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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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.
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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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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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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.
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.
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.
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.
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.
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.
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.
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.
Supplementary information
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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DOI: https://doi.org/10.1038/s41556-022-00949-1
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