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. 2006 Mar 17;281(11):7237-43.
doi: 10.1074/jbc.M510169200. Epub 2006 Jan 19.

Smoothened regulates activator and repressor functions of Hedgehog signaling via two distinct mechanisms

Stacey K Ogden  1 , David J Casso, Manuel Ascano Jr, Mark M Yore, Thomas B Kornberg, David J Robbins
Affiliations

Smoothened regulates activator and repressor functions of Hedgehog signaling via two distinct mechanisms

Stacey K Ogden et al. J Biol Chem. .

Abstract

The secreted protein Hedgehog (Hh) plays an important role in metazoan development and as a survival factor for many human tumors. In both cases, Hh signaling proceeds through the activation of the seven-transmembrane protein Smoothened (Smo), which is thought to convert the Gli family of transcription factors from transcriptional repressors to transcriptional activators. Here, we provide evidence that Smo signals to the Hh signaling complex, which consists of the kinesin-related protein Costal2 (Cos2), the protein kinase Fused (Fu), and the Drosophila Gli homolog cubitus interruptus (Ci), in two distinct manners. We show that many of the commonly observed molecular events following Hh signaling are not transmitted in a linear fashion but instead are activated through two signals that bifurcate at Smo to independently affect activator and repressor pools of Ci.

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Figures

FIGURE 1
FIGURE 1. CSBD uncouples target gene activation from Ci stabilization and Fu/Cos2 hyperphosphorylation
A, CSBD is a strong inhibitor of Hh-mediated target gene activation. Cl8 cells were transfected with a ptc-luciferase reporter construct and increasing amounts of plasmids expressing CSBD, SmoC, or empty vector control, in the presence or absence of a Hh expression vector. Percent expression relative to maximal Hh activation is indicated. ptc-luciferase expression levels were normalized to an act-renilla transfection control. Error bars indicate S.E. For all experiments, 1× corresponds to 250 ng of transfected DNA and 2× corresponds to 500 ng. B, Ci reverses CSBD-mediated repression. Cl8 cells were co-transfected with plasmids expressing Hh, CSBD and increasing amounts of Ci, or empty vector control. Relative ptc-luciferase expression levels were normalized to an act-renilla control. Error bars indicate S.E. For all experiments, 1× corresponds to 250 ng of transfected DNA and 2× corresponds to 500 ng. C, CSBD and SmoC have different effects on the molecular markers of Hh activation. Cl8 cells were co-transfected with increasing amounts of plasmids expressing CSBD (250 and 500 ng) or SmoC (250 and 500 ng) in the presence or absence of Hh. DNA content was normalized with empty vector. 1% Nonidet P-40 cell lysates were immunoblotted for Ci155, Fu, Cos2, Ci75, CSBD, SmoC, and kinesin. Hyperphosphorylated forms of Cos2/Fu are marked with bars (P, phosphorylated; U, unphosphorylated). The results shown are representative of at least three independent experiments. D, CSBD inhibits Hh-mediated activation of endogenous ptc. Cl8 cells were transfected with increasing amounts of a plasmid expressing CSBD in the presence or absence of Hh. 1% Nonidet P-40 cell lysates were immunoblotted for Ptc, Fu, CSBD, and kinesin. Hyperphosphorylated forms of Fu are marked with bars (P, phosphorylated; U, unphosphorylated). The results shown are representative of at least three independent experiments.
FIGURE 2
FIGURE 2. CSBD uncouples target gene activation from Ci stabilization in vivo
A–D, CSBD overexpression results in disruption of Hh signaling. Wings from wild type flies demonstrate normal patterning of longitudinal veins (LV) 1–5 (A). Wings from ptc-GAL4;UAS-HA-CSBD flies demonstrate mild LV3-LV4 fusions (arrow). Wings from ptc-GAL4 (B), UAS-Smo RNAi flies (C) demonstrate a mild fusion of LV3-LV4 (arrow). Overexpression of CSBD in Smo-RNAi flies triggers a near lethal phenotype. Wings of surviving ptc-GAL4, UAS-Smo RNAi; UAS-CSBD flies (D) demonstrate significantly decreased LV3– 4 spacing and more severe LV3– 4 fusions (arrows), indicating an enhanced loss of normal Hh signaling. E–F, CSBD disrupts Hh target gene activation without affecting Ci protein stabilization. Late third instar wing imaginal discs from wild type (E) and ptc-GAL4, UAS-Smo RNAi; UAS-HA-CSBD (F) flies were immunostained with anti-HA (blue, F), anti-Ci (red, E and F), and anti-En (green) antibodies as indicated. CSBD has little effect on Hh activated Ci stabilization in cells 3– 8 cell diameters from the A/P border (arrow) but does affect Ci* (arrowheads) and anterior expression of the high level target gene en (F″and F* bracket). For all discs, anterior is toward the left and dorsal is toward the top. G–H, CSBD does not alter dpp expression. Late third instar wing imaginal discs from wild type (G) and ptc-GAL4, UAS-Smo RNAi; UAS-HA-CSBD (H) flies were immunostained with anti-HA (blue, H), anti-Ci (red, G′and H″), and anti β-galactosidase (green, G and H′) antibodies as indicated. Although CSBD does alter Ci* expression in cells immediately adjacent to the A/P border (arrowheads), expression of the low level target gene dpp-lacZ is not affected. For all discs, anterior is toward the left and dorsal is toward the top.
FIGURE 3
FIGURE 3. CSBD blocks Ci activation by targeting Smo
A, HSC membrane release is unaffected by CSBD. Cl8 cells were co-transfected with plasmids expressing increasing amounts of HA-CSBD (lanes 3– 6 and 9–12; 500 ng (lanes 3, 4, 9, 10) and 1 μg (lanes 5, 6, 11, 12) CSBD expression vector) in the presence (lanes 7–12) or absence (lanes 1– 6) of a Hh expression vector. Hypotonic cell lysates were fractionated, and the resulting membrane pellets (P) and cytosolic soluble fractions (S) were analyzed by immunoblot. U, unphosphorylated; P, phosphorylated. *, the band appearing in the S fraction in the Smo blot is either a nonspecific band or a minor membrane contamination in the soluble fraction, as it does not reproducibly appear in this assay. The results shown are representative of a minimum of three independent experiments. B, CSBD inhibits Hh-mediated Smo accumulation in S2 cells. Myc-Smo was expressed in S2 cells plus or minus HA-CSBD (1 μg, lanes 2 and 4) and Hh expression vector (lanes 3 and 4). 1% Nonidet P-40 cell lysates were analyzed by immunoblot analysis. C–F, CSBD alters Hh-mediated Smo subcellular relocalization. S2 cells transfected with 1 μg of a plasmid expressing Smo in the presence (E and F) or absence (C and D) of 1 μg of HA-CSBD expression vector and Hh expression vector (D and F) were plated on concanavalin A-treated slides and immunostained for Smo (red) and HA-CSBD (green).
FIGURE 4
FIGURE 4. A model for separation of activator and repressor functions of Hh signaling
A, Cos2 functions as a scaffold in HSC-R and HSC-A. Hh attenuates HSC-R function by inhibiting Ci phosphorylation and its subsequent processing into a transcriptional repressor. Hh promotes the activity of HSC-A by stimulating the phosphorylation and stabilization of Smo, thereby allowing for the formation of additional Cos2-Smo complexes. Stabilized Smo translocates to the plasma membrane where it likely activates Ci. CSBD selectively inhibits HSC-A by blocking the relevant Cos2-Smo association. HSC-R is intact in the presence of CSBD, as its regulation by Hh does not depend on Cos2 cargo domain-Smo binding. B, model for the reciprocal activity of HSC-A and HSC-R in response to Hh. High level Hh stimulation promotes the high activity of HSC-A and low level activity of HSC-R. As the level of Hh stimulation decreases, HSC-A action decreases and HSC-R activity becomes predominant.
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References

    1. Aza-Blanc P, Ramirez-Weber FA, Laget MP, Schwartz C, Kornberg TB. Cell. 1997;89:1043–1053. - PubMed
    1. Sanchez-Herrero E, Couso JP, Capdevila J, Guerrero I. Mech Dev. 1996;55:159–170. - PubMed
    1. Wang QT, Holmgren RA. Development (Camb) 2000;127:3131–3139. - PubMed
    1. Ohlmeyer JT, Kalderon D. Nature. 1998;396:749–753. - PubMed
    1. Methot N, Basler K. Cell. 1999;96:819–831. - PubMed

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