UFMylation maintains tumour suppressor p53 stability by antagonizing its ubiquitination

Abstract

p53 is the most intensively studied tumour suppressor1. The regulation of p53 homeostasis is essential for its tumour-suppressive function2,3. Although p53 is regulated by an array of post-translational modifications, both during normal homeostasis and in stress-induced responses2,3,4, how p53 maintains its homeostasis remains unclear. UFMylation is a recently identified ubiquitin-like modification with essential biological functions5,6,7. Deficiency in this modification leads to embryonic lethality in mice and disease in humans8,9,10,11,12. Here, we report that p53 can be covalently modified by UFM1 and that this modification stabilizes p53 by antagonizing its ubiquitination and proteasome degradation. Mechanistically, UFL1, the UFM1 ligase6, competes with MDM2 to bind to p53 for its stabilization. Depletion of UFL1 or DDRGK1, the critical regulator of UFMylation6,13, decreases p53 stability and in turn promotes cell growth and tumour formation in vivo. Clinically, UFL1 and DDRGK1 expression are downregulated and positively correlated with levels of p53 in a high percentage of renal cell carcinomas. Our results identify UFMylation as a crucial post-translational modification for maintenance of p53 stability and tumour-suppressive function, and point to UFMylation as a promising therapeutic target in cancer.

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Fig. 1: Identification of p53 as a target for UFMylation.
Fig. 2: UFMylation stabilizes p53 by antagonizing its ubiquitination.
Fig. 3: UFMylation is required for p53 accumulation in the DNA damage response.
Fig. 4: UFMylation maintains the tumour-suppressive function of p53.
Fig. 5: UFL1 and DDRGK1 expression are downregulated and positively correlated with levels of p53 in RCC.

Data availability

All of the source data supporting the findings of this study are available within this paper and/or from the corresponding author upon reasonable request. The human kidney renal clear cell carcinoma data were derived from The Cancer Genome Atlas Research Network (http://cancergenome.nih.gov/). Source data are provided with this paper.

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Acknowledgements

We thank X. Li for providing the MDM2 and ubiquitin constructs, X. Xu for the UFM1 Lys-less construct and S. Bacchetti for critical reading of the manuscript. This work was supported by grants from the National Natural Science Foundation of China (31730020, 31871416, 81701380 and 31571409), Natural Science Foundation of Zhejiang Province of China (LY18C070001 and LY20H250002) and Hangzhou Science and Technology Bureau (20182014B01 and 20180533B27).

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Contributions

J.L., D.G. and Y.-S.C. conceived of and designed the experiments. J.L., D.G., M.D., J.Y., H.W., L.S., L.X., J.B., C.L., Q.L. and J.Z. carried out the experiments. J.L., D.G., J.M., Q.Z., X.W., M.W. and Y.-S.C. analysed and interpreted the data. J.L., D.G. and Y.-S.C. wrote the paper.

Corresponding author

Correspondence to Yu-Sheng Cong.

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Extended data

Extended Data Fig. 1 p53 interacts with UFL1 and DDRGK1.

a, Strategy for identification of binding proteins for UFL1 or DDRGK1. b, Proteins eluted from anti-Flag M2 affinity gel were subjected to SDS-PAGE followed by silver staining. c-e, Western blot analysis of the mutual interactions between p53, UFL1 and DDRGK1 in HEK293T cells by co-immunoprecipitation with anti-Flag M2 affinity gel. f, g, Western blot analysis of the mutual interactions between UFL1, DDRGK1 and p53 in HEK293T cells by co-immunoprecipitation with UFL1 (f) or DDRGK1 (g) antibody. Source data are available online. Source data

Extended Data Fig. 2 The effects of UBA5, UFC1, UFL1, and DDRGK1 on p53 ufmylation in cells.

a, Western blot analysis of p53 ufmylation in HEK293T cells expressed the ufmylation system components in various combinations as indicated. b-e, Western blot analysis of p53 ufmylation in HEK293T cells with depletion of UFL1 (b), UBA5 (c), UFC1 (d), or DDRGK1 (e), respectively as indicated. Source data are available online. Source data

Extended Data Fig. 3 p53 is ufmylated at Lys351, 357, 370 and 373 of C-terminal region.

a, The scheme diagram of p53-WT and its mutants. The Lys residues in p53 were replaced by Arg at the indicated positions. b, The ufmylation assay was performed to identity the ufmylation region of p53 in HEK293T cells expressed with p53-WT and its mutants as indicated in (a). c, Lys residues were replaced by Arg at indicated positions, and the ufmylation assay was performed in HEK293T cells. d, All Lys residues were replaced by Arg in p53 except one each of the five residues Lys351, 357, 370, 373 or 382 as indicated, and the ufmylation assay was performed in HEK293T cells. Source data are available online. Source data

Extended Data Fig. 4 Ufmylation maintains p53 stability.

a, b, Q-PCR analysis of the relative mRNA expression of p53 and p21 in HeLa cells with UFL1 depletion (a), or DDRGK1 depletion (b). Error bars indicate mean ± s.e.m., n=3 for biological replicates. c, d, p53 stability was examined by western blot in HeLa cells with UFL1 (c), or DDRGK1 (d) depletion. The cells were treated with 100 µg ml−1 CHX for the indicated times, and the graph represents the quantification of the p53 protein levels. e, f, Western blot analysis of p53 expression in HCT116 (e), and HeLa (f) cells with UFL1 or DDRGK1 depletion in presence of MG132 (20 µM, 8 h). Source data are available online. Source data

Extended Data Fig. 5 Ufmylation is required for p53 accumulation in DNA damage response.

a–d, Western blot analysis of p53 and p21 expression in HeLa cells with UFL1 or DDRGK1 depletion under DNA damage conditions (Doxo, 1 µM; Eto, 50µM) for indicated time. e, Western blot analysis of p53 and p21 expression in HCT116 p53+/+ and HCT116 p53−/− cells stably depletion of UFL1 or DDRGK1. f, Western blot analysis of p53 and p21 expression in HCT116 p53-/- cells stably expression of p53-WT or p53-4KR with UFL1 depletion. Source data are available online. Source data

Extended Data Fig. 6 UFL1 and DDRGK1 are downregulated in renal cell carcinoma.

a-c, Immunohistochemical staining of UFL1 (a), DDRGK1 (b) and p53 (c) expression in forty pairs of RCC and adjacent normal tissues in tissue array. d, Percentages of the RCC specimens (a-c) showed low or high UFL1, DDRGK1 and p53 expression. (N, Normal tissue; C, cancer tissue). P values by a Chi-square (χ2) test, UFL1, P=2.16*10-14; DDRGK1, P=3.76*10-7; p53, P=8.29*10-16. Source data are available online. Source data

Supplementary information

Reporting Summary

Supplementary Tables Supplementary Table 1: Mass spectrometry information—UFL1 enrichment. The immunoprecipitates of Flag-UFL1 were resolved by SDS-PAGE and subjected to mass spectrometry for analysis. The spectra and unique peptide for each protein, protein name, protein description and molecular weight are listed. Supplementary Table 2: Mass spectrometry information—DDRGK1 enrichment. The immunoprecipitates of Flag-DDRGK1 were resolved by SDS-PAGE and subjected to mass spectrometry for analysis. The spectra and unique peptide for each protein, protein name, protein description and molecular weight are listed. Supplementary Table 3: Mass spectrometry information—p53 enrichment. The immunoprecipitates of Flag-p53 were resolved by SDS-PAGE and subjected to mass spectrometry for analysis. The spectra and unique peptide for each protein, protein name, protein description and molecular weight are listed. Supplementary Table 4: Antibody information. Information on the antibodies used in this study is provided in detail, including source, catalogue number, species, clone number (if available) and dilutions in distinct application. Supplementary Table 5: Plasmid information. Information on the plasmids used in the study is provided. Supplementary Table 6: Oligo information. Information on primers, including those used in quantitative PCR and siRNA interference, is provided in detail, including source and sequence data. Supplementary Table 7: Patient characteristics. Information on human research participants in the study is provided in detail, including patient ID, age, gender, p53 genotype and disease characteristics.

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Liu, J., Guan, D., Dong, M. et al. UFMylation maintains tumour suppressor p53 stability by antagonizing its ubiquitination. Nat Cell Biol 22, 1056–1063 (2020). https://doi.org/10.1038/s41556-020-0559-z

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