PHOSPHORYLATION OF EUKARYOTIC PROTEIN-SYNTHESIS INITIATION-FACTOR 4E BY INSULIN-STIMULATED PROTAMINE KINASE

Insulin-stimulated protamine kinase (cPK) and protein kinase C (PKC) phosphorylated eukaryotic protein synthesis initiation factor 4E (eIF-4E) on serine and threonine residues located on an identical tryptic fragment as judged by two-dimensional phosphopeptide mapping. With cPK and PKC, the apparent K-m for eIF-4E was about 1.2 and 50 mu M, respectively. Relative to recombinant human eIF-4E, cPK exhibited about 100% and less than or equal to 5% activity with eIF-4E(S209A), and eIF-4E(T210A) respectively, and eIF-4E(S209A) was phosphorylated exclusively on threonines. Bovine kidney eIF 4E enhanced up to 1.8-fold globin synthesis in m(7)GTP-Sepharose-treated reticulocyte lysates. In contrast, following incubation with cPK, these eIF-4E preparations stimulated globin synthesis up to 6-fold. Compared to the dephosphorylation of the cPK-modified serine on elF-4E, reticulocyte lysates and highly purified protein phosphatase 2A exhibited marked preference for the cPK-modified threonine. The results indicate that cPK phosphorylates eIF-4E on Ser(209) and Thr(210), that the hydroxyl group or phosphorylation of Thr(210) is necessary for cPK to act on Ser(209), and that Ser(209) phosphorylation activates reticulocyte globin synthesis. The results suggest that cPK could contribute to the insulin-stimulated phosphorylation of eIF-4E, but that protein phosphatase 2A may confer the site specificity of this response.