PHOSPHORYLATION OF EIF-4E ON SERINE-209 BY PROTEIN-KINASE-C IS INHIBITED BY THE TRANSLATIONAL REPRESSORS, 4E-BINDING PROTEINS

Translation initiation in eukaryotes is facilitated by the mRNA 5' cap structure (m(7)GpppX, where X is any nucleotide) that binds the multi subunit initiation factor eIF4F through one of its subunits, eIF4E. eI F4E is a phosphoprotein whose phosphorylation state positively correla tes with cell growth. Protein kinase C phosphorylates eIF4E in vitro, and possibly in vivo. Using recombinant eIF4E incubated in vitro with purified protein kinase C and analyzed by solid-phase phosphopeptide s equencing in combination with high performance liquid chromatography c oupled to mass spectrometry, we demonstrated that the third amino acid of the peptide SGSTTK (Ser(209)) is the major site of phosphorylation . This finding is consistent with the newly assigned in vivo phosphory lation site of eIF4E (Joshi, B., Cai, A L., Keiper, B. D., Minich, W. B., Mendez, R., Beach, C. M., Stepinski, J., Stolarski, R., Darzynkiew icz, E., and Rhoads, R. E. (1995) J. Biol. Chem. 270, 14597-14603). A S209A mutation resulted in dramatically reduced phosphorylation, both in vitro and in vivo. Furthermore, the mutant protein was phosphorylat ed on threonine (most probably threonine 210) in vivo. Here we show th at in the presence of the recently characterized translational repress ors 4E-BP1 or 4E-BP2, phosphorylation of eIF4E by protein kinase C is strongly reduced. This suggests a two-step model for the phosphorylati on (and activation) of eIF4E by growth factors and hormones: first, di ssociation of eIF4E from 4E-BPs, followed by eIF4E phosphorylation.