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

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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).

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