Regulation of Smoothened Phosphorylation and High-Level Hedgehog Signaling Activity by a Plasma Membrane Associated Kinase.

RESEARCH ARTICLE Regulation of Smoothened Phosphorylation and High-Level Hedgehog Signaling Activity by a Plasma Membrane Associated Kinase Shuangxi Li1,2☯, Shuang Li1,2☯, Yuhong Han1,2, Chao Tong2¤, Bing Wang1,2, Yongbin Chen2,3, Jin Jiang1,2,4* 1 Department of Molecular Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America, 2 Department of Developmental Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America, 3 Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China, 4 Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America a11111 ☯ These authors contributed equally to this work. ¤ Current address: Life Sciences Institute and Innovation Center for Cell Biology, Zhejiang University, Hangzhou, China * OPEN ACCESS Citation: Li S, Li S, Han Y, Tong C, Wang B, Chen Y, et al. (2016) Regulation of Smoothened Phosphorylation and High-Level Hedgehog Signaling Activity by a Plasma Membrane Associated Kinase. PLoS Biol 14(6): e1002481. doi:10.1371/journal. pbio.1002481 Academic Editor: Konrad Basler, University of Zurich, SWITZERLAND Received: November 16, 2015 Accepted: May 12, 2016 Published: June 9, 2016 Copyright: © 2016 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: JJ received grants from the National Institute of General Medicine Sciences (GM061269, GM067045, GM118063) and the Welch Foundation (I-1603), and YC received grants from the National Natural Science Foundation of China (31328017, 81322030, and 31271579). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Abstract 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 finetunes 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. Author Summary 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 PLOS Biology | DOI:10.1371/journal.pbio.1002481 June 9, 2016 1 / 24 Smoothened Regulation by a Plasma Membrane-Associated Kinase Competing Interests: The authors have declared that no competing interests exist. Abbreviations: AEL, after egg laying; aPKC, atypical protein kinase C; Arr, Arrow; CFP, Cyan fluorescent protein; Ci, Cubitus interruptus; CK1, casein kinase 1; CK2, casein kinase 2; Cl8, Clone 8; CL-II, cluster II; C-tail, carboxyl-terminal intracellular tail; EYFP, enhanced yellow fluorescent protein; En, Engrailed; FRET, Fluorescence resonance energy transfer; Fu, Fused; GFP, green fluorescent protein; Gish, Gilgamesh; Gli, Glioma-associated oncogene homologue; GPCR, G protein coupled receptor; Gprk2/GRK2, G protein coupled receptor kinase 2; GST, Glutathione S-transferase; Hh, Hedgehog; mSmo, mammalian Smo; PCP, planar cell polarity; PKA, protein kinase A; Ptc, Patched; SAID, Smo auto-inhibitory domain; Ser, Serine; Shh, Sonic hedgehog; Smo, Smoothened; Thr, Threonine; Ub, ubiquitin; Wg, Wingless; YFP, Yellow fluorescent protein. 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. Introduction Hedgehog (Hh) signaling plays an essential role in embryonic development and adult tissue homeostasis, and its deregulation has been implicated in congenital diseases and cancers [1–6]. Hh exerts its biological influence through an intracellular signal transduction cascade that emanates from a G protein coupled receptor (GPCR)-family protein Smoothened (Smo) and culminates in the activation of the latent transcription factor Cubitus interruptus (Ci)/Gliomaassociated oncogene homologue (Gli) [1,3,7,8]. In the signaling off state, Smo is inhibited by a twelve-transmembrane protein Patched (Ptc). Binding of Hh to Ptc alleviates such inhibition, allowing Smo to be phosphorylated and accumulate on the cell surface in Drosophila or primary cilia in vertebrates, where Smo adopts an open and active conformation to relay the Hh signal to the intracellular signaling components [9–15]. Phosphorylation plays a critical role in the regulation of Smo conformation and subcel (...truncated)