Regulation of RIPK3- and RHIM-dependent Necroptosis by the Proteasome

J Biol Chem. 2016 Mar 11;291(11):5948-59. doi: 10.1074/jbc.M115.700997. Epub 2016 Jan 19.

Abstract

Receptor-interacting protein kinase 3 (RIPK3) is a serine/threonine kinase with essential function in necroptosis. The activity of RIPK3 is controlled by phosphorylation. Once activated, RIPK3 phosphorylates and activates the downstream effector mixed lineage kinase domain-like (MLKL) to induce necroptosis. In certain situations, RIPK3 has also been shown to promote apoptosis or cytokine expression in a necroptosis and kinase-independent manner. The ubiquitin-proteasome system is the major pathway for selective degradation of cellular proteins and thus has a critical role in many cellular processes such as cell survival and cell death. Clinically, proteasome inhibition has shown promise as an anti-cancer agent. Here we show that the proteasome inhibitors MG132 and bortezomib activate the RIPK3-MLKL necroptotic pathway in mouse fibroblasts as well as human leukemia cells. Unlike necroptosis induced by classical TNF-like cytokines, necroptosis induced by proteasome inhibitors does not require caspase inhibition. However, an intact RIP homotypic interaction motif (RHIM) is essential. Surprisingly, when recruitment of MLKL to RIPK3 is restricted, proteasome inhibitors induced RIPK3-dependent apoptosis. Proteasome inhibition led to accumulation of K48-linked ubiquitinated RIPK3, which was partially reduced when Lys-264 was mutated. Taken together, these results reveal the ubiquitin-proteasome system as a novel regulatory mechanism for RIPK3-dependent necroptosis.

Keywords: anticancer drug; apoptosis; cell death; necrosis (necrotic death); proteasome; ubiquitylation (ubiquitination).

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Antineoplastic Agents / pharmacology
  • Bortezomib / pharmacology
  • Cell Death / drug effects*
  • Cell Line
  • Cell Line, Tumor
  • Humans
  • Leupeptins / pharmacology*
  • Mice
  • Neoplasms / drug therapy
  • Neoplasms / metabolism
  • Proteasome Endopeptidase Complex / metabolism*
  • Proteasome Inhibitors / pharmacology*
  • Protein Kinases / metabolism
  • Receptor-Interacting Protein Serine-Threonine Kinases / metabolism*
  • Ubiquitination / drug effects

Substances

  • Antineoplastic Agents
  • Leupeptins
  • Proteasome Inhibitors
  • Bortezomib
  • MLKL protein, human
  • MLKL protein, mouse
  • Protein Kinases
  • RIPK3 protein, human
  • Receptor-Interacting Protein Serine-Threonine Kinases
  • Ripk3 protein, mouse
  • Proteasome Endopeptidase Complex
  • benzyloxycarbonylleucyl-leucyl-leucine aldehyde