Phosphorylation of the Smo tail is controlled by membrane localisation and is dispensable for clustering
Phosphorylation of the Smo tail is controlled by membrane localisation and is dispensable for clustering
复制标题
DOI：
10.1242/jcs.128926
复制
发表时间：
2013-10
影响因子：
4
通讯作者：
Adam P. Kupinski;I. Raabe;Marcus Michel;Divya Ail;L. Brusch;T. Weidemann;Christian Bökel
中科院分区：
文献类型：
--
作者：
Adam P. Kupinski;I. Raabe;Marcus Michel;Divya Ail;L. Brusch;T. Weidemann;Christian Bökel
摘要 Hedgehog (Hh) 信号级联是高度保守的，并参与整个进化过程中的发育和疾病。然而，与其他途径相比，我们对 Hh 信号转导机制的理解还非常不完整。在没有配体的情况下，Hh 受体 Patched (Ptc) 通过未知机制抑制关键信号转导器 Smoothened (Smo)。 Hh 与 Ptc 结合减轻了这种抑制，导致 Smo 重新分布到质膜、磷酸化和 Smo 胞质尾部打开，以及 Smo 寡聚化。然而，人们对这些事件的顺序和相互依赖性还知之甚少。我们对两种替代途径激活模式的 Smo 激活进行了数学建模和模拟，其中 Ptc 主要影响 Smo 定位或磷酸化。通过一种新颖的基于荧光的报告基因可视化 Smo 激活，使我们能够测试这些竞争模型。在这里，我们表明，在 Ptc 存在的情况下，Smo 定位到质膜足以使细胞质尾部磷酸化。使用荧光互相关光谱 (FCCS)，我们还证明，无论 Smo 磷酸化如何，Hh 使 Ptc 失活都会诱导 Smo 聚类。因此，我们的观察结果支持 Hh 信号转导模型，其中 Smo 亚细胞定位而不是磷酸化是 Ptc 功能的主要目标。
Summary The Hedgehog (Hh) signalling cascade is highly conserved and involved in development and disease throughout evolution. Nevertheless, in comparison with other pathways, our mechanistic understanding of Hh signal transduction is remarkably incomplete. In the absence of ligand, the Hh receptor Patched (Ptc) represses the key signal transducer Smoothened (Smo) through an unknown mechanism. Hh binding to Ptc alleviates this repression, causing Smo redistribution to the plasma membrane, phosphorylation and opening of the Smo cytoplasmic tail, and Smo oligomerisation. However, the order and interdependence of these events is as yet poorly understood. We have mathematically modelled and simulated Smo activation for two alternative modes of pathway activation, with Ptc primarily affecting either Smo localisation or phosphorylation. Visualising Smo activation through a novel, fluorescence-based reporter allowed us to test these competing models. Here, we show that Smo localisation to the plasma membrane is sufficient for phosphorylation of the cytoplasmic tail in the presence of Ptc. Using fluorescence cross-correlation spectroscopy (FCCS), we also demonstrate that inactivation of Ptc by Hh induces Smo clustering irrespective of Smo phosphorylation. Our observations therefore support a model of Hh signal transduction whereby Smo subcellular localisation and not phosphorylation is the primary target of Ptc function.