George Ana, Leahy Hannah, Zhou Jianhua, Morin Peter J
Geriatric Research, Education, and Clinical Center, Edith Nourse Rogers Memorial Veteran's Administration Hospital, Bedford, MA 01730-1114, USA.
Neurobiol Dis. 2007 Apr;26(1):125-33. doi: 10.1016/j.nbd.2006.12.004. Epub 2007 Jan 17.
Endosomal acidification and transport are essential functions in signal transduction. Recent data suggest that Wnt signaling requires intact endosomal transport machinery but the effects of endosomal acidification on Wnt signal transduction have not been evaluated. Here we report that bafilomycin, a specific inhibitor of the vacuolar proton ATPase that blocks endosomal acidification, inhibits canonical Wnt signal transduction initiated by Wnt ligand and partially inhibits signaling initiated by disheveled. Bafilomycin does not affect Tcf promoter activation by beta-catenin. These data indicate that endosomal acidification is necessary for Wnt signaling. To identify interactions between endosomal transport proteins and Wnt receptors, we performed a GST fusion protein pulldown experiment and identified a possible indirect interaction between the LRP6 intracellular domain and vacuolar protein sorting protein 35 (VPS35). We show that an N-terminal deletion mutant of VPS35 reduces canonical Wnt signaling in HEK-293 cells expressing exogenous Wnt-1. These data suggest that endosomal V-type ATPase activity and retromer trafficking proteins are functionally important in Wnt signal transduction.
内体酸化和运输是信号转导中的重要功能。最近的数据表明，Wnt信号传导需要完整的内体运输机制，但内体酸化对Wnt信号转导的影响尚未得到评估。在此我们报告，巴弗洛霉素是一种阻断内体酸化的液泡质子ATP酶特异性抑制剂，它抑制由Wnt配体引发的经典Wnt信号转导，并部分抑制由散乱蛋白引发的信号传导。巴弗洛霉素不影响β-连环蛋白对Tcf启动子的激活。这些数据表明内体酸化是Wnt信号传导所必需的。为了确定内体运输蛋白与Wnt受体之间的相互作用，我们进行了一项GST融合蛋白下拉实验，并确定了低密度脂蛋白受体相关蛋白6（LRP6）胞内结构域与液泡蛋白分选蛋白35（VPS35）之间可能存在间接相互作用。我们表明，VPS35的N端缺失突变体在表达外源性Wnt-1的HEK-293细胞中会降低经典Wnt信号传导。这些数据表明，内体V型ATP酶活性和回收体运输蛋白在Wnt信号转导中具有重要功能。
