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

The inhibition of protein synthesis that occurs upon phosphorylation o f the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) at serine 51 correlates with reduced guanine nucleotide exchange activit y of eIF-2B in vivo and inhibition of eIF-2B activity in vitro, althou gh it is not known if phosphorylation is the cause of the reduced eIF- 2B activity in vivo. To characterize the importance of eIF-2 alpha pho sphorylation in the regulation of eIF-2B activity, we studied the over expression of mutant eIF-2 alpha subunits in which serine 48 or 51 was replaced by an alanine (48A or 51A mutant). Previous studies demonstr ated that the 51A mutant was resistant to phosphorylation, whereas the 48A mutant was a substrate for phosphorylation. Additionally, express ion of either mutant partially protected Chinese hamster ovary (CHO) c ells from the inhibition of protein synthesis in response to heat shoc k 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, 1 993). In this study, we show that eIF-2B activity was inhibited in par ental CHO cell extracts upon addition of purified reticulocyte heme-re gulated inhibitor (HRI), an eIF-2 alpha kinase that phosphorylates Ser -51. Preincubation with purified HRI also reduced the eIF-2B activity in extracts from cells overexpressing wild-type eIF-2 alpha. In contra st, the eIF-2B activity was not readily inhibited in extracts from cel ls overexpressing either the eIF-2 alpha 48A or 51A mutant. In additio n, eIF-2B activity was decreased in extracts prepared from heat-shocke d cells overexpressing wild-type eIF-2 alpha, whereas the decrease in eIF-2B activity was less in heat-shocked cells overexpressing either m utant 48A or mutant 51A. While the phosphorylation at serine 51 in eIF -2 alpha impairs the eIF-2B activity, we propose that serine 48 acts t o maintain a high affinity between phosphorylated eIF-2 alpha and eIF- 2B, thereby inactivating eIF-2B activity. These findings support the h ypothesis that phosphorylation of eIF-2 alpha inhibits protein synthes is directly through reducing eIF-2B activity and emphasize the importa nce of both serine 48 and serine 51 in the interaction with eIF-2B and regulation of eIF-2B activity.