TI - Zfra physically interacts with TRADD , and binds to the N terminal first WW domain and C terminal SDR domain of WOX1 . AB - We have utilized a novel cytoplasm-based two hybrid analysis in mapping protein/protein interactions between domains and/or motifs [3,6,8] . Briefly , bait is expressed in the cytoplasm of yeast cells . When the bait physically interacts with a cell membrane-anchored target , mutant yeast cells grow in a selective medium at 37degC as a result of activation of Ras signal pathway [3,6,8] . We showed that Zfra interacted with WOX1 from yeast two hybrid library screening . To map the Zfra -binding domains in WOX1 , we determined that Zfra physically interacted with the N-terminal first WW domain of WOX1 ( Figure 6 ) . Alteration of the known phosphorylaTION site Tyr33 to Arg in this domain abrogated the binding ( Figure 6 ) , suggesting that Tyr33 PHOSphorylation is needed for WOX1 to interact with Zfra . During activation , WOX1 undergoes Tyr33 PHOSphorylation and nuclear translocation both in vitro and in vivo [3,6,8,9] . Zfra also bound the C-terminal SDR domain . This domain has a mitochondria-targeting region and Zfra did not bind to this region ( Figure 6 ) . In a negative control , empty vector versus empty vector was tested . In a positive control , self-binding of MafB is shown [3,6,8] ( Figure 6 ) . We also determined that Zfra physically interacted with the full-length TRADD but not RIP and WOX1 interacted with TRADD ( Figure 6 ) . Together , these observations suggest that Zfra and WOX1 may complex with TRADD , and this complex affects TNF -mediated cell death .