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Comparative Study
. 2003 Nov 11;13(22):1998-2003.
doi: 10.1016/j.cub.2003.10.004.

Identification of a functional interaction between the transmembrane protein Smoothened and the kinesin-related protein Costal2

Stacey K Ogden  1 , Manuel Ascano Jr, Melanie A Stegman, Liza M Suber, Joan E Hooper, David J Robbins
Affiliations
Comparative Study

Identification of a functional interaction between the transmembrane protein Smoothened and the kinesin-related protein Costal2

Stacey K Ogden et al. Curr Biol. .

Abstract

The hedgehog (Hh) family of morphogens plays important instructional roles in the development of numerous metazoan structures. Consistent with the role Hh homologs play in cell fate determination, aberrant Hh signaling results in numerous human pathologies. Hh signal transduction is initiated when Hh binds to its receptor Patched (Ptc), activating the transmembrane protein Smoothened (Smo). Smo transmits its activation signal to a microtubule-associated Hedgehog signaling complex (HSC). At a minimum, the HSC consists of the Kinesin-related protein Costal2 (Cos2), the protein kinase Fused (Fu), and the transcription factor Cubitus interruptus (Ci). In response to HSC activation, the ratio between repressor and activator forms of Ci is altered, determining the expression levels of various Hh target genes. The steps between Smo activation and signaling to the HSC have not been described. Here, we describe a functional interaction between Smo and Cos2, which is necessary for Hh signaling. We propose that this interaction is direct and allows for activation of Ci in response to Hh. This work fills in the last major gap in our understanding of the Hh signal transduction pathway by suggesting that no intermediate signal is required to connect Smo to the HSC.

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Figures

Figure 1
Figure 1
Smo Associates with the HSC via Direct Interaction with Cos2 (A) The HSC associates with endogenous Smo. Wild-type Drosophila embryos were lysed and immunoprecipitated with antibodies generated to either amino- (dN), carboxyl-terminal (dC) regions of Smo, or control antibody. Cos2 and Fu immunoprecipitating with Smo are indicated. (B) Smo and Cos2 colocalize in Drosophila S2 cells. Endogenous Smo and Cos2 in S2 cells were stained using dC anti-Smo (Santa Cruz) and 5D6 anti-Cos2 mAbs, respectively. (C) Smo and Cos2 associate directly. Matings were performed between indicated yeast strains, then streaked on selective media in numbered sectors as follows: (1) pGAD-SmoC and pGBDU-Cos2, (2) pGAD-Fu-tail and pGBDU-Cos2, (3) pGAD-Fu-tail and pGBDU-antiCos2, (4) pGAD-SmoC and pGBDU-antiCos2, (5) pGAD-antiCos2 and pGBDU-SmoC, and (6) pGAD-Cos2 and pGBDU-SmoC. “Anti” indicates antisense orientation of the indicated cDNA.
Figure 2
Figure 2
Smo-Cos2 Association Is Not Modified in Response to Hh (A) Smo and Cos2 association in S2 cells is Hh insensitive. Postnuclear lysates were prepared from S2 cells transfected with Hh expression (pDA-Flag-HhN) or control vector. Smo was immunoprecipitated from these lysates using Smo dC or dN antibodies, as indicated. Immunoprecipitates were probed for the presence of Fu and Cos2. (B) Smo-Cos2 association is Hh insensitive in vivo. Drosophila embryos representing normal (wt), repressed (ptc), or activated (hh) Hh signaling were generated by expressing Ptc or Hh. Lysates from 25 mg of embryos were immunoprecipitated with either α-Cos2 5D6 mAb (Cos2 IP), Smo antibody (Smo IP), or Fz antibody (Fz IP). Western blots were probed for Smo (N-terminal antibody), Fu, and Cos2. Each protein has multiple isoforms, with the slower-migrating forms due to phosphorylation [6, 25]. The unmodified form of each protein is favored in the absence of Hh signaling (and in ptc-overexpressing embryos), while the phosphorylated forms appear with Hh exposure (hh lanes). (C) Smo-Cos2 colocalization is similar across the wing imaginal disc. Wild-type wing imaginal discs were immunostained with antibodies directed against Smo (red) and Cos2 (green) (top), or Kinesin (red) and Cos2 (green) (bottom). For each panel, anterior is to the left, and dorsal is toward the top. The anterior/posterior boundary is marked by arrowheads. The colocalization of Kinesin or Fz1 (data not shown) was minimal, emphasizing the specificity of the Smo-Cos2 interaction. Low-magnification images are also provided for comparison; see Figure S1.
Figure 3
Figure 3
Cos2 Association with Smo Is Necessary for Hh Signal Transduction (A) Myr-SmoC diagram. Illustration of wild-type Smo and Myr-SmoC. Myr-SmoC contains an amino-terminal myristoylation signal, as well as a Myc epitope tag. (B) Myr-SmoC associates with Cos2. Myr-SmoC was immunoprecipitated, using anti-Myc beads (Sigma), from postnuclear lysates of Cl8 cells transfected with increasing amounts of pUAS-Myr-SmoC, as indicated. An IgG control immunoprecipitation was performed from the lysate of cells transfected with 0.5 μg of Myr-SmoC. Immunoprecipitates were separated by SDS-PAGE and immunoblotted for Cos2 and Fu (data not shown). (C) Myr-SmoC inhibits Hh activated transcription. 5 × 106 Cl8 cells were transfected with 0.1 μg ptcΔ136-Luc reporter, 0.02 μg pRL-TK transfection control, increasing amounts of pUAS-Myr-SmoC (0, 0.1, 0.2, and 0.5 μg) with pActGal4, and either 0.2 μg pDA-HhN-Flag (black columns) or a control vector (gray columns). Luciferase activity was normalized to a Renilla transfection control and is expressed relative to baseline ptcΔ136-Luc activity.
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