Regulation of Smoothened Phosphorylation and High-Level Hedgehog Signaling Activity by a Plasma Membrane Associated Kinase.
Regulation of Smoothened Phosphorylation and High-Level Hedgehog Signaling Activity by a Plasma Membrane Associated Kinase.
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DOI：
10.1371/journal.pbio.1002481
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发表时间：
2016-06
期刊：
影响因子：
9.8
通讯作者：
Jiang J
中科院分区：
文献类型：
--
作者：
Li S;Li S;Han Y;Tong C;Wang B;Chen Y;Jiang J
Hedgehog (Hh) 信号通过 G 蛋白偶联受体 (GPCR) 家族蛋白 Smoothened (Smo) 控制胚胎发育和成体组织稳态。受到刺激后，Smo 会积聚在果蝇的细胞表面或脊椎动物的初级纤毛上，这被认为对其激活和功能至关重要，但其潜在机制仍知之甚少。在这里，我们发现 Hh 刺激 Smo 与质膜相关激酶 Gilgamesh (Gish)/CK1γ 的结合，并且 Gish 通过磷酸化 Smo 羧基末端细胞内尾 (C-tail) 的近膜区域的 Ser/Thr 簇 (CL-II) 来微调 Hh 通路活性。我们发现 CL-II 磷酸化是由蛋白激酶 A (PKA) 介导的 Smo C 尾磷酸化促进的，并且依赖于 Gish 和 Smo 的细胞表面定位。与 CL-II 对于高阈值 Hh 靶基因表达至关重要一致，其磷酸化似乎需要比 Smo 上的 PKA 位点磷酸化更高水平的 Hh 或更长的时间暴露于相同水平的 Hh。此外，我们发现脊椎动物 CK1γ 定位于初级纤毛，促进 Smo 磷酸化和 Sonic Hedgehog (Shh) 通路激活。我们的研究揭示了一种保守机制，即 Hh 诱导 Smo 亚细胞定位的变化，以促进其与质膜定位激酶的关联并被质膜定位激酶激活，并为 Hh 形态发生素如何逐步激活 Smo 提供了新的见解。对果蝇和脊椎动物细胞的研究表明，质膜相关激酶 Gish/CK1γ 通过磷酸化 Smoothened 来促进 Hedgehog 信号的转导。分泌型糖蛋白 Hedgehog (Hh) 在果蝇和人类等物种的胚胎发育和成体组织稳态中发挥保守作用。 Hh 信号转导的失调会导致多种人类疾病，包括出生缺陷和癌症。七次跨膜蛋白 Smoothened (Smo) 是一种必需且保守的 Hh 信号转导器，但 Hh 如何刺激其活性仍不清楚。在这里，我们确定了一种质膜相关激酶 Gilgamesh (Gish)/CK1γ，作为 Hh 信号传导活性的正调节因子。我们发现 Gish 通过磷酸化 Smo C 端胞内尾部的特定位点来激活 Hh 信号传导。 Gish 对 Smo 的磷酸化是 Smo 最大激活所必需的，并且取决于 Gish 的膜结合和蛋白激酶 A (PKA) 先前对 Smo 的磷酸化。我们还发现，Hh 在到达质膜后刺激 Smo 与 Gish 的结合，从而促进其被 Gish 磷酸化。最后，我们提供证据表明 CK1γ 存在于哺乳动物的初级纤毛中，并且磷酸化 Smo 以激活 Hh 通路。我们的结果揭示了 Gish/CK1γ 在 Smo 磷酸化调节中的保守作用，并为 Hh 信号如何跨质膜转导的分子基础提供了新的见解。
Hedgehog (Hh) signaling controls embryonic development and adult tissue homeostasis through the G protein coupled receptor (GPCR)-family protein Smoothened (Smo). Upon stimulation, Smo accumulates on the cell surface in Drosophila or primary cilia in vertebrates, which is thought to be essential for its activation and function, but the underlying mechanisms remain poorly understood. Here we show that Hh stimulates the binding of Smo to a plasma membrane-associated kinase Gilgamesh (Gish)/CK1γ and that Gish fine-tunes Hh pathway activity by phosphorylating a Ser/Thr cluster (CL-II) in the juxtamembrane region of Smo carboxyl-terminal intracellular tail (C-tail). We find that CL-II phosphorylation is promoted by protein kinase A (PKA)-mediated phosphorylation of Smo C-tail and depends on cell surface localization of both Gish and Smo. Consistent with CL-II being critical for high-threshold Hh target gene expression, its phosphorylation appears to require higher levels of Hh or longer exposure to the same level of Hh than PKA-site phosphorylation on Smo. Furthermore, we find that vertebrate CK1γ is localized at the primary cilium to promote Smo phosphorylation and Sonic hedgehog (Shh) pathway activation. Our study reveals a conserved mechanism whereby Hh induces a change in Smo subcellular localization to promote its association with and activation by a plasma membrane localized kinase, and provides new insight into how Hh morphogen progressively activates Smo. A study of both fruit flies and vertebrate cells shows that the plasma membrane-associated kinase Gish/CK1γ promotes transduction of the Hedgehog signal by phosphorylating Smoothened. The secreted glycoprotein Hedgehog (Hh) plays a conserved role in embryonic development and adult tissue homeostasis in species ranging from Drosophila to humans. Deregulation of Hh signal transduction contributes to a wide range of human disorders, including birth defects and cancer. The seven-transmembrane protein Smoothened (Smo) is an obligatory and conserved Hh signal transducer, but how Hh stimulates its activity remains unclear. Here we identify a plasma membrane associated kinase, Gilgamesh (Gish)/CK1γ, as a positive regulator of the Hh signaling activity. We find that Gish activates Hh signaling by phosphorylating a specific site in the Smo C-terminal intracellular tail. Phosphorylation of Smo by Gish is required for maximal activation of Smo and depends on membrane association of Gish and prior phosphorylation of Smo by protein kinase A (PKA). We also find that Hh stimulates the association of Smo with Gish after it travels to the plasma membrane, thus facilitating its phosphorylation by Gish. Finally, we provide evidence that CK1γ is found at the primary cilium in mammals and phosphorylates Smo to activate the Hh pathway. Our results uncover a conserved role of Gish/CK1γ in the regulation of Smo phosphorylation and provide new insight into the molecular underpinning of how Hh signal is transduced across the plasma membrane.