【作者】 王萱；

【导师】 林圣彩；

【作者基本信息】 厦门大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 Daxx是一种重要的细胞死亡相关因子。近十年来的研究表明，Daxx通过其不同的功能结构域与多种参与细胞生理生化过程的蛋白相互作用而形成多蛋白复合体。这些多样的蛋白复合体，涉及了细胞增殖、生长与分化，细胞凋亡，转录调控以及病毒复制等许多细胞复杂的生命活动。尽管获得了大量实验数据，但是，对Daxx在诱导细胞凋亡的具体作用机理方面，至今仍存在一些争议。其中，有关系统揭示Daxx如何通过p53信号途径诱导细胞死亡的课题亟待研究。故此，本论文采用生物化学与分子生物学方法，系统阐明Daxx协同Axin／HIPK2／p53三聚复合体活化p53信号通路的分子机制及作用模型，从而强调出Daxx在促细胞凋亡方面的重要功能。本论文研究通过酵母双杂交实验发现了Daxx与Axin之间的相互作用。免疫共沉淀实验进一步验证了内源性的Daxx和Axin能在体内直接结合。并且，二者之间的这种相互作用，在细胞接受UV刺激条件下会明显增强。一系列实验结果均表明Daxx不能直接结合p53，但是Axin作为“桥梁”介导了Daxx与p53的结合。之后的研究证实了Daxx／Axin／p53复合体的作用，即尽管Daxx能够单独活化与HIPK2结合的p53，但Axin明显加强了这一作用，说明Daxx／Axin／HIPK2／p53复合体的形成能够导致p53活性的最大化，其功能相关性进一步被RNA干扰实验证明。而且，Daxx调节的细胞生长抑制依赖于功能性的Axin与p53，因为在克隆斑形成实验中，Daxx不能诱导Axin缺失细胞和p53缺失细胞的细胞死亡。敲除Daxx或Axin严重削弱了UV或p53诱导的凋亡，与Daxx／Axin／HIPK2／p53复合体能够诱导p53活性最大化的实验结论一致。有趣的是，Daxx和Axin似乎选择性地活化p53的靶向基因，强烈活化PUMA基因，而非p21和Bax基因。本论文还发现Daxx和Axin能够诱导细胞色素c的释放，这与两种蛋白均能诱导PUMA基因活化相吻合。所有这些结果显示出，Daxx能够调节Axin／HIPK2／p53复合体诱导细胞死亡，阐明了Daxx调控肿瘤抑制子p53活化以及发挥促凋亡功能的新机理，也提示出Daxx在DNA严重损伤条件下诱导细胞死亡的分子机制。Pirh2是一个RING结构域蛋白，先前已知它能够与p53相互作用，并作为其E3连接酶负调节p53的稳定性。在本论文研究中，我们通过酵母双杂交筛选，已确定Pirh2作为一个新的Axin相互作用蛋白。目前证据显示，Pirh2能够通过破坏基于Axin的p53活化复合体的形成而抑制UV刺激响应下的p53活化。通过结合Axin，Pirh2促使HIPK2从Axin复合体上解离，抑制了HIPK2蛋白激酶对p53的作用。不过，我们在研究中用Mdm2作为阳性对照，未发现Pirh2对p53显示出任何E3连接酶活性。当与Pirh2共转染时，p53的稳定水平并没有发生改变。并且，丧失E3活性的Pirh2突变体Pirh2 C145A未能削弱其对Axin或UV诱导下的p53第46位磷酸化的抑制功能。这些研究结果与先前的报道存在明显差异。此外，我们发现siRNA沉默Pirh2表达能够增强HIPK2与Axin的免疫共沉淀，以及Axin或UV诱导下的p53第46位磷酸化。结合之前的研究结果，我们发现在刺激响应下Pirh2能够通过基于Axin的活化复合体而调节p53信号通路，并不依赖于其E3连接酶活性。更多还原

【Abstract】 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.更多还原