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Drosophila Smoothened phosphorylation sites essential for Hedgehog signal transduction

Nature Cell Biology volume 7, pages 86–92 (2005)Cite this article

Abstract

The Hedgehog (Hh) signalling pathway is crucial for animal development and is aberrantly activated in several types of cancer1. In Drosophila melanogaster, Hh signalling regulates target gene expression through the transcription factor Cubitus interruptus (Ci). Together, Protein Kinase A, Casein Kinase 1 and Glycogen Synthase Kinase 3 silence the pathway in the absence of ligand by phosphorylating Ci at a defined cluster of sites, thereby promoting its proteolytic conversion to a transcriptional repressor (Ci-75)2,3. In the presence of Hh, Ci-155 is no longer converted to Ci-75 and its ability to activate transcription is potentiated. All Hh responses require the seven transmembrane domain protein Smoothened1,4, which itself becomes hyperphosphorylated during Hh signalling5. Here we show that a cluster of protein kinase A and protein kinase A-primed casein kinase 1 phosphorylation sites in Smoothened, similarly distributed to those regulating Ci, are essential for Smoothened to transduce a Hh signal and for normal regulation of Smoothened protein levels.

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Figure 1: HA–Smo transgene activity in embryos.
Figure 2: Dominant-negative activity of HA–SmoPKA and HA–SmoCK1.
Figure 3: Rescue activity of HA–Smo transgenes in wing discs.
Figure 4: PKA activity affects Fu phosphorylation.
Figure 5: CK1 inhibition reduces Smo activity but increases Smo levels in anterior cells.

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References

  1. Ingham, P. W. & McMahon, A. P. Hedgehog signaling in animal development: paradigms and principles. Genes Dev. 15, 3059–3087 (2001).

    Article  CAS  Google Scholar 

  2. Jia, J. et al. Shaggy/GSK3 antagonizes Hedgehog signalling by regulating Cubitus interruptus. Nature 416, 548–552 (2002).

    Article  CAS  Google Scholar 

  3. Price, M. A. & Kalderon, D. Proteolysis of the Hedgehog signaling effector Cubitus interruptus requires phosphorylation by Glycogen Synthase Kinase 3 and Casein Kinase 1. Cell 108, 823–835 (2002).

    Article  CAS  Google Scholar 

  4. Lum, L. & Beachy, P. A. The Hedgehog response network: sensors, switches, and routers. Science 304, 1755–1759 (2004).

    Article  CAS  Google Scholar 

  5. Denef, N., Neubuser, D., Perez, L. & Cohen, S. M. Hedgehog induces opposite changes in turnover and subcellular localization of patched and smoothened. Cell 102, 521–531 (2000).

    Article  CAS  Google Scholar 

  6. Chen, Y., Cardinaux, J. R., Goodman, R. H. & Smolik, S. M. Mutants of cubitus interruptus that are independent of PKA regulation are independent of hedgehog signaling. Development 126, 3607–3616 (1999).

    CAS  PubMed  Google Scholar 

  7. Price, M. A. & Kalderon, D. Proteolysis of cubitus interruptus in Drosophila requires phosphorylation by protein kinase A. Development 126, 4331–4339 (1999).

    CAS  Google Scholar 

  8. Wang, G., Wang, B. & Jiang, J. Protein kinase A antagonizes Hedgehog signaling by regulating both the activator and repressor forms of Cubitus interruptus. Genes Dev. 13, 2828–2837 (1999).

    Article  CAS  Google Scholar 

  9. Li, W., Ohlmeyer, J. T., Lane, M. E. & Kalderon, D. Function of protein kinase A in hedgehog signal transduction and Drosophila imaginal disc development. Cell 80, 553–562 (1995).

    Article  CAS  Google Scholar 

  10. Ohlmeyer, J. T. & Kalderon, D. Dual pathways for induction of wingless expression by protein kinase A and Hedgehog in Drosophila embryos. Genes Dev. 11, 2250–2258 (1997).

    Article  CAS  Google Scholar 

  11. Vervoort, M. hedgehog and wing development in Drosophila: a morphogen at work? Bioessays 22, 460–468 (2000).

    Article  CAS  Google Scholar 

  12. Chen, Y. & Struhl, G. Dual roles for patched in sequestering and transducing Hedgehog. Cell 87, 553–563 (1996).

    Article  CAS  Google Scholar 

  13. Crozatier, M., Glise, B. & Vincent, A. Connecting Hh, Dpp and EGF signalling in patterning of the Drosophila wing; the pivotal role of collier/knot in the AP organiser. Development 129, 4261–4269 (2002).

    CAS  PubMed  Google Scholar 

  14. Ingham, P. W. et al. Patched represses the Hedgehog signalling pathway by promoting modification of the Smoothened protein. Curr. Biol. 10, 1315–1318 (2000).

    Article  CAS  Google Scholar 

  15. Alcedo, J., Zou, Y. & Noll, M. Posttranscriptional regulation of smoothened is part of a self-correcting mechanism in the Hedgehog signaling system. Mol. Cell 6, 457–465 (2000).

    Article  CAS  Google Scholar 

  16. Lane, M. E. & Kalderon, D. Genetic investigation of cAMP-dependent protein kinase function in Drosophila development. Genes Dev. 7, 1229–1243 (1993).

    Article  CAS  Google Scholar 

  17. Ohlmeyer, J. T. & Kalderon, D. Hedgehog stimulates maturation of Cubitus interruptus into a labile transcriptional activator. Nature 396, 749–753 (1998).

    Article  CAS  Google Scholar 

  18. Therond, P. P., Knight, J. D., Kornberg, T. B. & Bishop, J. M. Phosphorylation of the fused protein kinase in response to signaling from hedgehog. Proc. Natl Acad. Sci. USA 93, 4224–4228 (1996).

    Article  CAS  Google Scholar 

  19. Ruel, L., Rodriguez, R., Gallet, A., Lavenant-Staccini, L. & Therond, P. P. Stability and association of Smoothened, Costal2 and Fused with Cubitus interruptus are regulated by Hedgehog. Nature Cell Biol. 5, 907–913 (2003).

    Article  CAS  Google Scholar 

  20. Glise, B., Jones, D. L. & Ingham, P. W. Notch and Wingless modulate the response of cells to Hedgehog signalling in the Drosophila wing. Dev. Biol. 248, 93–106 (2002).

    Article  CAS  Google Scholar 

  21. Nakano, Y. et al. Functional domains and sub-cellular distribution of the Hedgehog transducing protein Smoothened in Drosophila. Mech. Dev. 121, 507–518 (2004).

    Article  CAS  Google Scholar 

  22. Brinkworth, R. I., Breinl, R. A. & Kobe, B. Structural basis and prediction of substrate specificity in protein serine/threonine kinases. Proc. Natl Acad. Sci. USA 100, 74–79 (2003).

    Article  CAS  Google Scholar 

  23. Liu, C. et al. Control of β-catenin phosphorylation/degradation by a dual-kinase mechanism. Cell 108, 837–847 (2002).

    Article  CAS  Google Scholar 

  24. Lum, L. et al. Identification of Hedgehog pathway components by RNAi in Drosophila cultured cells. Science 299, 2039–2045 (2003).

    Article  CAS  Google Scholar 

  25. Kalderon, D. Similarities between the Hedgehog and Wnt signaling pathways. Trends Cell Biol. 12, 523–531 (2002).

    Article  CAS  Google Scholar 

  26. Dai, P., Akimaru, H. & Ishii, S. A hedgehog-responsive region in the Drosophila wing disc is defined by debra-mediated ubiquitination and lysosomal degradation of Ci. Dev. Cell 4, 917–928 (2003).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  28. Ogden, S. K. et al. Identification of a functional interaction between the transmembrane protein Smoothened and the Kinesin-related protein Costal2. Curr. Biol. 13, 1998–2003 (2003).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank M. Ciancanelli, J. Qiu, H. Tukachinsky, C. Vied, M. Crozatier, J. Hooper and D. Robbins for help with experiments and reagents. We especially thank C. Vied for providing Supplementary Information Fig. S2 and several panels for Supplementary Information Fig. S6, M. Smelkinson for Supplementary Information Fig. S1 and M. A. Price for permitting use of the unpublished CK1α RNAi transgene. This work was supported by funding from the National Institutes of Health.

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Authors and Affiliations

  1. Department of Biological Sciences, Columbia University, 1212 Amsterdam Avenue, New York, 10027, NY, USA

    Sergey Apionishev, Steven A. Marks & Daniel Kalderon

  2. Department of Genetics and Development, Center for Neurobiology and Behavior, College of Physicians and Surgeons, Columbia University, 701 West 168th Street, New York, 10032, NY, USA

    Natalya M. Katanayeva & Andrew Tomlinson

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  1. Sergey Apionishev
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Correspondence to Daniel Kalderon.

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The authors declare no competing financial interests.

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Apionishev, S., Katanayeva, N., Marks, S. et al. Drosophila Smoothened phosphorylation sites essential for Hedgehog signal transduction. Nat Cell Biol 7, 86–92 (2005). https://doi.org/10.1038/ncb1210

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