Biochemical analysis of the Nemo kinase and its role in modulating Wg signaling. A.Y. Zeng 1, D. Bessette 1, K. Charish 1, I. Mirkovic 1, C.J. Gottardi 2, E.M. Verheyen 1. 1) Dept. of Molecular Biology & Biochemistry, Simon Fraser University, Burnaby, BC, Canada; 2) Dept. of Cellular Biochemistry & Biophysics, Memorial Sloan Kettering Cancer Research Centre, New York, NY.
Drosophila Nemo is the founding member of an evolutionarily conserved novel family of serine/threonine protein kinases most similar to MAPKs. Biochemical and genetic studies in Drosophila, C. elegans and vertebrates have uncovered functional roles for Nemo homologs in several Wnt-dependent processes. Murine Nlk can phosphorylate TCF and inhibit the binding of the b-catenin/TCF transcriptional complex to DNA. C. elegans Lit-1 both requires Wrm-1 for its activity, and phosphorylates itself, POP-1 and Wrm-1. This regulates the cellular localisation of Pop-1 in what are considered to be two non-canonical variants of Wnt pathways in C. elegans. The TGFb activated kinase Tak1 has been shown to activate the kinase activity of Nemo homologs, and is currently the only known regulator of Nemo homologs.
We have previously shown genetic evidence that in Drosophila Nemo antagonizes the Wg pathway in wing development. To further investigate the role of Nemo in regulating Wg signaling, we have taken several approaches. We find that modulation of Nemo expression affects the Wg-dependent expression of wg and engrailed. In addition, we have carried out biochemical studies that demonstrate that Drosophila Nemo can bind to Armadillo and two vertebrate-catenins. We will present the results of kinase assays that are aimed at determining whether Nemo can autophosphorylate, as has been shown for Nlk, but not for Lit-1. We are also investigating what extracellular factors can activate the Nemo kinase activity, including previously characterized activators of MAPKs. Our studies will hopefully provide insight to the regulatory crosstalk between TGFb and Wnt signalling modulated by Nemo kinases.