Coordinated ubiquitination and phosphorylation of RIP1 regulates necroptotic cell death

Cell Death Differ. 2017 Jan;24(1):26-37. doi: 10.1038/cdd.2016.78. Epub 2016 Aug 12.


Proper regulation of cell death signaling is crucial for the maintenance of homeostasis and prevention of disease. A caspase-independent regulated form of cell death called necroptosis is rapidly emerging as an important mediator of a number of human pathologies including inflammatory bowel disease and ischemia-reperfusion organ injury. Activation of necroptotic signaling through TNF signaling or organ injury leads to the activation of kinases receptor-interacting protein kinases 1 and 3 (RIP1 and RIP3) and culminates in inflammatory cell death. We found that, in addition to phosphorylation, necroptotic cell death is regulated by ubiquitination of RIP1 in the necrosome. Necroptotic RIP1 ubiquitination requires RIP1 kinase activity, but not necroptotic mediators RIP3 and MLKL (mixed lineage kinase-like). Using immunoaffinity enrichment and mass spectrometry, we profiled numerous ubiquitination events on RIP1 that are triggered during necroptotic signaling. Mutation of a necroptosis-related ubiquitination site on RIP1 reduced necroptotic cell death and RIP1 ubiquitination and phosphorylation, and disrupted the assembly of RIP1 and RIP3 in the necrosome, suggesting that necroptotic RIP1 ubiquitination is important for maintaining RIP1 kinase activity in the necrosome complex. We also observed RIP1 ubiquitination in injured kidneys consistent with a physiological role of RIP1 ubiquitination in ischemia-reperfusion disease. Taken together, these data reveal that coordinated and interdependent RIP1 phosphorylation and ubiquitination within the necroptotic complex regulate necroptotic signaling and cell death.

MeSH terms

  • Animals
  • Apoptosis* / drug effects
  • CRISPR-Cas Systems / genetics
  • Cell Line
  • Creatinine / blood
  • HEK293 Cells
  • HT29 Cells
  • Humans
  • Kidney Diseases / etiology
  • Kidney Diseases / metabolism
  • Mice
  • Nuclear Pore Complex Proteins / deficiency
  • Nuclear Pore Complex Proteins / genetics
  • Nuclear Pore Complex Proteins / metabolism*
  • Oligopeptides / pharmacology
  • Phosphorylation / drug effects
  • Protein Kinases / metabolism
  • Protein Structure, Tertiary
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*
  • Receptor-Interacting Protein Serine-Threonine Kinases / metabolism
  • Reperfusion Injury / complications
  • Reperfusion Injury / pathology
  • Signal Transduction / drug effects
  • Tumor Necrosis Factor-alpha / pharmacology
  • Ubiquitination / drug effects


  • AGFG1 protein, human
  • BV6 peptide
  • Nuclear Pore Complex Proteins
  • Oligopeptides
  • RNA-Binding Proteins
  • Tumor Necrosis Factor-alpha
  • benzyloxycarbonyl-valyl-alanyl-aspartic acid
  • Creatinine
  • MLKL protein, human
  • Protein Kinases
  • RIPK3 protein, human
  • Receptor-Interacting Protein Serine-Threonine Kinases