Biochemical and structural studies of key components in the WNT signaling pathway

The canonical WNT signaling pathway plays an important role in embryonic development and certain cancer forms. The interactions of key components in this pathway are essential for many biological functions. beta-catenin is one of the central players which tightly regulates and interacts with numerous proteins including Adenomatous Polyposis Coli (APC), Tcf, E-cadherin and Axin.;APC is a critical tumor suppressor that regulates beta-catenin turnover and localization. APC gene mutations are associated with ∼85% of colorectal cancers. The interaction between APC and beta-catenin is critical for APC function. Previous biochemical studies indicate that beta-catenin could bind all of the three APC 15aa repeats and seven 20aa repeats. In order to reveal the structural basis of APC-beta-catenin interaction, I have analytically determined the affinity of beta-catenin to one 15aa repeat fragment and each of the seven 20 as repeats in both phosphorylated and unphosphorylated states. The results showed that different fragments have dramatically different binding affinities to beta-catenin. For every 20aa repeat, phosphorylation significantly increases its binding affinity for beta-catenin, suggesting phosphorylation has a critical regulatory role in APC function. The third 20aa repeat appeared to be the tightest binding site for beta-catenin among all the repeats. The fact that most APC mutations associated with colon cancers have lost the third 20aa repeat underlines its importance of APC-beta-catenin interaction in Wnt signaling and human diseases. In addition, CD and NMR studies demonstrated that the central region of APC is unstructured in the absence of beta-catenin and axin, and suggest that beta-catenin may interact with each of the APC 15aa and 20aa repeats independently.;In C.elegans, SYS-1 is a functional beta-catenin with no significant sequence similarity. Its discovery in 2005 added one new member to the already known beta-catenin family in C.elegans that includes BAR-1, HMP-2 and WRM-1. By determining the crystal structures of SYS-1 and its complex with POP-1, the C. elegans TCF homolog, I provided strong evidence that SYS-1 possesses signature features of canonical beta-catenin and the beta-catenin/TCF complex that could not be predicted from sequence analysis. Most importantly, SYS-1 bears 12 armadillo repeats and the SYS-1/POP-1 interface is anchored by a conserved salt-bridge, the "charged button". Based on structural information of SYS-1 and beta-catenin, I have modeled structures for three other C. elegans beta-catenins to predict the molecular basis of their distinct binding properties. Finally, we generated a phylogenetic tree, using the region of highest structural similarity between SYS-1 and beta-catenin, and found that SYS-1 clusters robustly within the beta-catenin clade. This finding confirmed our previous speculation that the novel SYS-1 protein belongs to the beta-catenin family and suggested that additional divergent beta-catenins await discovery.