Double-stranded RNA-dependent protein kinase phosphorylation of the alpha-subunit of eukaryotic translation initiation factor 2 mediates apoptosis

Area of Science:

• Cellular signaling
• Molecular biology
• Stress response pathways

Background:

• Cell stress signaling pathways involve complex molecular events.
• Phosphorylation of translation initiation factor 2 alpha (eIF2alpha) at Ser51 inhibits translation upon stress kinase activation.
• The precise role of eIF2alpha phosphorylation in determining cell fate remains to be fully elucidated.

Purpose of the Study:

• To investigate the hypothesis that eIF2alpha phosphorylation is a central death stimulus in eukaryotic cells.
• To determine if eIF2alpha phosphorylation is sufficient to induce apoptosis.
• To explore the divergent roles of different eIF2alpha kinases in cell fate determination.

Main Methods:

• Gene targeting in mouse embryo fibroblasts to create homozygous eIF2alpha mutants (Ser51 to Ala).
• Exposure of mutant and wild-type cells to apoptotic stimuli (dsRNA, TNF-alpha, serum deprivation).
• Analysis of caspase 3 activation and eIF2alpha phosphorylation in response to various treatments.

Main Results:

• Homozygous eIF2alpha mutant cells were resistant to apoptosis induced by dsRNA, TNF-alpha, and serum deprivation.
• TNF-alpha treatment induced eIF2alpha phosphorylation and caspase 3 activation via the PKR kinase.
• Expression of a phospho-mimetic eIF2alpha mutant (Ser51 to Asp) activated caspase 3, confirming sufficiency for apoptosis.
• PKR-mediated eIF2alpha phosphorylation promoted apoptosis, while PERK-mediated phosphorylation led to survival.

Conclusions:

• eIF2alpha phosphorylation at Ser51 is a critical signaling event that can act as a fundamental death signal.
• The ultimate outcome (death or survival) is dependent on the specific kinase involved and the overall cellular signaling context.
• Divergent cell fates, apoptosis or survival, can be mediated through phosphorylation at the same site within eIF2alpha.