Expression of mutant eukaryotic initiation factor 2 alpha subunit (eIF-2 alpha) reduces inhibition of guanine nucleotide exchange activity of eIF-2B mediated by eIF-2 alpha phosphorylation.

Abstract

The inhibition of protein synthesis that occurs upon phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2a) at serine 51 correlates with reduced guanine nucleotide exchange activity of eIF-2B in vivo and inhibition of eIF-2B activity in vitro, although it is not known if phosphorylation is the cause of the reduced eIF-2B activity in vivo. To characterize the importance of eIF-2a phosphorylation in the regulation of eIF-2B activity, we studied the overexpression of mutant eIF-2at subunits in which serine 48 or 51 was replaced by an alanine (48A or 51A mutant). Previous studies demonstrated that the 51A mutant was resistant to phosphorylation, whereas the 48A mutant was a substrate for phosphorylation. Additionally, expression of either mutant partially protected Chinese hamster ovary (CHO) cells from the inhibition of protein synthesis in response to heat shock treatment (P. Murtha-Riel, M. V. Davies, J. B. Scherer, S. Y. Choi, J. W. B. Hershey, and R. J. Kaufman, J. Biol. Chem. 268:12946-12951, 1993). In this study, we show that eIF-2B activity was inhibited in parental CHO cell extracts upon addition of purified reticulocyte heme-regulated inhibitor (HRI), an eIF-2ao kinase that phosphorylates Ser-51. Preincubation with purified HRI also reduced the eIF-2B activity in extracts from cells overexpressing wild-type eIF-2ot. In contrast, the eIF-2B activity was not readily inhibited in extracts from cells overexpressing either the eIF-2ot 48A or 51A mutant. In addition, eIF-2B activity was decreased in extracts prepared from heat-shocked cells overexpressing wild-type eIF-2a, whereas the decrease in eIF-2B activity was less in heat-shocked cells overexpressing either mutant 48A or mutant 51A. While the phosphorylation at serine 51 in eIF-2oa impairs the eIF-2B activity, we propose that serine 48 acts to maintain a high affinity between phosphorylated eIF-2a and eIF-2B, thereby inactivating eIF-2B activity. These findings support the hypothesis that phosphorylation of eIF-2ot inhibits protein synthesis directly through reducing eIF-2B activity and emphasize the importance of both serine 48 and serine 51 in the interaction with eIF-2B and regulation of eIF-2B activity.In the initiation step of protein synthesis in eukaryotic cells, the joining of the 48S preinitiation complex with the 60S subunit to form the 80S initiation complex is accompanied by the hydrolysis of GTP and the formation of a binary complex of eukaryotic initiation factor 2 (eIF-2) and GDP (eIF-2 GDP) (reviewed in references 15 and 29). The exchange of GTP for GDP in the eIF-2 * GDP complex is catalyzed by a multimeric protein factor called eIF-2B (previously called reversing factor or GEF) (1,22,26,33,41). The guanine nucleotide exchange is a prerequisite for eIF-2 to enter another initiation cycle and bind the initiator tRNA, MettRNAi. eIF-2 is a heterotrimer composed of the a, P, and y subunits. The activity of eIF-2 is an important control point in the regulation of polypeptide chain initiation and is regulated at least i...

An efficient in vitro translation system from mammalian cells lacking the translational inhibition caused by eIF2 phosphorylation

“…This mutant eIF2a is not a substrate for the eIF2 kinases in vivo and in vitro (Ramaiah et al 1994;Scheuner et al 2001). We show that extracts from A/A cells have a translational efficiency that is 30-fold greater than that of wild-type (S/S) extracts.…”

Phosphorylation of Eukaryotic Translation Initiation Factor 2 Mediates Apoptosis in Response to Activation of the Double-stranded RNA-dependent Protein Kinase

“…Cells were co-transfected with the pEDmtx r VA Ϫ empty vector and pSV 2 Neo, selected for G418 resistance, pooled, and used as a control. NIH3T3 cells transfected and selected for wild-type and S51A mutant eIF-2␣ expression were previously described (24). Cells were cultured at 37°C with 10% CO 2 in Dulbecco's modified essential medium containing 10% fetal bovine serum (FBS) and penicillin/streptomycin in 100-mm tissue culture dishes (for cell cycle and DNA fragmentation analysis) or on coverslips (for fluorescence microscopy).…”

“…The expression of wild-type and S51A mutant eIF-2␣ in these NIH3T3 cells was previously characterized (24). Immunoprecipitation of eIF-2␣ from [ 32 P]phosphate-labeled NIH3T3 cells that overexpress wild-type eIF-2␣ detected [ 32 P]phosphate incorporation into eIF-2␣ (Fig.…”

Defects in Translational Regulation Mediated by the α Subunit of Eukaryotic Initiation Factor 2 Inhibit Antiviral Activity and Facilitate the Malignant Transformation of Human Fibroblasts

“…To analyze the specific effects of eIF2␣ phosphorylation on the cell in the absence of other eIF2-independent stress signaling cascades, we established novel 3T3 L1 cell lines that inducibly express a FLAG-tagged phosphomimetic variant of eIF2␣, referred to as eIF2␣-S51D, or the phosphorylation-incompetent variant eIF2␣-S51A (61). Clonal 3T3 L1 cell populations were isolated which express the exogenous forms of eIF2␣ under the control of a DOX-repressible promoter (28).…”