In Hedgehog (Hh) signaling, the seven-transmembrane protein Smoothened (Smo) acts as a signal transducer that is regulated by phosphorylation, ubiquitination, and cell surface accumulation. However, it is not clear how Smo cell surface accumulation and intracellular trafficking are regulated. Here, we demonstrate that inactivation of Hrs by deletion or RNAi accumulates Smo in the late endosome that is marked by late endosome markers. Inactivation of Hrs enhances the wing defects caused by dominant-negative Smo. We show that Hrs promotes Smo ubiquitination, deleting the ubiquitin-interacting-motif (UIM) in Hrs abolishes the ability of Hrs to regulate Smo ubiquitination. However, the UIM domain neither recognizes the ubiquitinated Smo nor directly interacts with Smo. Hrs lacking UIM domain still downregulates Smo activity even though to a less extent. We have characterized that the N-terminus of Hrs directly interacts with the PKA/CK1 phosphorylation clusters to prevent Smo phosphorylation and activation, indicating an ubiquitin-independent regulation of Smo by Hrs. Finally, we found that knockdown of Tsg101 accumulates Smo that is co-localized with Hrs and other late endosome markers. Taken together, our data indicate that Hrs mediates Smo trafficking in the late endosome by not only promoting Smo ubiquitination but also blocking Smo phosphorylation.
References
[1]
Ingham PW, Nakano Y, Seger C (2011) Mechanisms and functions of Hedgehog signalling across the metazoa. Nat Rev Genet 12: 393–406.
[2]
Jiang J, Hui CC (2008) Hedgehog signaling in development and cancer. Dev Cell 15: 801–812.
[3]
Ingham PW, McMahon AP (2001) Hedgehog signaling in animal development: paradigms and principles. Genes Dev 15: 3059–3087.
[4]
Villavicencio EH, Walterhouse DO, Iannaccone PM (2000) The sonic hedgehog-patched-gli pathway in human development and disease. Am J Hum Genet 67: 1047–1054.
[5]
Taipale J, Beachy PA (2001) The Hedgehog and Wnt signalling pathways in cancer. Nature 411: 349–354.
[6]
Pasca di Magliano M, Hebrok M (2003) Hedgehog signalling in cancer formation and maintenance. Nat Rev Cancer 3: 903–911.
[7]
Jia J, Jiang J (2006) Decoding the Hedgehog signal in animal development. Cell Mol Life Sci 63: 1249–1265.
[8]
Zheng X, Mann RK, Sever N, Beachy PA (2010) Genetic and biochemical definition of the Hedgehog receptor. Genes Dev 24: 57–71.
[9]
Lum L, Beachy PA (2004) The Hedgehog response network: sensors, switches, and routers. Science 304: 1755–1759.
[10]
Hooper JE, Scott MP (2005) Communicating with Hedgehogs. Nat Rev Mol Cell Biol 6: 306–317.
[11]
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: 172–184.
[12]
Chen Y, Jiang J (2013) Decoding the phosphorylation code in Hedgehog signal transduction. Cell Res 23: 186–200.
[13]
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: e1001238.
[14]
Li S, Chen Y, Shi Q, Yue T, Wang B, et al. (2012) Hedgehog-regulated ubiquitination controls smoothened trafficking and cell surface expression in Drosophila. PLoS Biol 10: e1001239.
[15]
Nakano Y, Nystedt S, Shivdasani AA, Strutt H, Thomas C, et al. (2004) Functional domains and sub-cellular distribution of the Hedgehog transducing protein Smoothened in Drosophila. Mech Dev 121: 507–518.
[16]
Jekely G, Rorth P (2003) Hrs mediates downregulation of multiple signalling receptors in Drosophila. EMBO Rep 4: 1163–1168.
[17]
Rohatgi R, Milenkovic L, Scott MP (2007) Patched1 regulates hedgehog signaling at the primary cilium. Science 317: 372–376.
[18]
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: 3196–3201.
[19]
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: 2623–2628.
[20]
Wilson CW, Chuang PT (2010) Mechanism and evolution of cytosolic Hedgehog signal transduction. Development 137: 2079–2094.
[21]
Chen Y, Sasai N, Ma G, Yue T, Jia J, et al. (2011) Sonic Hedgehog dependent phosphorylation by CK1alpha and GRK2 is required for ciliary accumulation and activation of smoothened. PLoS Biol 9: e1001083.
[22]
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: 1045–1050.
[23]
Denef N, Neubuser D, Perez L, Cohen SM (2000) Hedgehog induces opposite changes in turnover and subcellular localization of patched and smoothened. Cell 102: 521–531.
[24]
Jia H, Liu Y, Xia R, Tong C, Yue T, et al. (2010) Casein kinase 2 promotes Hedgehog signaling by regulating both smoothened and Cubitus interruptus. J Biol Chem 285: 37218–37226.
[25]
Raiborg C, Rusten TE, Stenmark H (2003) Protein sorting into multivesicular endosomes. Curr Opin Cell Biol 15: 446–455.
[26]
Raiborg C, Stenmark H (2009) The ESCRT machinery in endosomal sorting of ubiquitylated membrane proteins. Nature 458: 445–452.
[27]
Jia H, Liu Y, Yan W, Jia J (2009) PP4 and PP2A regulate Hedgehog signaling by controlling Smo and Ci phosphorylation. Development 136: 307–316.
[28]
Robbins DJ, Fei DL, Riobo NA (2012) The Hedgehog signal transduction network. Sci Signal 5: re6.
[29]
Williams RL, Urbe S (2007) The emerging shape of the ESCRT machinery. Nat Rev Mol Cell Biol 8: 355–368.
[30]
Mukhopadhyay D, Riezman H (2007) Proteasome-independent functions of ubiquitin in endocytosis and signaling. Science 315: 201–205.
[31]
Polo S, Sigismund S, Faretta M, Guidi M, Capua MR, et al. (2002) A single motif responsible for ubiquitin recognition and monoubiquitination in endocytic proteins. Nature 416: 451–455.
[32]
Amit I, Yakir L, Katz M, Zwang Y, Marmor MD, et al. (2004) Tal, a Tsg101-specific E3 ubiquitin ligase, regulates receptor endocytosis and retrovirus budding. Genes Dev 18: 1737–1752.
[33]
Yang X, Mao F, Lv X, Zhang Z, Fu L, et al.. (2013) Drosophila Vps36 is involved in Hh signaling by regulating Smo trafficking. J Cell Sci.
[34]
Molnar C, Ruiz-Gomez A, Martin M, Rojo-Berciano S, Mayor F, et al. (2011) Role of the Drosophila non-visual ss-arrestin kurtz in hedgehog signalling. PLoS Genet 7: e1001335.
[35]
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: 2709–2720.
[36]
Chanut-Delalande H, Jung AC, Baer MM, Lin L, Payre F, et al. (2010) The Hrs/Stam complex acts as a positive and negative regulator of RTK signaling during Drosophila development. PLoS One 5: e10245.
