title: Regulation of guanine nucleotide exchange through phosphorylation of eukaryotic initiation factor eIF2alpha. Role of the alpha- and delta-subunits of eiF2b.
abstract: The guanine nucleotide exchange activity of eIF2B plays a key regulatory role in the translation initiation phase of protein synthesis. The activity is markedly inhibited when the substrate, i. e. eIF2, is phosphorylated on Ser51 of its alpha-subunit. Genetic studies in yeast implicate the alpha-, beta-, and delta-subunits of eIF2B in mediating the inhibition by substrate phosphorylation. However, the mechanism involved in the inhibition has not been defined biochemically. In the present study, we have coexpressed the five subunits of rat eIF2B in Sf9 cells using the baculovirus system and have purified the recombinant holoprotein to >90% homogeneity. We have also expressed and purified a four-subunit eIF2B complex lacking the alpha-subunit. Both the five- and four-subunit forms of eIF2B exhibit similar rates of guanine nucleotide exchange activity using unphosphorylated eIF2 as substrate. The five-subunit form is inhibited by preincubation with phosphorylated eIF2 (eIF2(alphaP)) and exhibits little exchange activity when eIF2(alphaP) is used as substrate. In contrast, eIF2B lacking the alpha-subunit is insensitive to inhibition by eIF2(alphaP) and is able to exchange guanine nucleotide using eIF2(alphaP) as substrate at a faster rate compared with five-subunit eIF2B. Finally, a double point mutation in the delta-subunit of eIF2B has been identified that results in insensitivity to inhibition by eIF2(alphaP) and exhibits little exchange activity when eIF2(alphaP) is used as substrate. The results provide the first direct biochemical evidence that the alpha- and delta-subunits of eIF2B are involved in mediating the effect of substrate phosphorylation.
authors.affiliation: Department of Cellular and Molecular Physiology, Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033, USA. skimball@psu.edu
authors.forename: S R
authors.initials: SR
authors.lastname: Kimball
authors.forename: J R
authors.initials: JR
authors.lastname: Fabian
authors.forename: G D
authors.initials: GD
authors.lastname: Pavitt
authors.forename: A G
authors.initials: AG
authors.lastname: Hinnebusch
authors.forename: L S
authors.initials: LS
authors.lastname: Jefferson
mesh_terms: D000818:Animals; D002460:Cell Line; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D020717:Eukaryotic Initiation Factor-2B; D020662:Guanine Nucleotide Exchange Factors; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera
chemical_list: D015852:Eukaryotic Initiation Factor-2; D020717:Eukaryotic Initiation Factor-2B; D020662:Guanine Nucleotide Exchange Factors; D011506:Proteins; D011994:Recombinant Proteins