Mechanism of translational control by partial phosphorylation of the alpha subunit of eukaryotic initiation factor 2.

Abstract

Catalysis of ternary complex formation by the GDP exchange factor (GEF), in the presence of Mg2+, is blocked by phosphorylation of the .alpha. subunit of the eukaryotic initiation factor 2 (eIF-2). This phosphorylation may interfere with the interaction between eIF-2 and GEF (then termed ESP). If so, inhibition should be related to the extent of phosphorylation. However, work in other laboratories indicated that in fully inhibited, heme-deficient lysates only 20-40% of the eIF-2 is phosphorylated. To understand the nature of the molecular lesion in eIF-2-.alpha. phosphorylation a system of pure components was used in which the rate of exchange of eIF-2-bound [3H]GDP with unlabeled GDP (via the reaction eIF-2.cntdot.GDP + GEF .dblarw. eIF-2.cntdot.GEF + GDP) was measured by using mixtures of eIF-2(.alpha.P).cntdot.[3H]GDP and eIF-2.cntdot.[3H]GDP in different proportions at constant concentration of eIF-2.cntdot.GEF. If, for example, the ratio of eIF-2.cntdot.GEF to total (phosphorylated and unphosphorylated) eIF-2.cntdot.[3H]GDP was 0.25, the exchange was maximally inhibited when the proportion of eIF-2(.alpha.P).cntdot.[3H]GDP in the mixture reached 25%. This suggests that the reaction stops because the available GEF is trapped in an inactive complex with eIF-2(.alpha.P). In the absence of free GEF, eIF-2 would not be able to recycle and initiation would come to a standstill when the available eIF-2 is tied up as eIF-2.cntdot.GDP. The trapping of GEF by eIF-2(.alpha.P) is strongly supported by the following observation. Incubation of eIF-2.cntdot.GEF with excess [3H]GDP leads to the formation of eIF-2.cntdot.[3H]GDP and free GEF and, if eIF-2(.alpha.32P).cntdot.GDP is also present, all of the GEF is converted to eIF-2(.alpha.32P).cntdot.GEF. Whereas the equilibrium of the reaction eIF-2.cntdot.GEF + GDP .dblarw. eIF-2.cntdot.GDP + GEF favors the formation of free GEF, the equlibrium of the reaction eIF-2(.alpha.P).cntdot.GDP + GEF .dblarw. eIF-2(.alpha.P).cntdot.GEF + GDP is in favor of the association of GEF to eIF-2(.alpha.P).