Phosphorylation of mammalian translation initiation factor 5 (elF5) in vitro and in vivo

Eukaryotic translation initiation factor 5 (elF5) interacts with the 40S initiation complex isolated a protein kinase from rabbit reticulocyte lysates on the basis of its ability to phosphorylate purified bacterially expressed recombinant rat elF5. Physical, biochemical and antigenic properties of this kinase identify it as casein kinase II (CK II). Mass spectrometric analysis of maximally in vitro phosphorylated elF5 localized the major phosphorylation sites at Ser-387 and Ser-388 near the C-terminus of elF5. These serine residues are embedded within a cluster of acidic amino acid residues and account for nearly 90% of the total in vitro elF5 phosphorylation. A minor phosphorylation site at Ser-174 was also observed. Alanine substitution mutagenesis at Ser-387 and Ser-388 of elF5 abolishes phosphorylation by the purified kinase as well as by crude reticulocyte lysates. The same mutations also abolish phosphorylation of elF5 when transfected into mammalian cells suggesting that CK II phosphorylates elF5 at these two serine residues in vivo as well.