Hierarchical phosphorylation of the translation inhibitor 4E-BP1

TLDR

Translation initiation is regulated by eIF4E‑binding proteins (4E‑BPs) that reversibly inhibit eIF4E binding; hypophosphorylated 4E‑BPs tightly bind eIF4E, whereas hyperphosphorylation induced by hormones, cytokines, or growth factors releases eIF4E. The study aimed to identify the serum‑responsive, rapamycin‑sensitive phosphorylation sites on 4E‑BP1. Using mass spectrometry combined with two‑dimensional isoelectric focusing/SDS‑PAGE and phosphospecific Western blotting, the authors mapped the phosphorylation sequence of 4E‑BP1 as Thr37/Thr46 → Thr70 → Ser65. They found that phosphorylation of Thr37/Thr46 is required for subsequent phosphorylation of Ser65 and Thr70, and that Ser65/Thr70 phosphorylation alone does not prevent eIF4E binding, indicating that multiple phosphorylation events are necessary for dissociation.

Abstract

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 factor 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, elicited 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 insensitive to serum deprivation and rapamycin treatment, and that phosphorylation of these residues is required for the subsequent phosphorylation of a set of unidentified serum-responsive sites. Here, using mass spectrometry, we identify 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 establish the order of 4E-BP1 phosphorylation in vivo; phosphorylation of Thr 37/Thr 46 is followed by Thr 70 phosphorylation, and Ser 65 is phosphorylated last. Finally, we show that phosphorylation of Ser 65 and Thr 70 alone is insufficient to block binding to eIF4E, indicating that a combination of phosphorylation events is necessary to dissociate 4E-BP1 from eIF4E.