title: Mutational analysis of TRAF6 reveals a conserved functional role of the RING dimerization interface and a potentially necessary but insufficient role of RING-dependent TRAF6 polyubiquitination towards NF-κB activation.
abstract: TRAF6 is an E3 ubiquitin ligase that plays a pivotal role in the activation of NF-κB by innate and adaptive immunity stimuli. TRAF6 consists of a highly conserved carboxyl terminal TRAF-C domain which is preceded by a coiled coil domain and an amino terminal region that contains a RING domain and a series of putative zinc-finger motifs. The TRAF-C domain contributes to TRAF6 oligomerization and mediates the interaction of TRAF6 with upstream signaling molecules whereas the RING domain comprises the core of the ubiquitin ligase catalytic domain. In order to identify structural elements that are important for TRAF6-induced NF-κB activation, mutational analysis of the TRAF-C and RING domains was performed. Alterations of highly conserved residues of the TRAF-C domain of TRAF6 did not affect significantly the ability of the protein to activate NF-κB. On the other hand a number of functionally important residues (L77, Q82, R88, F118, N121 and E126) for the activation of NF-κB were identified within the RING domain of TRAF6. Interestingly, several homologues of these residues in TRAF2 were shown to have a conserved functional role in TRAF2-induced NF-κB activation and lie at the dimerization interface of the RING domain. Finally, whereas alteration of Q82, R88 and F118 compromised both the K63-linked polyubiquitination of TRAF6 and its ability to activate NF-κB, alteration of L77, N121 and E126 diminished the NF-κB activating function of TRAF6 without affecting TRAF6 K63-linked polyubiquitination. Our results support a conserved functional role of the TRAF RING domain dimerization interface and a potentially necessary but insufficient role for RING-dependent TRAF6 K63-linked polyubiquitination towards NF-κB activation in cells.
authors.affiliation: School of Biology, Aristotle University of Thessaloniki, 54124, Thessaloniki, Greece.
authors.forename: Charilaos
authors.initials: C
authors.lastname: Megas
authors.forename: Eudoxia G
authors.initials: EG
authors.lastname: Hatzivassiliou
authors.forename: Qian
authors.initials: Q
authors.lastname: Yin
authors.forename: Elli
authors.initials: E
authors.lastname: Marinopoulou
authors.forename: Paul
authors.initials: P
authors.lastname: Hadweh
authors.forename: Dario A A
authors.initials: DA
authors.lastname: Vignali
authors.forename: George
authors.initials: G
authors.lastname: Mosialos
mesh_terms: D002460:Cell Line; D019281:Dimerization; D006801:Humans; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D047992:TNF Receptor-Associated Factor 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D048029:TNF Receptor-Associated Factor 6 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D016335:Zinc Fingers
chemical_list: D016328:NF-kappa B; D047992:TNF Receptor-Associated Factor 2; D048029:TNF Receptor-Associated Factor 6