Alterations in interprotein interactions between translation initiation factors assessed by fluorescence resonance energy transfer.
Interaction of the translational repressor 4E-BP1 with the mRNA cap binding protein eIF4E plays an important role in the regulation of translation initiation. This interaction is modulated by phosphorylation of 4E-BP1 on at least six residues. However, analysis of the functional importance of the individual phosphorylation sites is complicated by the lack of information about the kinases and phosphatases involved in modulating phosphorylation of each site. The goal of the present study was to establish a system whereby alterations in the interaction of 4E-BP1 with eIF4E could be easily and directly measured. In initial studies, both eIF4E and 4E-BP1 were expressed as recombinant proteins coupled to variants of green fluorescent protein (ECFP and EYFP, respectively). Addition of purified EYFP--4E-BP1 to ECFP--eIF4E caused both a decrease in emission intensity at 480 nm and an increase at 535 nm indicating that protein-protein interaction had occurred. The interaction was stoichiometric and was blocked by eIF4G. Phosphorylation of EYFP--4E-BP1 by the mitogen-activated protein kinase ERK2, but not by casein kinase CK-II, also attenuated the interaction. Results using proteins in which the fluorescent protein tag was located at either the N- or C-terminus suggested that, in the protein complex, the N-termini of the two proteins are in close spatial proximity, as are the C-termini. Overall, the results demonstrate that fluorescence resonance energy transfer between EYFP--4E-BP1 and ECFP--eIF4E is a valuable tool in directly measuring alterations in the interaction of the two proteins.
Kimball
S R
SR
Department of Cellular and Molecular Physiology, The Pennsylvania State University, College of Medicine, P.O. Box 850, Hershey, PA 17033, USA. skimball@psu.edu
Horetsky
R L
RL
D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D047390:Casein Kinase II; D004735:Energy Transfer; D039561:Eukaryotic Initiation Factor-4E; D039603:Eukaryotic Initiation Factor-4G; D005456:Fluorescent Dyes / Q000378:metabolism; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D002352:Carrier Proteins; D039561:Eukaryotic Initiation Factor-4E; D039603:Eukaryotic Initiation Factor-4G; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D010448:Peptide Initiation Factors; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D019950:Mitogen-Activated Protein Kinase 1