The mRNA cap-binding protein (eukaryotic initiation factor 4E [eIF4E]) binds the m 7 GpppN cap on mRNA, thereby initiating translation. eIF4E is essential and rate limiting for protein synthesis. Overexpression of eIF4E transforms cells, and mutations in eIF4E arrest cells in G 1 in cdc33 mutants. In this work, we identified the promoter region of the gene encoding eIF4E, because we previously identified eIF4E as a potential myc-regulated gene. In support of our previous data, a minimal, functional, 403-nucleotide promoter region of eIF4E was found to contain CACGTG E box repeats, and this core eIF4E promoter was myc responsive in cotransfections with c-myc. A direct role for myc in activating the eIF4E promoter was demonstrated by cotransfections with two dominant negative mutants of c-myc (Myc⌬TAD and Myc⌬BR) which equally suppressed promoter function. Furthermore, electrophoretic mobility shift assays demonstrated quantitative binding to the E box motifs that correlated with myc levels in the electrophoretic mobility shift assay extracts; supershift assays demonstrated max and USF binding to the same motif. cis mutations in the core or flank of the eIF4E E box simultaneously altered myc-max and USF binding and inactivated the promoter. Indeed, mutations of this E box inactivated the promoter in all cells tested, suggesting it is essential for expression of eIF4E. Furthermore, the GGCCACGTG(A/T)C(C/G) sequence is shared with other in vivo targets for c-myc, but unlike other targets, it is located in the immediate promoter region. Its critical function in the eIF4E promoter coupled with the known functional significance of eIF4E in growth regulation makes it a particularly interesting target for c-myc regulation.

Translation initiation is poised between global regulation of cell growth and specific regulation of cell division. The mRNA cap-binding protein (eIF4E) is a critical integrator of cell growth and division because it is ratelimiting for translation initiation and is also rate-limiting for G 1 progression. Translation initiation factor eIF4E is also oncogenic and a candidate target of c-myc. Recently, an activated inhibitory 4E-binding protein (4EBP) that blocks eIF4E was used to study its regulation of Drosophila growth. We adopted this approach in mammalian cells after identifying an autosensing mechanism that protects against increased levels of 4EBP1. Increased 4EBP1 induced a quantitative increase in the inactivated phosphorylated form of 4EBP1 in vitro and in vivo. To overcome this protective mechanism, we introduced alanine substitutions at four phosphorylation/ inactivation sites in 4EBP1 to constitutively activate a 4EBP to block eIF4E. Overexpression of activated 4EBP inhibited cell proliferation and completely blocked transformation by both eIF4E and c-myc, although it did not block all tested oncogenes. Surprisingly, expression of the activated 4EBP increased cell size and protein content. Activated 4EBP blocked both cell proliferation and c-myc transformation by inhibiting G 1 progression and increasing apoptosis, without decreasing protein synthesis. Our results identify mammalian eIF4E as rate-limiting for cell cycle progression before it regulates cell growth. It further identifies G 1 control by translation initiation factors as an essential genetic target of c-myc that is necessary for its ability to transform cells.

Regulation of the mRNA cap binding protein (eIF4E) is critical to the control of cellular proliferation since this protein is the rate-limiting factor in translation initiation and transforms fibroblasts and since eIF4E mutants arrest budding yeast in the G 1 phase of the cell cycle (cdc33). We previously demonstrated regulation of eIF4E by altered transcription of its mRNA in serum-stimulated fibroblasts and in response to c-myc. To identify additional factors regulating eIF4E transcription, we used linker-scanning constructs to characterize sites in the promoter of the eIF4E gene required for its expression. Promoter activity was dependent on sites at ؊5, ؊25, ؊45, and ؊75; the site at ؊75 included a previously described myc box. Electrophoretic mobility shift assays identified DNA-protein interactions at ؊25 and revealed a binding site (TTACCCCCCCTT) that is unique to the eIF4E promoter. Proteins of 68 and 97 kDa bound this site in UV cross-linking and Southwestern experiments. Levels of 4E regulatory factor activities correlated with c-Myc levels, eIF4E expression levels, and protein synthesis in differentiating U937 and HL60 cells, suggesting that these activities may function to regulate protein synthesis rates during differentiation. Since the eIF4E promoter lacked typical TATA and initiator elements, further studies of this novel initiator-homologous element should provide insights into mechanisms of transcription initiation and growth regulation.

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