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 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.
Posttranscriptional Control Group, Department of Biomolecular Sciences, UMIST, P. O. Box 88, Manchester M60 1QD, United Kingdom.
M M
MM
Karim
J M
JM
Hughes
J
J
Warwicker
G C
GC
Scheper
C G
CG
Proud
J E
JE
McCarthy
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002846:Chromatography, Affinity; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D008322:Mammals; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D014176:Protein Biosynthesis / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C087000:EIF4EBP1 protein, human; D010750:Phosphoproteins; D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid