AGC kinases regulate phosphorylation and activation of eukaryotic translation initiation factor 4B

Molecular Immunology Laboratory, Department of Immunology, University Medical Center Utrecht, Utrecht, The Netherlands.
Oncogene (Impact Factor: 8.46). 11/2008; 28(1):95-106. DOI: 10.1038/onc.2008.367
Source: PubMed


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.

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Available from: Fried J T Zwartkruis, Sep 19, 2014
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    • "The process is thought to be important for recruiting the 40S ribosome to the mRNA-eIF4F complex. eIF4B is phosphorylated at Ser422 by Akt directly (van Gorp et al., 2009) and by Ser406 and Ser422 in MEK and mTOR/p79S6K-dependent manner (Raught et al., 2004; Shahbazian et al., 2006). Phosphorylated eIF4B enhances eIF4A helicase activity, suggesting that mTORC2 also participates in translational control. "
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    ABSTRACT: Target of rapamycin (TOR) was first identified in yeast as a target molecule of rapamycin, an anti-fugal and immunosuppressant macrolide compound. In mammals, its orthologue is called mammalian TOR (mTOR). mTOR is a serine/threonine kinase that converges different extracellular stimuli, such as nutrients and growth factors, and diverges into several biochemical reactions, including translation, autophagy, transcription, and lipid synthesis among others. These biochemical reactions govern cell growth and cause cells to attain an anabolic state. Thus, the disruption of mTOR signaling is implicated in a wide array of diseases such as cancer, diabetes, and obesity. In the central nervous system, the mTOR signaling cascade is activated by nutrients, neurotrophic factors, and neurotransmitters that enhances protein (and possibly lipid) synthesis and suppresses autophagy. These processes contribute to normal neuronal growth by promoting their differentiation, neurite elongation and branching, and synaptic formation during development. Therefore, disruption of mTOR signaling may cause neuronal degeneration and abnormal neural development. While reduced mTOR signaling is associated with neurodegeneration, excess activation of mTOR signaling causes abnormal development of neurons and glia, leading to brain malformation. In this review, we first introduce the current state of molecular knowledge of mTOR complexes and signaling in general. We then describe mTOR activation in neurons, which leads to translational enhancement, and finally discuss the link between mTOR and normal/abnormal neuronal growth during development.
    Frontiers in Molecular Neuroscience 04/2014; 7(1):28. DOI:10.3389/fnmol.2014.00028 · 4.08 Impact Factor
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    • "Ser406 has also been shown to be phosphorylated by proproliferative and pro-survival protein kinase Pim-2 that is thought to regulate the cell cycle in an mTOR independent manner [46]. Interestingly, S406A substitution abrogates stimulatory effect of eIF4B overexpression in vivo similarly to S422A [44]. Although in early work [35] Ser406 was shown to be dispensable for phosphorylation of both Ser422 and the other site (the above mentioned undetermined, serum-independent one), it may nevertheless play an important role in manifestation of their effects in vivo. "
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    ABSTRACT: Eukaryotic translation initiation factor eIF4B is necessary for ribosomal scanning through structured mRNA leaders. In higher eukaryotes, eIF4B serves as a downstream effector of several signaling pathways. In response to mitogenic stimuli, eIF4B undergoes multiple phosphorylations which are thought to regulate its activity. Recently, Ser422 was identified as a predominant site for human eIF4B phosphorylation via several signaling pathways, and phosphomimetic amino acid substitutions S422D or S422E were shown to activate eIF4B in living cells. However, stimulatory role of these modifications has never been analyzed directly. Here, using both mammalian reconstituted translation initiation assay and complete cell-free translation system, we perform a comparison of recombinant eIF4B derivatives with the wild type recombinant protein, and do not find any difference in their activities. On the contrary, native eIF4B purified from HeLa cells reveals significantly higher activity in both assays. Thus, the effects of S422D and S422E substitutions on eIF4B activity in living cells observed previously either require some other protein modification(s), or may only be manifested in an intact cell. Our study raises the question on whether the phosphorylation of Ser422 is sufficient for eIF4B activation observed upon mitogenic stimulation.
    Biochimie 06/2012; 94(12). DOI:10.1016/j.biochi.2012.06.021 · 2.96 Impact Factor
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    • "We found that HuD itself is not a direct target of Akt1 (Figure 7). A prime candidate target is eIF4B, which can be phosphorylated by Akt1 (Supplementary Figure S4) (18–20) and can stimulate the helicase activity of eIF4A (21,22) and as a consequence translation. Thus, eIF4A activity might indirectly be enhanced by the action of Akt1 kinase via phosphorylation of an activator of eIF4A (e.g. "
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    ABSTRACT: The RNA binding protein HuD plays essential roles in neuronal development and plasticity. We have previously shown that HuD stimulates translation. Key for this enhancer function is the linker region and the poly(A) binding domain of HuD that are also critical for its function in neurite outgrowth. Here, we further explored the underlying molecular interactions and found that HuD but not the ubiquitously expressed HuR interacts directly with active Akt1. We identify that the linker region of HuD is required for this interaction. We also show by using chimeric mutants of HuD and HuR, which contain the reciprocal linker between RNA-binding domain 2 (RBD2) and RBD3, respectively, and by overexpressing a dominant negative mutant of Akt1 that the HuD-Akt1 interaction is functionally important, as it is required for the induction of neurite outgrowth in PC12 cells. These results suggest the model whereby RNA-bound HuD functions as an adapter to recruit Akt1 to trigger neurite outgrowth. These data might also help to explain how HuD enhances translation of mRNAs that encode proteins involved in neuronal development.
    Nucleic Acids Research 11/2011; 40(5):1944-53. DOI:10.1093/nar/gkr979 · 9.11 Impact Factor
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