Our group has shown that ultraviolet (UV) irradiation of fission yeast cells in G1 phase induces a delayed entry into S phase. More recently, we found that the cell cycle delay is accompanied by a general depression of translation and phosphorylation of the eukaryotic translation initiation factor eIF2α. Both the cell cycle delay and downregulation of translation are absolutely dependent on the Gcn2 kinase, which is known to phosphorylate eIF2α at Ser52 and thereby reduce global translation. However, the checkpoint is only partially dependent on eIF2α Ser52 phosphorylation, indicating that there must be more Gcn2 targets in fission yeast.
One attractive hypothesis is that other sites on eIF2α might be phosphorylated by Gcn2. In this work, intention was to investigate additional phosphorylation sites on eIF2α in response to UV irradiation. Using bioinformatics tools and comparisons with published observations on eIF2α in S. cerevisiae, wheat and brine shrimp, a number of potential phosphorylation sites were predicted. Indeed, some of these sites were shown to be phosphorylated in fission yeast in the presence of thiabendazole in a recent paper.
To confirm the predicted phosphorylation sites, eIF2α was tagged by an epitope tag. Then protein extracts were prepared from cells treated with UV irradiation as well as from untreated control cells and analyzed MS and 2D-Western. Preliminary results suggest that eIF2α is indeed phosphorylated on other sites, in addition to Ser52, after UV-irradiation. However, further studies are needed to fully understand eIF2α’s involvement in the downregulation of translation and in the G1/S delay after UV irradiation, as well as the exact role of Gcn2 in the observed modifications.