Methods for analyzing the evolutionary relationship of NF-κB proteins using free, web-driven bioinformatics and...

Phylogenetic analysis enables one to reconstruct the functional evolution of proteins. Current understanding of NF-κB signaling derives primarily from studies of a relatively small number of laboratory models-mainly vertebrates and insects-that represent a tiny fraction of animal evolution. As such, NF-κB has been the subject of limited phylogenetic analysis. The recent discovery of NF-κB proteins in "basal" marine animals (e.g., sponges, sea anemones, corals) and NF-κB-like proteins in non-metazoan lineages extends the origin of NF-κB signaling by several hundred million years and provides the opportunity to investigate the early evolution of this pathway using phylogenetic approaches. Here, we describe a combination of bioinformatic and phylogenetic analyses based on menu-driven, open-source computer programs that are readily accessible to molecular biologists without formal training in phylogenetic methods. These phylogenetically based comparisons of NF-κB proteins are powerful in that they reveal deep conservation and repeated instances of parallel evolution in the sequence and structure of NF-κB in distant animal groups, which suggest that important functional constraints limit the evolution of this protein.