Defining components of the ß-catenin destruction complex and exploring its regulation and mechanisms of action during development.

BACKGROUND: A subset of signaling pathways play exceptionally important roles in embryonic and post-embryonic development, and mis-regulation of these pathways occurs in most human cancers. One such pathway is the Wnt pathway. The primary mechanism keeping Wnt signaling off in the absence of ligand is regulated proteasomal destruction of the canonical Wnt effector ßcatenin (or its fly homolog Armadillo). A substantial body of evidence indicates that SCF(ßTrCP) mediates ßcat destruction, however, an essential role for Roc1 has not been demonstrated in this process, as would be predicted. In addition, other E3 ligases have also been proposed to destroy ßcat, suggesting that ßcat destruction may be regulated differently in different tissues. METHODOLOGY/PRINCIPAL FINDINGS: Here we used cultured Drosophila cells, human colon cancer cells, and Drosophila embryos and larvae to explore the machinery that targets Armadillo for destruction. Using RNAi in Drosophila S2 cells to examine which SCF components are essential for Armadillo destruction, we find that Roc1/Roc1a is essential for regulating Armadillo stability, and that in these cells the only F-box protein playing a detectable role is Slimb. Second, we find that while embryonic and larval Drosophila tissues use the same destruction complex proteins, the response of these tissues to destruction complex inactivation differs, with Armadillo levels more elevated in embryos. We provide evidence consistent with the possibility that this is due to differences in armadillo mRNA levels. Third, we find that there is no correlation between the ability of different APC2 mutant proteins to negatively regulate Armadillo levels, and their recently described function in positively-regulating Wnt signaling. Finally, we demonstrate that APC proteins lacking the N-terminal Armadillo-repeat domain cannot restore Armadillo destruction but retain residual function in negatively-regulating Wnt signaling. CONCLUSIONS/SIGNIFICANCE: We use these data to refine our model for how Wnt signaling is regulated during normal development.

A novel ecdysone receptor mediates steroid-regulated developmental events during the mid-third instar of Drosophila.

The larval salivary gland of Drosophila melanogaster synthesizes and secretes glue glycoproteins that cement developing animals to a solid surface during metamorphosis. The steroid hormone 20-hydroxyecdysone (20E) is an essential signaling molecule that modulates most of the physiological functions of the larval gland. At the end of larval development, it is known that 20E--signaling through a nuclear receptor heterodimer consisting of EcR and USP--induces the early and late puffing cascade of the polytene chromosomes and causes the exocytosis of stored glue granules into the lumen of the gland. It has also been reported that an earlier pulse of hormone induces the temporally and spatially specific transcriptional activation of the glue genes; however, the receptor responsible for triggering this response has not been characterized. Here we show that the coordinated expression of the glue genes midway through the third instar is mediated by 20E acting to induce genes of the Broad Complex (BRC) through a receptor that is not an EcR/USP heterodimer. This result is novel because it demonstrates for the first time that at least some 20E-mediated, mid-larval, developmental responses are controlled by an uncharacterized receptor that does not contain an RXR-like component.