M.S. in Biology 2009

Inappropriate activation of the Wingless (Wg)/Wnt signaling pathway occurs in many human cancers. Wg signaling stabilizes the key effector, Armadillo (Arm) which otherwise is a substrate for ubiquitin-mediated proteasomal destruction. A major advance in understanding regulation of Arm stability was the discovery that inactivation of the Drosophila F-box protein Slimb (mammalian ßTrCP) activates Wnt signaling and stabilizes Arm. This suggested that Arm is ubiquitinated by an SCF-class E3 ubiquitin ligase. SCF complexes consist of a Skp substrate adaptor, a Cullin scaffold, an F-box protein, and a RING-finger protein of the Roc1/Rbx1 family. Published data supports this model of SCF-Slimb-mediated ubiquitination of Arm, yet two sets of data suggest that Arm degradation is more complex. First, the major fly Roc1, Roc1a, is not essential for Arm degradation in wing imaginal discs, although it does mediate destruction of the Hedgehog effector Ci.

This suggests that a different Roc protein may contribute to the E3 ligase, Roc proteins may function redundantly, or E3 ligases containing other RING-finger proteins may target Arm for degradation in some tissues. The RING-finger protein Sina/Siah is one candidate, as Siah mediates p53-dependent Arm/ß-cat degradation. Second, published data suggest that loss of Slimb and Axin in larval tissues trigger high level accumulation of Arm, while our data suggest that loss of both APCs has a less dramatic effect on Arm levels, raising the possibility that multiple mechanisms of Arm regulation exist. We are exploring mechanisms of Arm regulation in Drosophila using three strategies. We are performing a small-scale RNAi screen of all Drosophila Skps, Cullins, Rocs, and F-box proteins in S2 cells to define the molecular components of the Arm E3 ligase. Second, we are evaluating potential redundancy and tissue-specific requirements of different RING-finger proteins. Finally, we are analyzing mitotic clones in the wing disc and brain to look at Arm accumulation after mutation of potential Arm E3 ligase components.