Raught B, Gingras AC, Gygi SP, Imataka H, Morino S, Gradi A et al.. Serum-stimulated, rapamycin-sensitive phosphorylation sites in the eukaryotic translation initiation factor 4GI. EMBO J 19: 434-444

Department of Biochemistry and McGill Cancer Centre, McGill University, 3655 Drummond, Montréal, Québec H3G 1Y6, Canada.
The EMBO Journal (Impact Factor: 10.43). 03/2000; 19(3). DOI: 10.1093/emboj/19.3.434
Source: PubMed Central

ABSTRACT The eukaryotic translation initiation factor 4G (eIF4G) proteins play a critical role in the recruitment of the translational machinery to mRNA. The eIF4Gs are phosphoproteins. However, the location of the phosphorylation sites, how phosphorylation of these proteins is modulated and the identity of the intracellular signaling pathways regulating eIF4G phosphorylation have not been established. In this report, two-dimensional phosphopeptide mapping demonstrates that the phosphorylation state of specific eIF4GI residues is altered by serum and mitogens. Phosphopeptides resolved by this method were mapped to the C–terminal one-third of the protein. Mass spectrometry and mutational analyses identified the serum-stimulated phosphorylation sites in this region as serines 1108, 1148 and 1192. Phosphoinositide–3–kinase (PI3K) inhibitors and rapamycin, an inhibitor of the kinase FRAP/mTOR (FKBP12–rapamycin-associated protein/mammalian target of rapamycin), prevent the serum-induced phosphorylation of these residues. Finally, the phosphorylation state of N–terminally truncated eIF4GI proteins acquires resistance to kinase inhibitor treatment. These data suggest that the kinases phosphorylating serines 1108, 1148 and 1192 are not directly downstream of PI3K and FRAP/mTOR, but that the accessibility of the C–terminus to kinases is modulated by this pathway(s).

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Available from: Nahum Sonenberg, Sep 26, 2015
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    • "Phosphorylated eIF4B enhances eIF4A helicase activity, suggesting that mTORC2 also participates in translational control. eIF4G phosphorylation at Ser1108, Ser1148, and Ser1192 is reportedly rapamycin-sensitive (Raught et al., 2000) (Figure 3, Upper panel). In addition, p70S6K phosphorylates eukaryotic elongation factor 2 kinase (eEF2K) and suppresses its activity (Wang et al., 2001), This causes the downregulation of eEF2 phosphorylation thus induces its activation (Figure 3, Lower panel). "
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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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    • "We also screened the Aplysia sequence for conservation of identified and studied phosphorylation sites in vertebrate eIF4Gs. Two of the major sites that have been examined, Ser 1108, a serum-activated and rapamycin sensitive site [14], and Ser 1232, an ERK site [15] (numbering is for human eIF4G1), are not conserved in Aplysia or in most other invertebrate species (data not shown). One identified site that is highly conserved in most invertebrates, a p21-activated kinase 2 (PAK2) phosphorylation site [16], is also absent in Aplysia, despite its presence in the limpet, Lottia. "
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    ABSTRACT: The rate-limiting step(s) of translation in the nervous system have not been clearly identified. We have been examining this question in the cell body of the Aplysia sensory neuron, where translational regulation is important for the regulation of synaptic strength. In the present study, we examined the role of the adaptor protein eIF4G. We cloned Aplysia eIF4G (Ap4G) and Ap4G contains all the standard metazoan eIF4G protein-protein interaction domains. Overexpressing Ap4G in Aplysia sensory neurons caused an increase in both cap-dependent and internal ribosome entry site (IRES)-dependent translation using a previously characterized bicistronic fluorescent reporter. Unexpectedly, measurement of overall translation using the methionine analog, L-azidohomoalanine, revealed that overexpression of Ap4G did not lead to an increase in overall translation rates. Indeed, the effect of Ap4G on the bicistronic reporter depended on the presence of an upstream open reading frame (uORF) in the 5' UTR encoded by the vector. We have previously shown that Mnk strongly decreased cap-dependent translation and this depended on a putative 4G binding domain. Here we extend these results showing that even in the absence of the uORF, overexpression of Mnk strongly decreases cap-dependent translation and this depends on the Mnk binding site in eIF4G. Similarly, an increase in cap-dependent translation seen with overexpression of elongation factor 2 kinase did not depend on the uORF. Overall, we show that eIF4G is rate limiting for translation of an mRNA encoding an uORF, but is not generally a rate-limiting step for translation.
    PLoS ONE 09/2013; 8(9):e74085. DOI:10.1371/journal.pone.0074085 · 3.23 Impact Factor
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    • "In contrast, Robalino [57] detected a constant level of eif41a in oocyte and early embryo. eif41a is involved in recruiting cytoplasmic mRNA and initiation translation [58], and higher levels of eif41a generally correlate with increased protein synthesis and cell growth [59]. To indicate MZT, the TATA box binding protein (tbp) was selected. "
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    ABSTRACT: Zygotic transcription in fish embryos initiates around the time of gastrulation, and all prior development is initiated and controlled by maternally derived messenger RNAs. Atlantic cod egg and embryo viability is variable, and it is hypothesized that the early development depends upon the feature of these maternal RNAs. Both the length and the presence of specific motifs in the 3'UTR of maternal RNAs are believed to regulate expression and stability of the maternal transcripts. Therefore, the aim of this study was to characterize the overall composition and 3'UTR structure of the most common maternal RNAs found in cod eggs and pre-zygotic embryos. 22229 Sanger-sequences were obtained from 3'-end sequenced cDNA libraries prepared from oocyte, 1-2 cell, blastula and gastrula stages. Quantitative PCR revealed that EST copy number below 9 did not reflect the gene expression profile. Consequently genes represented by less than 9 ESTs were excluded from downstream analyses, in addition to sequences with low-quality gene hits. This provided 12764 EST sequences, encoding 257 unique genes, for further analysis. Mitochondrial transcripts accounted for 45.9-50.6% of the transcripts isolated from the maternal stages, but only 12.2% of those present at the onset of zygotic transcription. 3'UTR length was predicted in nuclear sequences with poly-A tail, which identified 191 3'UTRs. Their characteristics indicated a more complex regulation of transcripts that are abundant prior to the onset of zygotic transcription. Maternal and stable transcripts had longer 3'UTR (mean 187.1 and 208.8 bp) and more 3'UTR isoforms (45.7 and 34.6%) compared to zygotic transcripts, where 15.4% had 3'UTR isoforms and the mean 3'UTR length was 76 bp. Also, diversity and the amount of putative polyadenylation motifs were higher in both maternal and stable transcripts. We report on the most pronounced processes in the maternally transferred cod transcriptome. Maternal stages are characterized by a rich abundance of mitochondrial transcripts. Maternal and stable transcripts display longer 3'UTRs with more variation of both polyadenylation motifs and 3'UTR isoforms. These data suggest that cod eggs possess a complex array of maternal RNAs which likely act to tightly regulate early developmental processes in the newly fertilized egg.
    BMC Genomics 09/2012; 13(1):443. DOI:10.1186/1471-2164-13-443 · 3.99 Impact Factor
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