Translation initiation factor eIF-4E binds to the eukaryotic mRNA 5' cap structure (m7GpppN, where N is any nucleotide), eIF-4E is a limiting factor in translation and plays a key role in regulation of translation. We have shown previously that overexpression of eIF-4E in rodent fibroblasts results in tumorigenic transformation. eIF-4E also exhibits mitogenic activity when microinjected into serum-starved NIH-3T3 cells. To understand the mechanisms by which eIF-4E exerts its mitogenic property, we examined the involvement of the Ras signaling pathway in this activity. Here, we report that Ras is activated in eIF-4E-overexpressing cells, as the proportion of GTP-bound Ras is increased. Overexpression of the negative effector of cellular Ras, GTPase activating protein, causes reversion of the transformed phenotype. Furthermore, we show that neutralizing antibodies to Ras, or a dominant-negative mutant of Ras, inhibit the mitogenic activity of eIF-4E. We conclude that eIF-4E exerts its mitogenic and oncogenic activities by the activation of Ras.

To identify proteins that can bind the 3 untranslated region (UTR) of hepatitis C virus (HCV) we screened human cDNA libraries using the Saccharomyces cerevisiae three-hybrid system. Screening with an RNA sequence derived from the 3-terminal 98 nucleotides (3X region) of an infectious clone of HCV (H77c) yielded clones of human ribosomal proteins L22, L3, S3, and mL3, a mitochondrial homologue of L3. We performed preliminary characterization of the binding between the 3X region and these proteins by a three-hybrid mating assay using mutant 3X sequences. We have further characterized the interaction between 3X and L22, since this protein is known to be associated with two small Epstein-Barr virus (EBV)-encoded RNA species (EBERs) which are abundantly produced in cells latently infected with EBV. The EBERs, which have similar predicted secondary structure to the HCV 3X, assemble into ribonucleoprotein particles that include L22 and La protein. To confirm that L22 binds HCV 3X we performed in vitro binding assays using recombinant L22 (expressed as a glutathione S-transferase [GST] fusion protein) together with a 3X riboprobe. The 3X region binds to the GST-L22 fusion protein (but not to GST alone), and this interaction is subject to competition with unlabeled 3X RNA. To establish the functional role played by L22 in internal ribosome entry site (IRES)-mediated translation of HCV sequences we performed translational analysis in HuH-7 cells using monocistronic and bicistronic reporter constructs. The relative amount of core-chloramphenicol acetyltransferase reporter protein translated under the control of the HCV IRES was stimulated in the presence of L22 and La when these proteins were supplied in trans.It is estimated that 170 million people worldwide are chronically infected with hepatitis C virus (HCV) (36). HCV infection is a leading cause of liver cirrhosis and hepatocellular carcinoma. As there is no vaccine or effective treatment available, HCV poses a significant threat to public health and there is thus an urgent need to understand the virus better and to develop vaccines and therapeutic agents (50).HCV, a member of the Flaviviridae, is an enveloped virus containing a single stranded, approximately 9.6-kb genomic RNA molecule of positive polarity (10). The genome contains a single open reading frame flanked by 5Ј untranslated regions (5ЈUTRs) and 3ЈUTRs. There are at least six genotypes of HCV whose sequences differ from each other by up to 30% over the complete genome, and the genotypes are grouped into subtypes according to sequence similarities (57). Recently, several infectious cDNA clones of HCV genome have been isolated (31, 68, 70). The genome encodes a polypeptide of approximately 3,010 amino acids which is cotranslationally processed by the host-and virus-encoded proteases to produce at least 10 mature proteins (11,40,50). In contrast to the recent progress made in understanding the genome organization of HCV, the proteolytic processing of the polyprotein and the biochemical characterization of th...

Eukaryotic initiation factor 4F (eIF-4F) is a three-subunit complex that binds the 5' cap structure (m7GpppX, where X is any nucleotide) of eukaryotic mRNAs. This factor facilitates ribosome binding by unwinding the secondary structure in the mRNA 5' noncoding region. The limiting component of the 4F complex is believed to be the 24-kDa cap-binding phosphoprotein, eIF-4E. In this report, we describe the phosphorylation of eIF-4E in response to expression of the tyrosine kinase oncoproteins pp6vs-c and pp60c-src527F' The results suggest that eIF-4E functions as a downstream target of the phosphorylation cascade induced by tyrosine-specific protein kinases as well as by effectors of the mitogenic response.

Translation initiation factor eIF-4E, which binds to the 5' cap structure of eukaryotic mRNAs, is believed to play an important role in the control of cell growth. Consistent with this, overexpression of eIF4E in fibroblasts results in their malignant transformation. The activity of eIF-4E is thought to be regulated by phosphorylation on a single serine residue (Ser-53). Treatment of rat pheochromocytoma (PC12) cells with nerve growth factor (NGF) strongly curtails their growth and causes their differentiation into cells that resemble sympatheticneurons. The present study shows that eIF-4E is rapidly phosphorylated in PC12 cells upon NGF treatment, resulting in a significant increase in the steady-state levels of the phosphorylated protein. In contrast, epidermal growth factor, a factor which elicits a weak mitogenic response in PC12 cells, did not significantly enhance eIF-4E phosphorylation. We also show that although the mitogen and tumor promoter, phorbol 12-myristate-13-acetate, is able to induce phosphorylation of eIF-4E in PC12 cells, the NGF-mediated increase is primarily a protein kinase C-independent response. The NGF-induced enhancement of eiF4E phosphorylation is abrogated in PC12 cells expressing a dominant inhibitory ras mutant (Ser-17 replaced by Asn), indicating that eIF-4E phosphorylation is dependent on a ras signalling pathway. As phosphorylation of eIF-4E effects translation initiation, these results suggest that NGF-mediated and ras-dependent eIF-4E phosphorylation may play a role in switching the pattern of gene expression during the differentiation of PC12 cells.Changes in translation rates play a significant role in the control of cell growth (for a recent review, see reference 31). Enhanced protein synthesis is obligatory for entry into and progression through the cell cycle (4, 5). Modulation of the rate of protein synthesis also occurs during heat shock (14, 51) and mitosis (17) and in response to cell growth modulators (60,63). These changes are effected primarily at the level of initiation, which is rate-limiting under most circumstances (34), and are thought to be governed by the phosphorylation state of key initiation factors (for a review, see reference 30).