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.

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Studies on various forms of synaptic plasticity have demonstrated a link between mRNA translation, learning and memory. Like memory, synaptic plasticity includes an early phase which depends on modification of pre-existing proteins, and a late phase that requires transcription and synthesis of new proteins 1,2 . Activation of post-synaptic targets appears to trigger the transcription of plasticityrelated genes. The new mRNAs are either translated in the soma or transported to synapses before translation. GCN2, a key protein kinase, regulates the initiation of translation. We now report a unique feature of hippocampal slices from GCN2 -/-mice: in CA1, a single 100 Hz train induces a strong and sustained long-term potentiation (late-LTP or L-LTP), which is transcription and translation dependent. In contrast, stimulation that elicits late-LTP in wild type slices, such as four 100 Hz trains or forskolin, fails to evoke L-LTP in GCN2 -/-slices. This aberrant synaptic plasticity is mirrored in the behavior of GCN2 -/-mice in the Morris water maze: after weak training, their spatial memory is enhanced, but it is impaired after more intense training. Activated GCN2 stimulates mRNA translation of ATF4, a CREB antagonist. Accordingly, in the hippocampus of GCN2 -/-mice, the expression of ATF4 is reduced and CREB activity is increased. Our study provides genetic, physiological, behavioral and molecular evidence that GCN2 regulates synaptic plasticity, as well as learning and memory through modulation of the ATF4/CREB pathway.Translation of eukaryotic mRNAs is primarily regulated at the level of initiation 3 . Binding of the initiator tRNA, Met-tRNA i Met , to the 40S subunit is facilitated by the initiation factor 2 (eIF2) which forms a ternary complex with GTP and Met-tRNA i Met . Although phosphorylation

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.

Mammalian translation initiation factor 4F (eIF4F) consists of three subunits, eIF4A, eIF4E, and eIF4G. eIF4G interacts directly with both eIF4A and eIF4E. The binding site for eIF4E is contained in the aminoterminal third of eIF4G, while the binding site for eIF4A was mapped to the carboxy-terminal third of the molecule. Here we show that human eIF4G possesses two separate eIF4A binding domains in the middle third (amino acids [aa] 478 to 883) and carboxy-terminal third (aa 884 to 1404) of the molecule. The amino acid sequence of the middle portion of eIF4G is well conserved between yeasts and humans. We show that mutations of conserved amino acid stretches in the middle domain abolish or reduce eIF4A binding as well as eIF3 binding. In addition, a separate and nonoverlapping eIF4A binding domain exists in the carboxy-terminal third (aa 1045 to 1404) of eIF4G, which is not present in yeast. The C-terminal two-thirds region (aa 457 to 1404) of eIF4G, containing both eIF4A binding sites, is required for stimulating translation. Neither one of the eIF4A binding domains alone activates translation. In contrast to eIF4G, human p97, a translation inhibitor with homology to eIF4G, binds eIF4A only through the amino-terminal proximal region, which is homologous to the middle domain of eIF4G.Eukaryotic translation initiation factor 4F (eIF4F) is a protein complex consisting of eIF4E, eIF4A, and eIF4G. eIF4E binds to the cap structure (m 7 GpppN, where N is any nucleotide) of the mRNA. eIF4A, an RNA-dependent ATPase and ATP-dependent RNA helicase, is thought to unwind the secondary structure of the 5Ј untranslated region of the mRNA to facilitate ribosome binding (for reviews, see references 16, 19, and 25). eIF4G serves as a scaffold for eIF4E and eIF4A to coordinate their functions. eIF4F exhibits a much higher RNA helicase activity than eIF4A alone (22), and dominant negative mutants of eIF4A abolish both cap-dependent and cap-independent translation (20). This is consistent with the idea that eIF4A is essential for translation of all mRNAs and that eIF4A recycles through the eIF4F complex to function in mRNA unwinding (20).Several members of the picornavirus family, including poliovirus, cause the cleavage of eIF4G into an N-terminal third (amino acids [aa] 1 to 479 for poliovirus) and a C-terminal two-thirds fragment (aa 480 to 1396) (3,7,14). Extensive digestion of eIF4G with the foot-and-mouth-disease virus L protease or rhinovirus 2A protease in vitro yields several smaller fragments. One of the fragments (aa 319 to 479) binds to eIF4E (10). Mader et al. (15) localized the eIF4E binding site more precisely to the amino acid sequence 413 KKRYDRE FLLGFQFIF 428 , which is well conserved between yeast and human eIF4Gs. The conserved eIF4E binding site is also found in eIF4E-binding proteins (4E-BPs) (15), and competition between eIF4G and 4E-BPs for eIF4E explains how 4E-BPs inhibit cap-dependent translation (6). Although the C-terminal third of human eIF4G was shown to contain the binding site for eIF4A (10), the y...