Hierarchical phosphorylation of the translation inhibitor 4E-BP1

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 translation initiation by binding to the translation fa ctor eIF4E, and preventing recruitment of the translation machinery to mRNA . The 4E-BPs inhibit translation in a reversible manner. Hypophosphorylated 4E-BPs interact avidly with eIF4E, whereas 4E-BP hyperphosphorylation, eli cited by stimulation of cells with hormones, cytokines, or growth factors, results in an abrogation of eIF4E-binding activity. We reported previously that phosphorylation of 4E-BP1 on Thr 37 and Thr 46 is relatively insensiti ve to serum deprivation and rapamycin treatment, and that phosphorylation o f these residues is required for the subsequent phosphorylation of a set of unidentified serum-responsive sites. Here, using mass spectrometry, we ide ntify the serum-responsive, rapamycin-sensitive sites as Ser 65 and Thr 70. Utilizing a novel combination of two-dimensional isoelectric focusing/SDS- PAGE and Western blotting with phosphospecific antibodies, we also establis h the order of 4E-BP1 phosphorylation in vivo; phosphorylation of Thr 37/Th r 46 is followed by Thr 770 phosphorylation, and Ser 65 is phosphorylated l ast. Finally, we show that phosphorylation of Set 65 and Thr 70 alone is in sufficient to block binding to eIF4E, indicating that a combination of phos phorylation events is necessary to dissociate 4E-BP1 from eIF4E.