Jia Hongge, Liu Yajuan, Yan Wei, Jia Jianhang
Sealy Center for Cancer Cell Biology, Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA.
Development. 2009 Jan;136(2):307-16. doi: 10.1242/dev.030015. Epub 2008 Dec 15.
The seven-transmembrane protein Smoothened (Smo) and Zn-finger transcription factor Ci/Gli are crucial components in Hedgehog (Hh) signal transduction that mediates a variety of processes in animal development. In Drosophila, multiple kinases have been identified to regulate Hh signaling by phosphorylating Smo and Ci; however, the phosphatase(s) involved remain obscured. Using an in vivo RNAi screen, we identified PP4 and PP2A as phosphatases that influence Hh signaling by regulating Smo and Ci, respectively. RNAi knockdown of PP4, but not of PP2A, elevates Smo phosphorylation and accumulation, leading to increased Hh signaling activity. Deletion of a PP4-interaction domain (amino acids 626-678) in Smo promotes Smo phosphorylation and signaling activity. We further find that PP4 regulates the Hh-induced Smo cell-surface accumulation. Mechanistically, we show that Hh downregulates Smo-PP4 interaction that is mediated by Cos2. We also provide evidence that PP2A is a Ci phosphatase. Inactivating PP2A regulatory subunit (Wdb) by RNAi or by loss-of-function mutation downregulates, whereas overexpressing regulatory subunit upregulates, the level and thus signaling activity of full-length Ci. Furthermore, we find that Wdb counteracts kinases to prevent Ci phosphorylation. Finally, we have obtained evidence that Wdb attenuates Ci processing probably by dephosphorylating Ci. Taken together, our results suggest that PP4 and PP2A are two phosphatases that act at different positions of the Hh signaling cascade.
七次跨膜蛋白Smoothened（Smo）和锌指转录因子Ci/Gli是刺猬信号通路（Hh）信号转导的关键组成部分，该信号通路介导动物发育中的多种过程。在果蝇中，已鉴定出多种激酶通过磷酸化Smo和Ci来调节Hh信号；然而，所涉及的磷酸酶仍不清楚。通过体内RNA干扰筛选，我们鉴定出PP4和PP2A分别作为通过调节Smo和Ci影响Hh信号的磷酸酶。RNA干扰敲低PP4而非PP2A会提高Smo磷酸化和积累，导致Hh信号活性增加。删除Smo中PP4相互作用结构域（氨基酸626 - 678）会促进Smo磷酸化和信号活性。我们进一步发现PP4调节Hh诱导的Smo细胞表面积累。从机制上讲，我们表明Hh下调由Cos2介导的Smo - PP4相互作用。我们还提供证据表明PP2A是Ci磷酸酶。通过RNA干扰或功能缺失突变使PP2A调节亚基（Wdb）失活会下调全长Ci的水平及其信号活性，而过度表达调节亚基则会上调。此外，我们发现Wdb可对抗激酶以防止Ci磷酸化。最后，我们获得证据表明Wdb可能通过使Ci去磷酸化来减弱Ci的加工过程。综上所述，我们的结果表明PP4和PP2A是在Hh信号级联反应不同位置起作用的两种磷酸酶。
