The role of CReP in reversing eIF2-alpha phosphorylation after ER stress

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER stress and leads to the unfolded protein response (UPR). During the unfolded protein response, reversible phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2- α) occurs. This phosphorylation event is necessary in the UPR to decrease rates of protein synthesis by regulating mRNA-specific translation. The reversal of eIF2-α phosphorylation occurs through cellular phosphatase complexes that contain a protein phosphatase 1 catalytic subunit (PP1) and substrate-specific regulatory subunit. Substratespecific regulatory subunits identified in mammals include PPP1R15A (GADD34), a growth arrest and DNA damage-inducible protein, and PPP1R15B (constitutive repressor of eIF2-α phosphorylation, CReP) which is constitutively expressed in the cell. Previous structure-function studies identified two regions that were required for PP1 binding in the Cterminus of GADD34. In addition to PP1 binding, N-terminal residue deletion studies of green fluorescent protein (GFP)-GADD34 showed that subcellular targeting of GADD34 to the ER, and consequently the alpha isoform of PP1, were necessary for reversal of eIF2-α phosphorylation. While structural homology is not present between the N-terminus of mammalian GADD34 and CReP, our imaging studies of human GFP-CReP (GFP-hCReP) Nterminal deletion constructs suggest that functional homology exists. We used confocal microscopy to study GFP-hCReP transfected HELA cells expressing full-length and various N-terminal deletion constructs of GFP-hCReP. Full-length GFP-hCReP showed perinuclear sublocalization patterns suggesting ER localization, homologous to subcellular localization patterns seen in full-length GADD34. GFP-hCReP Nterminal deletion constructs showed loss of subcellular perinuclear localization. As deletion lengths increased, GFP-hCReP showed increased diffuse cytoplasmic presence and eventually appeared throughout the nucleus of the HELA cells. Future studies analyzing eIF2-α phosphorylation will be done in the presence of GFP-hCReP N-terminal deletion constructs that lack ER targeting domains in order to determine if subcellular localization is required for their function