Regulation of Smoothened Phosphorylation and High-Level Hedgehog Signaling Activity by a Plasma Membrane Associated Kinase.
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.
Department of Molecular Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America.|Department of Developmental Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America.
Shuangxi
S
Li
Department of Molecular Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America.|Department of Developmental Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America.
Shuang
S
Li
Department of Molecular Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America.|Department of Developmental Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America.
Yuhong
0000-0001-8933-4637
Y
Han
Department of Developmental Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America.
Chao
C
Tong
Department of Molecular Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America.|Department of Developmental Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America.
Bing
B
Wang
Department of Developmental Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America.|Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China.
Yongbin
Y
Chen
Department of Molecular Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America.|Department of Developmental Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America.|Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America.
Jin
J
Jiang
D000818:Animals; D030801:Animals, Genetically Modified; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D047389:Casein Kinase I / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002923:Cilia / Q000235:genetics / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D053823:Hedgehog Proteins / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D009154:Mutation; D010766:Phosphorylation; D034622:RNA Interference; D015398:Signal Transduction; D000072086:Smoothened Receptor / Q000235:genetics / Q000378:metabolism*; D014921:Wings, Animal / Q000378:metabolism
D029721:Drosophila Proteins; D053823:Hedgehog Proteins; D008164:Luminescent Proteins; D000072086:Smoothened Receptor; C100664:smo protein, Drosophila; C077245:hh protein, Drosophila; D047389:Casein Kinase I; C479663:gish protein, Drosophila; D017868:Cyclic AMP-Dependent Protein Kinases