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

Members of the nuclear factor κB (NF-κB) family of transcription factors regulate many cellular functions. Activation of NF-κB signaling is commonly classified as occurring through canonical or noncanonical pathways. Most NF-κB-inducing stimuli, including the viral oncoprotein Tio, lead to a concerted activation of both NF-κB 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-κB 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-κB cascade. Furthermore, the Tio-TRAF3 interaction did not affect components of the canonical NF-κB signaling pathway or the expression of target genes; thus, Tio induced noncanonical NF-κB independently of crosstalk with the canonical pathway. Together, these data identify a distinct molecular mechanism of noncanonical NF-κB activation that should enable studies into the particular functions of this pathway.