J. Biol. Chem., Vol. 262, Issue 30, 14538-14543, 10, 1987

Phosphorylation of eukaryotic initiation factor 2 during physiological stresses which affect protein synthesis

KA Scorsone, R Panniers, AG Rowlands and EC Henshaw
Cancer Center, University of Rochester, New York 14642.

Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2) is a major mechanism regulating protein synthesis in rabbit reticulocytes. To determine whether phosphorylation of eIF-2 alpha is a likely regulatory mechanism in the Ehrlich cell, we have measured the percent of cellular eIF-2 alpha which is phosphorylated in cells exposed to heat shock, 2-deoxyglucose, or amino acid deprivation, conditions which rapidly decrease the concentration of 40 S initiation complexes and inhibit protein synthesis. eIF-2 alpha and eIf-2 alpha (P) were separated by isoelectric focusing and were detected by immunoblotting with a monoclonal antibody we developed for this purpose. Under the above three inhibitory conditions, phosphorylation of eIF-2 alpha increased rapidly, and this increase correlated in time with the rapid inhibition of protein synthesis. In heat-shocked cells which were returned to 37 degrees C, both phosphorylation and protein synthesis remained unchanged for 10 min and then returned toward control values slowly and in parallel. The close temporal correspondence between changes in protein synthesis and phosphorylation supports an important regulatory role for phosphorylation in protein synthesis. An increase of 25-35 percentage points, to 50-60% phosphorylation from control levels of 20-30% phosphorylation, correlated with an 80-100% inhibition of protein synthesis. This steep curve of inhibition is consistent with a mechanism in which eIF-2 alpha (P) saturates and inhibits the guanine-nucleotide exchange factor.
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