Receptor Modifications in Hedgehog Regulation

  • Qing Shi
  • Jin JiangEmail author
Part of the Topics in Medicinal Chemistry book series (TMC, volume 16)


The Hedgehog (Hh) signaling pathway is one of the highly conserved signaling cascades that control cell growth, cell fate, and pattern formation in species ranging from Drosophila to human. Smoothened (Smo), a G-protein-coupled-receptor (GPCR) family protein, serves as a core component to tranduce Hh signal across the cell membrane. Studying how the activity of Smo is regulated is a key to understand how Hh morphegen gradiant differentially induces target gene expression during normal animal development as well as how abnormal Hh signaling activity contributes to various human diseases. In this chapter, we focus on the regulation of Smo by its posttranslational modifications. By reviewing our current knowledge on how Hh ligands dynamically induce phosphorylation and ubiquitination of Smo and how these modifications coordinatedly modulate Smo function and downstream signaling events, we hope to inspire future exploration of comprehensive mechanisms underlying the regulation of Smo.


Hedgehog Phospohrylation Posttranslational modification Smo Smo binding proteins Ubiquitination 





Basal cell carcinoma


Cyan fluorescent protein


Ci activator


Ci repressor


Casein kinase 1


Casein kinase 2




Carboxyl-terminal cytoplasmic tail


Drosophila Smo


Deubiquitinating enzymes


An Ub-activation enzyme


An Ub-conjugating enzyme


An Ub ligase


Fluorescence resonance energy transfer






G-protein-coupled-receptor kinase 2


Glycogen synthase kinase 3






The third intracellular loop


Mammalian Smo




Protein kinase A


Protein phosphatase 1


Protein phosphatase 2A




Posttranslational modification


Arg to Ala


Smo auto-inhibitory domain




Yellow fluorescent protein



J.J. is supported by grants from National Institute of Health (GM061269 and GM067045), Cancer Prevention Research Institute of Texas (RP100561), and Welch Foundation (I-1603).


  1. 1.
    Chen MH, Li YJ, Kawakami T, Xu SM, Chuang PT (2004) Palmitoylation is required for the production of a soluble multimeric Hedgehog protein complex and long-range signaling in vertebrates. Genes Dev 18:641–659CrossRefGoogle Scholar
  2. 2.
    Panakova D, Sprong H, Marois E, Thiele C, Eaton S (2005) Lipoprotein particles are required for Hedgehog and Wingless signalling. Nature 435:58–65CrossRefGoogle Scholar
  3. 3.
    Zeng X, Goetz JA, Suber LM, Scott WJ Jr, Schreiner CM, Robbins DJ (2001) A freely diffusible form of Sonic hedgehog mediates long-range signalling. Nature 411:716–720CrossRefGoogle Scholar
  4. 4.
    Huang S, Zhang Z, Zhang C, Lv X, Zheng X, Chen Z, Sun L, Wang H, Zhu Y, Zhang J, Yang S, Lu Y, Sun Q, Tao Y, Liu F, Zhao Y, Chen D (2013) Activation of Smurf E3 ligase promoted by smoothened regulates hedgehog signaling through targeting patched turnover. PLoS Biol 11:e1001721CrossRefGoogle Scholar
  5. 5.
    Stone DM, Hynes M, Armanini M, Swanson TA, Gu Q, Johnson RL, Scott MP, Pennica D, Goddard A, Phillips H, Noll M, Hooper JE, de Sauvage F, Rosenthal A (1996) The tumour-suppressor gene patched encodes a candidate receptor for Sonic hedgehog. Nature 384:129–134CrossRefGoogle Scholar
  6. 6.
    Jiang J (2006) Regulation of Hh/Gli signaling by dual ubiquitin pathways. Cell Cycle 5:2457–2463CrossRefGoogle Scholar
  7. 7.
    Jiang J, Hui CC (2008) Hedgehog signaling in development and cancer. Dev Cell 15:801–812CrossRefGoogle Scholar
  8. 8.
    Wilson CW, Chuang PT (2010) Mechanism and evolution of cytosolic Hedgehog signal transduction. Development 137:2079–2094CrossRefGoogle Scholar
  9. 9.
    Chen Y, Jiang J (2013) Decoding the phosphorylation code in Hedgehog signal transduction. Cell Res 23:186–200CrossRefGoogle Scholar
  10. 10.
    Ingham PW, McMahon AP (2001) Hedgehog signaling in animal development: paradigms and principles. Genes Dev 15:3059–3087CrossRefGoogle Scholar
  11. 11.
    Nusslein-Volhard C, Wieschaus E (1980) Mutations affecting segment number and polarity in Drosophila. Nature 287:795–801CrossRefGoogle Scholar
  12. 12.
    Alcedo J, Ayzenzon M, Von Ohlen T, Noll M, Hooper JE (1996) The Drosophila smoothened gene encodes a seven-pass membrane protein, a putative receptor for the Hedgehog signal. Cell 86:221–232CrossRefGoogle Scholar
  13. 13.
    Van den Heuval M, Ingham PW (1996) Smoothened encodes a receptor-like serpentine protein required for hedgehog signalling. Nature 382:547–551CrossRefGoogle Scholar
  14. 14.
    Rudin CM, Hann CL, Laterra J, Yauch RL, Callahan CA, Fu L, Holcomb T, Stinson J, Gould SE, Coleman B, LoRusso PM, Von Hoff DD, de Sauvage FJ, Low JA (2009) Treatment of medulloblastoma with hedgehog pathway inhibitor GDC-0449. N Engl J Med 361:1173–1178CrossRefGoogle Scholar
  15. 15.
    Von Hoff DD, LoRusso PM, Rudin CM, Reddy JC, Yauch RL, Tibes R, Weiss GJ, Borad MJ, Hann CL, Brahmer JR, Mackey HM, Lum BL, Darbonne WC, Marsters JC Jr, de Sauvage FJ, Low JA (2009) Inhibition of the hedgehog pathway in advanced basal-cell carcinoma. N Engl J Med 361:1164–1172CrossRefGoogle Scholar
  16. 16.
    Low JA, de Sauvage FJ (2010) Clinical experience with Hedgehog pathway inhibitors. J Clin Oncol 28:5321–5326CrossRefGoogle Scholar
  17. 17.
    Kunstfeld R (2014) Smoothened inhibitors in the treatment of advanced basal cell carcinomas. Curr Opin Oncol 26:184–195CrossRefGoogle Scholar
  18. 18.
    Bidet M, Joubert O, Lacombe B, Ciantar M, Nehme R, Mollat P, Bretillon L, Faure H, Bittman R, Ruat M, Mus-Veteau I (2011) The hedgehog receptor patched is involved in cholesterol transport. PLoS One 6:e23834CrossRefGoogle Scholar
  19. 19.
    Taipale J, Cooper MK, Maiti T, Beachy PA (2002) Patched acts catalytically to suppress the activity of Smoothened. Nature 418:892–897CrossRefGoogle Scholar
  20. 20.
    Denef N, Neubuser D, Perez L, Cohen SM (2000) Hedgehog induces opposite changes in turnover and subcellular localization of patched and smoothened. Cell 102:521–531CrossRefGoogle Scholar
  21. 21.
    Jia J, Tong C, Wang B, Luo L, Jiang J (2004) Hedgehog signalling activity of smoothened requires phosphorylation by protein kinase a and casein kinase I. Nature 432:1045–1050CrossRefGoogle Scholar
  22. 22.
    Zhu AJ, Zheng L, Suyama K, Scott MP (2003) Altered localization of Drosophila Smoothened protein activates Hedgehog signal transduction. Genes Dev 17:1240–1252CrossRefGoogle Scholar
  23. 23.
    Corbit KC, Aanstad P, Singla V, Norman AR, Stainier DY, Reiter JF (2005) Vertebrate Smoothened functions at the primary cilium. Nature 437:1018–1021CrossRefGoogle Scholar
  24. 24.
    Rohatgi R, Milenkovic L, Scott MP (2007) Patched1 regulates hedgehog signaling at the primary cilium. Science 317:372–376CrossRefGoogle Scholar
  25. 25.
    Zhao Y, Tong C, Jiang J (2007) Hedgehog regulates smoothened activity by inducing a conformational switch. Nature 450:252–258CrossRefGoogle Scholar
  26. 26.
    Jia J, Tong C, Jiang J (2003) Smoothened transduces Hedgehog signal by physically interacting with Costal2/Fused complex through its C-terminal tail. Genes Dev 17:2709–2720CrossRefGoogle Scholar
  27. 27.
    Lum L, Zhang C, Oh S, Mann RK, von Kessler DP, Taipale J, Weis-Garcia F, Gong R, Wang B, Beachy PA (2003) Hedgehog signal transduction via Smoothened association with a cytoplasmic complex scaffolded by the atypical kinesin, Costal-2. Mol Cell 12:1261–1274CrossRefGoogle Scholar
  28. 28.
    Ogden SK, Ascano M Jr, Stegman MA, Suber LM, Hooper JE, Robbins DJ (2003) Identification of a functional interaction between the transmembrane protein Smoothened and the kinesin-related protein Costal2. Curr Biol 13:1998–2003CrossRefGoogle Scholar
  29. 29.
    Ruel L, Gallet A, Raisin S, Truchi A, Staccini-Lavenant L, Cervantes A, Therond PP (2007) Phosphorylation of the atypical kinesin Costal2 by the kinase fused induces the partial disassembly of the Smoothened-Fused-Costal2-Cubitus interruptus complex in Hedgehog signalling. Development 134:3677–3689CrossRefGoogle Scholar
  30. 30.
    Zhang W, Zhao Y, Tong C, Wang G, Wang B, Jia J, Jiang J (2005) Hedgehog-regulated costal2-kinase complexes control phosphorylation and proteolytic processing of cubitus interruptus. Dev Cell 8:267–278CrossRefGoogle Scholar
  31. 31.
    Shi Q, Li S, Jia J, Jiang J (2011) The Hedgehog-induced Smoothened conformational switch assembles a signaling complex that activates fused by promoting its dimerization and phosphorylation. Development 138:4219–4231CrossRefGoogle Scholar
  32. 32.
    Ohlmeyer JT, Kalderon D (1998) Hedgehog stimulates maturation of Cubitus interruptus into a labile transcriptional activator. Nature 396:749–753CrossRefGoogle Scholar
  33. 33.
    Cheung HO, Zhang X, Ribeiro A, Mo R, Makino S, Puviindran V, Law KK, Briscoe J, Hui CC (2009) The kinesin protein Kif7 is a critical regulator of Gli transcription factors in mammalian hedgehog signaling. Sci Signal 2:ra29Google Scholar
  34. 34.
    Liem KF Jr, He M, Ocbina PJ, Anderson KV (2009) Mouse Kif7/Costal2 is a cilia-associated protein that regulates Sonic hedgehog signaling. Proc Natl Acad Sci U S A 106:13377–13382CrossRefGoogle Scholar
  35. 35.
    Endoh-Yamagami S, Evangelista M, Wilson D, Wen X, Theunissen JW, Phamluong K, Davis M, Scales SJ, Solloway MJ, de Sauvage FJ, Peterson AS (2009) The mammalian Cos2 homolog Kif7 plays an essential role in modulating Hh signal transduction during development. Curr Biol 19:1320–1326CrossRefGoogle Scholar
  36. 36.
    Law KK, Makino S, Mo R, Zhang X, Puviindran V, Hui CC (2012) Antagonistic and cooperative actions of Kif7 and Sufu define graded intracellular Gli activities in Hedgehog signaling. PLoS One 7:e50193CrossRefGoogle Scholar
  37. 37.
    Maurya AK, Ben J, Zhao Z, Lee RT, Niah W, Ng AS, Iyu A, Yu W, Elworthy S, van Eeden FJ, Ingham PW (2013) Positive and negative regulation of Gli activity by Kif7 in the zebrafish embryo. PLoS Genet 9:e1003955CrossRefGoogle Scholar
  38. 38.
    Chen MH, Gao N, Kawakami T, Chuang PT (2005) Mice deficient in the fused homolog do not exhibit phenotypes indicative of perturbed hedgehog signaling during embryonic development. Mol Cell Biol 25:7042–7053CrossRefGoogle Scholar
  39. 39.
    Merchant M, Evangelista M, Luoh SM, Frantz GD, Chalasani S, Carano RA, van Hoy M, Ramirez J, Ogasawara AK, McFarland LM, Filvaroff EH, French DM, de Sauvage FJ (2005) Loss of the serine/threonine kinase fused results in postnatal growth defects and lethality due to progressive hydrocephalus. Mol Cell Biol 25:7054–7068CrossRefGoogle Scholar
  40. 40.
    Maloverjan A, Piirsoo M, Michelson P, Kogerman P, Osterlund T (2010) Identification of a novel serine/threonine kinase ULK3 as a positive regulator of Hedgehog pathway. Exp Cell Res 316:627–637CrossRefGoogle Scholar
  41. 41.
    Maloverjan A, Piirsoo M, Kasak L, Peil L, Osterlund T, Kogerman P (2010) Dual function of UNC-51-like kinase 3 (Ulk3) in the Sonic hedgehog signaling pathway. J Biol Chem 285:30079–30090CrossRefGoogle Scholar
  42. 42.
    Ogden SK, Fei DL, Schilling NS, Ahmed YF, Hwa J, Robbins DJ (2008) G protein Galpha(i) functions immediately downstream of Smoothened in Hedgehog signalling. Nature 456:967–970CrossRefGoogle Scholar
  43. 43.
    Low WC, Wang C, Pan Y, Huang XY, Chen JK, Wang B (2008) The decoupling of Smoothened from Galphai proteins has little effect on Gli3 protein processing and Hedgehog-regulated chick neural tube patterning. Dev Biol 321:188–196CrossRefGoogle Scholar
  44. 44.
    Cozzone AJ (1988) Protein phosphorylation in prokaryotes. Annu Rev Microbiol 42:97–125CrossRefGoogle Scholar
  45. 45.
    Stock JB, Ninfa AJ, Stock AM (1989) Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev 53:450–490Google Scholar
  46. 46.
    Chang C, Stewart RC (1998) The two-component system. Regulation of diverse signaling pathways in prokaryotes and eukaryotes. Plant Physiol 117:723–731CrossRefGoogle Scholar
  47. 47.
    Barford D, Das AK, Egloff MP (1998) The structure and mechanism of protein phosphatases: insights into catalysis and regulation. Annu Rev Biophys Biomol Struct 27:133–164CrossRefGoogle Scholar
  48. 48.
    Jiang J, Struhl G (1995) Protein kinase A and Hedgehog signalling in Drosophila limb development. Cell 80:563–572CrossRefGoogle Scholar
  49. 49.
    Li W, Ohlmeyer JT, Lane ME, Kalderon D (1995) Function of protein kinase A in hedghehog signal transduction and Drosophila imaginal disc development. Cell 80:553–562CrossRefGoogle Scholar
  50. 50.
    Wang G, Wang B, Jiang J (1999) Protein kinase A antagonizes Hedgehog signaling by regulating both the activator and repressor forms of Cubitus interruptus. Genes Dev 13:2828–2837CrossRefGoogle Scholar
  51. 51.
    Jia J, Zhang L, Zhang Q, Tong C, Wang B, Hou F, Amanai K, Jiang J (2005) Phosphorylation by double-time/CKIepsilon and CKIalpha targets cubitus interruptus for Slimb/beta-TRCP-mediated proteolytic processing. Dev Cell 9:819–830CrossRefGoogle Scholar
  52. 52.
    Price MA, Kalderon D (2002) Proteolysis of the Hedgehog signaling effector Cubitus interruptus requires phosphorylation by Glycogen Synthase Kinase 3 and Casein Kinase 1. Cell 108:823–835CrossRefGoogle Scholar
  53. 53.
    Ohlmeyer JT, Kalderon D (1997) Dual pathways for induction of wingless expression by protein kinase A and Hedgehog in Drosophila embryos. Genes Dev 11:2250–2258CrossRefGoogle Scholar
  54. 54.
    Apionishev S, Katanayeva NM, Marks SA, Kalderon D, Tomlinson A (2005) Drosophila Smoothened phosphorylation sites essential for Hedgehog signal transduction. Nat Cell Biol 7:86–92CrossRefGoogle Scholar
  55. 55.
    Zhang C, Williams EH, Guo Y, Lum L, Beachy PA (2004) Extensive phosphorylation of Smoothened in Hedgehog pathway activation. Proc Natl Acad Sci U S A 101:17900–17907CrossRefGoogle Scholar
  56. 56.
    Jia H, Liu Y, Xia R, Tong C, Yue T, Jiang J, Jia J (2010) Casein kinase 2 promotes Hedgehog signaling by regulating both smoothened and Cubitus interruptus. J Biol Chem 285:37218–37226CrossRefGoogle Scholar
  57. 57.
    Chen Y, Li S, Tong C, Zhao Y, Wang B, Liu Y, Jia J, Jiang J (2010) G protein-coupled receptor kinase 2 promotes high-level Hedgehog signaling by regulating the active state of Smo through kinase-dependent and kinase-independent mechanisms in Drosophila. Genes Dev 24:2054–2067CrossRefGoogle Scholar
  58. 58.
    Cheng S, Maier D, Neubueser D, Hipfner DR (2010) Regulation of smoothened by Drosophila G-protein-coupled receptor kinases. Dev Biol 337:99–109CrossRefGoogle Scholar
  59. 59.
    Chen W, Ren XR, Nelson CD, Barak LS, Chen JK, Beachy PA, de Sauvage F, Lefkowitz RJ (2004) Activity-dependent internalization of smoothened mediated by beta-arrestin 2 and GRK2. Science 306:2257–2260CrossRefGoogle Scholar
  60. 60.
    Meloni AR, Fralish GB, Kelly P, Salahpour A, Chen JK, Wechsler-Reya RJ, Lefkowitz RJ, Caron MG (2006) Smoothened signal transduction is promoted by G protein-coupled receptor kinase 2. Mol Cell Biol 26:7550–7560CrossRefGoogle Scholar
  61. 61.
    Chen Y, Sasai N, Ma G, Yue T, Jia J, Briscoe J, Jiang J (2011) Sonic Hedgehog dependent phosphorylation by CK1a and GRK2 is required for ciliary accumulation and activation of Smoothened. PLoS Biol 9:e1001083CrossRefGoogle Scholar
  62. 62.
    Evangelista M, Lim TY, Lee J, Parker L, Ashique A, Peterson AS, Ye W, Davis DP, de Sauvage FJ (2008) Kinome siRNA screen identifies regulators of ciliogenesis and hedgehog signal transduction. Sci Signal 1:ra7Google Scholar
  63. 63.
    Kovacs JJ, Whalen EJ, Liu R, Xiao K, Kim J, Chen M, Wang J, Chen W, Lefkowitz RJ (2008) Beta-arrestin-mediated localization of smoothened to the primary cilium. Science 320:1777–1781CrossRefGoogle Scholar
  64. 64.
    Rohatgi R, Milenkovic L, Corcoran RB, Scott MP (2009) Hedgehog signal transduction by Smoothened: pharmacologic evidence for a 2-step activation process. Proc Natl Acad Sci U S A 106:3196–3201CrossRefGoogle Scholar
  65. 65.
    Wang Y, Zhou Z, Walsh CT, McMahon AP (2009) Selective translocation of intracellular Smoothened to the primary cilium in response to Hedgehog pathway modulation. Proc Natl Acad Sci U S A 106:2623–2628CrossRefGoogle Scholar
  66. 66.
    Wilson CW, Chen MH, Chuang PT (2009) Smoothened adopts multiple active and inactive conformations capable of trafficking to the primary cilium. PLoS One 4:e5182CrossRefGoogle Scholar
  67. 67.
    Yang C, Chen W, Chen Y, Jiang J (2012) Smoothened transduces Hedgehog signal by forming a complex with Evc/Evc2. Cell Res 22:1593–1604CrossRefGoogle Scholar
  68. 68.
    Fan J, Liu Y, Jia J (2012) Hh-induced Smoothened conformational switch is mediated by differential phosphorylation at its C-terminal tail in a dose- and position-dependent manner. Dev Biol 366:172–184CrossRefGoogle Scholar
  69. 69.
    Su Y, Ospina JK, Zhang J, Michelson AP, Schoen AM, Zhu AJ (2011) Sequential phosphorylation of smoothened transduces graded hedgehog signaling. Sci Signal 4:ra43CrossRefGoogle Scholar
  70. 70.
    Xia R, Jia H, Fan J, Liu Y, Jia J (2012) USP8 promotes smoothened signaling by preventing its ubiquitination and changing its subcellular localization. PLoS Biol 10:e1001238CrossRefGoogle Scholar
  71. 71.
    Li S, Chen Y, Shi Q, Yue T, Wang B, Jiang J (2012) Hedgehog-regulated ubiquitination controls smoothened trafficking and cell surface expression in Drosophila. PLoS Biol 10:e1001239CrossRefGoogle Scholar
  72. 72.
    Komander D, Rape M (2012) The ubiquitin code. Annu Rev Biochem 81:203–229CrossRefGoogle Scholar
  73. 73.
    Miranda M, Sorkin A (2007) Regulation of receptors and transporters by ubiquitination: new insights into surprisingly similar mechanisms. Mol Interv 7:157–167CrossRefGoogle Scholar
  74. 74.
    Hicke L (2001) Protein regulation by monoubiquitin. Nat Rev Mol Cell Biol 2:195–201CrossRefGoogle Scholar
  75. 75.
    Reyes-Turcu FE, Ventii KH, Wilkinson KD (2009) Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes. Annu Rev Biochem 78:363–397CrossRefGoogle Scholar
  76. 76.
    Yang X, Mao F, Lv X, Zhang Z, Fu L, Lu Y, Wu W, Zhou Z, Zhang L, Zhao Y (2013) Drosophila Vps36 regulates Smo trafficking in Hedgehog signaling. J Cell Sci 126:4230–4238CrossRefGoogle Scholar
  77. 77.
    Liu Y, Cao X, Jiang J, Jia J (2007) Fused-Costal2 protein complex regulates Hedgehog-induced Smo phosphorylation and cell-surface accumulation. Genes Dev 21:1949–1963CrossRefGoogle Scholar
  78. 78.
    Shenoy SK, McDonald PH, Kohout TA, Lefkowitz RJ (2001) Regulation of receptor fate by ubiquitination of activated beta 2-adrenergic receptor and beta-arrestin. Science 294:1307–1313CrossRefGoogle Scholar
  79. 79.
    Shenoy SK, Xiao K, Venkataramanan V, Snyder PM, Freedman NJ, Weissman AM (2008) Nedd4 mediates agonist-dependent ubiquitination, lysosomal targeting, and degradation of the beta2-adrenergic receptor. J Biol Chem 283:22166–22176CrossRefGoogle Scholar
  80. 80.
    Molnar C, Ruiz-Gomez A, Martin M, Rojo-Berciano S, Mayor F, de Celis JF (2011) Role of the Drosophila non-visual ss-arrestin kurtz in hedgehog signalling. PLoS Genet 7:e1001335CrossRefGoogle Scholar
  81. 81.
    Molnar C, Holguin H, Mayor F Jr, Ruiz-Gomez A, de Celis JF (2007) The G protein-coupled receptor regulatory kinase GPRK2 participates in Hedgehog signaling in Drosophila. Proc Natl Acad Sci U S A 104:7963–7968CrossRefGoogle Scholar
  82. 82.
    Zhao Y, Tong C, Jiang J (2007) Transducing the Hedgehog signal across the plasma membrane. Fly (Austin) 1:333–336CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  1. 1.Department of Developmental BiologyUniversity of Texas Southwestern Medical Center at DallasDallasUSA
  2. 2.Department of pharmacologyUniversity of Texas Southwestern Medical Center at DallasDallasUSA

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