TI - Discussion and conclusion . AB - In this study we have shown that the small size Zfra regulates TNF -mediated cell death via directly binding with TRADD , NF-kappaB , JNK1 , p-ERK and WOX1 , and indirectly with FADD , RIP and p53 ( Figure 12 ) . Upon binding with p55 TNF receptor ( p55-TNFR ) , TNF engages in two signaling pathways for either protecting cells from death or committing cells to death . For initiating cell death , p55-TNFR recruits TRADD , FADD and RIP to generate a death-inducing signaling complex ( DISC ) (Figure 12A) . Caspase 8 is then recruited to the DISC and becomes activated , followed by activating downstream caspases to induce apoptosis at both the mitochondrial and nuclear levels [16 , 17] . We determined that Zfra interacts with TRADD but not FADD or RIP (Figure 12A) . Zfra affects the cytotoxic function of these death domain proteins via direct and indirect manners ( open arrow ) . Zfra physically interacts with WOX1 , and that WOX1 binds TRADD . Thus , Zfra and WOX1 are likely to be recruited to the DISC during TNF signaling ( Figure 12A ) . This assumption has yet to be validated by co-immunoprecipitation and confocal and immunoelectron microscopy . Previously we have shown that WOX1 enhances TNF cytotoxicity [3] . WOX1 also enhances the cytotoxic function of TRADD [3] . Here we determined that both WOX1 and Zfra counteract with each other in regulating apoptosis . Zfra either enhances or inhibits the function of death domain proteins . Thus , the driving force for committing cells to death in response to TNF is likely coming from a balanced and counter-balanced work among Zfra , WOX1 and DISC . In the TNF-initiated protective pathway , Zfra is shown to interact with TRADD , JNK1 and NF-kappaB ( Figure 12B ) . Thus , formation of Zfra-containing regulatory complexes probably occurs in the TNF signaling cascade . In Step 1 , at the membrane level , Zfra binds TRADD in the presence of TRADD , TRAF2 , RIP and WOX1 . In Step 2 , a trimolecular complex of Zfra/JNK1/WOX1 may form when JNK1 becomes activated by the upstream activated MEK . Zfra binds and counteracts the apoptotic function of JNK1 . Also , JNK1 counteracts the apoptotic function of WOX1 [6] . In Step 3 , MEK activates ERK , and that Zfra may bind and sequester ERK to the cytoplasm . In Step 4 , PHOSphorylation of IkappaBalpha by IKK causes degradation of IkappaBalpha and release of NF-kappaB for nuclear translocation . Again , Zfra is able to bind and sequester NF-kappaB in the cytoplasm . In Step 5 and 6 , p53 is a downstream effector of TNF signaling [8,22-24] . TNF induces NF-kappaB activation , and then NF-kappaB activates p53 [22] . The non-ankyrin C-terminus of IkappaBalpha physically interacts with cytosolic p53 to prevent degradation in vivo , and the complex dissociates in response to TNF and apoptotic stress [25] . p53 is functionally associated with WOX1 , and both proteins may induce apoptosis synergistically [3,4,6,8] . Thus , an in vivo complex of IkappaBalpha/p53/WOX1 is likely , and that Zfra may regulate the formation of this complex . We show that ectopic Zfra blocks UV light-induced p53 nuclear translocation or activation , suggesting a negative regulation for the cell death event . Ser46 -PHOSphorylated p53 is known to play a critical role in apoptosis [8,25-29] . That is , Zfra may prevent cell death by blocking the apoptotic function of Ser46 -PHOSphorylated p53 . The IkappaBalpha/p53/WOX1 or p53/WOX1 may translocate to the mitochondria . Whether this event is blocked by Zfra is unknown . Presence of two cysteine residues in the amino acid sequence of Zfra suggests the likely presence of a dimeric form in cells [10] . Also , whether Zfra covalently interacts with specific proteins remains to be established . As determined by GST-pull down analysis , both TNF and UV light are able to increase self-association of Zfra , supporting the presence of dimers in vivo [10] . Zfra is inducible under stress conditions [10] . We show that at low levels Zfra enhances cell growth , and yet overexpressed Zfra induces cell death . Ectopic Zfra also blocked adherence -independent growth of L929 fibroblasts , suggesting that it may act as a tumor suppressor to block cancer growth and progression . Zfra is most abundant in the spleen , whereas its role in regulating immune cell differentiation and functions is unknown . However , it is reasonable to suggest that Zfra -regulated TNF signaling is likely to play a role in the lymphocyte differentiation . We also determined that absent expression of Zfra is found in several breast and prostate cancer cell lines [10] , implying that Zfra deficiency may provide a growth advantage for cancer cells . Majority of zinc finger proteins participate in gene transcription and embryonic development [30-32] . Synthetic peptides have been shown promises in therapy to modulate the transcriptional activities of endogenous zinc finger proteins [33,34] . Shortening and modification of the naturally occurring Zfra may be of therapeutic value in controlling cancer cell growth and death . Zfra is distributed ubiquitously in cellular compartments . In response to TNF , a portion of cytosolic Zfra appears to translocate to the cell membrane area and binds TRADD . Death domain/death domain interaction is responsible for TRADD binding with p55-TNFR , FADD and RIP . Whether Zfra binds to the death domain is unknown . Sequence alignment analysis shows a common structural motif in TRADD , JNK1 , NF-kappaB , and the N-terminal WW domain and C-terminal SDR domain of WOX1 . Whether this motif interacts with Zfra remains to be established . WWOX/WOX1 is known to interact with p53 , p73 , JNK1 , AP-2gamma and ErbB4 [3 , 6 , 35-37] , suggesting its role of the regulation of gene transcription . Both in vivo and in vitro studies have shown that overexpressed WWOX1/WOX1 suppresses tumor growth in nude mice , and induces apoptosis in cultured cells [3,6,8,9,38,39] . Overexpressed WW and SDR/C domains induce cell death [3] . Most interestingly , the C-terminal tail of WOX1 is needed to work together with SDR in causing cell death . In stark contrast , non-invasive breast and prostate cancers may have upregulated expression of WWOX/WOX1 , Tyr33 PHOSphorylation and WWOXv2/WOX2 ( and other family proteins if present ) [40-42] . Downregulation of WOX1 and WOX2 promotes neurodegeneration in Alzheimer's disease , suggesting its protective role against neurodegeneration [43] . FOR (WWOX/WOX1) is shown to protect against the effects of ionizing radiation in Drosophila [44] . These observations support a pro-survival role of WOX1 in vivo [4] . In domain/domain mapping by yeast two hybrid analysis , Zfra was shown to bind the N-terminal Tyr33 -PHOSphorylated WW domain of WOX1 . Alteration of this PHOSphorylation site abolishes the binding interaction . Also , it interacts with the C-terminal SDR domain of WOX1 . Since Zfra is a small peptide , its simultaneous binding to these domains in WOX1 under physiologic and stress conditions is very likely . Our supporting evidence shows that stress stimuli induce PHOSphorylation of WOX1 at Tyr33 and Zfra at Ser8 , and that the Zfra/WOX1 complex co-translocates to the mitochondria to regulate cell death via the mitochondrial pathway . Also , in this study we found that Zfra enhances the apoptotic activity of SDR domain and the C-terminal tail of WOX1 . Overexpressed Zfra may nullify the apoptotic activities of WOX1 and p53 . An apparent mechanism is that overexpressed Zfra sequesters transcriptional factors in the cytoplasm . For example , ectopic Zfra restricts nuclear localization of endogenous WOX1 , p53 , ERK and NF-kappaB ( p65 ) , indicating that an upregulated level of Zfra may control the transcriptional system in cells under stress conditions . Taken together , we have identified a molecular pathway underlying Zfra regulation of TNF cytotoxic function . Zfra interacts with TRADD and binds downstream NF-kappaB , p53 , JNK1 and WOX1 of the TNF signal pathway , thereby either enhancing or restricting TNF -mediated cell death .