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Molecular and Cellular Biology, March 2004, p. 2012-2024, Vol. 24, No. 5
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.5.2012-2024.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Biochemical Characterization of the Drosophila Wingless Signaling Pathway Based on RNA Interference

Hiroko Matsubayashi,1,{dagger} Sonoka Sese,1,{dagger} Jong-Seo Lee,1,{ddagger} Tadaoki Shirakawa,1 Takeshi Iwatsubo,2 Taisuke Tomita,2 and Shin-ichi Yanagawa1*

Department of Viral Oncology, Institute for Virus Research, Kyoto University, Sakyo-Ku, Kyoto 606-85071,1 Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, University of Tokyo, Bunkyo-Ku, Tokyo 113-00332, Japan2

Received 2 July 2003/ Returned for modification 12 August 2003/ Accepted 25 November 2003

Regulation of Armadillo (Arm) protein levels through ubiquitin-mediated degradation plays a central role in the Wingless (Wg) signaling. Although zeste-white3 (Zw3)-mediated Arm phosphorylation has been implicated in its degradation, we have recently shown that casein kinase I{alpha} (CKI{alpha}) also phosphorylates Arm and induces its degradation. However, it remains unclear how CKI{alpha} and Zw3, as well as other components of the Arm degradation complex, regulate Arm phosphorylation in response to Wg. In particular, whether Wg signaling suppresses CKI{alpha}- or Zw3-mediated Arm phosphorylaytion in vivo is unknown. To clarify these issues, we performed a series of RNA interference (RNAi)-based analyses in Drosophila S2R+ cells by using antibodies that specifically recognize Arm phosphorylated at different serine residues. These analyses revealed that Arm phosphorylation at serine-56 and at threonine-52, serine-48, and serine-44, is mediated by CKI{alpha} and Zw3, respectively, and that Zw3-directed Arm phosphorylation requires CKI{alpha}-mediated priming phosphorylation. Daxin stimulates Zw3- but not CKI{alpha}-mediated Arm phosphorylation. Wg suppresses Zw3- but not CKI{alpha}-mediated Arm phosphorylation, indicating that a vital regulatory step in Wg signaling is Zw3-mediated Arm phosphorylation. In addition, further RNAi-based analyses of the other aspects of the Wg pathway clarified that Wg-induced Dishevelled phosphoylation is due to CKI{alpha} and that presenilin and protein kinase A play little part in the regulation of Arm protein levels in Drosophila tissue culture cells.

* Corresponding author. Mailing address: Department of Viral Oncology, Institute for Virus Research, Kyoto University, Shyo-goin-Kawahara-Cho, Sakyo-Ku, Kyoto 606-8507, Japan. Phone: 81-75-751-3996. Fax: 81-75-751-3995. E-mail: syanagaw{at}virus.kyoto-u.ac.jp.

{dagger} H.M. and S.S. contributed equally to this study.

{ddagger} Present address: Lab Frontier Co., Ewha University, 120-750 Seoul, Korea.

Molecular and Cellular Biology, March 2004, p. 2012-2024, Vol. 24, No. 5
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.5.2012-2024.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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