Regulation of the translational inhibitor 4E-BP1 by phosphorylation

In most instances, translation is regulated at the initiation phase, when a ribosome is recruited to the 5' end of an mRNA. The eIF4E-binding proteins (4E-BPs) interdict cap-dependent translation initiation by binding to the translation factor eIF4E, and preventing recruitment of the translation machinery to mRNA. The 4E-BPs do not inhibit cap-independent translation, such as that driven by picornavirus IRESes. The 4E-BPs inhibit translation in a reversible manner. Hypophosphorylated 4E-BPs interact avidly with eIF4E, whereas 4E-BP hyperphosphorylation, elicited by stimulation of cells with hormones, cytokines, or growth factors, results in an abrogation of eIF4E binding activity. Here I show that upon infection of cells with encephalomyocarditis virus (EMCV) or poliovirus, 4E-BP1 becomes dephosphorylated, and binds more strongly to eIF4E. This dephosphorylation could play a role in the shutoff induced by EMCV. Conversely, infection with adenovirus results in hyperphosphorylation of 4E-BPI and 4E-BP2 early in infection, at a time when viral protein synthesis proceeds via a cap-dependent mechanism. I also showed that the pathway leading to 4E-BP1 phosphorylation is dependent upon the activity of PI3 kinase, Akt/PKB and FRAP/mTOR. I then analyzed the phosphorylation of individual phosphorylation sites. Thr37 and Thr46 are phosphorylated by FRAP/mTOR immunoprecipitates. In vivo, a high stoichiometry of phosphorylation on these sites is observed in conditions of serum deprivation, or following treatment of cells with PI3K or FRAP/mTOR inhibitors. Phosphorylation on these sites is, however, required for the subsequent phosphorylation of the serum-sensitive sites, which I identified as Ser65 and Thr70. Phosphorylation of 4E-BP1 can be further ordered: phosphorylation of Thr37 and Thr46 precedes that of Thr70, which occurs prior to Ser65 phosphorylation. Phosphorylation of a combination of sites is required to mediate release from eIF4E, as phosphorylation of either Th