Analysis of Casein kinase I function in the Wnt signal-mediated regulation of Armadillo family protein degradation.

Wnt家族的分泌蛋白在许多基本的发育过程中起着关键作用。此外，Wnt通路的许多成分的突变，如APC、Axin、β-catenin，都是致癌的。已经证实，通过泛素介导的降解调节ARM蛋白水平在Wg信号转导中起着核心作用。虽然Zust White 3(Zw3)介导的ARM磷酸化与其降解有关，但我们最近发现酪蛋白激酶Ia(CKIα)也使ARM磷酸化并诱导其降解。因此，CKIα是典型的WNT/Wg信号通路的一个非常强的负调控因子，但目前尚不清楚CKIα和ZW3以及ARM降解复合体的其他成分如何调节ARM对Wg的磷酸化。特别是，Wg信号是否抑制CKIα或Zw3介导的体内手臂磷酸化尚不清楚。为了澄清这些问题，我们使用特异性识别不同丝氨酸残基磷酸化的手臂的抗体，在果蝇S2R+细胞中进行了一系列基于RNA干扰(RNAi)的分析。这些分析表明，丝氨酸56位、苏氨酸52位、丝氨酸48位和丝氨酸44位的手臂磷酸化分别由CKIα和ZW3介导，而ZW3直接的手臂磷酸化需要CKIα介导的启动磷酸化。大新可刺激ZW3-，但不能刺激CKIα介导的手臂磷酸化。WG抑制Zw3介导的ARM磷酸化，但不抑制CKIα介导的ARM磷酸化，这表明WG信号中一个重要的调节步骤是ZW3介导的ARM磷酸化。此外，进一步的基于RNA干扰的Wg途径的其他方面的分析表明，Wg诱导的凌乱的磷酸化是由于CKIα，并且早老素和蛋白激酶A在调节果蝇组织培养细胞的ARM蛋白水平中发挥的作用很小。

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.