Activated eIF4E-binding Protein Slows G1 Progression and Blocks Transformation by c-myc without Inhibiting Cell Growth

Abstract

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.

RhoE Inhibits 4E-BP1 Phosphorylation and eIF4E Function Impairing Cap-dependent Translation

“…Consistent with this, RhoE did not inhibit growth factor-induced cell size increase, another well characterized mTOR-regulated response. These results are in agreement with reports showing that eIF4E inhibition specifically affects cell proliferation and transformation, but not cell growth (27).…”

“…These results suggest that RhoE specifically blocks 4E-BP1, but not S6K/S6 phosphorylation, and thus is unlikely to inhibit mTOR. As an alternative functional readout for mTOR/S6K activation, we measured the increase in cell size in response to growth factor stimulation, because this pathway but not 4E-BP1/eIF4E is thought to be responsible for driving general protein synthesis and thus cellular growth (27). Although RhoE-expressing cells appeared to be slightly smaller than control cells, their cell size increased upon serum stimulation similarly to that of control cells (Fig.…”

Candidate tumor suppressor DDX3 RNA helicase specifically represses cap-dependent translation by acting as an eIF4E inhibitory protein

“…In contrast, sequestration of eIF4E levels can reverse the transformed phenotype. Conceivably, the eIF4E inhibitory protein 4E-BP1 has been reported to act as a tumor suppressor gene product (Rousseau et al, 1996;Lynch et al, 2004). Notably, many other tumor suppressors, such as p53 and Pdcd4 (programmed cell death 4), also exert their growth inhibitory effect by modulation of translation initiation in addition to their roles as a transcriptional regulator (Horton et al, 2002;Yang et al, 2003).…”

eIF4E – from translation to transformation

“…Ectopic expression of 4E-BP1 is pro-apoptotic in both normal as well as ras-transformed fibroblasts Li et al, 2002b). Finally, a constitutively active 4E-BP1 mutant in which the critical phosphorylation sites (Thr36, Thr45, Ser64 and Thr 69) are mutated to Ala, slows G1 progression and blocks c-myc induced transformation (Lynch et al, 2004).…”