A quantitative molecular model for modulation of mammalian translation by the eIF4E-binding protein 1

Translation initiation is a key point of regulation in eukaryotic gene expr ession. 4E-binding proteins (4E-BPs) inhibit initiation by blocking the ass ociation of eIF4E with eIF4G, two integral components of the mRNA cap-bindi ng complex. Phosphorylation of 4E-BP1 reduces its ability to bind to eIF4E and thereby to compete with eIF4G. A novel combination of biophysical and b iochemical tools was used to measure the impact, of phosphorylation and aci dic side chain substitution at each potentially modulatory site in 4E-BP1. For each individual site, we have analyzed the effects of modification on e IF4E binding using affinity chromatography and surface plasmon resonance an alysis, 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 th at modifications at the two sites immediately flanking the eIF4E-binding do main, Thr(46) and Ser(65), consistently have the most significant effects, and that phosphorylation of Ser(65) causes the greatest reduction in bindin g affinity. These results establish a quantitative framework that should co ntribute to understanding of the molecular interactions underlying 4E-BP1-m ediated translational regulation.