Anne-Claude Gingras

Human eukaryotic translation initiation factor 4G (eIF4G) recruits Mnk1 to phosphorylate eIF4E

Human eukaryotic translation initiation factor 4E (eIF4E) binds to the mRNA cap structure and interacts with eIF4G, which serves as a scaffold protein for the assembly of eIF4E and eIF4A to form the eIF4F complex. eIF4E is an important modulator of cell growth and proliferation. It is the least abundant component of the translation initiation machinery and its activity is modulated by phosphorylation and interaction with eIF4E‐binding proteins (4E‐BPs). One strong candidate for the eIF4E kinase is the recently cloned MAPK‐activated protein kinase, Mnk1, which phosphorylates eIF4E on its physiological site Ser209 in vitro. Here we report that Mnk1 is associated with the eIF4F complex via its interaction with the C‐terminal region of eIF4G. Moreover, the phosphorylation of an eIF4E mutant lacking eIF4G‐binding capability is severely impaired in cells. We propose a model whereby, in addition to its role in eIF4F assembly, eIF4G provides a docking site for Mnk1 to phosphorylate eIF4E. We also show that Mnk1 interacts with the C‐terminal region of the translational inhibitor p97, an eIF4G‐related protein that does not bind eIF4E, raising the possibility that p97 can block phosphorylation of eIF4E by sequestering Mnk1.

Cap-Dependent Translation Initiation in Eukaryotes Is Regulated by a Molecular Mimic of eIF4G

eIF4G uses a conserved Tyr-X-X-X-X-Leu-phi segment (where X is variable and phi is hydrophobic) to recognize eIF4E during cap-dependent translation initiation in eukaryotes. High-resolution X-ray crystallography and complementary biophysical methods have revealed that this eIF4E recognition motif undergoes a disorder-to-order transition, adopting an L-shaped, extended chain/alpha-helical conformation when it interacts with a phylogenetically invariant portion of the convex surface of eIF4E. Inhibitors of translation initiation known as eIF4E-binding proteins (4E-BPs) contain similar eIF4E recognition motifs. These molecules are molecular mimics of eIF4G, which act by occupying the same binding site on the convex dorsum of eIF4E and blocking assembly of the translation machinery. The implications of our results for translation initiation are discussed in detail, and a molecular mechanism for relief of translation inhibition following phosphorylation of the 4E-BPs is proposed.

Phosphorylation of eucaryotic translation initiation factor 4B Ser422 is modulated by S6 kinases.

The eucaryotic translation initiation factor 4B (eIF4B) stimulates the helicase activity of the DEAD box protein eIF4A to unwind inhibitory secondary structure in the 5′ untranslated region of eucaryotic mRNAs. Here, using phosphopeptide mapping and a phosphospecific antiserum, we identify a serum‐responsive eIF4B phosphorylation site, Ser422, located in an RNA‐binding region required for eIF4A helicase‐promoting activity. Ser422 phosphorylation appears to be regulated by the S6Ks: (a) Ser422 phosphorylation is sensitive to pharmacological inhibitors of phosphoinositide‐3 kinase and the mammalian target of rapamycin; (b) S6K1/S6K2 specifically phosphorylate Ser422 in vitro; and (c) rapamycin‐resistant S6Ks confer rapamycin resistance upon Ser422 phosphorylation in vivo. Substitution of Ser422 with Ala results in a loss of activity in an in vivo translation assay, indicating that phosphorylation of this site plays an important role in eIF4B function. We therefore propose that eIF4B may mediate some of the effects of the S6Ks on translation.

Structure of translation factor elF4E bound to m7GDP and interaction with 4E-binding protein

elF4E, the mRNA cap binding protein, is a master switch that controls eukaryotic translation. To be active, it must bind elF4G and form the elF4F complex, which also contains elF4A. Translation is downregulated by association of elF4E with 4E-BP, which occupies the elF4G binding site. Signalling events acting on 4E-BP cause it to dissociate from elF4E, and elF4E is then free to bind elF4G to form the active elF4F complex. We have solved the structure of the yeast elF4E/m7Gpp complex in a CHAPS micelle. We determined the position of the second nucleotide in a complex with m7GpppA, and identified the 4E-BP binding site. elF4E has a curved eight-stranded antiparallel β-sheet, decorated with three helices on the convex face and three smaller helices inserted in connecting loops. The m7G of the cap is intercalated into a stack of tryptophans in the concave face. The 4E-BP binding site is located in a region encompassing one edge of the β-sheet, the adjacent helix a2 and several regions of non-regular secondary structure. It is adjacent to, but does not overlap the cap-binding site.

Serum‐stimulated, rapamycin‐sensitive phosphorylation sites in the eukaryotic translation initiation factor 4GI

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).

mTOR signaling to translation.

Over the past few years, the target of rapamycin (TOR) pathway has been implicated in the control of translation, both in yeast and in higher eukaryotes. In this review, we provide an overview of translation in eukaryotes, and discuss the mechanisms and advantages of the regulation of translation. We then describe how the TOR pathway can modulate translation in yeast and in mammals, through the modulation of the phosphorylation of key translation components, and the regulation of the abundance of ribosomes and translation factors.

4E-BP3, a New Member of the Eukaryotic Initiation Factor 4E-binding Protein Family

Translation initiation in eukaryotes is mediated by the cap structure (m7GpppN, where N is any nucleotide) present at the 5′ end of all cellular mRNAs, except organellar. The cap is recognized by eukaryotic initiation factor 4F (eIF4F), which consists of three polypeptides, including eIF4E, the cap-binding protein subunit. The interaction of the cap with eIF4E facilitates the binding of the ribosome to the mRNA. eIF4E activity is regulated in part by two translational repressors, 4E-BP1 and 4E-BP2, which bind to it and prevent its assembly into eIF4F. We report here the isolation of 4E-BP3, a new member of the 4E-BP family. 4E-BP3 is homologous to 4E-BP1 and 4E-BP2, exhibiting 57 and 59% identity, respectively. The homology is most striking in the middle region of the protein, which contains the eIF4E binding motif and residues that are phosphorylated in 4E-BP1. 4E-BP3 is a heat stable protein that binds to eIF4E in vitro as well as in vivo. Further, 4E-BP3 overexpression specifically reduces eIF4E-dependent translation. The overlapping function and expression of the different 4E-BP family members imply that there is redundancy in this translational control mechanism, underscoring its importance.

Activation of GCN2 in UV-Irradiated Cells Inhibits Translation

BACKGROUND Mammalian cells subjected to ultraviolet (UV) irradiation actively repress DNA replication, transcription, and mRNA translation. While the effects of UV irradiation on DNA replication and transcription have been extensively studied, the mechanism(s) responsible for translational repression are poorly understood. RESULTS Here, we demonstrate that UV irradiation elicits phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) by activating the kinase GCN2 in a manner that does not require SAPK/JNK or p38 MAP kinase. GCN2-/- cells, and cells expressing nonphosphorylatable eIF2alpha as their only source of eIF2alpha protein, fail to repress translation in response to UV irradiation. CONCLUSIONS These results provide a mechanism for translation inhibition by UV irradiation and identify a hitherto unrecognized role for mammalian GCN2 as a mediator of the cellular response to UV stress.

mu-Opioid receptor activates signaling pathways implicated in cell survival and translational control.

The μ-opioid receptor mediates the analgesic and addictive properties of morphine. Despite the clinical importance of this G-protein-coupled receptor and many years of pharmacological research, few intracellular signaling mechanisms triggered by morphine and other μ-opioid agonists have been described. We report that μ-opioid agonists stimulate three different effectors of a phosphoinositide 3-kinase (PI3K)-dependent signaling cascade. By using a cell line stably transfected with the μ-opioid receptor cDNA, we show that the specific agonist [d-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO) stimulates the activity of Akt, a serine/threonine protein kinase implicated in protecting neurons from apoptosis. Activation of Akt by DAMGO correlates with its phosphorylation at serine 473. The selective PI3K inhibitors wortmannin and LY294002 blocked phosphorylation of this site, previously shown to be necessary for Akt enzymatic activity. DAMGO also stimulates the phosphorylation of two other downstream effectors of PI3K, the p70 S6 kinase and the repressors of mRNA translation, 4E-BP1 and 4E-BP2. Upon μ-opioid receptor stimulation, p70 S6 kinase is activated and phosphorylated at threonine 389 and at threonine 421/serine 424. Phosphorylation of p70 S6 kinase and 4E-BP1 is also repressed by PI3K inhibitors as well as by rapamycin, the selective inhibitor of FRAP/mTOR. Consistent with these findings, DAMGO-stimulated phosphorylation of 4E-BP1 impairs its ability to bind the translation initiation factor eIF-4E. These results demonstrate that the μ-opioid receptor activates signaling pathways associated with neuronal survival and translational control, two processes implicated in neuronal development and synaptic plasticity.

Eukaryotic Translation Initiation Factor 4E Availability Controls the Switch between Cap-Dependent and Internal Ribosomal Entry Site-Mediated Translation

ABSTRACT Translation of m7G-capped cellular mRNAs is initiated by recruitment of ribosomes to the 5′ end of mRNAs via eukaryotic translation initiation factor 4F (eIF4F), a heterotrimeric complex comprised of a cap-binding subunit (eIF4E) and an RNA helicase (eIF4A) bridged by a scaffolding molecule (eIF4G). Internal translation initiation bypasses the requirement for the cap and eIF4E and occurs on viral and cellular mRNAs containing internal ribosomal entry sites (IRESs). Here we demonstrate that eIF4E availability plays a critical role in the switch from cap-dependent to IRES-mediated translation in picornavirus-infected cells. When both capped and IRES-containing mRNAs are present (as in intact cells or in vitro translation extracts), a decrease in the amount of eIF4E associated with the eIF4F complex elicits a striking increase in IRES-mediated viral mRNA translation. This effect is not observed in translation extracts depleted of capped mRNAs, indicating that capped mRNAs compete with IRES-containing mRNAs for translation. These data explain numerous reported observations where viral mRNAs are preferentially translated during infection.