CHROMATOGRAPHIC RESOLUTION OF IN-VIVO PHOSPHORYLATED AND NONPHOSPHORYLATED EUKARYOTIC TRANSLATION INITIATION-FACTOR EIF-4E - INCREASED CAP AFFINITY OF THE PHOSPHORYLATED FORM

Eukaryotic translation initiation factor eIF-4E plays a central role i n the recognition of the 7-methylguanosine-containing cap structure of mRNA and the formation of initiation complexes during protein synthes is. eIF-4E exists in both phosphorylated and nonphosphorylated forms, and the primary site of phosphorylation has been identified. Previous studies have suggested that eIF-4E phosphorylation facilitates its par ticipation in protein synthesis. However, the biochemical basis for th e functional difference between the two forms of eIF-4E is unknown. To address this directly, we have developed a method for the separation of phosphorylated and nonphosphorylated eIF-4E from rabbit reticulocyt es by chromatography on rRNA-Sepharose. Using the resultant purified f orms, we have studied the protein's interaction with the cap analogs m (7)GTP and m(7)GpppG and with the cap of globin mRNA by fluorescence q uenching of tryptophan residues. It was found that phosphorylated eIF- 4E had 3- to 4-fold greater affinity for cap analogs and mRNA than non phosphorylated eIF-4E. The equilibrium binding constants (x 10(5), exp ressed as M(-1)) for the interaction of phosphorylated eIF-4E with m(7 )GTP, m(7)GpppG, and globin mRNA were 20.0 +/- 0.1, 16.4 +/- 0.1, and 31.0 +/- 0.1, respectively, whereas those for the nonphosphorylated fo rm were 5.5 +/- 0.4, 4.3 +/- 0.4, and 10.0 +/- 0.1, respectively. Trea tment with potato acid phosphatase converted the phosphorylated form t o the nonphosphorylated form and decreased the binding constant for m( 7)GTP by a factor of 3. The increased affinity for mRNA caps may accou nt for the in vivo and in vitro correlations between eIF-4E phosphoryl ation and accelerated protein synthesis and cell growth.