Alterations in interprotein interactions between translation initiation factors assessed by fluorescence resonance energy transfer

Scot R. Kimball, Rick L. Horetsky

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)797-806
Number of pages10
JournalInternational Journal of Biochemistry and Cell Biology
Volume33
Issue number8
DOIs
StatePublished - 2001

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Cell Biology

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