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.

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.

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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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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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