【作者】 卢再恋；

【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， 2009， 博士

【摘要】 Axin是一个多结构域的构架蛋白，它能通过与不同蛋白的相互作用调节不同的信号通路，其中包括Axin能通过促进β-catenin的降解下调Wnt信号；同源二聚化的Axin能通过MEKK1或MEKK4激活JNK磷酸化；Axin也能通过HIPK2激活p53第46位丝氨酸的磷酸化；最近的研究发现Axin还能通过Arkadia增加Smad7的降解增强TGF-β信号等。Axin通过对不同信号的调节参与调控生物个体发育、抑制肿瘤发生、参与细胞骨架重排等过程。鼠源Axin是由鼠的Fused（Fu）基因编码；Axin^Fu等位基因是由转座子IAP的插入产生的。Axin^Fu/Fu小鼠会出现胚胎致死、神经管分叉和不同程度尾巴卷曲等各种表型。目前的研究在Axin^Fu小鼠体内检测到了截短的Axin mRNA，但并没有相关突变蛋白的报道。Axin^Fu小鼠体内是不是真实存在Axin的突变蛋白，Axin^Fu小鼠的的表型是不是由于Axin突变的蛋白的存在引起的，如果是，又是通过怎样的途径引起的，这些问题目前还不清楚。本论文通过生物化学和分子生物学的方法，系统地阐明了Axin突变蛋白的存在及其在生物体内的重要功能。本论文首先用免疫沉淀的方法证明Axin^Fu小鼠体内确实存在Axin截短的突变体。该突变体只包含Axin第1-596个氨基酸，将其命名为Axin^Fu-NT。实验的研究发现Axin^Fu-NT能够像野生型Axin一样下调LEF-1和TOPFLASH的转录活性；与Wnt信号的主要调节因子的免疫共沉淀实验显示，Axin^Fu-NT能有效形成β-catenin的降解复合体；通过Axin和Axin^Fu-NTmRNA的斑马鱼胚胎注射实验也观察到β-catenin的靶向基因boz和tbx6都受到了类似程度的下调。这些说明Axin^Fu-NT对Wnt信号的调节与Axin没有明显的差异。本论文在研究过程中还发现Axin^Fu-NT不但不能激活JNK(这可能与Axin^Fu-NT缺失了Axin的C端有关)，还能强烈抑制Axin激活JNK。进一步的研究发现Axin^Fu-NT存在形成同源二聚化的结构域，Axin^Fu-NT通过该结构域能破坏Axin形成同源二聚体，从而起到抑制Axin激活JNK的作用；而且Axin^Fu-NT能竞争Axin与MEKK1或MEKK4结合，这可能是Axin^Fu-NT抑制Axin激活JNK的另一分子机制。在斑马鱼体内检测Axin^Fu-NT的生物学功能的实验也证明了Axin^Fu-NT能抑制β-catenin和JNK信号，从而获得了腹部化的表型。本论文还检测了Axin^Fu-NT对p53磷酸化的影响。实验的研究发现Axin^Fu-NT不影响p53-luc的转录活性但能强烈抑制Axin增强的p53-luc的转录活性；p53磷酸化的实验也证明了Axin^Fu-NT不能增强p53第46位丝氨酸的磷酸化(这可能与Axin^Fu-NT缺失了HIPK2的结合域有关)但能强烈抑制Axin促进p53第46位丝氨酸的磷酸化。Axin^Fu-NT缺失p53结合区域的突变体Axin^Fu-NTΔMID不干扰Axin促进p53第46位丝氨酸的磷酸化作用。因此，Axin^Fu-NT不能与HIPK2相互作用但能竞争Axin与p53结合可能是Axin^Fu-NT抑制Axin促进p53第46位丝氨酸的磷酸化作用的分子机制。与此相一致的是，Axin^Fu-NT能抑制Axin通过促进p53磷酸化引起的细胞凋亡。总之，Axin^Fu-NT影响了Axin在多条信号通路中的重要调节作用，Axin^Fu小鼠的表型可能是Axin对多条信号通路调节受到影响的共同作用结果。更多还原

【Abstract】 Axin is a multidomian scaffold protein, regulating many signaling pathwaysthrough interacting with different regulators. Axin can promote degradation ofβ-catenin to down-regualte Wnt signaling; homodimeric Axin can induce JNKactivation through MEKK1 or MEKK4; Axin can also up-regulate HIPK2-mediatedphosphorylation of p53 at Ser46. Through regulating multiple signaling pathways,Axin plays important roles in development, suppression of tumorgenesis, cytoskeletonrearrangement and so on. Axin was originally identified from the characterization ofthe mouse Fused （Fu） locus; the Axin^Fu allele is caused by the insertion of an IAPtransposon. Axin^Fu/Fu mice display varying phenotypes ranging from embryoniclethality to relatively normal adulthood with kinky tails. Aberrant mRNA species ofAxin from Axin^Fu mouse was previously identified; however, the mutant proteinproduct（s） had not been characterized. In particular, it was unclear how thephenotypes of Axin^Fu mouse are caused by the mutant protein products. In this thesis,the Axin mutant protein was identified by immunoprecipitation using brain extractsfrom Axin^Fu mice with specific antibodies. The mutant protein is a truncated Axincontaining amino acids 1-596, which is designated as Axin^Fu-NT. When tested forfunction, Axin^Fu-NT exhibits no difference in the inhibition of Wnt signaling comparedwith wild type Axin as determined by LEF or TOPFLASH reporter gene assay,interaction with key Wnt regulators or detection ofβ-catenin target genes inzebrafish embryos. However, Axin^Fu-NT was found to abolish Axin-mediatedactivation of JNK, which plays a critical role in dorsoventral patterning. Together withGST pull-down assay, it was found that Axin^Fu-NT contains a previouslyuncharacterized dimerization domain and can form a heterodimeric interaction withwildtype Axin; it was also found that Axin^Fu-NT can compete with Axin in binding toMEKK1 or MEKK4. Consistently, Axin^Fu-NT can ventralize zebrafish embryos byantagonizing JNK activation. In the same study, it was found that Axin^Fu-NT, whichcan interact with p53 but not HIPK2, can inhibit Axin-induced phoshorylation of p53at Ser46, but Axin^Fu-NT△MID, which was deleted of its p53 binding domain, has no effect on Axin-induced phoshorylation of p53 Ser46. It’s suggested that Axin ^Fu-NT canattenuate p53 activation by sequestering p53. Consistently, Axin^Fu-NT can abolishAxin-mediated cell apoptosis. In summary, Axin^Fu-NT can dominant negatively affectthe function of Axin in many signaling pathways, and the phenotypes of Axin^Fu micemay be caused by perturbation of more than one signaling pathway.更多还原