Biochemical analysis of Wnt/Wingless signal transduction pathway with tissue culture system

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The Wnt family of secretory protein plays pivotal roles in a number of basic developmental processes. Components of the Wnt/Wingless (Wg) signaling pathway are Frizzled, Dishevelled, Glycogen synthase kinase-38, Axin, APC tumor suppressor, β-catenin, and T-cell transcription factor. These genes conserved in vertebrates and invertebrates. Using cell culture assay for Wnt/Wg that we have established, we are analyzing biochemical interactions among these components of the Wnt/Wg pathway. Casein kinase I (CKI) was recently reported as a positive regulator of Wnt signaling in vertebrates and C. elegans (Peters, J. M. et al : Nature 1999 401 : 345-350) To elucidate the function of Drosophila CKI in the wingless (wg) pathway, we have disrupted its function by double-stranded RNA-mediated interference (RNAi). While previous studies mainly based on CKI overexpression, this is the first convincing loss-of-function approach of CKI. Surprisingly, CKI_α- or CKI_ε-RNAi markedly elevated the Armadillo (Arm) protein levels in Drosophila Schneider S2R+ cells, without affecting its mRNA levels. Pulse-chase analysis showed that CKI-RNAi indeed stabilizes Arm protein. Moreover Drosophila embryos injected with CKI_α-double-stranded RNA showed a naked cuticle phenotype, which is associated with activation of Wg signaling. These results indicate that CKI functions as a negative regulator of Wg/Arm signaling. Overexpression of CKI_α induced hyper-phosphorylation of both Arm and Dishevelled in S2R+ cells and conversely, CKI_α-RNAi reduced the amount of hyper-modified forms. His-tagged Arm was phosphorylated by CKI_α in vitro on a set of serine and threonine residues that are also phosphorylated by Zeste-white 3. Thus, we propose that CKI phosphorylates Arm and stimulates its degradation.