Visualization of AP-1–NF-κB ternary complexes in living cells by using a BiFC-based FRET

Abstract

Venus ͉ Cerulean ͉ YFP ͉ cross-talk ͉ NFAT P rotein-protein interactions play a pivotal role in mediating signal-transduction pathways and executing cellular functions. Defining how each protein interacts with all possible partners in cells provides insight into cellular roles of individual proteins. This task has become more demanding given that many interacting networks have been generated in the postgenome era (1, 2). Although a number of methods are available for protein interaction study, several fluorescent protein-based methods, such as FRET and bimolecular fluorescence complementation (BiFC), are most widely used because of direct visualization and easy operation (3-5).FRET is an assay that can measure the proximity or distance between a donor and an acceptor. If two fluorophores are fused to a pair of interacting proteins, a fraction of an excited donor fluorophore nonradiatively transfers energy to an acceptor molecule. The efficiency of the energy transfer is defined as the fraction of donor excitation events that results in energy transfer to an acceptor. Hence, FRET efficiency can be used as an indicator of protein-protein interactions (6). Although the most commonly used fluorescent proteins for FRET analysis are CFP and YFP (4-6), development of new fluorescent proteins with improved properties has significantly expanded our choice of selecting appropriate fluorescent proteins for live-cell imaging (4). For example, the YFP mutant Venus and the CFP mutant Cerulean have been developed, and these mutants have shown better properties for live-cell imaging (7-11).We previously used CFP and YFP to develop a BiFC assay and a multicolor BiFC assay for visualization of protein interactions in living cells (12,13). Over the past few years, these assays have been widely used for visualization of protein-protein interactions in living cells and in different model systems (3). Because the YFP-and CFP-based BiFC assays required a preincubation of cells at lower temperature before visualization of BiFC signals because of their sensitivity to higher temperatures, we recently improved the system and demonstrated that several newly developed fluorescent proteins, such as Venus and Cerulean, can be used for BiFC and multicolor BiFC analysis (14). The use of Venus and Cerulean not only allows for BiFC analysis under physiological conditions but also increases the signal output and the specificity (14).Although determination of individual interacting proteins by FRET and BiFC assays provides useful information for function of a pair of interacting proteins, proteins often form multiple protein complexes such as ternary complexes. In particular, many signaling proteins function as ternary complexes. These complexes include membrane-bound receptors, cytoplasmic signaling molecules, and transcriptional regulatory complexes in the nucleus. For example, the formation of ternary complexes of transcriptional regulatory proteins is critical for gene transcription. These complexes include cross-talk between different families...

TNF-induced oscillations in combinatorial transcription factor binding

“…Recent experiments using the BiFC-based FRET (BiFC-FRET) assay for visualization of complexes in living cells has shown that NF-κB and AP-1 (jun/fos) interact in the nucleus to form tertiary complexes [9]. In the TNF-stimulated state, we show using re-ChIP analysis that AP-1 factors and NF-κB combine to form a complex not seen in the basal state.…”

Fos Proteins Suppress Dextran Sulfate Sodium-Induced Colitis through Inhibition of NF-κB

“…The possible impaired expression and/or activation of TAK1 and Tpl-2 in Fra-1 Tg mice need to be further explored. The direct association between AP-1 and NF-kB might also be a mechanism (35). One intriguing possibility is that the AP-1 and NF-kB pathways are reciprocally regulated.…”

Fat-specific protein 27 modulates nuclear factor of activated T cells 5 and the cellular response to stress

“…The vectors pBiFC-VN173 and pBiFC-VC155 were kindly provided by Dr. Chang-Deng Hu from Purdue University ( 15 ). Fulllength mouse FSP27 cDNA was cloned into the EcoR I/ Xho I site of the pBiFC-VC155.…”