120 cells pooled from 3 independent experiments; 40 cells per experiment. h–k, MDA-MB-231 cells treated with 30 µM cisplatin for 24 h were processed for IF staining against p53 and pAktS473. The nuclear levels of p53 and pAktS473 were quantified. The low (L) and high (H) levels of nuclear p53 were determined based on the average of the p53 levels in the corresponding groups (i). The correlation between the nuclear p53 and pAktS473 was determined by Pearson’s r (j,k). N = 120 cells pooled from 3 independent experiments; 40 cells per experiment. l–o, A549 cells were transfected with Flag-tagged p53R175H and then 24 h later treated with 30 µM cisplatin for 4 h. Cells were then processed for IF staining of Flag-tagged p53R175H and pAktS473. The nuclear pAktS473 levels in Flag-negative and Flag-positive cells were quantified (m). The correlation between the nuclear levels of Flag-tagged p53R175H and pAktS473 was determined by Pearson’s r (n,o). N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. p, A549 wild-type and p53 KO cells were treated with 30 µM cisplatin for 24 h and analyzed by WB. Data shown represent 3 independent experiments. For all graph panels, data are represented as mean ± SD, p value denotes two-sided paired t-test. Scale bar: 5 µm.

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Extended Data Fig. 8 Stress-induced Akt, DNA-PK, and p300 phosphorylation by p53 is dependent on its PIPn-binding motif.

a–c, A549 p53 KO cells were transiently transfected with Flag-tagged p53R175H or the corresponding 6Q or 379Q mutant. 24 h later, cells were treated with 30 µM cisplatin for an additional 24 h. A549 WT cells were treated with 30 µM cisplatin or vehicle for 24 h. The cells were then fixed and processed for IF staining of p53, pAktT308, pDNA-PKS2056, and phospho-p300S1834. The nuclear pAktT308 (a), pDNA-PKS2056 (b), and phospho-p300S1834 (c) levels in A549 WT cells, p53 KO cells, and Flag-tagged p53R175H-positive cells normalized to A549 WT cells treated with cisplatin were quantified as indicated. N = 30 cells pooled from 3 independent experiments; 10 cells per experiment. Scale bar: 5 µm. d, A549 p53 KO cells were transiently transfected with Flag-tagged p53R175H, p53R248Q or their corresponding 6Q or 379Q mutant. 24 h later, cells were treated with 30 µM cisplatin or vehicle for an additional 24 h before being processed for crystal violet (CV) viability assay. Cell viability was normalized to untransfected cells treated with vehicle. N = 3 independent experiments. e,f, A549 p53 KO cells were transiently transfected with Flag-tagged mutant p53R175H, p53R248Q or their corresponding 6Q or 379Q mutant. 24 h later, cells were serum-starved for an additional 24 h and then scored for invasion through Laminin-coated transwell inserts with 8 μm pores using 10 ng/ml EGF as a chemoattractant for 16 h. The invading cells at the insert bottom were stained with crystal violet, imaged, and quantified based on the extracted dye using a plate reader. Invasion was normalized to untransfected cells treated with EGF. N = 3 independent experiments. Scale bar: 100 µm. For all panels, data are represented as mean ± SD, p value denotes two-sided paired t-test.

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Supplementary Table 1

The binding affinity of the nuclear p53–PIPn signalosome components with p53. The interaction of recombinant fluorescently labelled p53 and non-labelled signalosome components was quantitated by MST assay. A constant concentration of fluorescently labelled p53 (target, 5 nM) was incubated with increasing concentrations of non-labelled ligands to calculate the binding affinity. The binding affinities determined by MST are shown as indicated Kd values. MST was performed using a Monolith NT.115 pico, and the binding affinity was autogenerated by MO. Control v.1.6 software. Data are presented as the mean ± s.d; n = 3.

Supplementary Video 1

Three-dimensional (3D) reconstitution of the p53–pAKTS473 complex in the nucleoplasm of MDA-MB-231 cells under basal conditions. MDA-MB-231 cells were treated with vehicle for 24 h before being processed for PLA between p53 and pAKTS473 (red). Lamin A/C stained the nuclear envelopes (green), and the nuclei were counterstained with DAPI (blue). The z-stack images were taken with a Leica SP8 confocal microscope with each frame over a 0.2-μm thickness. The video of the 3D reconstitution of a representative cell from three independent experiments was generated using LASX.

Supplementary Video 2

3D reconstitution of the p53–pAKTS473 complex in the nucleoplasm of MDA-MB-231 cells under stress. MDA-MB-231 cells were treated with 30 µM cisplatin for 24 h before being processed for PLA between p53 and pAKTS473 (red). Lamin A/C stained the nuclear envelopes (green), and the nuclei were counterstained with DAPI (blue). The z-stack images were taken with a Leica SP8 confocal microscope with each frame over a 0.2-μm thickness. The video of the 3D reconstitution of a representative cell from three independent experiments was generated using LASX.

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Chen, M., Choi, S., Wen, T. et al. A p53–phosphoinositide signalosome regulates nuclear AKT activation. Nat Cell Biol 24, 1099–1113 (2022). https://doi.org/10.1038/s41556-022-00949-1

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