Abstract 1268: Identification of a novel tumor necrosis factor receptor-associated factor 6-binding partner that is a potential lysine-63 linked ubiquitination substrate

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an ubiquitin E3 ligase that catalyzes lysine-63 linked polyubiquitination of various signaling proteins. The recruitment and formation of different protein complexes with TRAF6 leads to activation of TRAF6-associated signaling and regulates cell proliferation and apoptosis. In this study, we search for novel TRAF6-binding partners that may have implication in TRAF6 signaling and cancer progression. Bioinformatic analysis identified a putative TRAF6-binding protein (TRAF6BP) that contains the consensus TRAF6-binding motif X-X-P-X-E-X-X-Ar/Ac-X and thereby TRAF6BP was subjected to further characterization. In accord with the bioinformatics prediction, coimmunoprecipitation analysis demonstrated the binding between TRAF6BP and TRAF6. TRAF6BP also interacts with other TRAF proteins including TRAF-2, −3, −5 but the interaction was much weaker than TRAF6. Domain mapping with TRAF6 deletion mutants identified the TRAF-C domain of TRAF6 is important for its interaction with TRAF6BP. In vivo ubiquitination assay further unveiled that TRAF6BP is a substrate of TRAF6 E3 ligase and underwent ubiquitination in the presence of TRAF6. Only the expression of full-length TRAF6, but not the TRAF-C deletion mutants of TRAF6 resulted in the ubiquitination of TRAF6BP. In addition, TRAF6BP ubiquitiation was abrogated when the lysine-63 ubiquitin mutant (K63R) but not the lysine-48 mutant (K48R) was used as the ubiquitin substrate. Together, we demonstrated for the first time that TRAF6BP is a novel TRAF6-binding protein and is a potential lysine-63 ubiquitination substrate of TRAF6; the TRAF6BP-TRAF6 interaction is essential for TRAF6 to ubiquitinate TRAF6BP. The significances of TRAF6BP-TRAF6 interactions and TRAF6BP ubiquitination in cancer progression and TRAF6 signaling are worthy to further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1268.