A Broadly Conserved G-Protein-Coupled Receptor Kinase Phosphorylation Mechanism Controls Smoothened Activity

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Hedgehog proteins are critical regulators of embryonic tissue growth and organization in species ranging from flies to humans. Binding of the secreted Hh protein to its receptor at the surface of cells triggers an intracellular signal that is initiated by Smoothened (Smo). Upon exposure of cells to Hh, Smo becomes active and signals through a series of downstream proteins to regulate gene expression. Although Smo proteins in flies and mammals are similar, the critical regions involved in activation and signal initiation differ between the two, implying that different mechanisms have evolved in different organisms. Using the fruit fly as a model organism, we identified regions in Smo that are phosphorylated by a protein kinase called Gprk2 to enhance Smo activity. These phosphorylation sites overlap with previously identified sites in mouse Smo and are conserved in Smo proteins in many animals. Phosphorylation at these sites regulates the recruitment of Costal2 to Smo, a critical step in signal initiation, through a region of the protein that is also highly conserved. Our results indicate that Gprk2 phosphorylation represents an evolutionarily ancient and conserved mechanism for regulating Smo activity, and suggest that Smo regulation and signaling are more similar between different species than previously thought.

Vyšlo v časopise: A Broadly Conserved G-Protein-Coupled Receptor Kinase Phosphorylation Mechanism Controls Smoothened Activity. PLoS Genet 10(7): e32767. doi:10.1371/journal.pgen.1004399
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1004399

Souhrn

Zdroje

1. ZhaoY, TongC, JiangJ (2007) Hedgehog regulates smoothened activity by inducing a conformational switch. Nature 450 : 252–258.

2. ChenY, SasaiN, MaG, YueT, JiaJ, et al. (2011) Sonic Hedgehog dependent phosphorylation by CK1alpha and GRK2 is required for ciliary accumulation and activation of smoothened. PLoS Biol 9: e1001083.

3. BriscoeJ, TherondPP (2013) The mechanisms of Hedgehog signalling and its roles in development and disease. Nat Rev Mol Cell Biol 14 : 416–429.

4. SuY, OspinaJK, ZhangJ, MichelsonAP, SchoenAM, et al. (2011) Sequential phosphorylation of smoothened transduces graded hedgehog signaling. Sci Signal 4: ra43.

5. JiaJ, TongC, WangB, LuoL, JiangJ (2004) Hedgehog signalling activity of Smoothened requires phosphorylation by protein kinase A and casein kinase I. Nature 432 : 1045–1050.

6. ZhangC, WilliamsEH, GuoY, LumL, BeachyPA (2004) Extensive phosphorylation of Smoothened in Hedgehog pathway activation. Proc Natl Acad Sci U S A 101 : 17900–17907.

7. ApionishevS, KatanayevaNM, MarksSA, KalderonD, TomlinsonA (2005) Drosophila Smoothened phosphorylation sites essential for Hedgehog signal transduction. Nat Cell Biol 7 : 86–92.

8. JiaJ, TongC, JiangJ (2003) Smoothened transduces Hedgehog signal by physically interacting with Costal2/Fused complex through its C-terminal tail. Genes Dev 17 : 2709–2720.

9. LumL, ZhangC, OhS, MannRK, von KesslerDP, et al. (2003) Hedgehog signal transduction via Smoothened association with a cytoplasmic complex scaffolded by the atypical kinesin, Costal-2. Mol Cell 12 : 1261–1274.

10. YangC, ChenW, ChenY, JiangJ (2012) Smoothened transduces Hedgehog signal by forming a complex with Evc/Evc2. Cell Res 22 : 1593–1604.

11. WangG, AmanaiK, WangB, JiangJ (2000) Interactions with Costal2 and suppressor of fused regulate nuclear translocation and activity of cubitus interruptus. Genes Dev 14 : 2893–2905.

12. WangQT, HolmgrenRA (2000) Nuclear import of cubitus interruptus is regulated by hedgehog via a mechanism distinct from Ci stabilization and Ci activation. Development 127 : 3131–3139.

13. CheungHO, ZhangX, RibeiroA, MoR, MakinoS, et al. (2009) The kinesin protein Kif7 is a critical regulator of Gli transcription factors in mammalian hedgehog signaling. Sci Signal 2: ra29.

14. ShiQ, LiS, JiaJ, JiangJ (2011) The Hedgehog-induced Smoothened conformational switch assembles a signaling complex that activates Fused by promoting its dimerization and phosphorylation. Development 138 : 4219–4231.

15. InghamPW, NakanoY, SegerC (2011) Mechanisms and functions of Hedgehog signalling across the metazoa. Nat Rev Genet 12 : 393–406.

16. ChengS, MaierD, NeubueserD, HipfnerDR (2010) Regulation of Smoothened by Drosophila G-protein-coupled receptor kinases. Dev Biol 337 : 99–109.

17. MolnarC, HolguinH, MayorFJr, Ruiz-GomezA, de CelisJF (2007) The G protein-coupled receptor regulatory kinase GPRK2 participates in Hedgehog signaling in Drosophila. Proc Natl Acad Sci U S A 104 : 7963–7968.

18. ChenY, LiS, TongC, ZhaoY, WangB, et al. (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 : 2054–2067.

19. ChengS, MaierD, HipfnerDR (2012) Drosophila G-protein-coupled receptor kinase 2 regulates cAMP-dependent Hedgehog signaling. Development 139 : 85–94.

20. KinoshitaE, Kinoshita-KikutaE, TakiyamaK, KoikeT (2006) Phosphate-binding tag, a new tool to visualize phosphorylated proteins. Mol Cell Proteomics 5 : 749–757.

21. NeilsonKA, AliNA, MuralidharanS, MirzaeiM, MarianiM, et al. (2011) Less label, more free: approaches in label-free quantitative mass spectrometry. Proteomics 11 : 535–553.

22. ChenCH, von KesslerDP, ParkW, WangB, MaY, et al. (1999) Nuclear trafficking of Cubitus interruptus in the transcriptional regulation of Hedgehog target gene expression. Cell 98 : 305–316.

23. PearlmanSM, SerberZ, FerrellJEJr (2011) A mechanism for the evolution of phosphorylation sites. Cell 147 : 934–946.

24. HuangCC, TesmerJJ (2011) Recognition in the face of diversity: interactions of heterotrimeric G proteins and G protein-coupled receptor (GPCR) kinases with activated GPCRs. J Biol Chem 286 : 7715–7721.

25. BischofJ, MaedaRK, HedigerM, KarchF, BaslerK (2007) An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proc Natl Acad Sci U S A 104 : 3312–3317.

26. DenefN, NeubuserD, PerezL, CohenSM (2000) Hedgehog induces opposite changes in turnover and subcellular localization of patched and smoothened. Cell 102 : 521–531.

27. ClaingA, LaporteSA, CaronMG, LefkowitzRJ (2002) Endocytosis of G protein-coupled receptors: roles of G protein-coupled receptor kinases and beta-arrestin proteins. Prog Neurobiol 66 : 61–79.

28. ChenY, JiangJ (2013) Decoding the phosphorylation code in Hedgehog signal transduction. Cell Res 23 : 186–200.

29. NoblesKN, XiaoK, AhnS, ShuklaAK, LamCM, et al. (2011) Distinct phosphorylation sites on the beta(2)-adrenergic receptor establish a barcode that encodes differential functions of beta-arrestin. Sci Signal 4: ra51.

30. McDowellJH, NawrockiJP, HargravePA (1993) Phosphorylation sites in bovine rhodopsin. Biochemistry 32 : 4968–4974.

31. BusilloJM, ArmandoS, SenguptaR, MeucciO, BouvierM, et al. (2010) Site-specific phosphorylation of CXCR4 is dynamically regulated by multiple kinases and results in differential modulation of CXCR4 signaling. J Biol Chem 285 : 7805–7817.

32. ProssnitzER, KimCM, BenovicJL, YeRD (1995) Phosphorylation of the N-formyl peptide receptor carboxyl terminus by the G protein-coupled receptor kinase, GRK2. J Biol Chem 270 : 1130–1137.

33. TanJ, OhK, BurgessJ, HipfnerDR, BrillJA (2014) PI4KIIIalpha is required for cortical integrity and cell polarity during Drosophila oogenesis. J Cell Sci [Epub ahead of print.].

34. XiaR, JiaH, FanJ, LiuY, JiaJ (2012) USP8 promotes smoothened signaling by preventing its ubiquitination and changing its subcellular localization. PLoS Biol 10: e1001238.

35. DrakeMT, ShenoySK, LefkowitzRJ (2006) Trafficking of G protein-coupled receptors. Circ Res 99 : 570–582.

36. LiS, ChenY, ShiQ, YueT, WangB, et al. (2012) Hedgehog-regulated ubiquitination controls smoothened trafficking and cell surface expression in Drosophila. PLoS Biol 10: e1001239.

37. CorcoranRB, ScottMP (2006) Oxysterols stimulate Sonic hedgehog signal transduction and proliferation of medulloblastoma cells. Proc Natl Acad Sci U S A 103 : 8408–8413.

38. ReiterE, LefkowitzRJ (2006) GRKs and beta-arrestins: roles in receptor silencing, trafficking and signaling. Trends Endocrinol Metab 17 : 159–165.

39. ClaretS, SanialM, PlessisA (2007) Evidence for a novel feedback loop in the Hedgehog pathway involving Smoothened and Fused. Curr Biol 17 : 1326–1333.

40. ZhouQ, KalderonD (2011) Hedgehog activates fused through phosphorylation to elicit a full spectrum of pathway responses. Dev Cell 20 : 802–814.

41. CheliusD, BondarenkoPV (2002) Quantitative profiling of proteins in complex mixtures using liquid chromatography and mass spectrometry. J Proteome Res 1 : 317–323.