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Detection of Necroptosis by Phospho-RIPK3 Immunohistochemical Labeling

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Programmed Necrosis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1857))

Abstract

Activation of the kinase RIPK3 (receptor interacting protein kinase 3) is a hallmark of cells dying by necroptosis. RIPK3 phosphorylates both itself and the pseudokinase MLKL (mixed lineage kinase-like) resulting in MLKL translocation to membranes and cell lysis. Antibodies recognizing RIPK3 autophosphorylation or the RIPK3-dependent phosphorylation sites on MLKL have therefore been used to monitor necroptosis induction. Here we describe immunohistochemical labeling for autophosphorylated mouse RIPK3 as a means of detecting cells undergoing necroptosis in mouse tissues.

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References

  1. Sun L, Wang H, Wang Z, He S, Chen S, Liao D, Wang L, Yan J, Liu W, Lei X, Wang X (2012) Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 148(1–2):213–227. https://doi.org/10.1016/j.cell.2011.11.031

    Article  PubMed  CAS  Google Scholar 

  2. Chen W, Zhou Z, Li L, Zhong CQ, Zheng X, Wu X, Zhang Y, Ma H, Huang D, Li W, Xia Z, Han J (2013) Diverse sequence determinants control human and mouse receptor interacting protein 3 (RIP3) and mixed lineage kinase domain-like (MLKL) interaction in necroptotic signaling. J Biol Chem 288(23):16247–16261. https://doi.org/10.1074/jbc.M112.435545

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. Murphy JM, Czabotar PE, Hildebrand JM, Lucet IS, Zhang JG, Alvarez-Diaz S, Lewis R, Lalaoui N, Metcalf D, Webb AI, Young SN, Varghese LN, Tannahill GM, Hatchell EC, Majewski IJ, Okamoto T, Dobson RC, Hilton DJ, Babon JJ, Nicola NA, Strasser A, Silke J, Alexander WS (2013) The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism. Immunity 39(3):443–453. https://doi.org/10.1016/j.immuni.2013.06.018

    Article  PubMed  CAS  Google Scholar 

  4. Rodriguez DA, Weinlich R, Brown S, Guy C, Fitzgerald P, Dillon CP, Oberst A, Quarato G, Low J, Cripps JG, Chen T, Green DR (2016) Characterization of RIPK3-mediated phosphorylation of the activation loop of MLKL during necroptosis. Cell Death Differ 23(1):76–88. https://doi.org/10.1038/cdd.2015.70

    Article  PubMed  CAS  Google Scholar 

  5. Wang H, Sun L, Su L, Rizo J, Liu L, Wang LF, Wang FS, Wang X (2014) Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3. Mol Cell 54(1):133–146. https://doi.org/10.1016/j.molcel.2014.03.003

    Article  PubMed  CAS  Google Scholar 

  6. Mulay SR, Desai J, Kumar SV, Eberhard JN, Thomasova D, Romoli S, Grigorescu M, Kulkarni OP, Popper B, Vielhauer V, Zuchtriegel G, Reichel C, Brasen JH, Romagnani P, Bilyy R, Munoz LE, Herrmann M, Liapis H, Krautwald S, Linkermann A, Anders HJ (2016) Cytotoxicity of crystals involves RIPK3-MLKL-mediated necroptosis. Nat Commun 7:10274. https://doi.org/10.1038/ncomms10274

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Newton K, Wickliffe KE, Maltzman A, Dugger DL, Strasser A, Pham VC, Lill JR, Roose-Girma M, Warming S, Solon M, Ngu H, Webster JD, Dixit VM (2016) RIPK1 inhibits ZBP1-driven necroptosis during development. Nature 540(7631):129–133. https://doi.org/10.1038/nature20559

    Article  PubMed  CAS  Google Scholar 

  8. Newton K, Dugger DL, Wickliffe KE, Kapoor N, de Almagro MC, Vucic D, Komuves L, Ferrando RE, French DM, Webster J, Roose-Girma M, Warming S, Dixit VM (2014) Activity of protein kinase RIPK3 determines whether cells die by necroptosis or apoptosis. Science 343(6177):1357–1360. https://doi.org/10.1126/science.1249361

    Article  PubMed  CAS  Google Scholar 

  9. Kaiser WJ, Upton JW, Long AB, Livingston-Rosanoff D, Daley-Bauer LP, Hakem R, Caspary T, Mocarski ES (2011) RIP3 mediates the embryonic lethality of caspase-8-deficient mice. Nature 471(7338):368–372. https://doi.org/10.1038/nature09857

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Oberst A, Dillon CP, Weinlich R, McCormick LL, Fitzgerald P, Pop C, Hakem R, Salvesen GS, Green DR (2011) Catalytic activity of the caspase-8-FLIP(L) complex inhibits RIPK3-dependent necrosis. Nature 471(7338):363–367. https://doi.org/10.1038/nature09852

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Alvarez-Diaz S, Dillon CP, Lalaoui N, Tanzer MC, Rodriguez DA, Lin A, Lebois M, Hakem R, Josefsson EC, O'Reilly LA, Silke J, Alexander WS, Green DR, Strasser A (2016) The Pseudokinase MLKL and the kinase RIPK3 have distinct roles in autoimmune disease caused by loss of death-receptor-induced apoptosis. Immunity 45(3):513–526. https://doi.org/10.1016/j.immuni.2016.07.016

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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Correspondence to Kim Newton .

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Webster, J.D., Solon, M., Haller, S., Newton, K. (2018). Detection of Necroptosis by Phospho-RIPK3 Immunohistochemical Labeling. In: Ting, A. (eds) Programmed Necrosis. Methods in Molecular Biology, vol 1857. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8754-2_15

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  • DOI: https://doi.org/10.1007/978-1-4939-8754-2_15

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8753-5

  • Online ISBN: 978-1-4939-8754-2

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