A Quantitative Molecular Model for Modulation of Mammalian Translation by the eIF4E-binding Protein 1

Abstract

Translation initiation is a key point of regulation in eukaryotic gene expression. 4E-binding proteins (4E-BPs) inhibit initiation by blocking the association of eIF4E with eIF4G, two integral components of the mRNA cap-binding complex. Phosphorylation of 4E-BP1 reduces its ability to bind to eIF4E and thereby to compete with eIF4G. A novel combination of biophysical and biochemical tools was used to measure the impact of phosphorylation and acidic side chain substitution at each potentially modulatory site in 4E-BP1. For each individual site, we have analyzed the effects of modification on eIF4E binding using affinity chromatography and surface plasmon resonance analysis, and on the regulatory function of the 4E-BP1 protein using a yeast in vivo model system and a mammalian in vitro translation assay. We find that modifications at the two sites immediately flanking the eIF4E-binding domain, Thr 46 and Ser 65 , consistently have the most significant effects, and that phosphorylation of Ser 65 causes the greatest reduction in binding affinity. These results establish a quantitative framework that should contribute to understanding of the molecular interactions underlying 4E-BP1-mediated translational regulation.

Regulation of the phosphorylation and integrity of protein synthesis initiation factor eIF4GI and the translational repressor 4E-BP1 by p53

“…Recently, several reports have shown that p53 is able to induce or activate novel protein phosphatases (Li et al, 2003;Ueda et al, 2003;Yin et al, 2003) but whether any of these act on 4E-BP1 remains to be established. It may be significant that the greatest p53-induced loss of phosphate from 4E-BP1 in vivo appears to involve Ser 64 , since this site can have a large influence on the binding of the protein to eIF4E (Mothe-Satney et al, 2000b;Karim et al, 2001) (although this conclusion has been challenged; Ferguson et al, 2003). 4E-BP1 is phosphorylated in a hierarchical fashion, with Ser 64 being the last site to be modified when cells are stimulated by serum (Gingras et al, 1999a(Gingras et al, , 2001a.…”

“…Hypophosphorylated 4E-BP1 binds to eIF4E in competition with eIF4GI or II, thus decreasing the availability of the eIF4F complex for initiation of translation (Marcotrigiano et al, 1999;Karim et al, 2001). We have reported previously that when cells expressing the Val 135 mutant form of p53 are incubated at 321C to activate the tumour suppressor, extensive 4E-BP1 dephosphorylation occurs.…”

“…Since phosphorylation at Ser 64 has been shown to be particularly important in preventing the binding of 4E-BP1 to eIF4E, at least in vitro (Mothe-Satney et al, 2000b;Karim et al, 2001), we isolated eIF4E by chromatography on m 7 GTP-Sepharose 4B beads and determined the levels of proteins associated with it by immunoblotting. The beads were added to cell extracts prepared from Pro 190 and Val 135 cells at various times after the shift to 321C to determine if the amount of eIF4GI and 4E-BP1 bound to eIF4E changed following p53 activation.…”

TNF-α impairs heart and skeletal muscle protein synthesis by altering translation initiation

“…Although both phosphorylated and nonphosphorylated eIF-4E bind to the mRNA cap structure, phosphorylation of eIF-4E enhances the affinity of the factor for the m 7 GTP cap by severalfold (39). Furthermore, in vitro studies have demonstrated that increases in eIF-4E phosphorylation are proportional to increases in the rate of translation (23). Conversely, a decrease in eIF-4E phosphorylation has been correlated with a reduction in protein synthesis after viral infection (13).…”

“…Phosphorylation of 4E-BP1 greatly decreases the affinity of 4E-BP1 for eIF-4E and leads to dissociation of the 4E-BP1 ⅐ eIF-4E complex (23). This permits the formation of a competent eIF-4F complex and the stimulation of initiation.…”