Chip controls necroptosis through ubiquitylation- and lysosome-dependent degradation of ripk3

ABSTRACT Receptor-interacting protein kinase 3 (RIPK3) functions as a key regulator of necroptosis. Here, we report that the RIPK3 expression level is negatively regulated by CHIP (carboxyl

terminus of Hsp70-interacting protein; also known as STUB1) E3 ligase-mediated ubiquitylation. _Chip_−/− mouse embryonic fibroblasts and CHIP-depleted L929 and HT-29 cells exhibited higher

levels of RIPK3 expression, resulting in increased sensitivity to necroptosis induced by TNF (also known as TNFα). These phenomena are due to the CHIP-mediated ubiquitylation of RIPK3, which

leads to its lysosomal degradation. Interestingly, RIPK1 expression is also negatively regulated by CHIP-mediated ubiquitylation, validating the major role of CHIP in necrosome formation

and sensitivity to TNF-mediated necroptosis. _Chip_−/− mice (C57BL/6) exhibit inflammation in the thymus and massive cell death and disintegration in the small intestinal tract, and die

within a few weeks after birth. These phenotypes are rescued by crossing with _Ripk3_−/− mice. These results imply that CHIP is a bona fide negative regulator of the RIPK1–RIPK3 necrosome

formation leading to desensitization of TNF-mediated necroptosis. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution

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about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS UBIQUITINATION OF RIPK1 REGULATES ITS ACTIVATION MEDIATED BY TNFR1 AND

TLRS SIGNALING IN DISTINCT MANNERS Article Open access 11 December 2020 RIPK1 DEPHOSPHORYLATION AND KINASE ACTIVATION BY PPP1R3G/PP1Γ PROMOTE APOPTOSIS AND NECROPTOSIS Article Open access 03

December 2021 RIPK3 SIGNALING AND ITS ROLE IN REGULATED CELL DEATH AND DISEASES Article Open access 29 April 2024 REFERENCES * Vanden Berghe, T., Linkermann, A., Jouan-Lanhouet, S.,

Walczak, H. & Vandenabeele, P. Regulated necrosis: the expanding network of non-apoptotic cell death pathways. _Nat. Rev. Mol. Cell Biol._ 15, 135–147 (2014). CAS  PubMed  Google Scholar

  * Galluzzi, L. et al. Essential versus accessory aspects of cell death: recommendations of the NCCD 2015. _Cell Death Differ._ 22, 58–73 (2015). CAS  PubMed  Google Scholar  * Mocarski, E.

S., Kaiser, W. J., Livingston-Rosanoff, D., Upton, J. W. & Daley-Bauer, L. P. True grit: programmed necrosis in antiviral host defense, inflammation, and immunogenicity. _J. Immunol._

192, 2019–2026 (2014). CAS  PubMed  Google Scholar  * Linkermann, A. & Green, D. R. Necroptosis. _N. Engl. J. Med._ 370, 455–465 (2014). CAS  PubMed  PubMed Central  Google Scholar  *

Murphy, J. M. & Silke, J. Ars Moriendi; the art of dying well—new insights into the molecular pathways of necroptotic cell death. _EMBO Rep._ 15, 155–164 (2014). CAS  PubMed  PubMed

Central  Google Scholar  * Cho, Y. S. et al. Phosphorylation-driven assembly of the RIP1–RIP3 complex regulates programmed necrosis and virus-induced inflammation. _Cell_ 137, 1112–1123

(2009). CAS  PubMed  PubMed Central  Google Scholar  * He, S. et al. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-α. _Cell_ 137, 1100–1111 (2009). CAS 

PubMed  Google Scholar  * Zhang, D. W. et al. RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. _Science_ 325, 332–336 (2009). CAS  PubMed

  Google Scholar  * Sun, L. et al. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. _Cell_ 148, 213–227 (2012). CAS  PubMed  Google Scholar  *

Murphy, J. M. et al. The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism. _Immunity_ 39, 443–453 (2013). CAS  PubMed  Google Scholar  * Wu, J. et al. Mlkl knockout

mice demonstrate the indispensable role of Mlkl in necroptosis. _Cell Res._ 23, 994–1006 (2013). CAS  PubMed  PubMed Central  Google Scholar  * Remijsen, Q. et al. Depletion of RIPK3 or MLKL

blocks TNF-driven necroptosis and switches towards a delayed RIPK1 kinase-dependent apoptosis. _Cell Death Dis._ 5, e1004 (2014). CAS  PubMed  PubMed Central  Google Scholar  * Li, J. et

al. The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis. _Cell_ 150, 339–350 (2012). CAS  PubMed  PubMed Central  Google Scholar  *

Dondelinger, Y. et al. MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates. _Cell Rep._ 7, 971–981 (2014). CAS  PubMed  Google Scholar  * Wang, H. et al.

Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3. _Mol. Cell_ 54, 133–146 (2014). CAS  PubMed  Google Scholar  * Cai, Z. et al.

Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis. _Nat. Cell Biol._ 16, 55–65 (2014). CAS  PubMed  Google Scholar  * Chen, X. et al.

Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death. _Cell Res._ 24, 105–121 (2014). CAS  PubMed  Google Scholar  * Wu, X. N. et al.

Distinct roles of RIP1–RIP3 hetero- and RIP3–RIP3 homo-interaction in mediating necroptosis. _Cell Death Differ._ 21, 1709–1720 (2014). CAS  PubMed  PubMed Central  Google Scholar  * Cook,

W. D. et al. RIPK1- and RIPK3-induced cell death mode is determined by target availability. _Cell Death Differ._ 21, 1600–1612 (2014). CAS  PubMed  PubMed Central  Google Scholar  * Chen, W.

et al. Ppm1b negatively regulates necroptosis through dephosphorylating Rip3. _Nat. Cell Biol._ 17, 434–444 (2015). CAS  PubMed  PubMed Central  Google Scholar  * Ahmed, S. F. et al. The

chaperone-assisted E3 ligase C terminus of Hsc70-interacting protein (CHIP) targets PTEN for proteasomal degradation. _J. Biol. Chem._ 287, 15996–16006 (2012). CAS  PubMed  PubMed Central 

Google Scholar  * Esser, C., Scheffner, M. & Hohfeld, J. The chaperone-associated ubiquitin ligase CHIP is able to target p53 for proteasomal degradation. _J. Biol. Chem._ 280,

27443–27448 (2005). CAS  PubMed  Google Scholar  * Ko, H. R. et al. P42 Ebp1 regulates the proteasomal degradation of the p85 regulatory subunit of PI3K by recruiting a chaperone-E3 ligase

complex HSP70/CHIP. _Cell Death Dis._ 5, e1131 (2014). CAS  PubMed  PubMed Central  Google Scholar  * Lee, E. W., Seo, J., Jeong, M., Lee, S. & Song, J. The roles of FADD in extrinsic

apoptosis and necroptosis. _BMB Rep._ 45, 496–508 (2012). CAS  PubMed  Google Scholar  * Vanden Berghe, T., Kalai, M., van Loo, G., Declercq, W. & Vandenabeele, P. Disruption of HSP90

function reverts tumor necrosis factor-induced necrosis to apoptosis. _J. Biol. Chem._ 278, 5622–5629 (2003). CAS  PubMed  Google Scholar  * Park, S. Y., Shim, J. H. & Cho, Y. S.

Distinctive roles of receptor-interacting protein kinases 1 and 3 in caspase-independent cell death of L929. _Cell Biochem. Funct._ 32, 62–69 (2014). CAS  PubMed  Google Scholar  *

Petrucelli, L. et al. CHIP and Hsp70 regulate tau ubiquitination, degradation and aggregation. _Hum. Mol. Genet._ 13, 703–714 (2004). CAS  PubMed  Google Scholar  * Mollah, S. et al.

Targeted mass spectrometric strategy for global mapping of ubiquitination on proteins. _Rapid Commun. Mass Spectrom._ 21, 3357–3364 (2007). CAS  PubMed  Google Scholar  * Kim, W. et al.

Systematic and quantitative assessment of the ubiquitin-modified proteome. _Mol. Cell_ 44, 325–340 (2011). CAS  PubMed  PubMed Central  Google Scholar  * Sahara, N. et al. _In vivo_ evidence

of CHIP up-regulation attenuating tau aggregation. _J. Neurochem._ 94, 1254–1263 (2005). CAS  PubMed  Google Scholar  * Gunther, C. et al. Caspase-8 regulates TNF-α-induced epithelial

necroptosis and terminal ileitis. _Nature_ 477, 335–339 (2011). PubMed  PubMed Central  Google Scholar  * Kaiser, W. J. et al. RIP3 mediates the embryonic lethality of caspase-8-deficient

mice. _Nature_ 471, 368–372 (2011). CAS  PubMed  PubMed Central  Google Scholar  * Oberst, A. et al. Catalytic activity of the caspase-8-FLIP(L) complex inhibits RIPK3-dependent necrosis.

_Nature_ 471, 363–367 (2011). CAS  PubMed  PubMed Central  Google Scholar  * Welz, P. S. et al. FADD prevents RIP3-mediated epithelial cell necrosis and chronic intestinal inflammation.

_Nature_ 477, 330–334 (2011). CAS  PubMed  Google Scholar  * Zhang, H. et al. Functional complementation between FADD and RIP1 in embryos and lymphocytes. _Nature_ 471, 373–376 (2011). CAS 

PubMed  PubMed Central  Google Scholar  * Dillon, C. P. et al. RIPK1 blocks early postnatal lethality mediated by caspase-8 and RIPK3. _Cell_ 157, 1189–1202 (2014). CAS  PubMed  PubMed

Central  Google Scholar  * Newton, K. et al. Activity of protein kinase RIPK3 determines whether cells die by necroptosis or apoptosis. _Science_ 343, 1357–1360 (2014). CAS  PubMed  Google

Scholar  * Rickard, J. A. et al. RIPK1 regulates RIPK3-MLKL-driven systemic inflammation and emergency hematopoiesis. _Cell_ 157, 1175–1188 (2014). CAS  PubMed  Google Scholar  * Dannappel,

M. et al. RIPK1 maintains epithelial homeostasis by inhibiting apoptosis and necroptosis. _Nature_ 513, 90–94 (2014). CAS  PubMed  PubMed Central  Google Scholar  * Takahashi, N. et al.

RIPK1 ensures intestinal homeostasis by protecting the epithelium against apoptosis. _Nature_ 513, 95–99 (2014). CAS  PubMed  Google Scholar  * Mandal, P. et al. RIP3 induces apoptosis

independent of pronecrotic kinase activity. _Mol. Cell_ 56, 481–495 (2014). CAS  PubMed  PubMed Central  Google Scholar  * Orozco, S. et al. RIPK1 both positively and negatively regulates

RIPK3 oligomerization and necroptosis. _Cell Death Differ._ 21, 1511–1521 (2014). CAS  PubMed  PubMed Central  Google Scholar  * Duprez, L. et al. RIP kinase-dependent necrosis drives lethal

systemic inflammatory response syndrome. _Immunity_ 35, 908–918 (2011). CAS  PubMed  Google Scholar  * Onizawa, M. et al. The ubiquitin-modifying enzyme A20 restricts ubiquitination of the

kinase RIPK3 and protects cells from necroptosis. _Nat. Immunol._ 16, 618–627 (2015). CAS  PubMed  PubMed Central  Google Scholar  * Petersen, S. L. et al. TRAF2 is a biologically important

necroptosis suppressor. _Cell Death Differ._ 22, 1846–1857 (2015). CAS  PubMed  PubMed Central  Google Scholar  * Kang, T. B., Yang, S. H., Toth, B., Kovalenko, A. & Wallach, D.

Caspase-8 blocks kinase RIPK3-mediated activation of the NLRP3 inflammasome. _Immunity_ 38, 27–40 (2013). CAS  PubMed  Google Scholar  * Lawlor, K. E. et al. RIPK3 promotes cell death and

NLRP3 inflammasome activation in the absence of MLKL. _Nat. Commun._ 6, 6282 (2015). CAS  PubMed  Google Scholar  * Vince, J. E. et al. Inhibitor of apoptosis proteins limit RIP3

kinase-dependent interleukin-1 activation. _Immunity_ 36, 215–227 (2012). CAS  PubMed  Google Scholar  * Moriwaki, K. et al. The Necroptosis Adaptor RIPK3 promotes injury-induced cytokine

expression and tissue repair. _Immunity_ 41, 567–578 (2014). CAS  PubMed  PubMed Central  Google Scholar  * Humphries, F., Yang, S., Wang, B. & Moynagh, P. N. RIP kinases: key decision

makers in cell death and innate immunity. _Cell Death Differ._ 22, 225–236 (2015). CAS  PubMed  Google Scholar  * Silke, J., Rickard, J. A. & Gerlic, M. The diverse role of RIP kinases

in necroptosis and inflammation. _Nat. Immunol._ 16, 689–697 (2015). CAS  PubMed  Google Scholar  * Min, J. N. et al. CHIP deficiency decreases longevity, with accelerated aging phenotypes

accompanied by altered protein quality control. _Mol. Cell. Biol._ 28, 4018–4025 (2008). CAS  PubMed  PubMed Central  Google Scholar  * Vanden Berghe, T. et al. Passenger mutations confound

interpretation of all genetically modified congenic mice. _Immunity_ 43, 200–209 (2015). CAS  PubMed  Google Scholar  * Schisler, J. C. et al. CHIP protects against cardiac pressure overload

through regulation of AMPK. _J. Clin. Invest._ 123, 3588–3599 (2013). CAS  PubMed  PubMed Central  Google Scholar  * Lee, I. H. et al. Ahnak functions as a tumor suppressor via modulation

of TGF_β_/Smad signaling pathway. _Oncogene_ 33, 4675–4684 (2014). CAS  PubMed  PubMed Central  Google Scholar  * Oh, W. et al. PML-IV functions as a negative regulator of telomerase by

interacting with TERT. _J Cell Sci._ 122, 2613–2622 (2009). CAS  PubMed  Google Scholar  * Lee, E. W. et al. USP11-dependent selective cIAP2 deubiquitylation and stabilization determine

sensitivity to Smac mimetics. _Cell Death Differ._ 22, 1463–1476 (2015). CAS  PubMed  PubMed Central  Google Scholar  * Lee, E. W. et al. Differential regulation of p53 and p21 by MKRN1 E3

ligase controls cell cycle arrest and apoptosis. _EMBO J._ 28, 2100–2113 (2009). CAS  PubMed  PubMed Central  Google Scholar  * Lee, E. W. et al. Ubiquitination and degradation of the FADD

adaptor protein regulate death receptor-mediated apoptosis and necroptosis. _Nat. Commun._ 3, 978 (2012). PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank S. Murata for

the _Chip_−/− mice and V. M. Dixit for the _Ripk3_−/− mice. We thank the Cancer Metabolism Interest Group (cMIG) led by NCC Korea for discussion and advice. This research was supported by a

grant from the National R&D Program for Cancer Control, Ministry of Health & Welfare, Republic of Korea (NCC-1420300) (to J.Song) and by a grant from the National Research Foundation

of Korea (NRF) funded by the Ministry of Education (2012R1A6A3A04040105) (to E.-W.L.) and by the Ministry of Science, ICT and Future Planning (NRF-2015R1A3A2066581) (to J.Song). Research in

the Vandenabeele group is supported by Flemish grants (Research Foundation Flanders: FWO G.0875.11, FWO G.0973.11, FWO G.0A45.12N, FWO G.0787.13N, FWO G0E04.16N), a Methusalem grant

(BOF16/MET_V/007), Ghent University grants (MRP, GROUP-ID consortium), a grant from the Foundation against Cancer (F94), and grants from VIB. Additionally, this research was partly supported

by the BK21 Plus project of the National Research Foundation of Korea Grant (to J.Seo, M.J., H.-K.L., D.S., J.-H.K. and S.Y.H.) and by the grants for KMPC (Korea Mouse Phenotype Center) (to

J.K.S.) from National Research Foundation of Korea (NRF), and C.L. acknowledges institutional support by KIST. AUTHOR INFORMATION Author notes * Jinho Seo and Eun-Woo Lee: These authors

contributed equally to this work. AUTHORS AND AFFILIATIONS * Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Korea Jinho Seo, Eun-Woo

Lee, Daehyeon Seong, Jihye Shin, Manhyung Jeong, Hae-Kyung Lee, Jung-Hoon Kim, Su Yeon Han & Jaewhan Song * Laboratory of Developmental Biology and Genetics, College of Veterinary

Medicine, BK21Plus Program for Creative Veterinary Science Research, Interdisciplinary Program for Bioinformatics, BIO-MAX institute, Korea Mouse Phenotyping Center, Seoul National

University, Seoul 151-742, South Korea Hyerim Sung & Je Kyung Seong * Department of Biomedical Molecular Biology, Ghent University, Technologiepark 927, 9052 Zwijnaarde-Ghent, Belgium

Yves Dondelinger & Peter Vandenabeele * Inflammation Research Center, VIB, Technologiepark 927, 9052 Zwijnaarde-Ghent, Belgium Yves Dondelinger & Peter Vandenabeele * BRI, Korea

Institute of Science and Technology, Seoul 136-791, Korea Jihye Shin & Cheolju Lee Authors * Jinho Seo View author publications You can also search for this author inPubMed Google

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author inPubMed Google Scholar * Jihye Shin View author publications You can also search for this author inPubMed Google Scholar * Manhyung Jeong View author publications You can also

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publications You can also search for this author inPubMed Google Scholar * Je Kyung Seong View author publications You can also search for this author inPubMed Google Scholar * Peter

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Scholar CONTRIBUTIONS J.Seo, E.-W.L. and J.Song conceived and designed the study. J.Seo, E.-W.L., D.S. and M.J. performed most of the experiments. J.Shin and C.L. performed the mass

spectrometry analysis. J.Seo, H.S., D.S., H.-K.L., J.-H.K., S.Y.H. and J.K.S. performed the animal experiments and analysis. Y.D. and P.V. discussed the results, conceived some experiments,

and provided critical reagents and comments. J.Seo, E.-W.L. and J.Song wrote the manuscript. CORRESPONDING AUTHOR Correspondence to Jaewhan Song. ETHICS DECLARATIONS COMPETING INTERESTS The

authors declare no competing financial interests. INTEGRATED SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURE 1 ABLATION OF CHIP INCREASES RIPK3-MEDIATED NECROPTOSIS IN HUMAN HT-29 AND MOUSE

L929 CELLS. (A) HT-29 cells were transfected with the indicated siRNAs for 48 h and then treated with 30 ng/mL human TNF_α_ (T), 5 μg/mL cycloheximide (C), and 30 μM z-VAD-fmk (Z) in the

presence or absence of 30 μM Nec-1 (N) for 8 h. The cells were stained with Annexin V and 7-AAD, and then analysed by FACS. Representative plots of data are shown in left panel, and means ±

S.D. from _n_ = 3 independent experiments are presented in right panel. (B,C) HT-29 cells were transfected with siRNA against CHIP and/or RIPK3 and treated as in (A) for 10 h. Knockdown

efficiency was confirmed by WB (B) and necrotic cells were detected by FACS (C). Representative plots of data are shown in left panel, and means ± S.D. from _n_ = 3 independent experiments

are presented in right panel (C). (D) L929 cells were transfected with the indicated siRNAs for 48 h and then treated with 5 ng/mL TNF_α_ and 10 μM z-VAD-fmk in the presence or absence of

Nec-1 for 2.5 h. The cells were stained with Annexin V and analysed by FACS. Representative plots of data from 3 independent experiments are shown in left panel and summarized in right

panel. (E) L929 cells were transfected and treated as described in (D). Cell loss were determined using the Cell Titer Glo Luminescent Cell Viability Assay Kit. (F–H) L929 cells were

transfected with the indicated siRNAs and treated with TNF_α_ and z-VAD. The knockdown efficiencies of each siRNAs were shown by WB (F). Necrotic cells were determined by Annexin V staining

and analysed by FACS (G). Cell loss was also determined as described above (H). (I,J) L929 cells stably expressing retroviral human CHIP (human CHIP shares all amino acids except for one

amino acid with mouse CHIP) were transfected with the indicated siRNAs for 48 h. Cells were then treated with or without TNF_α_ and z-VAD for 2.5 h. Expression of CHIP was analysed by WB

(I). Cell death was determined by Annexin V staining (J). Data in (D,E,G,H,J) are means ± S, D.; _n_ = 3 independent experiments (J). _P_ values were determined by unpaired two-tailed

Student’s _t_-test. (*_P_ < 0.05, **_P_ < 0.01, ***_P_ < 0.001). Raw data from independent experiments are provided in Supplementary Table 1. SUPPLEMENTARY FIGURE 2 CHIP DOES NOT

AFFECT TNF-MEDIATED APOPTOSIS. (A) Primary _Chip_+/+ and _Chip_−/−MEFs were treated as indicated (T: mTNF_α_, 30 ng/mL; C: Cycloheximide, 2 μg/mL; Z: z-VAD-fmk, 30 μM; N: necrostatin-1, 30 

μM) for 6 h or 12 h. The cells were stained with Annexin V and 7-AAD, and then analysed by FACS. Representative plots of data from _n_ = 3 independent experiments are shown in left panel,

and means ± S.D. are presented in right panel. (B) L929 cells were transfected with CHIP siRNA and treated as indicated for 2.5 h. Dead cells were determined by FACS analysis using Annexin V

and 7-AAD staining. Representative plots of data from _n_ = 3 independent experiments are shown in left panel, and means ± S.D. are presented in right panel. (C) HT-29 cells were

transfected with CHIP siRNA and treated as indicated (T: hTNF_α_, 30 ng/mL; C: Cycloheximide, 5 μg/mL; Z: z-VAD-fmk, 30 μM; N: necrostatin-1, 30 μM) for 10 h or 12 h. The cells were stained

with Annexin V and 7-AAD, and then analysed via FACS. Representative plots of data from _n_ = 3 independent experiments are shown in left panel, and means ± S.D. are presented in right

panel. _P_ values were determined by unpaired two-tailed Student’s _t_-test. (*_P_ < 0.05, **_P_ < 0.01, ***_P_ < 0.001). Raw data from independent experiments are provided in

Supplementary Table 1. SUPPLEMENTARY FIGURE 3 CHIP DEPLETION FACILITATES NECROSOME FORMATION BY STABILISING RIPK3 AND RIPK1 PROTEINS. (A,B) HT-29 and L929 cells transfected with the siRNA

against human CHIP or mouse CHIP for 48 h, respectively. HT-29 and L929 cells were then treated with hTNF, CHX, and zVAD (TCZ) or mTNF and CHX (TZ), respectively, as indicated. Cells were

lysed with lysis buffer and immunoprecipitated with anti-Caspase-8 (A) or FADD (B). Associated proteins were detected by WB using the indicated antibodies. (C–E) HT-29 and L929 cells were

transfected with control siRNA or siCHIP for 48 h. Expression of cell death related proteins were determined by WB in HT-29 and L929 cells with CHIP knockdown (C). Relative amounts of RIPK3

and RIPK1 were calculated and shown after normalising to Actin. mRNA levels were also measured by qRT-PCR (D,E). The data represent means ± S.D.; _n_ = 3 independent experiments; **_P_ <

0.01, ***_P_ < 0.001 (D, E). (F) The stability of RIPK1 and RIPK3 in CHIP-depleted L929 cells was measured by treating the cells with cycloheximide for the indicated period, followed by

WB analysis. Representative results of three independent experiments are shown. Relative amounts of RIPK3 and RIPK1 were calculated after normalising to Actin, and are shown in the graph.

Half-lives of RIPK3 and RIPK1 were also calculated and presented in the graph. SUPPLEMENTARY FIGURE 4 CHIP BINDS TO THE KINASE AND RHIM DOMAINS OF RIPK3 AND RIPK1, RESPECTIVELY. (A,B) 293FT

cells were transfected with plasmids expressing FLAG–CHIP and HA–RIPK3 or HA–RIPK1. The cell lysates were immunoprecipitated using an anti-FLAG or -HA antibodies, followed by WB using

anti-RIPK3, -RIPK1 and -FLAG antibodies. (C,D) L929 cells were immunoprecipitated using an anti-RIPK3 (C) or –RIPK1 (D) antibody. Interactions between endogenous CHIP and RIPK3 or RIPK1 were

then analysed by WB. (E,F) Bacterially produced GST and GST-CHIP were used for the _in vitro_ binding analysis. HA-RIPK3 or HA-RIPK1 was produced using the TNT T7 reticulocyte system. GST

or GST-CHIP was incubated with HA-RIPK3 or HA-RIPK1, and the sample was precipitated using Glutathione Sepharose beads. The interaction between CHIP and RIPKs was analysed by WB using

anti-RIPK3 and anti-RIPK1 antibodies. The asterisk indicates N-terminal cleaved form of RIPK3 because anti-HA antibody did not detect the lower band. Only full-length RIPK3 bound to

GST-CHIP, suggesting the N-terminal region of RIPK3 is required for the interaction with CHIP. (G,H) 293FT cells were transfected with the indicated plasmids. The cell lysates were

immunoprecipitated using an anti-Myc antibody, and the interaction between these two proteins was analysed by WB. (I,J) 293FT cells were transfected with a combination of plasmids expressing

of CHIP deficient mutants and RIPK3 or RIPK1. The cell lysates were immunoprecipitated using the indicated antibodies and then analysed via western blotting. (K,L) 293FT cells were

transfected with a combination of CHIP wild type and RIPK3 or RIPK1 domain expressing plasmids and then immunoprecipitated using the indicated antibodies. The interaction between the

peptides was analysed via western blotting using the indicated antibodies. (M) A schematic model of the interactions among RIPK1, RIPK3 and CHIP. SUPPLEMENTARY FIGURE 5 CHIP IS NOT INVOLVED

IN THE GELDANAMYCIN-MEDIATED DEGRADATION OF RIPK3 OR RIPK1 AND INDUCES THE DEGRADATION OF MUTANTS RIPK3 AND RIPK1 DEFECTIVE IN AGGRESOME FORMATION. (A) L929 cells were transfected with

siCtrl or siCHIP and then treated with 1 μM geldanamycin for 16 h. The proteins were detected by WB using the indicated antibodies. (B,C) H1299 cells were co-transfected with plasmids

expressing of FLAG-CHIP, HA-RIPK3 or HA-RIPK1 with GST, a transfection control, and then treated with geldanamycin for 12 h. The cells were analysed by WB. (D) H1299 cells were transfected

with GFP-RIPK3 or GFP-RIPK1 in the absence or presence of FLAG-CHIP or FLAG-CHIP H260Q mutant and then treated with E64d + Pepstatin A for 16 h. The lysosomes and nucleus were stained using

LysoTracker (red) and DAPI (blue). The cells were analysed via fluorescence microscopy. (E) H1299 cells were transfected with GFP-RIPK3 or GFP-RIPK1 in the absence or presence of WT or H260Q

mutant of FLAG-CHIP and then treated with E64d-Pepstatin A. The cells were stained with an anti-FLAG antibody (red). (F) H1299 cells were transfected with GFP-RIPK3 or GFP-RIPK1 in the

presence of FLAG-CHIP WT or mutant and then treated with E64d-Pepstatin A. Cells were stained with LysoTracker (red) and an anti-FLAG antibody (blue). (G) H1299 cells were transfected with

the indicated plasmids and then treated with E64d-Pepstatin A. Cells were stained with LysoTracker (red) and an anti-FLAG antibody (blue). (H) H1299 cells were transfected with HA-RIPK3

V460P in the absence and presence of FLAG-CHIP, followed by WB using anti-HA, -FLAG and-GST antibodies. (I) H1299 cells were transfected with HA-RIPK1 I539D in the absence and presence of

FLAG-CHIP, followed by WB using anti-HA, -FLAG and-GST antibodies. In D–G, a representative image is shown in the Fig. 5 and means from two independent experiments are presented in the

graph. In each independent experiment, _n_ = 100 cells were counted. All scale bars are 10 μm. SUPPLEMENTARY FIGURE 6 LYSINES OF 55 AND 363 OF RIPK3 ARE RESPONSIBLE TO CHIP DEPLETION-INDUCED

RIPK3 UP-REGULATION. (A) 293FT cells were transfected with plasmids expressing the HA-RIPK3 WT or 4KR, K55⋅363R, K89⋅501R mutants in the absence or presence of Myc-CHIP. The lysates were

immunoprecipitated using an anti-Myc antibody and then analysed via western blotting. (B) H1299 cells were transfected with HA-RIPK3 WT or K55⋅363R in the absence and presence of FLAG-CHIP,

and treated with CHX as indicated. The proteins were detected by WB using anti-HA, -FLAG, -Actin antibodies, quantified by Image J, and are shown in the graph. Half-life of RIPK3 are

presented in the graph. (C) H1299 cells were transfected with plasmids expressing GFP/RIPK3 K55⋅363R or K89⋅501R mutants in the absence or presence of FLAG-CHIP. Transfected cells were

treated with E64d-Pepstatin A and LysoTracker for lysosome staining. Stained cells were analysed using fluorescence microscopy. A representative image is shown in the upper panel and means

from two independent experiments are presented in the lower panel. In each independent experiment, _n_ = 100 cells were counted. (D) Degradation of WT and single point mutant RIPK3 by CHIP

was determined by WB in H1299 cells. (E,F) HeLa cells stably transfected with pBabe-RIPK3 WT, K55⋅363R and an empty vector were transfected with the indicated siRNAs for 48 h. Cells were

treated with 10 ng/mL hTNF_α_, 5 μg/mL cycloheximide, and 20 μM z-VAD-fmk (TCZ) for 4 h (E). Cell loss was determined by Cell Titer Glo Luminescent Cell Viability Assay Kit (E), and protein

levels were determined by western blot (F). Data are means ± S.D. _n_ = 3 independent experiments (*_P_ < 0.05, **_P_ < 0.01). Raw data from independent experiments are provided in

Supplementary Table 1. All scale bars are 10 μm. SUPPLEMENTARY FIGURE 7 THE K571⋅604⋅627R (3KR) RIPK1 ARE RESISTANT TO DEGRADATION AND LYSOSOMAL LOCALISATION BY CHIP. (A) H1299 cells were

transfected with HA-RIPK1 mutants with or without FLAG-CHIP as indicated. Degradation of each mutant by CHIP were determined by WB. (B) The amino acid sequence alignment of human and mouse

RIPK1. (C) 293FT cells were transfected with plasmids expressing the HA-RIPK1 WT or 3KR mutant in the absence or presence of Myc-CHIP. The lysates were immunoprecipitated using an anti-Myc

antibody and then analysed via western blotting. (D) 293FT cells transfected with the plasmid expression HA-RIPK1 (WT and 3KR) and His-Ub with or without FLAG-CHIP in the presence and

absence of E64d-Pepstatin A. A pull-down assay was performed using Ni2+-NTA beads. The level of RIPK1 ubiquitylation was determined by WB using an anti-RIPK1 antibody. (E) H1299 cells were

transfected with a combination of GFP/RIPK1 WT or 3KR and FLAG-CHIP and were treated with E64d/Pepstatin A and LysoTracker. The transfected cells were stained using an anti-FLAG antibody and

then analysed via fluorescence microscopy. A representative image is shown in the left panel and means from two independent experiments are presented in the right panel. In each independent

experiment, _n_ = 100 cells were counted. All scale bars are 10 μm. SUPPLEMENTARY FIGURE 8 THE ANALYSIS OF _CHIP_−/− AND CHIP−/− RIPK3−/− MICE IN SPLEEN, THYMUS AND SMALL INTESTINE. (A)

Genomic DNA was prepared via phenol-extraction. The genotypes were determined via PCR using specific primers for each gene. (B) The expected and observed frequencies and the ratio of each

genotype in the littermates from the crosses of _Chip_+/− x _Chip_+/− mice and _Chip_+/−_Ripk3_−/− x _Chip_+/−_Ripk3_−/− mice. (C) Representative spleen pictures and weight of WT (_n_ = 13;

male = 4, female = 9), _Chip_−/− (_n_ = 8; male = 3, female = 5) and DKO (_n_ = 9; male = 3, female = 6) at 4 weeks of age. Data are means ± S.D.; _n_ = number of mice analysed; *_P_ <

0.05. Raw data are presented in Supplementary Table 1. (D) Representative pictures of WT (_n_ = 3; female = 3) and DKO (_n_ = 3; female = 3) thymus and spleen at 16 weeks of age. n means

number of mice analysed. (E) Representative H&E-stained of sections of WT and DKO small intestine at 16 weeks of age (_n_ = 3 for each genotype). Scale bars, 100 μm. SUPPLEMENTARY

INFORMATION SUPPLEMENTARY INFORMATION Supplementary Information (PDF 18363 kb) SUPPLEMENTARY TABLE 1 Supplementary Information (XLSX 45 kb) RIGHTS AND PERMISSIONS Reprints and permissions

ABOUT THIS ARTICLE CITE THIS ARTICLE Seo, J., Lee, EW., Sung, H. _et al._ CHIP controls necroptosis through ubiquitylation- and lysosome-dependent degradation of RIPK3. _Nat Cell Biol_ 18,

291–302 (2016). https://doi.org/10.1038/ncb3314 Download citation * Received: 18 September 2015 * Accepted: 18 January 2016 * Published: 22 February 2016 * Issue Date: March 2016 * DOI:

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