Noncanonical NF-κB activation by the oncoprotein Tio occurs through a nonconserved TRAF3-binding motif.

Members of the nuclear factor kappa B (NF-kappa B) family of transcription factors regulate many cellular functions. Activation of NF-kB signaling is commonly classified as occurring through canonical or noncanonical pathways. Most NF- kappa B-inducing stimuli, including the viral oncoprotein Tio, lead to a concerted activation of both NF-kappa B pathways; however, extensive crosstalk at multiple levels between these signaling cascades restricts the ability to discriminate between the canonical and the noncanonical effects. We showed that noncanonical NF- kB activation by Tio depends on a distinct sequence motif that directly recruits tumor necrosis factor receptor-associated factor 3 (TRAF3). Through its TRAF3-binding motif, Tio triggered a ubiquitin-independent depletion of TRAF3 from the cytosol, which prevented TRAF3 from inhibiting signaling through the noncanonical NF-kappa B cascade. Furthermore, the Tio-TRAF3 interaction did not affect components of the canonical NF-kappa B signaling pathway or the expression of target genes; thus, Tio induced noncanonical NF-kappa B independently of crosstalk with the canonical pathway. Together, these data identify a distinct molecular mechanism of noncanonical NF- kB activation that should enable studies into the particular functions of this pathway.