Hierarchical phosphorylation of the translation inhibitor 4E-BP1

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.

Heterocellular OSM-OSMR signalling reprograms fibroblasts to promote pancreatic cancer growth and metastasis

“…6b ). For example, PCC–PSC–MØ-activated MAPK1/3 can phosphorylate a number of downstream phosphoproteins, such as RPS6KA1 (RSK1), in turn phosphorylating GSK-3β, and 4E-BP1, which are also regulated by AKT 49 , 50 . Notably, both SHC1-engaged SRC and gp130-activated JAK2 signals increased the phosphorylation of STAT3 [Y705] (Fig.…”

Overexpressed Smurf1 is degraded in glioblastoma cells through autophagy in a p62‐dependent manner

“…It further confirms that Smurf1 plays an important role in the PI3K/Akt signaling pathway, and autophagic degradation of Smurf1 inhibits the transduction of the PI3K/Akt signaling pathway in GB cells. Previously, the level of phosphor‐4E‐BP1 Thr37/46 did not change with the treatment of rapamycin, because Thr37 and Thr46 sites of 4E‐BP1 are not sensitive to rapamycin in glioma cells [ 39 , 40 ]. We also used mammalian target of rapamycin inhibitor rapamycin to treat the LN229 cells.…”

Dysregulation of mTOR Signaling after Brain Ischemia

“…Additionally, mTORC1 phosphorylates 4EBPs mainly at Thr 37/46 and Ser 65 . It has been proposed that hierarchical phosphorylation of 4EBPs may be crucial in the fine regulation of translational processes mediated by mTORC1 [ 60 , 61 , 62 ]. P70S6K is the other main substrate of mTORC1 related to protein synthesis.…”