Regulation of the Unfolded Protein Response by eIF2Bδ Isoforms

Abstract

Cells respond to a variety of stresses, including unfolded proteins in the endoplasmic reticulum (ER), by phosphorylating a subunit of translation initiation factor eIF2, eIF2␣. eIF2␣ phosphorylation inactivates the eIF2B complex. The inactivation of eIF2B not only suppresses the initiation of protein translation but paradoxically up-regulates the translation and expression of transcription factor ATF-4. Both of these processes are important for the cellular response to ER stress, also termed the unfolded protein response. Here we demonstrate that cellular response resulting from eIF2␣ phosphorylation is attenuated in several cancer cell lines. The deficiency of the unfolded protein response in these cells correlates with the expression of a specific isoform of a regulatory eIF2B subunit, eIF2B␦ variant 1 (V1). Replacement of total eIF2B␦ with V1 renders cells insensitive to eIF2␣ phosphorylation; specifically, they neither up-regulate ATF-4 and ATF-4 targets nor suppress protein translation. Expression of variant 2 eIF2B␦ in ER stress response-deficient cells restores the stress response. Our data suggest that V1 does not interact with the eIF2 complex, a requisite for eIF2B inhibition by eIF2␣ phosphorylation. Together, these data delineate a novel physiological mechanism to regulate the ER stress response with a large potential impact on a variety of diseases that result in ER stress.

Phosphorylation of eIF2α triggered by mTORC1 inhibition and PP6C activation is required for autophagy and is aberrant in PP6C-mutated melanoma

“…Rapamycin treatment also led to a time-dependent increase in eIF2α phosphorylation in primary mouse and human fibroblasts (Fig 1B). As we and others have previously noted, the extent and kinetics of tunicamycin-induced eIF2α phosphorylation varied amongst cell lines (19), as did rapamycin-induced eIF2α phosphorylation. However, in most cases the extent of rapamycin treated was similar to the extent of eIF2α phosphorylation noted with ER stress.…”

Fluorescence Amplification Method for Forward Genetic Discovery of Factors in Human mRNA Degradation

“…Of these top 24 highly-enriched sgRNAs, 13 target well-established NMD factors (Kurosaki and Maquat, 2016): UPF1 (2 out of 4 sgRNAs present in the library [Table 1, rows 1 and 11]), SMG7 (2 out of 4 sgRNAs [rows 2 and 3]), SMG6 (3 out of 4 sgRNAs [rows 5, 7, and 14]), SMG5 (3 out of 6 sgRNAs [rows 6, 8, and 18]), UPF2 (1 out of 4 sgRNAs [row 13]), and a known human NMD regulator EIF2B4 (Gardner, 2008; Martin et al, 2010) (2 out of 5 sgRNAs [rows 10 and 23]) (Table 1). We therefore demonstrate successful unbiased identification of NMD factors and regulators in a forward genetic screen performed directly in human cells.…”

“…As an example of successful forward genetic screening for NMD factors in human cells, we report identification of (i) known major NMD components (Kurosaki and Maquat, 2016): UPF1, UPF2, SMG5, SMG6, and SMG7; (ii) a known NMD regulator EIF2B4 (Gardner, 2008; Martin et al, 2010); as well as (iii) 11 candidate genes. Our method of fluorescence amplification coupled with sequential rounds of enrichment for functional sgRNAs provides a platform for forward genetic discovery of key components and modulators of the human mRNA degradation machinery.…”

Identification and Characterization of Small Molecules That Inhibit Nonsense-Mediated RNA Decay and Suppress Nonsense p53 Mutations

“…U2OS, Hela, 293, BJhTert, PBMC, MDA231, DU145, T98G and HCT116 cells were grown as previously described (29–30). N417 cells were grown in RPMI containing 10% FBS.…”

“…Immunoblots were prepared as previously described (29) and membranes were probed with antibodies against p53 (Abcam Inc, ab26), SMG1 (Santa Cruz Biotechnology, Inc. 6-RE13), UPF1/Rent1 (SC H-300), SMG7 (A302-170A, Bethyl Laboratories, Inc). Immunoprecipitations were performed as described (10).…”