Advertisement

Necrosis pp 31-42 | Cite as

Activity Assays for Receptor-Interacting Protein Kinase 1:A Key Regulator of Necroptosis

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1004)

Abstract

Necroptosis is a novel form of regulated non-apoptotic cell death, which displays morphological features of necrosis. The kinase activity of receptor-interacting protein kinase 1 (RIP1) is a critical component in signaling for necroptosis. The development of assays to evaluate RIP1 kinase activity is important in the further development of existing and novel inhibitors of necroptosis. Here, we describe RIP1 protein expression and purification from mammalian and insect cells as well as two in vitro kinase assays to detect RIP1 kinase activity and inhibition.

Key words

RIP1 kinase Necroptosis In vitro kinase assay HTRF KinEASE assay Necrostatins 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

The authors thank Elizabeth E. Smith and J. Tres Brazell for technical help. This work was supported by a grant from the NIGMS/NIH to A.D. (GM084205).

References

  1. 1.
    Dunai Z, Bauer PI, Mihalik R (2011) Necroptosis: biochemical, physiological and pathological aspects. Pathol Oncol Res 17(4):791–800CrossRefGoogle Scholar
  2. 2.
    Galluzzi L, Vanden Berghe T, Vanlangenakker N et al (2011) Programmed necrosis from molecules to health and disease. Int Rev Cell Mol Biol 289:1–35CrossRefGoogle Scholar
  3. 3.
    Degterev A, Huang Z, Boyce M et al (2005) Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol 1:112–119CrossRefGoogle Scholar
  4. 4.
    Cho YS, Challa S, Moquin D et al (2009) Phosphorylation-driven assembly of the ­RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell 137:1112–1123CrossRefGoogle Scholar
  5. 5.
    Zhang DW, Shao J, Lin J et al (2009) RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science 325:332–336CrossRefGoogle Scholar
  6. 6.
    He S, Wang L, Miao L et al (2009) Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell 137:1100–1111CrossRefGoogle Scholar
  7. 7.
    Vandenabeele P, Galluzzi L, Vanden Berghe T et al (2010) Molecular mechanisms of necroptosis: an ordered cellular explosion. Nat Rev Mol Cell Biol 11:700–714CrossRefGoogle Scholar
  8. 8.
    Degterev A, Hitomi J, Germscheid M et al (2008) Identification of RIP1 kinase as a ­specific cellular target of necrostatins. Nat Chem Biol 4:313–321CrossRefGoogle Scholar
  9. 9.
    Rosenbaum DM, Degterev A, David J et al (2010) Necroptosis, a novel form of caspase-independent cell death, contributes to neuronal damage in a retinal ischemia-reperfusion injury model. J Neurosci Res 88:1569–1576PubMedGoogle Scholar
  10. 10.
    Linkermann A, Brasen JH, Himmerkus N et al (2012) Rip1 (Receptor-interacting protein kinase 1) mediates necroptosis and contributes to renal ischemia/reperfusion injury. Kidney Int 81:751–761CrossRefGoogle Scholar
  11. 11.
    Smith CC, Davidson SM, Lim SY et al (2007) Necrostatin: a potentially novel cardioprotective agent? Cardiovasc Drugs Ther 21:227–233CrossRefGoogle Scholar
  12. 12.
    You Z, Savitz SI, Yang J et al (2008) Necrostatin-1 reduces histopathology and improves functional outcome after controlled cortical impact in mice. J Cereb Blood Flow Metab 28:1564–1573CrossRefGoogle Scholar
  13. 13.
    Zhu S, Zhang Y, Bai G et al (2011) Necrostatin-1 ameliorates symptoms in R6/2 transgenic mouse model of Huntington’s ­disease. Cell Death Dis 2:e115CrossRefGoogle Scholar
  14. 14.
    Duprez L, Takahashi N, Van Hauwermeiren F et al (2011) RIP kinase-dependent necrosis drives lethal systemic inflammatory response syndrome. Immunity 35:908–918CrossRefGoogle Scholar
  15. 15.
    Gunther C, Martini E, Wittkopf N et al (2011) Caspase-8 regulates TNF-alpha-induced epithelial necroptosis and terminal ileitis. Nature 477:335–339CrossRefGoogle Scholar
  16. 16.
    Sun X, Yin J, Starovasnik MA et al (2002) Identification of a novel homotypic interaction motif required for the phosphorylation of receptor-interacting protein (RIP) by RIP3. J Biol Chem 277:9505–9511CrossRefGoogle Scholar
  17. 17.
    Vandenabeele P, Declercq W, Van Herreweghe F et al (2010) The role of the kinases RIP1 and RIP3 in TNF-induced necrosis. Sci Signal 3:re4CrossRefGoogle Scholar
  18. 18.
    Christofferson DE, Yuan J (2010) Necroptosis as an alternative form of programmed cell death. Curr Opin Cell Biol 22:263–268CrossRefGoogle Scholar
  19. 19.
    Maki JL, Smith EE, Teng X et al (2012) Fluorescence polarization assay for inhibitors of the kinase domain of receptor interacting protein 1. Anal Biochem 427:164–174CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  1. 1.Department of Biochemistry, School of MedicineTufts UniversityBostonUSA

Personalised recommendations

Cite protocol

Buy options