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A nuclear phosphoinositide kinase complex regulates p53

Abstract

The tumour suppressor p53 (encoded by TP53) protects the genome against cellular stress and is frequently mutated in cancer. Mutant p53 acquires gain-of-function oncogenic activities that are dependent on its enhanced stability. However, the mechanisms by which nuclear p53 is stabilized are poorly understood. Here, we demonstrate that the stability of stress-induced wild-type and mutant p53 is regulated by the type I phosphatidylinositol phosphate kinase (PIPKI-α (also known as PIP5K1A)) and its product phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). Nuclear PIPKI-α binds to p53 upon stress, resulting in the production and association of PtdIns(4,5)P2 with p53. PtdIns(4,5)P2 binding promotes the interaction between p53 and the small heat shock proteins HSP27 (also known as HSPB1) and αB-crystallin (also known as HSPB5), which stabilize nuclear p53. Moreover, inhibition of PIPKI-α or PtdIns(4,5)P2 association results in p53 destabilization. Our results point to a previously unrecognized role of nuclear phosphoinositide signalling in regulating p53 stability and implicate this pathway as a promising therapeutic target in cancer.

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Fig. 1: PtdIns(4,5)P2 generation by PIPKI-α is required for stress-induced wild-type and mutant p53 stability.
Fig. 2: PIPKI-α associates with p53 in the nucleus.
Fig. 3: PtdIns(4,5)P2 specifically binds to p53 and the PtdIns(4,5)P2-associated p53 localizes in subnuclear compartments.
Fig. 4: PtdIns(4,5)P2-bound p53 localizes in subnuclear compartments distinct from the nuclear membrane.
Fig. 5: PtdIns(4,5)P2 binding controls the stability of stress-induced wild-type p53 and mutant p53.
Fig. 6: The sHSP HSP27 associates with nuclear p53.
Fig. 7: PtdIns(4,5)P2 binding to p53 promotes its interaction with sHSPs in vivo.
Fig. 8: PtdIns(4,5)P2 binding to p53 controls sHSP binding to p53 in vitro.

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Data availability

The mass spectrometry data have been deposited in the ProteomeXchange with the primary accession code PXD012463. Source data for Figs. 1a–e, 2c,e,g, 3a,b,e,g, 5a–d,f,g, 6g,i, 7a–c,e and 8a,c–e and Supplementary Figs. 1a, 2d, 3h,m, 4g,i,k, 5b,d,f, 6a,d, 7b,e and 8b–d have been provided as Supplementary Table 1. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

References

  1. Balla, T. Phosphoinositides: tiny lipids with giant impact on cell regulation. Physiol. Rev. 93, 1019–1137 (2013).

    Article  CAS  Google Scholar 

  2. Choi, S., Thapa, N., Tan, X., Hedman, A. C. & Anderson, R. A. PIP kinases define PI4,5P2 signaling specificity by association with effectors. Biochim. Biophys. Acta 1851, 711–723 (2015).

    Article  CAS  Google Scholar 

  3. Anderson, R. A., Boronenkov, I. V., Doughman, S. D., Kunz, J. & Loijens, J. C. Phosphatidylinositol phosphate kinases, a multifaceted family of signaling enzymes. J. Biol. Chem. 274, 9907–9910 (1999).

    Article  CAS  Google Scholar 

  4. 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  Google Scholar 

  5. 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  Google Scholar 

  6. 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  Google Scholar 

  7. Dai, C. & Gu, W. p53 post-translational modification: deregulated in tumorigenesis. Trends Mol. Med. 16, 528–536 (2010).

    Article  CAS  Google Scholar 

  8. Bieging, K. T., Mello, S. S. & Attardi, L. D. Unravelling mechanisms of p53-mediated tumour suppression. Nat. Rev. Cancer 14, 359–370 (2014).

    Article  CAS  Google Scholar 

  9. Kruiswijk, F., Labuschagne, C. F. & Vousden, K. H. p53 in survival, death and metabolic health: a lifeguard with a licence to kill. Nat. Rev. Mol. Cell Biol. 16, 393–405 (2015).

    Article  CAS  Google Scholar 

  10. Freed-Pastor, W. A. & Prives, C. Mutant p53: one name, many proteins. Genes Dev. 26, 1268–1286 (2012).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  13. Emerling, B. M. et al. Depletion of a putatively druggable class of phosphatidylinositol kinases inhibits growth of p53-null tumors. Cell 155, 844–857 (2013).

    Article  CAS  Google Scholar 

  14. 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  Google Scholar 

  15. Wiech, M. et al. Molecular mechanism of mutant p53 stabilization: the role of HSP70 and MDM2. PLoS One 7, e51426 (2012).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. Vakifahmetoglu-Norberg, H. et al. Chaperone-mediated autophagy degrades mutant p53. Genes Dev. 27, 1718–1730 (2013).

    Article  CAS  Google Scholar 

  18. Liu, J. et al. Beclin1 controls the levels of p53 by regulating the deubiquitination activity of USP10 and USP13. Cell 147, 223–234 (2011).

    Article  CAS  Google Scholar 

  19. Brooks, C. L. & Gu, W. p53 regulation by ubiquitin. FEBS Lett. 585, 2803–2809 (2011).

    Article  CAS  Google Scholar 

  20. Kruse, J. P. & Gu, W. Modes of p53 regulation. Cell 137, 609–622 (2009).

    Article  CAS  Google Scholar 

  21. Heck, J. N. et al. A conspicuous connection: structure defines function for the phosphatidylinositol-phosphate kinase family. Crit. Rev. Biochem. Mol. Biol. 42, 15–39 (2007).

    Article  CAS  Google Scholar 

  22. Choi, S., Houdek, X. & Anderson, R. A. Phosphoinositide 3-kinase pathways and autophagy require phosphatidylinositol phosphate kinases. Adv. Biol. Regul. 68, 31–38 (2018).

    Article  CAS  Google Scholar 

  23. Alexandrova, E. M. et al. Improving survival by exploiting tumour dependence on stabilized mutant p53 for treatment. Nature 523, 352–356 (2015).

    Article  CAS  Google Scholar 

  24. Fredriksson, S. et al. Protein detection using proximity-dependent DNA ligation assays. Nat. Biotechnol. 20, 473–477 (2002).

    Article  CAS  Google Scholar 

  25. Lam, F., Cladiere, D., Guillaume, C., Wassmann, K. & Bolte, S. Super-resolution for everybody: an image processing workflow to obtain high-resolution images with a standard confocal microscope. Methods 115, 17–27 (2016).

    Article  Google Scholar 

  26. Choi, S. et al. IQGAP1 is a novel phosphatidylinositol 4,5 bisphosphate effector in regulation of directional cell migration. EMBO J. 32, 2617–2630 (2013).

    Article  CAS  Google Scholar 

  27. Klein, D. E., Lee, A., Frank, D. W., Marks, M. S. & Lemmon, M. A. The pleckstrin homology domains of dynamin isoforms require oligomerization for high affinity phosphoinositide binding. J. Biol. Chem. 273, 27725–27733 (1998).

    Article  CAS  Google Scholar 

  28. Rohacs, T., Chen, J., Prestwich, G. D. & Logothetis, D. E. Distinct specificities of inwardly rectifying K+ channels for phosphoinositides. J. Biol. Chem. 274, 36065–36072 (1999).

    Article  CAS  Google Scholar 

  29. Balla, T. & Varnai, P. Visualization of cellular phosphoinositide pools with GFP-fused protein-domains.Curr. Protoc. Cell Biol. 42, 24.4.1–24.4.27 (2009).

    Article  Google Scholar 

  30. Varnai, P. & Balla, T. Visualization of phosphoinositides that bind pleckstrin homology domains: calcium- and agonist-induced dynamic changes and relationship to myo-[3H]inositol-labeled phosphoinositide pools. J. Cell Biol. 143, 501–510 (1998).

    Article  CAS  Google Scholar 

  31. Lewis, A. E. et al. Identification of nuclear phosphatidylinositol 4,5-bisphosphate-interacting proteins by neomycin extraction. Mol. Cell. Proteomics 10, M110 003376 (2011).

    Article  Google Scholar 

  32. Chatterjee, A. et al. U-box-type ubiquitin E4 ligase, UFD2a attenuates cisplatin mediated degradation of ΔNp63α. Cell Cycle 7, 1231–1237 (2008).

    Article  CAS  Google Scholar 

  33. Murphy, L. O., Smith, S., Chen, R. H., Fingar, D. C. & Blenis, J. Molecular interpretation of ERK signal duration by immediate early gene products. Nat. Cell Biol. 4, 556–564 (2002).

    Article  CAS  Google Scholar 

  34. Fukami, K. et al. Antibody to phosphatidylinositol 4,5-bisphosphate inhibits oncogene-induced mitogenesis. Proc. Natl Acad. Sci. USA 85, 9057–9061 (1988).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  36. Shah, Z. H. et al. Nuclear phosphoinositides and their impact on nuclear functions. FEBS J. 280, 6295–6310 (2013).

    Article  CAS  Google Scholar 

  37. Xu, Q. et al. Phosphatidylinositol phosphate kinase PIPKIγ and phosphatase INPP5E coordinate initiation of ciliogenesis. Nat. Commun. 7, 10777 (2016).

    Article  CAS  Google Scholar 

  38. Humbert, M. C. et al. ARL13B, PDE6D, and CEP164 form a functional network for INPP5E ciliary targeting. Proc. Natl Acad. Sci. USA 109, 19691–19696 (2012).

    Article  CAS  Google Scholar 

  39. Balla, T. & Varnai, P. Visualizing cellular phosphoinositide pools with GFP-fused protein-modules. Sci. STKE 2002, pl3 (2002).

    PubMed  Google Scholar 

  40. 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  Google Scholar 

  41. Xu, J. et al. Gain of function of mutant p53 by coaggregation with multiple tumor suppressors. Nat. Chem. Biol. 7, 285–295 (2011).

    Article  CAS  Google Scholar 

  42. Peng, Y., Chen, L., Li, C., Lu, W. & Chen, J. Inhibition of MDM2 by HSP90 contributes to mutant p53 stabilization. J. Biol. Chem. 276, 40583–40590 (2001).

    Article  CAS  Google Scholar 

  43. Li, D. et al. Functional inactivation of endogenous MDM2 and CHIP by HSP90 causes aberrant stabilization of mutant p53 in human cancer cells. Mol. Cancer Res. 9, 577–588 (2011).

    Article  CAS  Google Scholar 

  44. Arrigo, A. P. et al. Hsp27 (HspB1) and αB-crystallin (HspB5) as therapeutic targets. FEBS Lett. 581, 3665–3674 (2007).

    Article  CAS  Google Scholar 

  45. Venkatakrishnan, C. D. et al. HSP27 regulates p53 transcriptional activity in doxorubicin-treated fibroblasts and cardiac H9c2 cells: p21 upregulation and G2/M phase cell cycle arrest. Am. J. Physiol. Heart Circ. Physiol. 294, H1736–H1744 (2008).

    Article  CAS  Google Scholar 

  46. Adhikari, A. S., Sridhar Rao, K., Rangaraj, N., Parnaik, V. K. & Mohan Rao, C. Heat stress-induced localization of small heat shock proteins in mouse myoblasts: intranuclear lamin A/C speckles as target for αB-crystallin and Hsp25. Exp. Cell Res. 299, 393–403 (2004).

    Article  CAS  Google Scholar 

  47. Watanabe, G. et al. αB-crystallin: a novel p53-target gene required for p53-dependent apoptosis. Cancer Sci. 100, 2368–2375 (2009).

    Article  CAS  Google Scholar 

  48. Moyano, J. V. et al. αB-crystallin is a novel oncoprotein that predicts poor clinical outcome in breast cancer. J. Clin. Invest. 116, 261–270 (2006).

    Article  CAS  Google Scholar 

  49. Koletsa, T. et al. αB-crystallin is a marker of aggressive breast cancer behavior but does not independently predict for patient outcome: a combined analysis of two randomized studies. BMC Clin. Pathol. 14, 28 (2014).

    Article  Google Scholar 

  50. Hanahan, D. & Weinberg, R. A. Hallmarks of cancer: the next generation. Cell 144, 646–674 (2011).

    Article  CAS  Google Scholar 

  51. Marchenko, N. D. et al. Stress-mediated nuclear stabilization of p53 is regulated by ubiquitination and importin-α3 binding. Cell Death Differ. 17, 255–267 (2009).

    Article  Google Scholar 

  52. Passinen, S., Valkila, J., Manninen, T., Syvala, H. & Ylikomi, T. The C-terminal half of Hsp90 is responsible for its cytoplasmic localization. Eur. J. Biochem. 268, 5337–5342 (2001).

    Article  CAS  Google Scholar 

  53. Friedler, A., Veprintsev, D. B., Freund, S. M., Von Glos, K. I. & Fersht, A. R. Modulation of binding of DNA to the C-terminal domain of p53 by acetylation. Structure 13, 629–636 (2005).

    Article  CAS  Google Scholar 

  54. Laptenko, O. et al. The p53 C terminus controls site-specific DNA binding and promotes structural changes within the central DNA binding domain. Mol. Cell 57, 1034–1046 (2015).

    Article  CAS  Google Scholar 

  55. Cino, E. A., Soares, I. N., Pedrote, M. M., de Oliveira, G. A. & Silva, J. L. Aggregation tendencies in the p53 family are modulated by backbone hydrogen bonds. Sci. Rep. 6, 32535 (2016).

    Article  CAS  Google Scholar 

  56. Ano Bom, A. P. et al. Mutant p53 aggregates into prion-like amyloid oligomers and fibrils: implications for cancer. J. Biol. Chem. 287, 28152–28162 (2012).

    Article  CAS  Google Scholar 

  57. Malin, D., Petrovic, V., Strekalova, E., Sharma, B. & Cryns, V. L. αB-crystallin: portrait of a malignant chaperone as a cancer therapeutic target. Pharmacol. Ther. 160, 1–10 (2016).

    Article  CAS  Google Scholar 

  58. Bakthisaran, R., Tangirala, R. & Rao Ch, M. Small heat shock proteins: role in cellular functions and pathology. Biochim. Biophys. Acta 1854, 291–319 (2015).

    Article  CAS  Google Scholar 

  59. Arrigo, A. P. & Gibert, B. HspB1, HspB5 and HspB4 in human cancers: potent oncogenic role of some of their client proteins. Cancers (Basel) 6, 333–365 (2014).

    Article  Google Scholar 

  60. van den, I. P., Wheelock, R., Prescott, A., Russell, P. & Quinlan, R. A. Nuclear speckle localisation of the small heat shock protein αB-crystallin and its inhibition by the R120G cardiomyopathy-linked mutation. Exp. Cell Res. 287, 249–261 (2003).

    Article  Google Scholar 

  61. Sottile, M. L. & Nadin, S. B. Heat shock proteins and DNA repair mechanisms: an updated overview. Cell Stress Chaperones 23, 303–315 (2018).

    Article  CAS  Google Scholar 

  62. Fukami, K., Endo, T., Imamura, M. & Takenawa, T. α-Actinin and vinculin are PIP2-binding proteins involved in signaling by tyrosine kinase. J. Biol. Chem. 269, 1518–1522 (1994).

    CAS  PubMed  Google Scholar 

  63. Naguib, A. et al. p53 mutations change phosphatidylinositol acyl chain composition. Cell Rep. 10, 8–19 (2015).

    Article  CAS  Google Scholar 

  64. Shulga, Y. V., Anderson, R. A., Topham, M. K. & Epand, R. M. Phosphatidylinositol-4-phosphate 5-kinase isoforms exhibit acyl chain selectivity for both substrate and lipid activator. J. Biol. Chem. 287, 35953–35963 (2012).

    Article  CAS  Google Scholar 

  65. Waugh, M. G. Amplification of chromosome 1q genes encoding the phosphoinositide signalling enzymes PI4KB, AKT3, PIP5K1A and PI3KC2B in breast cancer. J. Cancer 5, 790–796 (2014).

    Article  CAS  Google Scholar 

  66. Prudovsky, I., Vary, C. P., Markaki, Y., Olins, A. L. & Olins, D. E. Phosphatidylserine colocalizes with epichromatin in interphase nuclei and mitotic chromosomes. Nucleus 3, 200–210 (2012).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  68. Peng, Y. et al. Stabilization of the MDM2 oncoprotein by mutant p53. J. Biol. Chem. 276, 6874–6878 (2001).

    Article  CAS  Google Scholar 

  69. Ran, F. A. et al. Genome engineering using the CRISPR–Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Addgene and J. Chen (H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA) for the p53 constructs. We are also grateful to J. L. Persson (Lund University) and K. Fukami (Tokyo University of Pharmacy and Life Sciences) for generously sharing ISA-2011B and the KT10 antibody, respectively. We also thank members of the R.A.A. and V.L.C. laboratories for helpful discussions. This work was supported in part by a National Institutes of Health grant GM114386 (R.A.A.), Department of Defense Breast Cancer Research Program grants W81XWH-17-1-0258 (R.A.A.) and W81XWH-17-1-0259 (V.L.C.) and a grant from the Breast Cancer Research Foundation (V.L.C.).

Authors contributions

S.C., M.C., V.L.C. and R.A.A. designed the experiments. S.C. and M.C. performed the experiments. S.C., M.C., V.L.C. and R.A.A. wrote the manuscript.

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Correspondence to Richard A. Anderson.

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Integrated supplementary information

Supplementary Figure 1 PIPKIα depletion or pharmacological inhibition reduces mutant p53 protein levels.

(a) siRNAs against PIPKIα were transiently transfected in the indicated cells for 72 h. Protein expression was analyzed by IB, p53 blots were quantified and the graph is shown as mean ± SD of n = 3 independent experiments. Scrambled siRNAs (siCon) were used as a negative control. Two-sided paired Student t-tests were used for statistical analysis. Statistical source data can be found in Supplementary Table 1. (b) SkBr3 cells stably expressing shRNA against a scrambled sequence or PIPKIα were fixed and immunostained with PIPKIα and p53 antibody. Representative data of three independent experiments were shown. Scale bar, 30 μm. (c) MDA-MB-231 cells were treated with vehicle control or 50 μM ISA-2011B for the indicated time. Cell lysates were analyzed by IB with the indicated antibodies. Representative data of three independent experiments were shown. Unprocessed images of the blots in a and c are shown in Supplementary Figure 9. (d) A431 cells were treated with vehicle control or 50 μM ISA-2011B for 72 h. Cells were fixed and immunostained with PIPKIα and p53 antibody. DAPI was used to stain nucleic acids. Representative data of three independent experiments were shown. Scale bar, 30 μm.

Supplementary Figure 2 PIPKIα associates with the p53 complex in subnuclear compartments.

(a and b) The indicated cells were treated with 30 μM cisplatin for 24 h. Endogenous PIPKIα was IP’ed and the associated proteins were analyzed by IB. Representative data of three independent experiments were shown. Unprocessed images of the blots are shown in Supplementary Figure 9. (c and d) A549 cells were treated with 30 μM cisplatin for 24 h before processed for IF staining of PIPKIα. DAPI was used to stain nucleic acids. The images were taken with a Leica SP8 confocal microscope. The mean intensity of the green channels in the nuclear region (DAPI positive) was quantified by LAS X (Leica). The green signal from cisplatin-treated group was normalized to the untreated control. The experiments were repeated three times and the graph is shown as mean ± SD of n = 10 cells from one representative experiment. Scale bar, 5 μm. Statistical source data can be found in Supplementary Table 1. (e and f) A549 cells were treated with 30 μM cisplatin for 24 h before processed for PLA between PIPKIα and p53. DAPI was used to stain nucleic acids. The z-stack images were taken with a Leica SP8 confocal microscope with each frame over a 0.2 μm thickness. The 3D view was generated by ImageJ. Scale bar, 5 μm. Representative data of three independent experiments were shown.

Supplementary Figure 3 PI4,5P2 accumulates in the nucleus in response to genotoxic stress and associates with p53.

(a) Schematic representation of p53 domains (top). 1 or 6 basic residues in the CTD were mutated to glutamine (Q) to generated PI4,5P2-binding defective mutant R379Q or 6Q. TAD, transactivation domain; PRD, proline-rich domain; DBD, DNA-binding domain; TD, tetramerization domain; CTD, C-terminal regulatory domain. (b) The indicated cells were treated with 30 μM cisplatin for 24 h. Endogenous p53 was IP’ed and IP samples were eluted with 0.2 M glycine (pH 2.5). Elution was analyzed by IB with an anti-PI4,5P2 KT10 antibody. Representative data of three independent experiments were shown. Unprocessed images of the blots are shown in Supplementary Figure 9. (c to h) A549 cells were treated with 30 μM cisplatin for 24 h before processed for IF staining of the indicated phosphoinositides. DAPI was used to stain nucleic acids. The images were taken with a Leica SP8 confocal microscope. The mean intensity of the green channels in the nuclear region (DAPI positive) was quantified by LAS X (Leica). The green signal from cisplatin-treated group was normalized to its untreated control. The experiments were repeated three times and the graph is shown as mean ± SD of n = 10 cells from one representative experiment. Scale bar, 5 μm. Statistical source data can be found in Supplementary Table 1. (i to m) A549 cells were treated with 30 μM cisplatin for 24 h before processed for PLA between p53 and the indicated phosphoinositides. DAPI was used to stain nucleic acids. The images were taken by Leica SP8 confocal microscope. The red PLA signal was quantified by LAS X (Leica) and the graph is shown as mean ± SD of n = 10 cells from one representative experiment. Scale bar, 5 μm. The experiments were repeated three times. Two-sided paired Student t-tests were used for statistical analysis. Statistical source data can be found in Supplementary Table 1.

Supplementary Figure 4 PI4,5P2 and p53 colocalize in nuclear speckles and DNA damage sites.

(a and b) A549 cells were treated with 30 μM cisplatin for 24 h before processed for IF staining of PI4,5P2 and p53. An anti-PS antibody was used to stain the nuclear membrane. The z-stack images were taken with a Leica SP8 confocal microscope with each frame over a 0.2 μm thickness. The 3D view was generated by ImageJ. Scale bar, 5 μm. The experiments were repeated three times. (c and d) A549 cells were treated with 30 μM cisplatin for 24 h before processed for PLA between PI4,5P2 and p53. An anti-PS antibody was used to stain the nuclear membrane. DAPI was used to stain nucleic acids. The z-stack images were taken with a Leica SP8 confocal microscope with each frame over a 0.2 μm thickness. The 3D view was generated by ImageJ. Scale bar, 5 μm. The experiments were repeated three times. (e) A549 cells were treated with 30 μM cisplatin for 24 h before processed for IF staining of the indicated molecules. The images were taken with a Leica SP8 confocal microscope. Scale bar, 5 μm. The experiments were repeated three times. (f to k) A549 cells were treated with 30 μM cisplatin for 24 h before processed for IF staining of the indicated molecules. The images were taken with a Leica SP8 confocal microscope and processed by ImageJ. White arrows indicate the colocalized signals. The experiments were repeated three times and the graph is shown as mean ± SD of n = 10 cells from one representative experiment. Scale bar, 5 μm. Two-sided paired Student t-tests were used for statistical analysis. Statistical source data can be found in Supplementary Table 1.

Supplementary Figure 5 PI4,5P2 binding controls the nuclear localization of p53.

(a to f) A549 cells were transfected with the indicated siRNAs for 48 h and then treated with 30 μM cisplatin for 24 h before processed for IF staining of the indicated molecules. The images were taken with a Leica SP8 confocal microscope and processed by ImageJ. White arrows indicate the colocalized signals. The experiments were repeated three times and the graph is shown as mean ± SD of n = 10 cells from one representative experiment. Scale bar, 5 μm. Two-sided paired Student t-tests were used for statistical analysis. Statistical source data can be found in Supplementary Table 1. (g) Flag-p53 constructs were co-transfected with HA-PIPKIα in HEK293 cells. Flag-p53 proteins were immunoprecipitated and the associated PIPKIα was analyzed by IB with an anti-HA antibody. Representative data of three independent experiments were shown. Unprocessed images of the blots are shown in Supplementary Figure 9.

Supplementary Figure 6 HSP27 is a novel binding partner of nuclear p53.

(a) Cells were treated with 30 μM cisplatin for 24 h, and cytoplasmic (Cy) and nuclear (Nu) proteins were fractionated. p53 was IP’ed and IP samples were eluted with 0.2 M glycine (pH 2.5). Elution was analyzed by IB with the indicated molecules and the graph is shown as mean ± SD of n = 3 independent experiments. Two-sided paired Student t-tests were used for statistical analysis. Statistical source data can be found in Supplementary Table 1. Unprocessed images of the blots are shown in Supplementary Figure 9. (b) The indicated p53 constructs were transiently transfected in p53-null H1299 cells for 48 h. p53 was IP’ed and IP samples were eluted with 0.2 M glycine (pH 2.5). Elution was analyzed by mass spectrometry. The table shows a selected list of detected proteins. The experiments were repeated twice. (c and d) A549 cells were treated with 30 μM cisplatin for 24 h before processed for IF staining of HSP27. DAPI was used to stain nucleic acids. The images were taken with a Leica SP8 confocal microscope. The mean intensity of the green channels in the nuclear region (DAPI positive) was quantified by LAS X (Leica). The green signal from cisplatin-treated group was normalized to the untreated control. The experiments were repeated three times and the graph is shown as mean ± SD of n = 10 cells from one representative experiment. Scale bar, 5 μm. Statistical source data can be found in Supplementary Table 1. (e and f) A549 cells were treated with 30 μM cisplatin for 24 h before processed for PLA between HSP27 and p53. DAPI was used to stain nucleic acids. The z-stack images were taken with a Leica SP8 confocal microscope with each frame over a 0.2 μm thickness. The 3D view was generated by ImageJ. Scale bar, 5 μm. Representative data of three independent experiments were shown.

Supplementary Figure 7 Small heat shock proteins are required for mutant p53 stability.

(a and b) p53-null H1299 cells were transiently transfected with the indicated p53 constructs for 24 and then treated with 30 μM cisplatin for 24 h. Cells were fixed and processed for IF staining against HSP27 and p53. DAPI was used to stain nucleic acids. The images were taken with a Leica SP8 confocal microscope and processed by ImageJ. White arrows indicate the colocalized signals. The experiments were repeated three times and the graph is shown as mean ± SD of n = 10 cells from one representative experiment. Scale bar, 5 μm. (c and d) Recombinant His-PIPKIα was incubated with untagged HSP27 or His-αB-Crystallin. HSP27 and His-αB-Crystallin were pulled down with an anti-HSP27 and an anti-αB-Crystallin antibody, respectively. Associated His-PIPKIα was detected with an anti-PIPKIα antibody. Representative data of three independent experiments were shown. (e) MCF7 cells were transfected with PIPKIα siRNA for 48 h. Cells were treated with 10 μM cisplatin for an additional 16 h. Protein expression was analyzed by IB, p53 blots were quantified and the graph is shown as mean ± SD of n = 3 independent experiments. (f) In the indicated breast cancer cells, PIPKIα or αB-Crystallin was knocked down with siRNAs for 72 h. Protein expression was analyzed by IB. Representative data of three independent experiments were shown. (g) αB-Crystallin was IP’ed in MDA-MB-468 cells and the associated proteins analyzed by IB. Representative data of three independent experiments were shown. Two-sided paired Student t-tests were used for statistical analysis in b and e. Statistical source data for b and e can be found in Supplementary Table 1. Unprocessed images of the blots in c-g are shown in Supplementary Figure 9.

Supplementary Figure 8 A point mutant of p53 that diminishes PI4,5P2 binding reduces p53 stability.

(a) 0.1 μM GST-p53 and 0.5 μM untagged HSP27 were incubated with 1 μM PI, PI4P, or PI4,5P2. GST-p53 was pulled down and the associated HSP27 was analyzed by IB. Representative data of three independent experiments were shown. (b) The indicated p53 constructs were transiently expressed in p53-null H1299 cells for 24 h. p53 was immunoprecipitated and eluted with 0.1 M glycine (pH 2.5) and the associated molecules were analyzed by IB. The graph is shown as mean ± SD of n = 3 independent experiments. (c) In PC3 cells (p53-null), the indicated mutant p53 constructs were stably expressed and analyzed by IB. The graph is shown as mean ± SD of n = 3 independent experiments. (d) The indicated p53 constructs were transfected in p53-null H1299 cells for 24 h. Endogenous HSP27 was immunoprecipitated and the associated p53 was analyzed by IB. The graph is shown as mean ± SD of n = 3 independent experiments. Two-sided paired Student t-tests were used for statistical analysis in b-d. Statistical source data for b-d can be found in Supplementary Table 1. Unprocessed images of the blots in a-d are shown in Supplementary Figure 9.

Supplementary Figure 9

Unprocessed gel images for Fig. 1a. Unprocessed gel images for Fig. 1c. Unprocessed gel images for Figs. 1d, 1e. Unprocessed gel images Fig. 2a. Unprocessed gel images Fig. 2b. Unprocessed gel images for Fig. 2c. Unprocessed gel images for Fig. 1a. Unprocessed gel images for Fig. 3b. Unprocessed gel images for Fig. 3c. Unprocessed gel images for Figs. 5a, 5c. Unprocessed gel images for Fig. 5d. Unprocessed gel images for Fig. 6a. Unprocessed gel images for Fig. 6b. Unprocessed gel images for Figs. 6c, 6d. Unprocessed gel images for Fig. 6e. Unprocessed gel images for Fig. 7a. Unprocessed gel images for Fig. 7c. Unprocessed gel images for Fig. 7g. Unprocessed gel images for Fig. 8a. Unprocessed gel images for Fig. 8b. Unprocessed gel images for Figs. 8d, 8e. Unprocessed gel images for Figs. 8f, 8g. Unprocessed gel images for Figs. 8h, 8i. Unprocessed gel images for Supplementary Fig. 1a, 1c. Unprocessed gel images for Supplementary Fig. 2a, 2b. Unprocessed gel images for Supplementary Fig. 3b. Unprocessed gel images for Supplementary Fig. 5g. Unprocessed gel images for Supplementary Fig. 6a. Unprocessed gel images for Supplementary Fig. 7c, 7d. Unprocessed gel images for Supplementary Fig. 7e. Unprocessed gel images for Supplementary Fig. 7f. Unprocessed gel images for Supplementary Fig. 7g. Unprocessed gel images for Supplementary Fig. 8a, 8c. Unprocessed gel images for Supplementary Fig. 8b, 8d.

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Supplementary Figures 1–9 and legend for Supplementary Table 1.

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Choi, S., Chen, M., Cryns, V.L. et al. A nuclear phosphoinositide kinase complex regulates p53. Nat Cell Biol 21, 462–475 (2019). https://doi.org/10.1038/s41556-019-0297-2

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