| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2004: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2003: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
The Wnt family of secretory protein plays pivotal roles in a number of basic developmental processes. In addition, numerous mutations in components of the Wnt pathway, such as apc, axin, β-catenin, are oncogenic. It is well established that regulation of Arm protein levels through ubiquitin-mediated degradation plays a central role in the Wingless (Wg) signaling. Although Zeste White 3(Zw3)-mediated Arm phosphorylation has been implicated in its degradation, we have recently shown that Casein kinase Ia(CKIα) also phosphorylates Arm and induces its degradation. Thus, CKIα is functioning as a very strong negative regulator of the canonical Wnt/Wg signaling pathway.
But it remains unclear how CKIα 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 CKIα- 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 using antibodies that specifically recognize Arm phosphorylated at different serine residues. These analyses revealed that Arm phosphorylation at serine56, and at threonine52, serine48, and serine44, is mediated by CKIα and Zw3, respectively and that Zw3-directed Arm phosphorylation requires CKIα-mediated priming phosphorylation. Daxin stimulates Zw3- but not CKIα-mediated Arm phosphorylation. Wg suppresses Zw3- but not CKIα-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 CKIα and that Presenilin and Protein kinaseA play little part in the regulation of Arm protein levels in Drosophila tissue culture cells.
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