Abstract
The auto-phosphorylation of murine receptor-interacting protein 3 (Rip3) on Thr 231 and Ser 232 in the necrosome is required to trigger necroptosis. However, how Rip3 phosphorylation is regulated is still largely unknown. Here we identified protein phosphatase 1B (Ppm1b) as a Rip3 phosphatase and found that Ppm1b restricts necroptosis in two settings: spontaneous necroptosis caused by Rip3 auto-phosphorylation in resting cells, and tumour necrosis factor-α (TNF)-induced necroptosis in cultured cells. We revealed that Ppm1b selectively suppresses necroptosis through the dephosphorylation of Rip3, which then prevents the recruitment of mixed lineage kinase domain-like protein (Mlkl) to the necrosome. We further showed that Ppm1b deficiency (Ppm1bd/d) in mice enhanced TNF-induced death in a Rip3-dependent manner, and the role of Ppm1b in inhibiting necroptosis was evidenced by elevated Rip3 phosphorylation and tissue damage in the caecum of TNF-treated Ppm1bd/d mice. These data indicate that Ppm1b negatively regulates necroptosis through dephosphorylating Rip3 in vitro and in vivo.
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Change history
19 March 2015
In the version of this Article originally published online the molecular mass of Ppm1b-S (isoform 2) should have read Mr43K. This has been corrected in all versions of the Article.
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Acknowledgements
We thank S. Tamura and M. Ohnishi for Ppm1b+/− mice. This work was supported by the National Basic Research Program of China (973 Program; 2015CB553800), the National Scientific and Technological Major Project (2013ZX10002-002), the National Natural Science Foundation of China (91429301, 31420103910, 31330047, 91029304, 31221065, and 31090360), the Hi-Tech Research and Development Program of China (863 program; 2012AA02A201), the 111 Project (B12001), the Science and Technology Foundation of Xiamen (No. 3502Z20130027), the National Science Foundation of China for Fostering Talents in Basic Research (Grant No.J1310027) and the Open Research Fund of State Key Laboratory of Cellular Stress Biology, Xiamen University. This research was also partly supported by grants from MOST (2012CB966600) and NIH (R01GM63773, R01AR053591, R01CA108454). The collaborative research is also supported by the special funds for Innovation Center for Cell Biology from Zhejiang University and Xiamen University.
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W.C., J.W., L.L., Zhengmo Z., J.R., Y.L., X-H.F. and J.H. carried out the experiments. F.C., C.Y., Zhenru Z., S.S.S., X.Z., Zhirong Z., C-Q.Z., H.W., M.X. and X.L. helped to prepare cell lines, generated the ppm1b gene-trap line, provided reagents and mass spectrum analysis. J.H. contributed to the overall design of the project. W.C. and J.H. interpreted the data and wrote the manuscript.
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Supplementary Figure 1 Ppm1b interacts with Rip3.
(a) Ppm1b interacts with Rip3 in resting stage in L929 cells. Rip3-KO–Flag–Rip3 L929 cells were lysed and the cell lysates were immunoprecipitated with anti-HA (IP: HA) and anti-Flag (IP: Flag) antibodies. Both the cell lysate (Input) and immunoprecipitates were analysed by immunoblotting with indicated antibodies. The vertical line represents a splice mark. The samples were obtained and processed in the same experiment, and the gels/blots were processed in parallel. (b) Both Ppm1b-L and Ppm1b-S co-immunoprecipitate with Rip3 in 293T cells. Myc-Rip3 was coexpressed with Flag-tagged Ppm1b-L, Ppm1b-S or empty vector. Then co-immunoprecipitation experiments were performed with anti-Flag antibody. Both the immunoprecipitates and lysates were analysed by immunoblotting with indicated antibodies. (c) Ppm1b selectively interacts with Rip3 but not the other necrosome components in 293T cells. Flag–Ppm1b-L was coexpressed with HA-tagged proteins as indicated. Co-immunoprecipitaton experiments were performed with anti-HA antibody and analysed as in (b). (d) Schematic of the domain structures of Ppm1b-L and Ppm1b-S and Rip3. (e) The phosphatase domain of Ppm1b interacts with Rip3. Myc-Rip3 was coexpressed with Flag-tagged full length and truncated Ppm1b in 293T cells. Then co-immunoprecipitation experiments were performed as in (b). Ppm1b-L-PD: Ppm1b-L phosphatase domain; Ppm1b-L-CD: Ppm1b-L C-terminal domain. (f) The kinase domain of Rip3 interacts with Ppm1b. Flag–Ppm1b-L-PD was coexpressed with Myc-tagged full length and truncated Rip3 in 293T cells. Then co-immunoprecipitation experiments were performed with anti-Myc antibody and analysed as in (b). Rip3-KD: Rip3 kinase domain; Rip3-CD: Rip3 C-terminal domain. Data shown were representative of two or more independent experiments. Uncropped images of blots are shown in Supplementary Fig. 7.
Supplementary Figure 2 Related to Fig. 2.
(a) The protein amounts in Ppm1b knocked down and control L929 cells were analysed by immunoblotting with indicated antibodies. Related to Fig. 2a. (b) Ppm1b-S, Ppm1b-L and both isoforms were knocked down with isoform-specific shRNAs in L929 cells. Left, 48 h later, spontaneous cell death was measured. Right, the protein amounts were analysed by immunoblotting with indicated antibodies. Results shown were mean ± s.e.m.;n = 3,000 cells pooled from 3 independent experiments. (c) The protein amounts in Ppm1b knocked down and control cells were analysed by immunoblotting with indicated antibodies. Related to Fig. 2c. (d) The protein amounts in WT and Rip3 KO L929 cells knocked down with shPpm1b or control shRNAs were analysed by immunoblotting with indicated antibodies. Related to Fig. 2f. (e) The protein amounts in WT and Rip3 KO mouse peritoneal macrophage cells knocked down with shPpm1b or control shRNAs were analysed by immunoblotting with indicated antibodies. Related to Fig. 2g. For Figure a, c, d, e, data shown were representative of two or more independent experiments. The asterisk (∗) denotes a nonspecific band. Statistics source data for this figure can be found in Supplementary Table 2. Uncropped images of blots are shown in Supplementary Fig. 7.
Supplementary Figure 3 Related to Fig. 3.
(a) Ppm1b was knocked down in Rip3 KO L929 cells reconstituted with Rip3-WT, Rip3 phosphorylation-deficient mutant (Rip3-2A) mutant and Rip3 RHIM domain mutant (Rip3RHIM). The cells were lysed and subjected to immunoblotting with indicated antibodies. Related to Fig. 3b. (b) Ppm1b was knocked down in WT and Rip1KO L929 cells. The cells were lysed and subjected to immunoblotting with indicated antibodies. The vertical line represents a splice mark. The spliced images were from the same blot. Related to Fig. 3c. (c) WT Rip3 and Rip3RHIM were introduced into Rip1 KO and WT L929 cells by lentiviral vectors. The cells were lysed and subjected to immunoblotting with indicated antibodies. Related to Fig. 3d. (d) Ppm1b was knocked down in WT and MlklKO L929 cells. The cells were lysed and subjected to immunoblotting with indicated antibodies. Related to Fig. 3h. (e) TNFR1 KO and WT L929 cells were infected with lentivirus encoding shPpm1b or control. Left, spontaneous cell death was analysed 48 h later. n = 3,000 cells pooled from three independent experiments. Right, TNFR1 KO and WT L929 cells were treated with or without TNF for 12 h. Cell death was analysed. n = 3 independent experiments. Results shown were mean ± s.e.m.;##P < 0.01;###P < 0.001. For Figure a-d, data shown were representative of two or more independent experiments. The asterisk (∗) denotes a nonspecific band. Statistics source data for this figure can be found in Supplementary Table 2. Uncropped images of blots are shown in Supplementary Fig. 7.
Supplementary Figure 4 Ppm1b targets TNF-induced necroptosis but not apoptosis.
(a,b) L929 cells were infected with lentivirus encoding shPpm1b or control, or not infected (Mock). 48 h later, (a) the cells were treated with TNF (10 ng ml−1) for indicated time periods and cell death was analysed by flow cytometer; (b) the cells were treated with different doses of TNF as indicated for 6 h and cell death was analysed. n = 3 independent experiments. (c) Ppm1b does not affect TNF-induced apoptosis in NIH3T3-A cells. Ppm1b was knocked down in NIH3T3-A cells. Left, 48 h later, the cells were treated with or without TNF (100 ng ml−1) for 24 h and cell death was analysed. n = 3 independent experiments. Right, the cell lysates were analysed by immunoblotting with indicated antibodies. (d) Both Ppm1b-L and S isoforms restrict TNF-induced necroptosis in L929. Ppm1b-S, Ppm1b-L or both isoforms were knocked down with isoform specific shRNAs in L929 cells. 48 h later, the cells were treated with or without TNF (10 ng ml−1) for 6 h and cell death was measured. n = 3 independent experiments. (e) Ppm1b KO L929 cells were infected with different doses of lentivirus encoding Ppm1b-L and Ppm1b-S as indicated, or not infected (Mock infection). Then the cells were treated with TNF (10 ng ml−1) for 6 h and cell death was analysed. n = 3 independent experiments. (f) Ppm1b does not affect the TNF-induced p65 and p38 phosphorylation. Ppm1b was knocked down with shRNA in L929 cells. 48 h later, the cells were treated with TNF (10 ng ml−1) for indicated time. The cell lysates were analysed by immunoblotting with indicated antibodies. (g) The Ppm1b-β-geo fusion mRNA sequences in Ppm1bd/d mice were determined by 3’ RACE. For Figure a-e, results shown were mean ± s.e.m.;#P < 0.05;##P < 0.01;###P < 0.001; NS: no significant difference. For Figure f, data shown were representative of two independent experiments. The asterisk (∗) denotes a nonspecific band. Statistics source data for this figure can be found in Supplementary Table 2. Uncropped images of blots are shown in Supplementary Fig. 7.
Supplementary Figure 5 The regulation of necroptosis by Ppm1b is independent of NF-κB pathway.
(a) HeLa cells were infected with lentivirus encoding sh-hPpm1b or control. 48 h later, the cells were treated with hTNF (30 ng ml−1) for indicated time and subjected to immunoblotting with indicated antibodies. (b) HT29 cells were analysed as in (a). (c) L929 cells were infected with lentivirus encoding shPpm1b or control, or not infected (Mock). 48 h later, the cells were treated with TNF for indicated time and the IL-6 level of the cell culture supernatant was analysed by ELISA. n = 3 independent experiments. (d) Left, L929 cells were infected with lentivirus encoding shPpm1b or control, or not infected (Mock). 5 h after infection, the cells were treated with DMSO, TPCA-1 (1μM) or IMD 0354 (5μM). 48 h later, spontaneous cell death was analysed. Middle, IKKβ KO and WT L929 cells were infected with lentivirus encoding shPpm1b or control, or not infected. 48 h later, spontaneous cell death was analysed. Right, the IKKβ protein amount in IKKβ KO and WT L929 cells was determined by immunoblotting. n = 3,000 cells pooled from 3 independent experiments. (e) Left, the cells were treated as in (d, left), except that the cells were treated with TNF for 5 h and the TNF-induced cell death rather than spontaneous cell death was analysed 48 h after infection. Right, the cells were treated as in (d, middle), except that the cells were treated with TNF for 5 h and TNF-induced cell death rather than spontaneous cell death was analysed 48 h after infection. n = 3 independent experiments. (f) L929 cells were pretreated with DMSO, TPCA-1 or IMD 0354 for 2 h followed by treatment with TNF for indicated time. The IL-6 level of the cell culture supernatant was analysed by ELISA. n = 3 independent experiments. (g) L929 cells were infected with lentivirus encoding shPpm1b or control, or not infected. 48 h later, the cells were pretreated with DMSO or 5z-7 (1 μm) for 2 h followed by treatment with TNF for 5 h. Then the cell death was analysed. n = 3 independent experiments. (h) HeLa cells stably expressing human Rip3 (HeLa-hRip3) were analysed as in (a). (i) HeLa-hRip3 cells were infected with lentivirus encoding sh-hPpm1b or control, or not infected. Spontaneous cell death and hTNF (30 ng ml−1) + Smac mimetic (100 nM) + zVAD (20 μM) (TSZ)-induced cell death were analysed as in (d, left) and (e, left). n = 3 independent experiments. (j) Littermates of Ppm1bd/d and WT mice were injected with TNF (15 μg) via the tail vein. Then the serum IL-6 level was analysed by ELISA at different time points as indicated. n = 4 mice for each group in single experiment where two independent experiments were performed to assess reproducibility. ND: not detectable. For Figure c-g, i, j, results shown were mean ± s.e.m.;#P < 0.05;##P < 0.01;###P < 0.001;. For Figure a, b, h, data shown were representative of two or more independent experiments. Statistics source data for this figure can be found in Supplementary Table 2. Uncropped images of blots are shown in Supplementary Fig. 7.
Supplementary Figure 6 TNF induces tissue damage in different organs of WT and Rip3−/− mice.
Littermates of WT and Rip3−/− mice were injected with TNF (15 μg) via the tail vein for indicated time. The sections of kidney were analysed by PAS staining while those of the other organs were analysed by H&E staining. The representative images were shown (n = 5 mice of each genotype at 12 h time point; n = 3 mice of each genotype at 0 h time point.). Scale bar, 50 or 100 μm as indicated.
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Chen, W., Wu, J., Li, L. et al. Ppm1b negatively regulates necroptosis through dephosphorylating Rip3. Nat Cell Biol 17, 434–444 (2015). https://doi.org/10.1038/ncb3120
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DOI: https://doi.org/10.1038/ncb3120
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