Brian Raught

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.

A NOVEL FUNCTIONAL HUMAN EUKARYOTIC TRANSLATION INITIATION FACTOR 4G

ABSTRACT Mammalian eukaryotic translation initiation factor 4F (eIF4F) is a cap-binding protein complex consisting of three subunits: eIF4E, eIF4A, and eIF4G. In yeast and plants, two related eIF4G species are encoded by two different genes. To date, however, only one functional eIF4G polypeptide, referred to here as eIF4GI, has been identified in mammals. Here we describe the discovery and functional characterization of a closely related homolog, referred to as eIF4GII. eIF4GI and eIF4GII share 46% identity at the amino acid level and possess an overall similarity of 56%. The homology is particularly high in certain regions of the central and carboxy portions, while the amino-terminal regions are more divergent. Far-Western analysis and coimmunoprecipitation experiments were used to demonstrate that eIF4GII directly interacts with eIF4E, eIF4A, and eIF3. eIF4GII, like eIF4GI, is also cleaved upon picornavirus infection. eIF4GII restores cap-dependent translation in a reticulocyte lysate which had been pretreated with rhinovirus 2A to cleave endogenous eIF4G. Finally, eIF4GII exists as a complex with eIF4E in HeLa cells, because eIF4GII and eIF4E can be purified together by cap affinity chromatography. Taken together, our findings indicate that eIF4GII is a functional homolog of eIF4GI. These results may have important implications for the understanding of the mechanism of shutoff of host protein synthesis following picornavirus infection.

eIF4E activity is regulated at multiple levels.

A key regulatory step in translation is initiation, or the recruitment of the translational machinery to the 5 end of mRNA. The 5 terminus of most mRNAs is demarcated by a m7GpppN cap (where m is a methyl group, and N is any nucleotide). The m7 cap is essential for the translation of most mRNAs, as it directs the translational machinery to the 5 end of the mRNA via its interaction with the cap binding protein, the eukaryotic translation initiation factor 4E (eIF4E). eIF4E is the limiting initiation factor in most cells. Thus, eIF4E activity plays a principal role in determining global translation rates. Consistent with this role, eIF4E is required for cell cycle progression, exhibits anti-apoptotic activity, and, when overexpressed, transforms cells. This review focuses upon the various mechanisms utilized in the regulation of eIF4E activity. (1) eIF4E is regulated transcriptionally; it is one of the few identified transcriptional targets of c-myc. (2) eIF4E is phosphorylated following activation of the MNK1 kinase, a substrate of the ERK and p38 MAPKs. The recent determination of the three-dimensional structure of eIF4E bound to a m7 cap analog has provided insight into the mechanisms involved in the regulation of the eIF4E-cap and eIF4E-mRNA interactions. As suggested by the crystal structure, phosphorylation of eIF4E may enhance its affinity for mRNA. (3) eIF4E is also regulated through binding to a family of translational repressor proteins. Interaction with the 4E-BPs prevents the incorporation of eIF4E into an active translation initiation complex, and thus, inhibits cap-dependent translation. This inhibitory interaction is relieved following phosphorylation of the 4E-BPs by a PI3K-dependent pathway, involving signalling by the anti-apoptotic kinase Akt/PKB, as well as FRAP/mTOR.

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.

The major mRNA‐associated protein YB‐1 is a potent 5′ cap‐dependent mRNA stabilizer

mRNA silencing and storage play an important role in gene expression under diverse circumstances, such as throughout early metazoan development and in response to many types of environmental stress. Here we demonstrate that the major mRNA‐associated protein YB‐1, also termed p50, is a potent cap‐dependent mRNA stabilizer. YB‐1 addition or overexpression dramatically increases mRNA stability in vitro and in vivo, whereas YB‐1 depletion results in accelerated mRNA decay. The cold shock domain of YB‐1 is responsible for the mRNA stabilizing activity, and a blocked mRNA 5′ end is required for YB‐1‐mediated stabilization. Significantly, exogenously added YB‐1 destabilizes the interaction of the cap binding protein, eIF4E, with the mRNA cap structure. Conversely, sequestration of eIF4E from the cap increases the association of endogenous YB‐1 with mRNA at or near the cap, and significantly enhances mRNA stability. These data support a model whereby down‐regulation of eIF4E activity or increasing the YB‐1 mRNA binding activity or concentration in cells activates a general default pathway for mRNA stabilization.

Activation of GCN2 in UV-Irradiated Cells Inhibits Translation

BACKGROUNDnMammalian 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.nnnRESULTSnHere, 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.nnnCONCLUSIONSnThese 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.

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.

Control of Translation by the Target of Rapamycin Proteins

Regulation of translation rates, the frequency with which a given mRNA is translated, plays an important role in the control of cell growth and differentiation. Translational control is exerted in most instances at the initiation phase, a rate-limiting step during which the ribosome is recruited to mRNA. Initiation is a complex process mediated by many translation initiation factors (at least 30 polypeptides), and the regulation of translation initiation factor activity involves modulation of gene expression, binding to other factors or repressors, proteolytic cleavage and changes in phosphorylation state. It has been known for some time that the phosphorylation state of various translation factors/inhibitors (and other proteins required for translation, such as ribosomal proteins) is modulated in response to hormonal/mitogenic signals and environmental or nutritional stresses, but the identity of the signaling pathways involved in translational regulation are only beginning to emerge. In this review, we describe a signaling module involved in translational control both in yeast and in mammalian cells, the TOR (or FRAP/mTOR) signaling pathway. In mammals, this pathway regulates the activity of several translation factors (eIF4B and eIF4GI), translation inhibitors (the 4E-BPs), and the ribosomal S6 kinases (S6K1 and 2). In yeast, inhibition of Tor activity leads to polysomal disaggregation and G 1 cell cycle arrest.