Transcription to Trafficking: Novel Regulation of Wnt/Wingless (Wg) Pathway by Protein Phosphatases

Development from a single cell into a multicellular entity is controlled by the intricate regulation of different cell signalling molecules. Wnt/Wingless (Wg) is one such evolutionarily conserved molecule that plays a critical role in cell fate specification, tissue patterning and organ development. Aberrant signalling leads to many developmental defects and cancer. The Wg pathway is regulated by reversible phosphorylation both in its silent and active states. Although several studies have shown the role of various kinases and phosphatases in regulating distinct steps of the Wg pathway, the entire cascade of events that regulate the pathway still remains elusive. To identify novel regulators of the Wg pathway, we performed an in vivo RNAi screen in the wing disc of Drosophila. This screen identified several new kinase and phosphatase modulators of the Wg pathway. Further characterization uncovered two proteins, the endosomal protein Myopic (Mop) and the serine threonine phosphatase Protein Phosphatase 4 (PP4), which are essential for Wg pathway activity. Knockdown of mop caused Wg protein accumulation in both the Wg secreting and receiving cells. Loss of Mop caused reduced Wg secretion due to the accumulation of Wg and Wntless in endosomes of secreting cells. The defective secretion and aberrant accumulation of Wg was rescued by overexpression of the endosomal protein Hrs. The vertebrate homolog of Mop, HDPTP has similar roles in regulating Wnt trafficking in mammalian cells. In Wg receiving cells, mop knockdown causes accumulation of the Frizzled receptor in early endosomes. Loss of hrs phenocopies this effect on Fz. Histochemical and genetic analyses suggested that Mop protects Hrs from lysosomal degradation by both promoting its deubiquitination by Ubpy and inhibiting the ubiquitin ligase Cbl. Thus, Mop stabilises Hrs in the endosomes, which promotes trafficking of Wg pathway components both in the signal sending and receiving cells. My work provides useful insight on how Mop-Hrs-Ubpy regulates the endosomal trafficking and signalling output of the Wg pathway. The serine threonine phosphatase PP4 also plays an important role in the Wg pathway. PP4 influences Notch pathway-driven wg transcription. Knockdown of PP4 affects expression of Notch pathway components and impairs growth of Drosophila appendages. These defects were rescued by the overexpression of nuclear Notch. Together, these studies provide the first evidence implicating a role for Mop and PP4 in trafficking and transcription of Wg.