TNFAIP8: A New Effector for Galpha(i) Coupling to Reduce Cell Death and Induce Cell Transformation

Galpha(i)-coupled receptors comprise a diverse family of receptors that induce transformation by largely unknown mechanisms. We previously found that the Galpha(i)-coupled dopamine-D2short (D2S) receptor transforms Balb-D2S cells via G alpha i3. To identify new G alpha i effectors, a yeast two-hybrid screen was done using constitutively active G alpha i3-Q204L as bait, and tumor necrosis factor-alpha (TNF alpha)-induced protein 8 (TNFAIP8, SCC-S2/NDED/GG2-I) was identified. In contrast, TNFAIP8-related TIPE1 and TIPE2 showed a very weak interaction with G alpha i3. In yeast mating, in vitro pull-down, co-immunoprecipitation and bioluminescence resonance energy transfer (BRET) assays, TNFAIP8 preferentially interacted with activated G alpha i proteins, consistent with direct G alpha i-INFAIP8 coupling. Overexpression or depletion of TNFAIP8 using antisense constructs in Balb-D2S cells did not affect D2S-induced signaling to G alpha i-dependent inhibition of cAMP. In contrast, antisense depletion of TNFAIP8 completely inhibited spontaneous and D2S-induced foci formation, consistent with a role for TNFAIP8 in G alpha i-dependent transformation. To address possible mechanisms, the effect of D2S signaling via TNFAIP8 on TNF alpha action was examined. D2S receptor activation inhibited TNF alpha-induced cell death in Balb-D2S cells, but not in cells depleted of TNFAIP8. However, depletion of TNFAIP8 did not prevent D2S-induced inhibition of TNF alpha-mediated caspase activation, suggesting that D2S/TNFAIP8-induced protection from TNF alpha-induced cell death is caspase-independent. The data suggest that G alpha i-TNFAIP8-mediated rescue of pre-oncogenic cells enhances progression to oncogenic transformation, providing a selective target to inhibit cellular transformation. J. Cell. Physiol. 225: 865-874, 2010. (C) 2010 Wiley-Liss, Inc.