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

Insulin-stimulated protamine kinase (cPK) and protein kinase C (PKC) p hosphorylated eukaryotic protein synthesis initiation factor 4E (eIF-4 E) on serine and threonine residues located on an identical tryptic fr agment as judged by two-dimensional phosphopeptide mapping. With cPK a nd PKC, the apparent K-m for eIF-4E was about 1.2 and 50 mu M, respect ively. 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(T 210A) respectively, and eIF-4E(S209A) was phosphorylated exclusively o n threonines. Bovine kidney eIF 4E enhanced up to 1.8-fold globin synt hesis in m(7)GTP-Sepharose-treated reticulocyte lysates. In contrast, following incubation with cPK, these eIF-4E preparations stimulated gl obin synthesis up to 6-fold. Compared to the dephosphorylation of the cPK-modified serine on elF-4E, reticulocyte lysates and highly purifie d protein phosphatase 2A exhibited marked preference for the cPK-modif ied threonine. The results indicate that cPK phosphorylates eIF-4E on Ser(209) and Thr(210), that the hydroxyl group or phosphorylation of T hr(210) is necessary for cPK to act on Ser(209), and that Ser(209) pho sphorylation activates reticulocyte globin synthesis. The results sugg est that cPK could contribute to the insulin-stimulated phosphorylatio n of eIF-4E, but that protein phosphatase 2A may confer the site speci ficity of this response.