Skip to main content
SpringerLink
Log in

Ci/Gli Phosphorylation by the Fused/Ulk Family Kinases

  • Protocol
  • First Online:
Hedgehog Signaling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2374))

Abstract

Hedgehog (Hh) signaling culminates in the conversion of the latent transcription factor Cubitus interruptus (Ci)/Gli from a repressor form (CiR/GliR) into an activator form (CiA/GliA). While sequential phosphorylation of Ci/Gli by protein kinase A(PKA), glycogen synthase kinase 3 (GSK3), and casein kinase 1 (CK1) is essential for its proteolytic processing that generates CiR/GliR, sequential phosphorylation of Ci/Gli by the Fused (Fu)/Unc-51 like kinase (Ulk) family kinases Fu/Ulk3/Stk36 and CK1 contributes to the formation of CiA/GliA. Fu/Ulk3/Stk36-mediated phosphorylation of Ci/Gli is stimulated by Hh, leading to altered interaction between Ci/Gli and the Hh pathway repressor Sufu. Here we describe both in vitro and in vivo assays that determine Ci/Gli phosphorylation by the Fu/Ulk family kinases and its regulation by Hh.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Jiang J, Hui CC (2008) Hedgehog signaling in development and cancer. Dev Cell 15(6):801–812

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Briscoe J, Therond PP (2013) The mechanisms of Hedgehog signalling and its roles in development and disease. Nat Rev Mol Cell Biol 14(7):418–431. https://doi.org/10.1038/nrm3598

    Article  CAS  Google Scholar 

  3. Nieuwenhuis E, Hui CC (2005) Hedgehog signaling and congenital malformations. Clin Genet 67(3):193–208

    Article  CAS  PubMed  Google Scholar 

  4. Jia J, Jiang J (2006) Decoding the Hedgehog signal in animal development. Cell Mol Life Sci 63(11):1249–1265

    Article  CAS  PubMed  Google Scholar 

  5. Jiang J, Struhl G (1995) Protein kinase A and hedgehog signaling in Drosophila limb development. Cell 80(4):563–572. https://doi.org/10.1016/0092-8674(95)90510-3

    Article  CAS  PubMed  Google Scholar 

  6. Li W, Ohlmeyer JT, Lane ME, Kalderon D (1995) Function of protein kinase A in hedghehog signal transduction and Drosophila imaginal disc development. Cell 80:553–562

    Article  CAS  PubMed  Google Scholar 

  7. Jiang J, Struhl G (1998) Regulation of the Hedgehog and wingless signalling pathways by the F- box/WD40-repeat protein Slimb. Nature 391(6666):493–496

    Article  CAS  PubMed  Google Scholar 

  8. Wang B, Fallon JF, Beachy PA (2000) Hedgehog-regulated processing of Gli3 produces an anterior/posterior repressor gradient in the developing vertebrate limb. Cell 100(4):423–434

    Article  CAS  PubMed  Google Scholar 

  9. Jia J, Amanai K, Wang G, Tang J, Wang B, Jiang J (2002) Shaggy/GSK3 antagonizes Hedgehog signalling by regulating cubitus interruptus. Nature 416(6880):548–552

    Article  CAS  PubMed  Google Scholar 

  10. 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(6):823–835

    Article  CAS  PubMed  Google Scholar 

  11. 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(6):819–830

    Article  CAS  PubMed  Google Scholar 

  12. Chen Y, Jiang J (2013) Decoding the phosphorylation code in Hedgehog signal transduction. Cell Res 23(2):186–200. https://doi.org/10.1038/cr.2013.10

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Jiang J (2002) Degrading Ci: who is Cul-pable? Genes Dev 16(18):2315–2321

    Article  CAS  PubMed  Google Scholar 

  14. 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(2):267–278

    Article  CAS  PubMed  Google Scholar 

  15. Zhou Z, Yao X, Li S, Xiong Y, Dong X, Zhao Y, Jiang J, Zhang Q (2015) Deubiquitination of Ci/Gli by Usp7/HAUSP regulates Hedgehog signaling. Dev Cell 34(1):58–72

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Ohlmeyer JT, Kalderon D (1998) Hedgehog stimulates maturation of cubitus interruptus into a labile transcriptional activator. Nature 396(6713):749–753. https://doi.org/10.1038/25533

    Article  CAS  PubMed  Google Scholar 

  17. Wang G, Amanai K, Wang B, Jiang J (2000) Interactions with Costal2 and suppressor of fused regulate nuclear translocation and activity of cubitus interruptus. Genes Dev 14(22):2893–2905

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Zhang Y, Fu L, Qi X, Zhang Z, Xia Y, Jia J, Jiang J, Zhao Y, Wu G (2013) Structural insight into the mutual recognition and regulation between suppressor of fused and Gli/Ci. Nat Commun 4:2608. https://doi.org/10.1038/ncomms3608

    Article  CAS  PubMed  Google Scholar 

  19. Shi Q, Han Y, Jiang J (2014) Suppressor of fused impedes Ci/Gli nuclear import by opposing Trn/Kapbeta2 in Hedgehog signaling. J Cell Sci 127(Pt 5):1092–1103. https://doi.org/10.1242/jcs.142828

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Han Y, Shi Q, Jiang J (2015) Multisite interaction with Sufu regulates Ci/Gli activity through distinct mechanisms in Hh signal transduction. Proc Natl Acad Sci U S A 112(20):6383–6388. https://doi.org/10.1073/pnas.1421628112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Liu C, Zhou Z, Yao X, Chen P, Sun M, Su M, Chang C, Yan J, Jiang J, Zhang Q (2014) Hedgehog signaling downregulates suppressor of fused through the HIB/SPOP-Crn axis in Drosophila. Cell Res 24(5):595–609. https://doi.org/10.1038/cr.2014.29

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Shi Q, Li S, Li S, Jiang A, Chen Y, Jiang J (2014) Hedgehog-induced phosphorylation by CK1 sustains the activity of Ci/Gli activator. Proc Natl Acad Sci U S A 111(52):E5651–E5660. https://doi.org/10.1073/pnas.1416652111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Zhang Q, Zhang L, Wang B, Ou CY, Chien CT, Jiang J (2006) A hedgehog-induced BTB protein modulates hedgehog signaling by degrading Ci/Gli transcription factor. Dev Cell 10(6):719–729

    Article  CAS  PubMed  Google Scholar 

  24. Zhang Q, Shi Q, Chen Y, Yue T, Li S, Wang B, Jiang J (2009) Multiple Ser/Thr-rich degrons mediate the degradation of Ci/Gli by the Cul3-HIB/SPOP E3 ubiquitin ligase. Proc Natl Acad Sci U S A 106(50):21191–21196. https://doi.org/10.1073/pnas.0912008106

    Article  PubMed  PubMed Central  Google Scholar 

  25. Preat T, Therond P, Limbourg-Bouchon B, Pham A, Tricoire H, Busson D, Lamour-Isnard C (1993) Segmental polarity in Drosophila melanogaster: genetic dissection of fused in a suppressor of fused background reveals interaction with costal-2. Genetics 135(4):1047–1062

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. 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(19):4219–4231. . dev.067959 [pii]. https://doi.org/10.1242/dev.067959

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Zhou Q, Kalderon D (2011) Hedgehog activates fused through phosphorylation to elicit a full spectrum of pathway responses. Dev Cell 20(6):802–814. S1534-5807(11)00174-2 [pii]. https://doi.org/10.1016/j.devcel.2011.04.020

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Zhang Y, Mao F, Lu Y, Wu W, Zhang L, Zhao Y (2011) Transduction of the Hedgehog signal through the dimerization of fused and the nuclear translocation of cubitus interruptus. Cell Res 21(10):1436–1451. https://doi.org/10.1038/cr.2011.136

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Han Y, Wang B, Cho YS, Zhu J, Wu J, Chen Y, Jiang J (2019) Phosphorylation of Ci/Gli by fused family kinases promotes Hedgehog signaling. Dev Cell 50(5):610–626.e614. https://doi.org/10.1016/j.devcel.2019.06.008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Han Y, Xiong Y, Shi X, Wu J, Zhao Y, Jiang J (2017) Regulation of Gli ciliary localization and Hedgehog signaling by the PY-NLS/karyopherin-beta2 nuclear import system. PLoS Biol 15(8):e2002063. https://doi.org/10.1371/journal.pbio.2002063

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This work was supported by grants from the National Institutes of Health (R35GM118063) and Welch Foundation (I-1603) to J.J.

Author information

Authors and Affiliations

  1. Department of Molecular Biology, UT Southwestern Medical Center at Dallas, Dallas, TX, USA

    Yuhong Han & Jin Jiang

Authors
  1. Yuhong Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jin Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jin Jiang .

Editor information

Editors and Affiliations

  1. Department of Molecular Genetics and Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Xiaochun Li

Rights and permissions

Reprints and permissions

Copyright information

© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Han, Y., Jiang, J. (2022). Ci/Gli Phosphorylation by the Fused/Ulk Family Kinases. In: Li, X. (eds) Hedgehog Signaling. Methods in Molecular Biology, vol 2374. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1701-4_19

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1701-4_19

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1700-7

  • Online ISBN: 978-1-0716-1701-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation