Identification and characterization of proteins interacting with Traf4, an enigmatic p53 target

Tumor necrosis factor receptor associated factor 4 (Traf4) mRNA expression is upregulated in various breast tumors and tumor cell lines. We previously identified Traf4 as a p53 target gene, and showed overexpression of Traf4 inhibited colony formation. However, basal Traf4 expression in cell lines does not appear to be dependent entirely on p53. To address the putative function of Traf4, a yeast 2-hybrid screen and coimmunoprecipitation/mass spectrometry experiments were performed to identify Traf4-interacting proteins. A yeast 2-hybrid using full length Traf4 as bait yielded several candidate interacting proteins including beta-catenin, GRIM19, PSMC3, p62 and dynamin. Although all of these proteins are novel interactors for Traf4, PSMC3 and p62 have been previously demonstrated to interact with the related protein, Traf6. Traf4 appears to enhance beta-catenin related transcription as well as to provide some protection of beta-catenin protein levels from p53-mediated degradation although a direct interaction was not observed in mammalian cells. To obtain interacting proteins using a more physiologically relevant environment, we immunoprecipitated Flag-tagged Traf4 followed by mass spectrometry which identified other novel Traf4 interacting proteins including Eg5, PRMT5, and MYH-9.