A microtiter plate assay for assessing the interaction of eukaryotic initiation factor eIF4E with eIF4G and eIF4E binding protein-1.
Modulation of interactions among proteins is an important mechanism for regulating both the subcellular location and the function of proteins. An example of the importance of protein-protein interaction is the reversible association of eukaryotic initiation factor eIF4E with the eIF4E binding proteins 4E-BP1 and eIF4G. When bound to 4E-BP1, eIF4E cannot bind to eIF4G to form the active eIF4F complex, an event that is required for the binding of mRNA to the ribosome. Thus, association of eIF4E with 4E-BP1 represses mRNA translation by preventing the binding of mRNA to the ribosome. Previous studies have measured the amount of 4E-BP1 or eIF4G bound to eIF4E by either affinity chromatography or immunoprecipitation of eIF4E followed by Western blot analysis for quantitation of 4E-BP1 and eIF4G. Both of these techniques have significant limitations. In the present study, we describe a microtiter plate-based assay for quantitation of the amount of 4E-BP1 and eIF4G bound to eIF4E that obviates many of the limitations of the earlier approaches. It also has the advantage that absolute amounts of the individual proteins can be easily estimated. The approach should be applicable to the study of a wide variety of protein-protein interactions.
Kimball
Scot R
SR
Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA. skimball@psu.edu
Horetsky
Rick L
RL
Jefferson
Leonard S
LS
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins; D039561:Eukaryotic Initiation Factor-4E / Q000378:metabolism*; D039603:Eukaryotic Initiation Factor-4G / Q000378:metabolism*; D039642:Eukaryotic Initiation Factors; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D032446:Myoblasts; D010750:Phosphoproteins / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods*; D011817:Rabbits; D051381:Rats
D048868:Adaptor Proteins, Signal Transducing; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C499527:Eif4ebp1 protein, mouse; C499526:Eif4ebp1 protein, rat; D039561:Eukaryotic Initiation Factor-4E; D039603:Eukaryotic Initiation Factor-4G; D039642:Eukaryotic Initiation Factors; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins