Phosphorylation of the alpha-subunit of the eukaryotic initiation factor-2 (eIF2alpha) reduces protein synthesis and enhances apoptosis in response to proteasome inhibition

J Biol Chem. 2005 Apr 8;280(14):14189-202. doi: 10.1074/jbc.M413660200. Epub 2005 Jan 31.

Abstract

Protein ubiquitination and subsequent degradation by the proteasome are important mechanisms regulating cell cycle, growth and differentiation, and apoptosis. Recent studies in cancer therapy suggest that drugs that disrupt the ubiquitin/proteasome pathway induce apoptosis and sensitize malignant cells and tumors to conventional chemotherapy. In this study we addressed the role of phosphorylation of the alpha-subunit eukaryotic initiation factor-2 (eIF2), and its attendant regulation of gene expression, in the cellular stress response to proteasome inhibition. Phosphorylation of eIF2alpha in mouse embryo fibroblast (MEF) cells subjected to proteasome inhibition leads to a significant reduction in protein synthesis, concomitant with induced expression of the bZIP transcription regulator, ATF4, and its target gene CHOP/GADD153. The primary eIF2alpha kinase activated by exposure of these fibroblast cells to proteasome inhibition is GCN2 (EIF2AK4), which has a central role in the recognition of cytoplasmic stress signals. Endoplasmic reticulum (ER) stress is not effectively induced in MEF cells subjected to proteasome inhibition, with minimal activation of the ER stress sensory proteins, eIF2alpha kinase PEK (PERK/EIF2AK3), IRE1 protein kinase and the transcription regulator ATF6 following up to 6 h of proteasome inhibitor treatment. Loss of eIF2alpha phosphorylation thwarts caspase activation and delays apoptosis. Central to this pro-apoptotic function of eIF2alpha kinases during proteasome inhibition is the transcriptional regulator CHOP, as deletion of CHOP in MEF cells impedes apoptosis. We conclude that eIF2alpha kinases are integral to cellular stress pathways induced by proteasome inhibitors, and may be central to the efficacy of anticancer drugs that target the ubiquitin/proteasome pathway.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Activating Transcription Factor 4
  • Animals
  • Antineoplastic Agents / metabolism
  • Apoptosis / physiology*
  • CCAAT-Enhancer-Binding Proteins / genetics
  • CCAAT-Enhancer-Binding Proteins / metabolism
  • Cells, Cultured
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Eukaryotic Initiation Factor-2 / genetics
  • Eukaryotic Initiation Factor-2 / metabolism*
  • Fibroblasts / cytology
  • Fibroblasts / metabolism
  • Gene Expression Regulation
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Mice
  • Mice, Knockout
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Phosphorylation
  • Proteasome Endopeptidase Complex / metabolism
  • Proteasome Inhibitors*
  • Protein Biosynthesis*
  • Protein Kinases / genetics
  • Protein Kinases / metabolism
  • Protein Serine-Threonine Kinases / genetics
  • Protein Serine-Threonine Kinases / metabolism
  • Protein Subunits / genetics
  • Protein Subunits / metabolism*
  • RNA Splicing
  • Regulatory Factor X Transcription Factors
  • Trans-Activators / genetics
  • Trans-Activators / metabolism
  • Transcription Factor CHOP
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Ubiquitin / metabolism

Substances

  • Antineoplastic Agents
  • Atf4 protein, mouse
  • CCAAT-Enhancer-Binding Proteins
  • DNA-Binding Proteins
  • Ddit3 protein, mouse
  • Eukaryotic Initiation Factor-2
  • Membrane Proteins
  • Nuclear Proteins
  • Proteasome Inhibitors
  • Protein Subunits
  • Regulatory Factor X Transcription Factors
  • Trans-Activators
  • Transcription Factors
  • Ubiquitin
  • Activating Transcription Factor 4
  • Transcription Factor CHOP
  • Protein Kinases
  • Ern2 protein, mouse
  • Eif2ak4 protein, mouse
  • Protein Serine-Threonine Kinases
  • Proteasome Endopeptidase Complex