Mechanism For Cell Death Induced By Axin-based P53 Activation Complex

Axin and p53 are tumor suppressors,controlling cell growth,apoptosis,anddevelopment.Axin has emerged as a major scaffold for many pathway,including Wntpathway,TGFβpathway and JNK signaling.p53 is positively or negatively regulatedby numerous stress signals including UV light,chemicals,and oncogenic stresses.The p53 activation mediated by many posttranslational modifications on multiple sites,and even in different combinations.Remarkably,depletion of Axin can attenuate p53signaling as assayed for p53-dependent transcription of reporter genes and apoptosisin response to UV light.Daxx,a death domain associated protein,has been implicatedin proapoptosis,antiapoptosis,and transcriptional regulation.Although it is clear thatDaxx may exert multiple functions,the underlying mechanisms remain far from clear.In fact,many initial reports remain controversial.A potential anti-apoptotic functionof Daxx came from Daxx knockout mouse embryos which display increased globalapoptosis.In contrast,pro-apoptotic functions of it have been obtained with tumor cellor transformed cells treated with diverse stimuli,such as UV,TGFβ,hydrogenperoxide,interferon-γand arsenite trioxide.In my present study,I show that Axin,originally identified for its scaffolding role to controlβ-catenin levels in Wntsignaling,strongly associates with Daxx at endogenous levels.Upon UV irradiation,Axin is translocated into the nucleus whereby it forms a large complex with Daxx andtwo pools of p53 (one directly bound to Axin,and the other occupied by HIPK2).Axin serves as a tethering factor for Daxx to associate with the tumor suppressor p53,and cooperates with Daxx,but not DaxxΔAxin,which is unable to interact with Axin,to stimulate HIPK2-mediated Ser46 phosphorylation and transcriptional activity ofp53.The Daxx/Axin/HIPK2/p53 complex formation is also enhanced by UV irradiation which indicates that the complex is physiologically regulated.Interestingly,Axin and Daxx seem to selectively activate certain target genes of p53,with strongactivation of PUMA,but not p21 or Bax.Daxx stimulated p53 transcriptional activitywas significantly diminished by small interfering RNA against Axin;Daxx fails toinhibit colony formation in Axin^-/- cells.Moreover,UV-induced cell death wasattenuated by the knockdown of Axin and Daxx.All these results show that Daxxcooperates with Axin to stimulate p53,and implicate a direct role for Axin,HIPK2,and p53 in the proapoptotic function of Daxx.Pirh2,a RING-domain protein,was previously shown to interact with p53 andnegatively regulate its stability as an E3 ligase.Here I found that Pirh2 exerts itsinhibitory role by modulating the dynamic assembly of the Axin-based p53 activationcomplex.Pirh2 interacted with Axin that is a positive p53 regulator by stimulatingHIPK2-catalyzed phosphorylation at Ser46.Pirh2 strongly abrogated p53 function inboth transcriptional activation and apoptosis induction;however,I found that Pirh2did not exhibit any ubiquitin E3 ligase activity towards p53,and that its p53-bindingactivity is not required for p53 inhibition.Rather,Pirh2 could effectively preventinteraction of HIPK2 with the complex of Axin and p53,rendering the Axin-boundp53 unphosphorylated.Similarly,Pirh2 could reduce the levels of p53co-immunoprecipitated with HIPK2.Consistently,Pirh2 siRNA resulted in higherlevels of HIPK2 co-immunoprecipitated with Axin,and increased Axin- orUV-induced p53 phosphorylation.Thus,my studies have established that Pirh2 is apotent negative regulator for the Axin-based p53 activation network and implicated itin the UV-triggered p53 signaling independently of its E3-ligase activity,and haveemphasized the critical role of Axin as a scaffold for p53 activation in stress responses.In sum,my studies have revealed several aspects of mechanism for Axin regulatingp53 pathway.My finding will provide new insights into how the the Axin-based p53activation complex to induce maximal activation of p53 and to regulate cell death.Pirh2 as a new interacting protein with Axin is a negatively regulator for the complex.