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

被引:68
作者
Karim, MM
Hughes, JMX
Warwicker, J
Scheper, GC
Proud, CG
McCarthy, JEG
机构
[1] Univ Manchester, Dept Biomol Sci, Posttranscript Control Grp, Manchester M60 1QD, Lancs, England
[2] Univ Dundee, Sch Life Sci, Dundee DD1 5EH, Scotland
关键词
D O I
10.1074/jbc.M011068200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:20750 / 20757
页数:8
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