Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2003 Nov 1;17(21):2709-20.
doi: 10.1101/gad.1136603.

Smoothened transduces Hedgehog signal by physically interacting with Costal2/Fused complex through its C-terminal tail

Jianhang Jia  1 , Chao Tong, Jin Jiang
Affiliations

Smoothened transduces Hedgehog signal by physically interacting with Costal2/Fused complex through its C-terminal tail

Jianhang Jia et al. Genes Dev. .

Abstract

The Hedgehog (Hh) family of secreted proteins controls many aspects of growth and patterning in animal development. The seven-transmembrane protein Smoothened (Smo) transduces the Hh signal in both vertebrates and invertebrates; however, the mechanism of its action remains unknown. We found that Smo lacking its C-terminal tail (C-tail) is inactive, whereas membrane-tethered Smo C-tail has constitutive albeit low levels of Hh signaling activity. Smo physically interacts with Costal2 (Cos2) and Fused (Fu) through its C-tail. Deletion of the Cos2/Fu-binding domain from Smo abolishes its signaling activity. Moreover, overexpressing Cos2 mutants that fail to bind Fu and Ci but retain Smo-binding activity blocks Hh signaling. Taken together, our results suggest that Smo transduces the Hh signal by physically interacting with the Cos2/Fu protein complex.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Tagged Smo and its deletion mutants. Filled and gray boxes indicate the transmembrane domains and myristoylation signal (Myr), respectively. Filled, gray, and open triangles indicate the position where GFP, Myc, or Flag tag was inserted, respectively. The amino acid residues that demarcate each deletion mutant are indicated. Individual constructs were assayed for Hh signaling activity by overexpression using the UAS/Gal4 system in developing wing. Ectopic activity was scored when full-length Ci was stabilized and dpp-lacZ was activated in A-compartment cells away from the A/P compartment boundary. Rescue activity was scored when expressing a given Smo derivative can restore ptc-lacZ in smo mutant clones at the A/P boundary and/or restore naked cuticles in smo zygotic null embryos. Cos2/Fu binding was determined by coimmunoprecipitation. (NA) Not accessed.
Figure 2.
Figure 2.
The Smo C-tail is essential for Hh signaling. Wing discs expressing UAS-GFP-Smo (A-A″) or UAS-GFP-SmoΔCT by MS1096 (B-B″) were immunostained to show the expression of GFP (green in A,B), Ci155 (red in A′,B′), and dpp-lacZ (blue in A″,B″). Wing discs containing smo mutant clones and expressing UAS-GFP-Smo (C-C″) or UAS-GFP-SmoΔCT by MS1096 (D-D″) were immunostained to show the expression of ptc-lacZ (green) and CD2 (red). Smo mutant clones are recognized by the lack of CD2 expression (arrows). (E-I) Cuticles were prepared from embryos of the following genotypes growing at 18°C: wild type (E), smo3/smo3 (F), smo3/smo3; prd-Gal4/UAS-GFP-Smo (G), smo3/smo3; prd-Gal4/UAS-GFP-SmoΔCT (H), and smo3/smo3; prd-Gal4/UAS-Myc-Smo (I). The wild-type embryo exhibits alternate naked cuticles and denticles (E), whereas the smo3 homozygote exhibits the typical zygotic null phenotype with a lawn of denticles covering most of the ventral surface (F). Naked cuticles (indicated by arrows) are restored in alternate segments of smo3 homozygotes expressing either UAS-GFPSmo (G) or UAS-Myc-Smo (I), but not in smo3 homozygotes expressing UAS-GFP-SmoΔCT (H).
Figure 3.
Figure 3.
Constitutive Hh signaling activity associated with the Smo C-tail. Wing discs expressing UAS-Sev-SmoCT (A-A″) or UAS-Myr-SmoCT by act > CD2 > Gal4 (B-B″) were stained for Ci155 (red), Flag (green), and dpp-lacZ (blue). A-compartment cells expressing either Sev-SmoCT or Myr-SmoCT (recognized by Flag staining) accumulate high levels of Ci155 and activate dpp-lacZ in a cell-autonomous fashion. Wild-type wing disc (C) or wing disc expressing Myr-SmoCT by act > CD2 > Gal4 (D-D″) were treated with LMB, followed by immunostaining for Ci155 (red) and Arm (green). In wild-type discs, Ci155 is detected in the nucleus near the A/P border (arrowheads in C). Ci155 translocates into the nucleus in anteriorly situated cells expressing Myr-SmoCT (arrows in D,D′). The inset in D′ shows a high-magnification view of anteriorly situated cells expressing Myr-SmoCT. (E,E′) A wing disc carrying an smo mutant clone and expressing MS1096/UAS-Myr-SmoCT was immunostained to show CD2 expression (green) and the accumulation of Ci155 (red). smo mutant cells (marked by the lack of CD2 expression) expressing Myr-SmoCT accumulate high levels of Ci155. (F) A-compartment cells expressing both UAS-Myr-SmoCT and UAS-ptc by act > CD2 > Gal4 accumulate high levels of Ci155 (arrows). Expression from UAS-ptc was confirmed by staining with an anti-Ptc antibody (data not shown). (G-G″) A wing disc expressing MS1096/UAS-Myr-SmoCT and containing smo3 clones was immunostained to show the expression of dpp-lacZ (red) and CD2 (green). smo3 mutant cells are marked by the lack of CD2 expression. Overexpressing Myr-SmoCT activates dpp-lacZ in smo3 mutant cells (arrows). (H-H″) A wing disc expressing UAS-Myr-Smo730-1035 under the control of act > CD2 > Gal4 was stained for Ci155 (red), Flag (green), and dpp-lacZ (blue). A-compartment cells expressing Myr-Smo730-1035 accumulate high levels of Ci155 and activate dpp-lacZ.
Figure 4.
Figure 4.
Myr-SmoCT does not fully activate the Hh pathway and interferes with endogenous Smo. (A) ptc-lacZ expression in a wild-type wing disc. Wild-type (B) or Su(fu)LP homozygous (C) wing discs expressing UAS-Myr-SmoCT by act > CD2 > Gal4 were immunostained to show the expression of CD2 (green) and ptc-lacZ (red). Myr-SmoCT-expressing cells are recognized by the lack of CD2 staining. Myc-SmoCT only activates low levels of ptc-lacZ in anteriormost cells (arrow in B). (C) In contrast, most A-compartment cells expressing Myr-SmoCT activate high levels of ptc-lacZ in Su(fu) homozygous discs. (D-E″) Wing disc expressing a strong line of Myr-SmoCT (UAS-Myr-SmoCTS) alone (D-D″) or in conjunction with GFP-Smo (E-E″) were immunostained to show the expression of ptc-lacZ (red), Flag (green), and En (blue). High levels of Myr-SmoCT inhibit the expression of ptc-lacZ and en in A-compartment cells near the A/P boundary (arrows in D,D″), which is reversed by coexpressing GFP-Smo (arrows in E,E″).
Figure 5.
Figure 5.
Smo binds to the Cos2/Fu complex through its C-tail. (A) HA-tagged Cos2 and its deletion mutants. The microtubule-binding (MB) and coiled-coil (CC) domains are indicated by the black and gray boxes, respectively. The Ci- and Fu-binding domains are demarcated by lines above the diagram. (B,C) S2 cells were transfected with Myc-Smo or Myc-SmoΔCT expressing constructs with (B) or without (C) Cos2-, Fu-, and Ci-expressing constructs. The blank expressing vector pUAST was used as a control. Cell extracts were immunoprecipitated (IP) with anti-Myc antibody. Immunoprecipitates and 5% of cell lysates were analyzed by immunoblotting (WB) with indicated antibodies. Myc-Smo but not Myc-SmoΔCT pulled down overexpressed as well as endogenous Cos2 and Fu. Of note, overexpressed Myc-Smo and Myc-SmoΔCT exhibit slow mobility (indicated by asterisks). A similar observation was made with overexpressed vertebrate Smo (Stone et al. 1996). (D) Cell extracts were prepared from 400 wing discs expressing UAS-Myc-Smo, UAS-Myc-SmoΔCT, or UAS-Myc-Smo in conjunction with UAS-Hh under the control of MS1096. Wing disc extracts were immunoprecipitated with anti-Myc antibody, followed by immunoblotting with indicated antibodies. Five percent of lysates were analyzed by direct Western with Cos2 or Fu antibody. Myc-Smo but not Myc-SmoΔCT pulled down endogenous Cos2 and Fu. Ectopic Hh appears to stimulate phosphorylation of Fu bound to Myc-Smo, as indicated by slower mobility. Myc-Smo and Myc-SmoΔCT are indicated by asterisks. (E) S2 cells were transfected with the indicated Flag-tagged Smo constructs. Cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting with anti-Cos2 (top), anti-Fu (middle), or anti-Flag (bottom) antibodies. Asterisks indicate the position of proteins expressed from corresponding Smo constructs. Arrows indicate IgG. (F) S2 cells were transfected with Myc-Smo in conjunction with various HA-tagged Cos2 deletion mutants. Cell extracts were immunoprecipitated with anti-Myc antibody, followed by immunoblotting with anti-HA antibody. Five percent of cell lysates were also subjected to Western blotting with anti-HA antibody (bottom). Asterisks indicate the position of HA-tagged Cos2 proteins expressed from corresponding constructs (bottom) or immunoprecipitated with Myc-Smo (top). Of note, HA-Cos2MB immunoprecipitated with Myc-Smo runs very closely to IgG.
Figure 6.
Figure 6.
Blockage of Hh signaling by Cos2 deletion mutants. (A,A′) A wing disc expressing UAS-HA-Cos2ΔN2 by act > CD2 > Gal4 was immunostained for ptc-lacZ (green) and HA (red). Cos2ΔN2 blocks the expression of ptc-lacZ near the A/P border (arrows). (B) A wing disc expressing UAS-HACos2CC by act > CD2 > Gal4. Cos2CC failed to suppress ptc-lacZ expression at the A/P compartment boundary (arrows). (C-C″) A wing disc expressing both UAS-HA-Cos2ΔN2 and UAS-GFP-Smo by act > CD2 > Gal4 was immunostained to show the expression of HA (red), ptc-lacZ (green), and GFP (blue). Coexpression of GFP-Smo with Cos2ΔN2 restores the expression of ptc-lacZ near the A/P border (arrows). (D-D″) A wing disc expressing UAS-Flag-Cos2CT1 by MS1096 was stained to show the expression of Flag (red), ptc-lacZ (green), and En (blue). High levels of Cos2CT1 suppress the expression of ptc-lacZ and en (arrows). A wild-type adult wing (E) or adult wing expressing UAS-Flag-Cos2CT1 by MS1096 (F). Overexpressing Cos2CT1 results in wing phenotypes similar to those caused by the fu mutation.
Figure 7.
Figure 7.
Signaling by Smo and its C-tail in response to different thresholds of Hh. (A) In the absence of Hh, Ptc prevents cell surface accumulation of Smo. In addition, the Smo C-tail may adopt a “closed” conformation that prevents it from binding to Cos2/Fu. Inside the cell, the full-length Ci (Ci155) forms a complex with Cos2, Fu, and Su(fu). The majority of Ci155 undergoes proteolytic processing to generate the repressor form (Ci75). Ci processing also requires the activity of PKA, GSK3, CKI, and SCFslimb (Jiang 2002). (B) The inhibition of Smo by Ptc is partially alleviated by low levels of Hh (indicated by broken line), leading to an increase of Smo on the cell surface. In addition, the Smo C-tail may adopt an “open” conformation, which allows Smo to bind the Cos2/Fu complex and inhibit its Ci-processing activity. Under this condition, Ci75 is not produced and dpp is derepressed. However, the transcriptional activity of Ci155 is still suppressed by Su(fu). (C) High levels of Hh completely block Ptc, resulting in a further increase in Smo signaling activity. Hyperactive forms of Smo (indicated by two asterisks) stimulate the phosphorylation and activity of bound Fu (indicated by asterisk), which in turn antagonizes Su(fu) to activate Ci155, leading to the expression of ptc and en. (D) Overexpressed membrane-tethered SmoCT binds the Cos2/Fu complex and inhibits Ci processing independently of Hh and endogenous Smo. However, the activity of Ci155 is still blocked by Su(fu). (E) In the presence of Hh, overexpressed Myr-SmoCT competes with activated Smo for binding to Cos2/Fu and prevents it from activating Fu. (F) Increasing the amount of full-length Smo by overexpressing GFP-Smo restores Smo**/Cos2/Fu interaction, allowing Smo** to activate Fu, which in turn stimulates Ci155.
See this image and copyright information in PMC

References

    1. Alcedo J., Ayzenzon, M., Von Ohlen, T., Noll, M., and Hooper, J.E. 1996. The Drosophila smoothened gene encodes a seven-pass membrane protein, a putative receptor for the Hedgehog signal. Cell 86: 221-232. - PubMed
    1. Alexandre C., Jacinto, A., and Ingham, P.W. 1996. Transcriptional activation of Hedgehog target genes in Drosophila is mediated directly by the Cubitus interruptus protein, a member of the GLI family of the zinc finger DNA-binding proteins. Genes & Dev. 10: 2003-2013. - PubMed
    1. Amanai K. and Jiang, J. 2001. Distinct roles of Central missing and Dispatched in sending the Hedgehog signal. Development 128: 5119-5127. - PubMed
    1. Ascano M., Nybakken, K.E., Sosinski, J., Stegman, M.A., and Robbins, D.J. 2002. The carboxyl-terminal domain of the protein kinase fused can function as a dominant inhibitor of hedgehog signaling. Mol. Cell. Biol. 22: 1555-1566. - PMC - PubMed
    1. Aza-Blanc P., Ramirez-Weber, F., Laget, M., Schwartz, C., and Kornberg, T. 1997. Proteolysis that is inhibited by Hedgehog targets Cubitus interruptus protein to the nucleus and converts it to a repressor. Cell 89: 1043-1053. - PubMed

Publication types