The Molecular Mechanisms That Axin^Fu-NT Encoded By Axin^Fu Allele Effects On Function Of Wildtype Axin

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.