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

A quantitative molecular model for modulation of mammalian translation by the eIF4E-binding protein 1
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, Thr46 and Ser65, consistently have the most significant effects, and that phosphorylation of Ser65 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.
0021-9258
20750-20757
Karim, M.M.
b435bd0d-c71d-4b66-80e5-61f9c27c9fa5
Hughes, J.M.X.
ea3ec87b-d54e-410a-b293-5a05e41c89ea
Warwicker, J.
688afc70-9000-4ccd-a4d5-c9887569d424
Scheper, G.C.
f82821cd-4256-4df9-943b-31760b4aa176
Proud, C.G.
c2cc50f9-4565-4d59-9dfc-aa70b9268a6e
McCarthy, J.E.G.
1596463a-c22c-4b19-8ed9-7e6f62603ce7
Karim, M.M.
b435bd0d-c71d-4b66-80e5-61f9c27c9fa5
Hughes, J.M.X.
ea3ec87b-d54e-410a-b293-5a05e41c89ea
Warwicker, J.
688afc70-9000-4ccd-a4d5-c9887569d424
Scheper, G.C.
f82821cd-4256-4df9-943b-31760b4aa176
Proud, C.G.
c2cc50f9-4565-4d59-9dfc-aa70b9268a6e
McCarthy, J.E.G.
1596463a-c22c-4b19-8ed9-7e6f62603ce7

Karim, M.M., Hughes, J.M.X., Warwicker, J., Scheper, G.C., Proud, C.G. and McCarthy, J.E.G. (2001) A quantitative molecular model for modulation of mammalian translation by the eIF4E-binding protein 1. The Journal of Biological Chemistry, 276 (23), 20750-20757. (doi:10.1074/jbc.M011068200).

Record type: Article

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, Thr46 and Ser65, consistently have the most significant effects, and that phosphorylation of Ser65 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.

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Published date: 1 June 2001

Identifiers

Local EPrints ID: 56703
URI: http://eprints.soton.ac.uk/id/eprint/56703
ISSN: 0021-9258
PURE UUID: e840d460-1501-4817-8d68-def7821d28c1

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Date deposited: 07 Aug 2008
Last modified: 15 Mar 2024 11:03

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Contributors

Author: M.M. Karim
Author: J.M.X. Hughes
Author: J. Warwicker
Author: G.C. Scheper
Author: C.G. Proud
Author: J.E.G. McCarthy

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