S6K1 phosphorylates eIF4B in vitro on a phosphopeptide comigrating with the endogenous serum-stimulated phosphopeptide 2. (A) Recombinant eIF4B was labeled in vitro with S6K1; or (B) immunoprecipitated from ³²P-labeled 293 cells, and subjected to phosphopeptide mapping. (C) The in vitro- and in vivo-labeled samples were then mixed, and subjected to phosphopeptide mapping.

Ser422 is phosphorylated in vivo, and is phosphorylated in vitro by S6K1. Recombinant (A) wt and (B) Ser422Thr eIF4B proteins were phosphorylated in vitro with S6K1, and then subjected to phosphopeptide mapping. Similarly, Flag-tagged eIF4B (C) wt, (D) Ser406Ala and (E) Ser422Thr proteins were expressed in 293T cells, ³²P-labeled in vivo and subjected to phosphopeptide mapping.

S6K1 mutants confer rapamycin resistance to eIF4B phosphorylation in vivo. Wt Flag-eIF4B was cotransfected into 293 cells with empty vector (A, B), wt S6K1 (C, D) or two different rapamycin-resistant S6K1 proteins (E–H), as indicated. Cells were starved of serum for 36 h, and then stimulated with serum for 30 min (A, C, E, G), or pretreated for 30 min with rapamycin (B, D, F, H) prior to serum stimulation. Flag-eIF4B was isolated and subjected to phosphopeptide mapping, as above.

(A) Schematic representation of His₆-eIF4B fragments. Six contiguous hexahistidine-tagged fragments comprised of ∼100 residues each were generated. The RRM, DRYG domain and ARM RNA-binding domain are indicated. (B) Recombinant fragments were purified by Ni²⁺ affinity chromatography, and then subjected to SDS–PAGE and Coomassie blue staining. (C) Equal amounts of the same fragments were phosphorylated by S6K1 in vitro. Lane numbers indicate eIF4B fragment number. The arrow indicates autophosphorylated S6K1.

Ser422 phosphorylation (or phosphorylation mimic) is required to inhibit translation. 293T cells were cotransfected with a reporter construct encoding human GH and various Flag eIF4B constructs. (A; top) Northern blot of total RNA probed with a human GH DNA segment. (Middle panel) Western analysis using the anti-Flag antibody. (Bottom panel) Western analysis using an anti-hGH antiserum. (B) Polysome profile analysis of human GH mRNA from cells transiently transfected with pS16-GH and Flag-eIF4B S422A (top panel) or eIF4B S422E (bottom panel). Locations in the gradient of 40S, 60S and 80S ribosomes, and polysomes are indicated.

Phosphorylation of a single major eIF4B peptide is modulated by serum, and is sensitive to pharmacological inhibitors of PI3K and mTOR. Phosphopeptide mapping of endogenous eIF4B isolated from 293 cells, (A) starved of serum for 36 h; (B) starved of serum, and then serum stimulated for 30 min; (C) starved of serum, pretreated with 5 μM LY294002 for 30 min, and then serum stimulated for 30 min; or (D) starved of serum, pretreated with 50 nM rapamycin for 30 min, and then serum stimulated for 30 min. The two major phosphopeptides are numbered, and the loading origin is indicated by an arrow. The position of the diminished phosphopeptide 2 is indicated with a dashed circle. A vertical streaked signal near the loading origin is also observed, but this signal does not appear to be responsive to serum stimulation nor sensitive to kinase inhibitors.

A phosphospecific antiserum confirms that phosphorylation of Ser422 is serum responsive, and is sensitive to rapamycin treatment and leucine deprivation. (A) Cells were starved of serum (odd-numbered lanes), or starved of serum and then serum stimulated (even-numbered lanes). Lysates were subjected to Western analysis using the phosphospecific eIF4B Ser422 antiserum (lanes 1 and 2). Preincubation of the antiserum with the phosphopeptide against which it was raised (lanes 3 and 4), or with the same peptide containing an unphosphorylated Ser422 (lanes 5 and 6). eIF4B expression levels were not affected by these treatments (lanes 7 and 8). (B) Cells were pretreated with rapamycin, and subjected to Western analysis using the phosphospecific antiserum (top panel) and antisera directed against total eIF4B (middle panel), as above. The same lysates were subjected to Western analysis using a phosphospecific antibody directed against S6K1 Thr389 (lower panel). (C) Cells were subjected to leucine-free (but otherwise complete) culture media for the indicated time periods. Lysates were subjected to Western blotting using the eIF4B Ser422 phosphospecific antiserum (top panel), a phosphorylation-independent antiserum directed against eIF4B (middle panel) or a phosphospecific antiserum directed against ribosomal S6 protein Ser235/236 (lower panel).