Sonic Hedgehog dependent phosphorylation by CK1α and GRK2 is required for ciliary accumulation and activation of smoothened.
Sonic Hedgehog dependent phosphorylation by CK1α and GRK2 is required for ciliary accumulation and activation of smoothened.
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CK1α和GRK2的声音刺猬依赖性磷酸化是纤毛积累和平滑激活所必需的。
DOI：
10.1371/journal.pbio.1001083
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发表时间：
2011-06
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：
Chen Y;Sasai N;Ma G;Yue T;Jia J;Briscoe J;Jiang J
Hedgehog（Hh）信号通过GPCR样蛋白Smoothened（Smo）调节胚胎发育和成体组织稳态，但脊椎动物Smo如何被激活仍知之甚少。在果蝇中，Hh依赖性磷酸化激活Smo。脊椎动物中是否也是这种情况尚不清楚，由于脊椎动物和果蝇Smo（dSmo）之间的显着序列差异和初级纤毛参与脊椎动物Hh信号传导。在这里，我们证明了哺乳动物Smo（mSmo）是通过CK1 α和GRK2对其羧基末端尾部的多位点磷酸化来激活的。mSmo的磷酸化诱导其活性构象，同时促进其纤毛积聚。我们证明，分级Hh信号诱导mSmo磷酸化水平的增加，微调其纤毛定位，构象和活性。我们发现，mSmo磷酸化诱导其激动剂和致癌突变，但被其拮抗剂环巴胺，和有效的mSmo磷酸化依赖于驱动蛋白-II纤毛电机。此外，我们提供的证据表明，Hh信号招募CK1 α启动mSmo磷酸化，磷酸化进一步增加CK1 α和GRK2与mSmo的结合，形成放大和/或维持mSmo磷酸化的正反馈回路。因此，尽管在它们的一级序列和它们的亚细胞运输方面存在分歧，mSmo和dSmo采用类似的机制来激活它们。Hedgehog（Hh）信号通路控制着从果蝇到人类的胚胎发育和成年期的稳态，其功能障碍与多种人类疾病有关。Hh信号由十二跨膜受体接收，并由七跨膜蛋白Smoothened（Smo）在细胞内传递。脊椎动物Smo如何被激活以传递Hh信号仍然知之甚少。在这里，我们调查的哺乳动物Smo（mSmo）激活的分子机制，并发现它是类似的描述果蝇Smo，尽管它们之间的显着序列差异。我们发现mSmo通过丝氨酸/苏氨酸激酶CK1 α和GRK2在多个位点的磷酸化被激活。我们提供的证据表明，音速刺猬（嘘，最好的研究的三种哺乳动物途径配体）可以调节mSmo这些激酶的可访问性和磷酸化促进纤毛积累这种跨膜蛋白在其活性构象。此外，增加浓度的Shh诱导mSmo磷酸化的进行性增加，其微调mSmo活性。因此，我们的研究结果提供了新的见解脊椎动物Hh信号转导的生化机制，并揭示了一个保守的模式Smo激活。
Hedgehog (Hh) signaling regulates embryonic development and adult tissue homeostasis through the GPCR-like protein Smoothened (Smo), but how vertebrate Smo is activated remains poorly understood. In Drosophila, Hh dependent phosphorylation activates Smo. Whether this is also the case in vertebrates is unclear, owing to the marked sequence divergence between vertebrate and Drosophila Smo (dSmo) and the involvement of primary cilia in vertebrate Hh signaling. Here we demonstrate that mammalian Smo (mSmo) is activated through multi-site phosphorylation of its carboxyl-terminal tail by CK1α and GRK2. Phosphorylation of mSmo induces its active conformation and simultaneously promotes its ciliary accumulation. We demonstrate that graded Hh signals induce increasing levels of mSmo phosphorylation that fine-tune its ciliary localization, conformation, and activity. We show that mSmo phosphorylation is induced by its agonists and oncogenic mutations but is blocked by its antagonist cyclopamine, and efficient mSmo phosphorylation depends on the kinesin-II ciliary motor. Furthermore, we provide evidence that Hh signaling recruits CK1α to initiate mSmo phosphorylation, and phosphorylation further increases the binding of CK1α and GRK2 to mSmo, forming a positive feedback loop that amplifies and/or sustains mSmo phosphorylation. Hence, despite divergence in their primary sequences and their subcellular trafficking, mSmo and dSmo employ analogous mechanisms for their activation. Hedgehog (Hh) signaling governs embryonic development and adult homeostasis in species ranging from Drosophila to human, and its malfunction has been implicated in a wide range of human disorders. Hh signal is received by the twelve-transmembrane receptor Patched and transmitted intracellularly by the seven-transmembrane protein Smoothened (Smo). How vertebrate Smo is activated in order to transmit the Hh signal remains poorly understood. Here we investigate the molecular mechanism of mammalian Smo (mSmo) activation and find it is similar to that described for Drosophila Smo despite the marked sequence divergence between them. We show that mSmo is activated via phosphorylation at multiple sites by the serine/threonine kinases CK1α and GRK2. We provide evidence that Sonic hedgehog (Shh; the best studied of the three mammalian pathway ligands) can regulate the accessibility of mSmo to these kinases and that phosphorylation promotes the ciliary accumulation of this transmembrane protein in its active conformation. Moreover, increasing concentrations of Shh induce a progressive increase in mSmo phosphorylation that fine-tunes mSmo activity. Thus, our results provide novel insights into the biochemical mechanism of vertebrate Hh signal transduction and reveal a conserved mode of Smo activation.
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