1. ¹Molecular Immunology Laboratory, Department of Immunology, University Medical Center Utrecht, Utrecht, The Netherlands
2. ²Department of Endocrinology and Metabolic Diseases, University Medical Center Utrecht, Utrecht, The Netherlands
3. ³Leibniz Institute for Age Research–Fritz Lipmann Institute, Beutenbergstr, Jena, Germany
4. ⁴Department of Physiological Chemistry, Centre for Biomedical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands
5. ⁵Department of Biological Chemistry, School of Medicine, University of California, Davis, CA, USA
6. ⁶Department of Pediatric Immunology, University Medical Center Utrecht, Utrecht, The Netherlands

Correspondence: Dr PJ Coffer, Molecular Immunology Laboratory, Department of Immunology (KC.02.085.2), University Medical Center, Lundlaan 6, 3584 CA Utrecht, The Netherlands. E-mail: P.J.Coffer@umcutrecht.nl

⁷Current address: Netherlands Forensic Institute, the Hague, The Netherlands.

⁸These authors contributed equally to this work.

Received 10 June 2008; Revised 27 August 2008; Accepted 1 September 2008; Published online 6 October 2008.

Abstract

Eukaryotic translation initiation factor 4B (eIF4B) plays a critical role during the initiation of protein synthesis and its activity can be regulated by multiple phosphorylation events. In a search for novel protein kinase B (PKB/c-akt) substrates, we identified eIF4B as a potential target. Using an in vitro kinase assay, we found that PKB can directly phosphorylate eIF4B on serine 422 (ser422). Activation of a conditional PKB mutant, interleukin-3 (IL-3) or insulin stimulation resulted in PKB-dependent phosphorylation of this residue in vivo. This was prevented by pretreatment of cells with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 or pharmacological inhibition of PKB. Pretreatment of cells with rapamycin, inhibiting mTOR or U0126 to inhibit MEK, had little effect on eIF4B ser422 phosphorylation. In contrast, following amino-acid refeeding, eIF4B ser422 phosphorylation was found to be mammalian target of rapamycin (mTOR)-dependent. We further identified eIF4B ser406 as a novel mitogen-regulated phosphorylation site. Insulin-induced phosphorylation of eIF4B ser406 was dependent on both MEK and mTOR activity. Utilizing a novel translational control luciferase assay, we could further demonstrate that phosphorylation of ser406 or ser422 is essential for optimal translational activity of eIF4B. These data provide novel insights into complex multikinase regulation of eIF4B phosphorylation and reveal an important mechanism by which PKB can regulate translation, potentially critical for the transforming capacity of this AGC kinase family member.