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A PKA inhibitor motif within SMOOTHENED controls Hedgehog signal transduction

Nature Structural & Molecular Biology volume 29, pages 990–999 (2022)Cite this article

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Abstract

The Hedgehog (Hh) cascade is central to development, tissue homeostasis and cancer. A pivotal step in Hh signal transduction is the activation of glioma-associated (GLI) transcription factors by the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO). How SMO activates GLI remains unclear. Here we show that SMO uses a decoy substrate sequence to physically block the active site of the cAMP-dependent protein kinase (PKA) catalytic subunit (PKA-C) and extinguish its enzymatic activity. As a result, GLI is released from phosphorylation-induced inhibition. Using a combination of in vitro, cellular and organismal models, we demonstrate that interfering with SMO-PKA pseudosubstrate interactions prevents Hh signal transduction. The mechanism uncovered echoes one used by the Wnt cascade, revealing an unexpected similarity in how these two essential developmental and cancer pathways signal intracellularly. More broadly, our findings define a mode of GPCR-PKA communication that may be harnessed by a range of membrane receptors and kinases.

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Fig. 1: SMO binds PKA-C as a pseudosubstrate.
Fig. 2: SMO induces changes in the amide fingerprint of PKA-C.
Fig. 3: SMO inhibits PKA-C enzymatic activity.
Fig. 4: The SMO PKI motif is required for Hh signal transduction.
Fig. 5: SMO colocalizes with endogenous PKA-C in primary cilia.
Fig. 6: The SMO PKI motif is required for Hh signal transduction.
Fig. 7: An avidity-based mechanism for SMO inhibition of PKA-C.

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Source data, including uncropped gels and western blots, are provided with this paper. All unique biological materials are available on request from the authors.

References

  1. Briscoe, J. & Therond, P. P. The mechanisms of Hedgehog signalling and its roles in development and disease. Nat. Rev. Mol. Cell Biol. 14, 416–429 (2013).

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  3. Ingham, P. W., Nakano, Y. & Seger, C. Mechanisms and functions of Hedgehog signalling across the metazoa. Nat. Rev. Genet. 12, 393–406 (2011).

    Article  CAS  PubMed  Google Scholar 

  4. Kong, J. H., Siebold, C. & Rohatgi, R. Biochemical mechanisms of vertebrate hedgehog signaling. Development 146, dev166892 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Muenke, M. & Beachy, P. A. Genetics of ventral forebrain development and holoprosencephaly. Curr. Opin. Genet Dev. 10, 262–269 (2000).

    Article  CAS  PubMed  Google Scholar 

  6. Digilio, M. C. et al. Atrioventricular canal defect and genetic syndromes: the unifying role of Sonic Hedgehog. Clin. Genet 95, 268–276 (2019).

    Article  CAS  PubMed  Google Scholar 

  7. Anderson, E., Peluso, S., Lettice, L. A. & Hill, R. E. Human limb abnormalities caused by disruption of Hedgehog signaling. Trends Genet. 28, 364–373 (2012).

    Article  CAS  PubMed  Google Scholar 

  8. Pak, E. & Segal, R. A. Hedgehog signal transduction: key players, oncogenic drivers, and cancer therapy. Dev. Cell 38, 333–344 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Wu, F., Zhang, Y., Sun, B., McMahon, A. P. & Wang, Y. Hedgehog signaling: from basic biology to cancer therapy. Cell Chem. Biol. 24, 252–280 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Hui, C. C. & Angers, S. Gli proteins in development and disease. Annu. Rev. Cell Dev. Biol. 27, 513–537 (2011).

    Article  CAS  PubMed  Google Scholar 

  11. Deshpande, I. et al. Smoothened stimulation by membrane sterols drives Hedgehog pathway activity. Nature 571, 284–288 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Huang, P. et al. Structural basis of Smoothened activation in Hedgehog signaling. Cell 174, 312–324 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Byrne, E. F. X. et al. Structural basis of Smoothened regulation by its extracellular domains. Nature 535, 517–522 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Qi, X., Friedberg, L., De Bose-Boyd, R., Long, T. & Li, X. Sterols in an intramolecular channel of Smoothened mediate Hedgehog signaling. Nat. Chem. Biol. 16, 1368–1375 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Wang, B., Fallon, J. F. & Beachy, P. A. Hedgehog-regulated processing of GLI3 produces an anterior/posterior repressor gradient in the developing vertebrate limb. Cell 100, 423–434 (2000).

    Article  CAS  PubMed  Google Scholar 

  16. Humke, E. W., Dorn, K. V., Milenkovic, L., Scott, M. P. & Rohatgi, R. The output of Hedgehog signaling is controlled by the dynamic association between Suppressor of Fused and the GLI proteins. Genes Dev. 24, 670–682 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Niewiadomski, P. et al. GLI protein activity is controlled by multisite phosphorylation in vertebrate Hedgehog signaling. Cell Rep. 6, 168–181 (2014).

    Article  CAS  PubMed  Google Scholar 

  18. Li, J. et al. PKA-mediated GLI2 and GLI3 phosphorylation is inhibited by Hedgehog signaling in cilia and reduced in Talpid3 mutant. Dev. Biol. 429, 147–157 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Nachury, M. V. & Mick, D. U. Establishing and regulating the composition of cilia for signal transduction. Nat. Rev. Mol. Cell Biol. 20, 389–405 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Reiter, J. F. & Leroux, M. R. Genes and molecular pathways underpinning ciliopathies. Nat. Rev. Mol. Cell Biol. 18, 533–547 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Gigante, E. D. & Caspary, T. Signaling in the primary cilium through the lens of the Hedgehog pathway. Wiley Interdiscip. Rev. Dev. Biol. 9, e377 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  22. Lefkowitz, R. J. The superfamily of heptahelical receptors. Nat. Cell Biol. 2, E133–E136 (2000).

    Article  CAS  PubMed  Google Scholar 

  23. Pierce, K. L., Premont, R. T. & Lefkowitz, R. J. Seven-transmembrane receptors. Nat. Rev. Mol. Cell Biol. 3, 639–650 (2002).

    Article  CAS  PubMed  Google Scholar 

  24. Walker-Gray, R., Stengel, F. & Gold, M. G. Mechanisms for restraining cAMP-dependent protein kinase revealed by subunit quantitation and cross-linking approaches. Proc. Natl Acad. Sci. USA 114, 10414–10419 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Johnson, D. A., Leathers, V. L., Martinez, A. M., Walsh, D. A. & Fletcher, W. H. Fluorescence resonance energy transfer within a heterochromatic cAMP-dependent protein kinase holoenzyme under equilibrium conditions: new insights into the conformational changes that result in cAMP-dependent activation. Biochemistry 32, 6402–6410 (1993).

    Article  CAS  PubMed  Google Scholar 

  26. Yang, S., Fletcher, W. H. & Johnson, D. A. Regulation of cAMP-dependent protein kinase: enzyme activation without dissociation. Biochemistry 34, 6267–6271 (1995).

    Article  CAS  PubMed  Google Scholar 

  27. Kopperud, R. et al. Formation of inactive cAMP-saturated holoenzyme of cAMP-dependent protein kinase under physiological conditions. J. Biol. Chem. 277, 13443–13448 (2002).

    Article  CAS  PubMed  Google Scholar 

  28. Smith, F. D. et al. Local protein kinase A action proceeds through intact holoenzymes. Science 356, 1288–1293 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Arveseth, C. D. et al. Smoothened transduces hedgehog signals via activity-dependent sequestration of PKA catalytic subunits. PLoS Biol. 19, e3001191 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Taylor, S. S., Ilouz, R., Zhang, P. & Kornev, A. P. Assembly of allosteric macromolecular switches: lessons from PKA. Nat. Rev. Mol. Cell Biol. 13, 646–658 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Dalton, G. D. & Dewey, W. L. Protein kinase inhibitor peptide (PKI): a family of endogenous neuropeptides that modulate neuronal cAMP-dependent protein kinase function. Neuropeptides 40, 23–34 (2006).

    Article  CAS  PubMed  Google Scholar 

  32. Soberg, K. & Skalhegg, B. S. The molecular basis for specificity at the level of the protein kinase a catalytic subunit. Front Endocrinol. (Lausanne) 9, 538 (2018).

    Article  Google Scholar 

  33. Taylor, S. S., Zhang, P., Steichen, J. M., Keshwani, M. M. & Kornev, A. P. PKA: lessons learned after twenty years. Biochim. Biophys. Acta 1834, 1271–1278 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Kemp, B. E. & Pearson, R. B. Protein kinase recognition sequence motifs. Trends Biochem. Sci. 15, 342–346 (1990).

    Article  CAS  PubMed  Google Scholar 

  35. Scott, J. D., Fischer, E. H., Demaille, J. G. & Krebs, E. G. Identification of an inhibitory region of the heat-stable protein inhibitor of the cAMP-dependent protein kinase. Proc. Natl Acad. Sci. USA 82, 4379–4383 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Cheng, H. C., van Patten, S. M., Smith, A. J. & Walsh, D. A. An active twenty-amino-acid-residue peptide derived from the inhibitor protein of the cyclic AMP-dependent protein kinase. Biochem. J. 231, 655–661 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Corbin, J. D. et al. Studies on the properties and mode of action of the purified regulatory subunit of bovine heart adenosine 3′:5′-monophosphate-dependent protein kinase. J. Biol. Chem. 253, 3997–4003 (1978).

    Article  CAS  PubMed  Google Scholar 

  38. Scott, J. D., Glaccum, M. B., Fischer, E. H. & Krebs, E. G. Primary-structure requirements for inhibition by the heat-stable inhibitor of the cAMP-dependent protein kinase. Proc. Natl Acad. Sci. USA 83, 1613–1616 (1986).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Glass, D. B., Cheng, H. C., Mende-Mueller, L., Reed, J. & Walsh, D. A. Primary structural determinants essential for potent inhibition of cAMP-dependent protein kinase by inhibitory peptides corresponding to the active portion of the heat-stable inhibitor protein. J. Biol. Chem. 264, 8802–8810 (1989).

    Article  CAS  PubMed  Google Scholar 

  40. Knighton, D. R. et al. Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science 253, 414–420 (1991).

    Article  CAS  PubMed  Google Scholar 

  41. Knighton, D. R. et al. Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science 253, 407–414 (1991).

    Article  CAS  PubMed  Google Scholar 

  42. Johnson, D. A., Akamine, P., Radzio-Andzelm, E., Madhusudan, M. & Taylor, S. S. Dynamics of cAMP-dependent protein kinase. Chem. Rev. 101, 2243–2270 (2001).

    Article  CAS  PubMed  Google Scholar 

  43. Olivieri, C. et al. Multi-state recognition pathway of the intrinsically disordered protein kinase inhibitor by protein kinase A. eLife 9, e55607 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Kim, J. et al. The role of ciliary trafficking in Hedgehog receptor signaling. Sci. Signal 8, ra55 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Whitehouse, S. & Walsh, D. A. Mg X ATP2-dependent interaction of the inhibitor protein of the cAMP-dependent protein kinase with the catalytic subunit. J. Biol. Chem. 258, 3682–3692 (1983).

    Article  CAS  PubMed  Google Scholar 

  46. Knape, M. J. et al. Divalent metal ions control activity and inhibition of protein kinases. Metallomics 9, 1576–1584 (2017).

    Article  CAS  PubMed  Google Scholar 

  47. Cook, P. F. et al. Adenosine cyclic 3′,5′-monophosphate dependent protein kinase: kinetic mechanism for the bovine skeletal muscle catalytic subunit. Biochemistry 21, 5794–5799 (1982).

    Article  CAS  PubMed  Google Scholar 

  48. Marullo, S. & Bouvier, M. Resonance energy transfer approaches in molecular pharmacology and beyond. Trends Pharmacol. Sci. 28, 362–365 (2007).

    Article  CAS  PubMed  Google Scholar 

  49. Machleidt, T. et al. NanoBRET–a novel BRET platform for the analysis of protein-protein interactions. ACS Chem. Biol. 10, 1797–1804 (2015).

    Article  CAS  PubMed  Google Scholar 

  50. Mick, D. U. et al. Proteomics of primary cilia by proximity labeling. Dev. Cell 35, 497–512 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Desai, P. B., Stuck, M. W., Lv, B. & Pazour, G. J. Ubiquitin links Smoothened to intraflagellar transport to regulate Hedgehog signaling. J. Cell Biol. 219, e201912104 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Pal, K. et al. Smoothened determines beta-arrestin-mediated removal of the G protein-coupled receptor Gpr161 from the primary cilium. J. Cell Biol. 212, 861–875 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Rohatgi, R., Milenkovic, L., Corcoran, R. B. & Scott, M. P. Hedgehog signal transduction by Smoothened: pharmacologic evidence for a 2-step activation process. Proc. Natl Acad. Sci. USA 106, 3196–3201 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Kim, J., Kato, M. & Beachy, P. A. Gli2 trafficking links Hedgehog-dependent activation of Smoothened in the primary cilium to transcriptional activation in the nucleus. Proc. Natl Acad. Sci. USA 106, 21666–21671 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Wilson, C. W., Chen, M. H. & Chuang, P. T. Smoothened adopts multiple active and inactive conformations capable of trafficking to the primary cilium. PLoS ONE 4, e5182 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  56. Shaywitz, A. J. & Greenberg, M. E. CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals. Annu. Rev. Biochem. 68, 821–861 (1999).

    Article  CAS  PubMed  Google Scholar 

  57. Knape, M. J. et al. Divalent Metal Ions Mg2+ and Ca2+ have distinct effects on protein kinase a activity and regulation. ACS Chem. Biol. 10, 2303–2315 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Varjosalo, M., Li, S. P. & Taipale, J. Divergence of hedgehog signal transduction mechanism between Drosophila and mammals. Dev. Cell 10, 177–186 (2006).

    Article  CAS  PubMed  Google Scholar 

  59. Myers, B. R., Neahring, L., Zhang, Y., Roberts, K. J. & Beachy, P. A. Rapid, direct activity assays for Smoothened reveal Hedgehog pathway regulation by membrane cholesterol and extracellular sodium. Proc. Natl Acad. Sci. USA 114, E11141–E11150 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Tuson, M., He, M. & Anderson, K. V. Protein kinase A acts at the basal body of the primary cilium to prevent Gli2 activation and ventralization of the mouse neural tube. Development 138, 4921–4930 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Lipinski, R. J., Bijlsma, M. F., Gipp, J. J., Podhaizer, D. J. & Bushman, W. Establishment and characterization of immortalized Gli-null mouse embryonic fibroblast cell lines. BMC Cell Biol. 9, 49 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  62. Chen, W. et al. Activity-dependent internalization of Smoothened mediated by beta-arrestin 2 and GRK2. Science 306, 2257–2260 (2004).

    Article  CAS  PubMed  Google Scholar 

  63. Wolff, C., Roy, S. & Ingham, P. W. Multiple muscle cell identities induced by distinct levels and timing of hedgehog activity in the zebrafish embryo. Curr. Biol. 13, 1169–1181 (2003).

    Article  CAS  PubMed  Google Scholar 

  64. Zhao, Z. et al. An essential role for Grk2 in Hedgehog signalling downstream of Smoothened. EMBO Rep. 17, 739–752 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Eccles, R. L. et al. Bimodal antagonism of PKA signalling by ARHGAP36. Nat. Commun. 7, 12963 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Sastri, M., Barraclough, D. M., Carmichael, P. T. & Taylor, S. S. A-kinase-interacting protein localizes protein kinase A in the nucleus. Proc. Natl Acad. Sci. USA 102, 349–354 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Taipale, J. et al. Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine. Nature 406, 1005–1009 (2000).

    Article  CAS  PubMed  Google Scholar 

  68. Corbit, K. C. et al. Vertebrate Smoothened functions at the primary cilium. Nature 437, 1018–1021 (2005).

    Article  CAS  PubMed  Google Scholar 

  69. Rohatgi, R., Milenkovic, L. & Scott, M. P. Patched1 regulates hedgehog signaling at the primary cilium. Science 317, 372–376 (2007).

    Article  CAS  PubMed  Google Scholar 

  70. Xie, J. et al. Activating Smoothened mutations in sporadic basal-cell carcinoma. Nature 391, 90–92 (1998).

    Article  CAS  PubMed  Google Scholar 

  71. Sweeney, R. T. et al. Identification of recurrent SMO and BRAF mutations in ameloblastomas. Nat. Genet. 46, 722–725 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Yogurtcu, O. N. & Johnson, M. E. Cytosolic proteins can exploit membrane localization to trigger functional assembly. PLoS Comput. Biol. 14, e1006031 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  73. Riobo, N. A., Saucy, B., Dilizio, C. & Manning, D. R. Activation of heterotrimeric G proteins by Smoothened. Proc. Natl Acad. Sci. USA 103, 12607–12612 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Shen, F., Cheng, L., Douglas, A. E., Riobo, N. A. & Manning, D. R. Smoothened is a fully competent activator of the heterotrimeric G protein Gi. Mol. Pharmacol. 83, 691–697 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Mukhopadhyay, S. et al. The ciliary G-protein-coupled receptor Gpr161 negatively regulates the Sonic Hedgehog pathway via cAMP signaling. Cell 152, 210–223 (2013).

    Article  CAS  PubMed  Google Scholar 

  76. Hwang, S.H. et al. The G protein-coupled receptor Gpr161 regulates forelimb formation, limb patterning and skeletal morphogenesis in a primary cilium-dependent manner. Development 145, dev154054 (2018).

  77. Shimada, I. S. et al. Basal suppression of the Sonic Hedgehog pathway by the G-protein-coupled receptor Gpr161 restricts medulloblastoma pathogenesis. Cell Rep. 22, 1169–1184 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Regard, J. B. et al. Activation of Hedgehog signaling by loss of GNAS causes heterotopic ossification. Nat. Med. 19, 1505–1512 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Moore, B. S. et al. Cilia have high cAMP levels that are inhibited by Sonic Hedgehog-regulated calcium dynamics. Proc. Natl Acad. Sci. USA 113, 13069–13074 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Low, W. C. et al. The decoupling of Smoothened from Galphai proteins has little effect on Gli3 protein processing and Hedgehog-regulated chick neural tube patterning. Dev. Biol. 321, 188–196 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Tschaikner, P., Enzler, F., Torres-Quesada, O., Aanstad, P. & Stefan, E. Hedgehog and Gpr161: regulating cAMP signaling in the primary cilium. Cells 9, 118 (2020).

  82. Pusapati, G. V. et al. G protein-coupled receptors control the sensitivity of cells to the morphogen Sonic Hedgehog. Sci. Signal 11, eaao5749 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  83. Meloni, A. R. et al. Smoothened signal transduction is promoted by G protein-coupled receptor kinase 2. Mol. Cell. Biol. 26, 7550–7560 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Hammerschmidt, M., Bitgood, M. J. & McMahon, A. P. Protein kinase A is a common negative regulator of Hedgehog signaling in the vertebrate embryo. Genes Dev. 10, 647–658 (1996).

    Article  CAS  PubMed  Google Scholar 

  85. Concordet, J. P. et al. Spatial regulation of a zebrafish patched homologue reflects the roles of Sonic Hedgehog and protein kinase A in neural tube and somite patterning. Development 122, 2835–2846 (1996).

    Article  CAS  PubMed  Google Scholar 

  86. Barresi, M. J., Stickney, H. L. & Devoto, S. H. The zebrafish slow-muscle-omitted gene product is required for Hedgehog signal transduction and the development of slow muscle identity. Development 127, 2189–2199 (2000).

    Article  CAS  PubMed  Google Scholar 

  87. MacDonald, B. T., Tamai, K. & He, X. Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev. Cell 17, 9–26 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Metcalfe, C. & Bienz, M. Inhibition of GSK3 by Wnt signalling–two contrasting models. J. Cell Sci. 124, 3537–3544 (2011).

    Article  CAS  PubMed  Google Scholar 

  89. Nusse, R. & Clevers, H. Wnt/beta-Catenin signaling, disease, and emerging therapeutic modalities. Cell 169, 985–999 (2017).

    Article  CAS  PubMed  Google Scholar 

  90. Steinhart, Z. & Angers, S. Wnt signaling in development and tissue homeostasis. Development 145, dev146589 (2018).

    Article  PubMed  Google Scholar 

  91. Piao, S. et al. Direct inhibition of GSK3beta by the phosphorylated cytoplasmic domain of LRP6 in Wnt/beta-catenin signaling. PLoS ONE 3, e4046 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  92. Cselenyi, C. S. et al. LRP6 transduces a canonical Wnt signal independently of Axin degradation by inhibiting GSK3’s phosphorylation of beta-catenin. Proc. Natl Acad. Sci. USA 105, 8032–8037 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Wu, G., Huang, H., Garcia Abreu, J. & He, X. Inhibition of GSK3 phosphorylation of beta-catenin via phosphorylated PPPSPXS motifs of Wnt coreceptor LRP6. PLoS ONE 4, e4926 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  94. Stamos, J. L., Chu, M. L., Enos, M. D., Shah, N. & Weis, W. I. Structural basis of GSK-3 inhibition by N-terminal phosphorylation and by the Wnt receptor LRP6. eLife 3, e01998 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  95. Ocbina, P. J., Tuson, M. & Anderson, K. V. Primary cilia are not required for normal canonical Wnt signaling in the mouse embryo. PLoS ONE 4, e6839 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  96. Hauser, A. S., Attwood, M. M., Rask-Andersen, M., Schioth, H. B. & Gloriam, D. E. Trends in GPCR drug discovery: new agents, targets and indications. Nat. Rev. Drug Discov. 16, 829–842 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. Oprea, T. I. et al. Unexplored therapeutic opportunities in the human genome. Nat. Rev. Drug Discov. 17, 317–332 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  98. Han, P., Sonati, P., Rubin, C. & Michaeli, T. PDE7A1, a cAMP-specific phosphodiesterase, inhibits cAMP-dependent protein kinase by a direct interaction with C. J. Biol. Chem. 281, 15050–15057 (2006).

    Article  CAS  PubMed  Google Scholar 

  99. Kim, J. et al. Dysfunctional conformational dynamics of protein kinase A induced by a lethal mutant of phospholamban hinder phosphorylation. Proc. Natl Acad. Sci. USA 112, 3716–3721 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Olivieri, C. et al. Defective internal allosteric network imparts dysfunctional ATP/substrate-binding cooperativity in oncogenic chimera of protein kinase A. Commun. Biol. 4, 321 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  101. Zimmermann, B., Schweinsberg, S., Drewianka, S. & Herberg, F. W. Effect of metal ions on high-affinity binding of pseudosubstrate inhibitors to PKA. Biochem. J. 413, 93–101 (2008).

    Article  CAS  PubMed  Google Scholar 

  102. Myers, B. R. et al. Hedgehog pathway modulation by multiple lipid binding sites on the Smoothened effector of signal response. Dev. Cell 26, 346–357 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  103. Lu, T. W. et al. Two PKA RIalpha holoenzyme states define ATP as an isoform-specific orthosteric inhibitor that competes with the allosteric activator, cAMP. Proc. Natl Acad. Sci. USA 116, 16347–16356 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  104. Olsen, S. R. & Uhler, M. D. Affinity purification of the C alpha and C beta isoforms of the catalytic subunit of cAMP-dependent protein kinase. J. Biol. Chem. 264, 18662–18666 (1989).

    Article  CAS  PubMed  Google Scholar 

  105. Walker, C. et al. Cushing’s syndrome driver mutation disrupts protein kinase A allosteric network, altering both regulation and substrate specificity. Sci. Adv. 5, eaaw9298 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. Walsh, D. A. & Ashby, C. D. Protein kinases: aspects of their regulation and diversity. Recent Prog. Horm. Res 29, 329–359 (1973).

    CAS  PubMed  Google Scholar 

  107. Manu, V. S., Olivieri, C., Pavuluri, K. & Veglia, G. Design and applications of water irradiation devoid RF pulses for ultra-high field biomolecular NMR spectroscopy. Phys. Chem. Chem. Phys. 24, 18477–18481 (2022).

    Article  CAS  PubMed  Google Scholar 

  108. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    Article  CAS  PubMed  Google Scholar 

  109. Lee, W., Tonelli, M. & Markley, J. L. NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy. Bioinformatics 31, 1325–1327 (2015).

    Article  PubMed  Google Scholar 

  110. Williamson, M. P. Using chemical shift perturbation to characterise ligand binding. Prog. Nucl. Magn. Reson. Spectrosc. 73, 1–16 (2013).

    Article  CAS  PubMed  Google Scholar 

  111. Hansen, J. N., Rassmann, S., Stuven, B., Jurisch-Yaksi, N. & Wachten, D. CiliaQ: a simple, open-source software for automated quantification of ciliary morphology and fluorescence in 2D, 3D, and 4D images. Eur. Phys. J. E Soft Matter 44, 18 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank J. Zalatan for making us aware of the parallels between SMO/PKA-C regulation in the Hh pathway and LRP/GSK-3β regulation in the Wnt pathway. We thank S. Lusk and K. Kwan (University of Utah) for providing smo-null zebrafish (smohi1640), and D. Klatt Shaw and D. Grunwald (University of Utah) for sharing advice and reagents regarding zebrafish immunohistochemistry. We thank J. Müller and S. Kasten (University of Kassel) for excellent technical assistance. We thank the Johnson Foundation Structural Biology and Biophysics Core at the Perelman School of Medicine (University of Pennsylvania, Philadelphia, PA, USA) for performing multi-angle light scattering coupled with size-exclusion chromatography analyses. We thank D. Julius, S. Nakielny, A. Manglik, K. Basham and M. He for providing feedback on the manuscript. B.R.M. acknowledges support from the 5 for the Fight Foundation (award no. 6000-32705) and a Cancer Center Support Grant Pilot Project Fund from the Huntsman Cancer Institute (award no. 200206). This work was supported by the DFG (the German Research Foundation) grant no. GRK2749/1 (F.W.H.) and National Institutes of Health grant nos. R01GM100310-08 (G.V.), 1R35GM130389 (S.S.T.), 1R03TR002947 (S.S.T.) and 1R35GM133672 (B.R.M.).

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Author notes

  1. Corvin D. Arveseth

    Present address: Washington University School of Medicine, St. Louis, MO, USA

  2. Jacob L. Capener

    Present address: Biological and Biomedical Sciences Program, University of North Carolina, Chapel Hill, NC, USA

  3. These authors contributed equally: John T. Happ, Corvin D. Arveseth, Jessica Bruystens, Daniela Bertinetti, Isaac B. Nelson, Cristina Olivieri.

Authors and Affiliations

  1. Department of Oncological Sciences, Department of Biochemistry, and Department of Bioengineering, University of Utah School of Medicine, Salt Lake City, UT, USA

    John T. Happ, Corvin D. Arveseth, Isaac B. Nelson, Danielle S. Hedeen, Ju-Fen Zhu, Jacob L. Capener & Benjamin R. Myers

  2. Department of Pharmacology, University of California, San Diego, La Jolla, CA, USA

    Jessica Bruystens, C. C. King & Susan S. Taylor

  3. Institute for Biology, Department of Biochemistry, University of Kassel, Kassel, Germany

    Daniela Bertinetti, Jan W. Bröckel & Friedrich W. Herberg

  4. Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, USA

    Cristina Olivieri & Gianluigi Veglia

  5. Department of Molecular and Cell Biology, School of Natural Sciences, University of California, Merced, CA, USA

    Jingyi Zhang & Xuecai Ge

  6. Department of Neurobiology, University of California, San Diego, La Jolla, CA, USA

    Lily Vu

  7. Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA

    C. C. King & Susan S. Taylor

  8. Instituto de Investigaciones Biomédicas ‘Alberto Sols,’ Consejo Superior de Investigaciones Científicas (CSIC), Universidad Autónoma de Madrid, Madrid, Spain

    Victor L. Ruiz-Perez

  9. CIBER de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III (ISCIII), Madrid, Spain

    Victor L. Ruiz-Perez

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  1. John T. Happ
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Contributions

J.T.H. designed, executed and interpreted CREB and GLI reporter assays. C.D.A. designed, executed and interpreted HEK293 BRET assays. J.B. designed, executed and interpreted fluorescence polarization studies and peptide array studies. D.B., J.W.B. and F.W.H. designed, executed and interpreted in vitro PKA-C activity assays and SPR studies. I.B.N. developed SMO pCT purification approaches and purified this domain for in vitro PKA-C activity assays. C.O. designed, executed and interpreted NMR studies. J.Z. designed, executed and interpreted all NIH3T3 imaging studies, under supervision from X.G. D.S.H. designed, executed and interpreted zebrafish embryology studies. J.-F.Z. designed, executed and interpreted coimmunoprecipitation and IMCD3 BRET assays. J.L.C. designed, executed and interpreted HEK293 confocal imaging studies. L.V. performed initial fluorescence polarization studies. C.C.K. collaborated with J.B. to develop SMO peptide arrays. V.L.R.-P. provided advice and guidance on mutagenesis experiments to disrupt SMO-PKA-C interactions. S.S.T. and B.R.M. conceived the project. G.V., F.W.H., S.S.T. and B.R.M. interpreted data and provided overall project supervision. B.R.M. performed IMCD3 ciliary imaging studies and wrote the manuscript with assistance from J.T.H., C.D.A. and I.B.N.

Corresponding authors

Correspondence to Susan S. Taylor or Benjamin R. Myers.

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The authors declare no competing interests.

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Nature Structural & Molecular Biology thanks Philip Ingham and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Carolina Perdigoto, Beth Moorefield and Anke Sparmann in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Sequence alignment of SMO PKI motif.

Extended alignment of a portion of the pCT from the indicated SMO orthologs, with key PKI motif residues colored as in Fig. 1a.

Extended Data Fig. 2 Additional binding and peptide array studies, and SPR sensorgrams.

a, Fluorescence polarization assays using mouse PKA-Cɑ, performed as in Fig. 1b. Triplicate points from representative experiments are shown. b, Peptide array, performed as in Fig. 1c, but with individual residues in the human SMO PKI motif mutated to alanine. c, SPR sensorgram for 625 nM PKA-Cɑ binding to GST-tagged wild-type (blue) or WRR mutant (purple) SMO pCT, or a PKIɑ positive control (red), in the presence of ATP and MgCl2. d, Exemplary steady-state analysis of binding interactions between human PKA-Cɑ and a recombinant wild-type SMO pCT, with a KD of 703 + /− 0.003 nM (dotted line) as assessed by SPR. This measurement was made three times, resulting in a mean KD value of 752 + /− 34 nM. e, SPR sensorgram, performed as in c, but with ATP and MgCl2 omitted from the buffer. PKA-Cɑ was present at 2.5 µM. Note that although removal of ATP and MgCl2 does not completely eliminate steady-state binding to the PKIɑ positive control, it dramatically accelerates the dissociation rate, as expected.

Source data

Extended Data Fig. 3 Raw NMR spectra for wild-type SMO peptide binding to PKA-Cα at varying kinase:peptide ratios.

2D NMR spectra from an experiment in which SMO peptide was titrated into ATPγN-bound PKA-Cα at the indicated kinase:peptide ratios. Upper left panel represents an overlay of all three spectra to highlight the concentration dependence of the SMO peptide-induced changes observed in each spectrum. Black spectrum represents ATPγN-bound PKA-Cα without SMO peptide (see also Extended Data Figs. 4, 5). In the overlay plot, a box denotes one example of a peak (likely corresponding to an unassigned tryptophan residue) that changes linearly according to SMO peptide concentration (magnified in the inset at left).

Extended Data Fig. 4 Raw NMR spectra for WRR mutant peptide binding to PKA-Cα, as shown in Fig. 2a.

a, The WRR mutant SMO peptide was titrated into ATPγN-bound PKA-Cα at the indicated kinase:peptide ratios. ATPγN-bound PKA-Cα without peptide (dark green spectrum, upper left) is shown for reference. See Extended Data Fig. 5 for raw NMR spectrum corresponding to PKA-Cα:wild-type SMO peptide at 1:6 ratio. b, Overlay of the spectra in a.

Extended Data Fig. 5 Raw NMR spectra for wild-type SMO peptide binding to PKA-Cα, as shown in Fig. 2a, and PKIα(5-24)-induced displacement of SMO peptide from PKA-Cα, as shown in Fig. 2c.

a, 2D NMR spectra for ATPγN-bound PKA-Cα, either alone (left) or with SMO peptide added at a 1:6 kinase:peptide ratio (right). b, 2D NMR spectra for titration of PKIα(5-24) peptide into the SMO peptide:ATPγN:PKA-Cα complex at the indicated kinase: PKIα(5-24) ratios. Upper left panel represents an overlay of the individual spectra to highlight the concentration dependence of the PKIα(5-24) peptide-induced effects.

Extended Data Fig. 6 Coimmunoprecipitation studies and ciliary colocalization studies to assess SMO / PKA-C interactions.

a, Coimmunoprecipitation of PKA-Cɑ-YFP with the indicated FLAG-tagged wild-type or mutant SMO constructs was assessed using FLAG chromatography from lysates of transfected HEK293 cells. Data shown are representative of two independent experiments. b, Left, Colocalization of FLAG-tagged wild-type or mutant SMO674 (magenta) with mNeonGreen-tagged PKA-Cɑ (green) in ciliated IMCD3 cells stably expressing both constructs and treated with the SMO agonist SAG21k. Cilia are marked by the SMO (FLAG-647) stain. mNeonGreen-tagged Nbβ2AR80 (which does not bind SMO29) serves as a negative control. 3D reconstructions from Z-stacks of confocal live-cell images are shown. Right, quantification of microscopy studies with the median represented by a dashed line and the upper and lower quartiles indicated by dotted lines (n = 142–244 cilia per condition). P < 0.0001 (****). See Supplementary Table 1 for full statistical analysis.

Source data

Extended Data Fig. 7 Characterization of NIH3T3 cell line expressing epitope-tagged SMO.

The NIH3T3 cell line used in Fig. 5 exhibited trace amounts of SMO in cilia under vehicle (‘Ctrl’) -treated conditions, and a dramatic accumulation of SMO in cilia under SAG-treated conditions. SMO (magenta) is visualized using an anti-V5 antibody, and cilia are visualized using an anti-Arl13b antibody (green). Scale bar = 10 µm. Data shown are representative of two independent experiments.

Extended Data Fig. 8 Controls for SMO / PKA-C binding, colocalization, and signaling studies.

a, Expression levels of SMO constructs in Fig. 3a, assessed by whole-cell nanoluc measurements. Data represent the mean + /− standard deviation, n = three biologically independent samples. NS = not significant. b, Surface levels of N-terminally FLAG-tagged wild-type or mutant SMO674 constructs were quantified via expression in HEK293 cells followed by FLAG staining and flow cytometry. Mock-infected cells stained with FLAG antibody (red) serve as a negative control. A representative histogram is shown. The % of FLAG-positive cells (that is, those to the right of the vertical dashed line) are: 0.3 + /− 0.3% (Ctrl); 95.9 + /− 0.9% (wt), 96.0 + /− 2.5% (WRR); 93.4 + /− 4.2% (A635S); values represent the mean + /− standard deviation from two biologically independent samples. See Supplementary Table 1 for full statistical analysis, and Supplementary Figure 2 for gating strategy. c, Ciliary localization in IMCD3 cells of myc-tagged wild-type or mutant SMO proteins (magenta). Cilia were visualized with Arl13b antibody (green). Scale bar = 5 µm. Data shown are representative of two independent experiments. d, GRK2/3-dependent phosphorylation of FLAG-tagged wild-type or mutant SMO674 constructs was determined via expression in HEK293 cells treated with or without the GRK2/3 inhibitor cmpd101, followed by FLAG purification. Levels of total and phosphorylated SMO were assessed by Stain Free imaging and ProQ Diamond fluorescence, respectively. SMO566, which is not phosphorylated by GRK2/3 (as it does not contain the C-tail and therefore lacks all previously mapped physiological GRK2/3 phosphorylation sites), serves as a negative control. Data shown are representative of two independent experiments.

Source data

Extended Data Fig. 9 Complete data set from SMO C-tail peptide array studies.

a, The same SMO tiled peptide array from Fig. 7c, but including the sequences of all positive hits in each array cluster. b, Complete human SMO C-tail sequence used to create the peptide array. In a,b, the SMO PKI motif identified in the pCT is indicated in red. Key residues in this PKI motif, along with ones in the candidate PKI motif in the dCT, are colored as in Fig. 1a.

Source data

Extended Data Fig. 10 Similarity between signal transduction mechanisms in the Hh and Wnt pathways.

Schematic diagram of transmembrane signal transduction in the Hh (left) and Wnt (right) pathways. During Hh signal transduction, active SMO is phosphorylated on its cytoplasmic tail by GRK2/3, triggering membrane sequestration and inhibition of PKA-C, and ultimately stabilization and activation of GLI. During Wnt signal transduction, active LRP5/6 is phosphorylated on its cytoplasmic tail by glycogen synthase kinase (GSK)-3β and casein kinase (CK)-1ɑ, triggering membrane sequestration and inhibition of GSK-3β, and ultimately stabilization and activation of β-catenin. Note that this is a simplified and highly schematized diagram and is not intended to be comprehensive; many other components of both pathways (for example, the destruction complex in which GSK-3β and β-catenin reside) are omitted in order to highlight mechanistic similarities between the underlying transmembrane signaling mechanisms.

Supplementary information

Supplementary Information

Supplementary Tables 1–3, Figs. 1 and 2, Discussion and Source Data for Supplementary Fig. 1.

Reporting Summary.

Source data

Source Data Fig. 1

Uncropped peptide array blots and raw data from fluorescence polarization and SPR studies.

Source Data Fig. 2

Raw data (list + CSP files) from NMR studies (also applies to Extended Data Figs. 3–5).

Source Data Fig. 3

Raw data from PKA-C in vitro enzymatic activity assays.

Source Data Fig. 4

Raw data from BRET studies and SMO-PKA-C colocalization studies in HEK293 cells

Source Data Fig. 5

Raw data from BRET studies and SMO-PKA-C colocalization studies in NIH3T3 cells.

Source Data Fig. 6

Raw data from CREB and GLI transcriptional reporter assays.

Source Data Fig. 7

Uncropped peptide array blots and raw data from BRET studies.

Source Data Extended Data Fig. 2

Uncropped peptide array blots and raw data from fluorescence polarization and SPR studies.

Source Data Extended Data Fig. 6

Uncropped protein gels, western blots and raw data from SMO-PKA-C colocalization studies in IMCD3 cells.

Source Data Extended Data Fig. 8

Uncropped protein gels and raw data from BRET studies.

Source Data Extended Data Fig. 9

Uncropped peptide array blots.

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Happ, J.T., Arveseth, C.D., Bruystens, J. et al. A PKA inhibitor motif within SMOOTHENED controls Hedgehog signal transduction. Nat Struct Mol Biol 29, 990–999 (2022). https://doi.org/10.1038/s41594-022-00838-z

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