An in vivo kinome and phosphatome RNAi screen in the Drosophila wing imaginal disc identifies a novel regulator of Wnt/Wg secretion. Tirthadipa Pradhan, Sharan Swarup, Esther Verheyen. Simon Fraser Unversity, Burnaby, Canada.

   Wingless (Wnt/Wg) proteins are secreted molecules which act in an evolutionary conserved pathway to regulate cell proliferation and cell fate specification. The key step in the pathway is the regulation of the levels of cytoplasmic -catenin. -catenin acts as a transcriptional regulator, which upon pathway activation accumulates in the cytoplasm and subsequently translocates to the nucleus where it interacts with the Tcf/Lef family of transcription factors to direct target gene expression. In the absence of the Wnt/Wg, the levels of -catenin are kept low in the cytoplasm through constitutive degradation via a protein destruction complex composed of Axin, Adenomatous Polyposis Coli (APC), Glycogen synthase kinase-3 (GSK-3), Casein kinase1 (CK1).
   Phosphorylation events are known to regulate multiple steps of the Wnt/Wg pathway. The key components such as -catenin, Dishevelled, LRP5/6, APC, Axin and TCF are phosphorylated in the pathway. The ubiquitous kinases GSK-3 and CKI and phosphatases such as PP1 and PP2 regulate multiple steps of these phosphorylations by distinct mechanisms. However the significance of most of these phophorylation events is not well understood. To fill the gap in our knowledge we did an in vivo RNAi screen in the Drosophila wing imaginal disc. Our screen has yielded a number of novel regulators of the Wnt/Wg pathway. Subsequent characterization of the one of the phosphatases by loss of function and overexpression analysis revealed its novel role in Wnt/Wg secretion. We found that in its absence Wg gets trapped in the secreting cells. Furthermore, loss of this phosphatase causes reduction in Wntless (Wls) levels in vivo. We are in the process of performing further genetic and biochemical interaction studies with the members of Wnt/Wg secretion machinery. Taken together, our data provides new insight into novel regulators of Wnt/Wg pathway and a better understanding of Wnt/Wg secretion.