Smoothened transduces Hedgehog signal by forming a complex with Evc/Evc2

Abstract

Hedgehog (Hh) signaling plays pivotal roles in embryonic development and adult tissue homeostasis in species ranging from Drosophila to mammals. The Hh signal is transduced by Smoothened (Smo), a seven-transmembrane protein related to G protein coupled receptors. Despite a conserved mechanism by which Hh activates Smo in Drosophila and mammals, how mammalian Hh signal is transduced from Smo to the Gli transcription factors is poorly understood. Here, we provide evidence that two ciliary proteins, Evc and Evc2, the products of human disease genes responsible for the Ellis-van Creveld syndrome, act downstream of Smo to transduce the Hh signal. We found that loss of Evc/Evc2 does not affect Sonic Hedgehog-induced Smo phosphorylation and ciliary localization but impedes Hh pathway activation mediated by constitutively active forms of Smo. Evc/Evc2 are dispensable for the constitutive Gli activity in Sufu(-/-) cells, suggesting that Evc/Evc2 act upstream of Sufu to promote Gli activation. Furthermore, we demonstrated that Hh stimulates binding of Evc/Evc2 to Smo depending on phosphorylation of the Smo C-terminal intracellular tail and that the binding is abolished in Kif3a(-/-) cilium-deficient cells. We propose that Hh activates Smo by inducing its phosphorylation, which recruits Evc/Evc2 to activate Gli proteins by antagonizing Sufu in the primary cilia.

Figure 1

Evc and Evc2 are positive regulators in Hh signaling pathway. (A, B) Representative Gli-luciferase assay in NIH3T3 cells transfected with Smo and shRNAs against Evc/Evc2, respectively, and treated with or without Shh-conditioned medium (A) or SAG (B). Individual Evc/Evc2 shRNA knockdown efficiency was verified by real-time PCR indicated by the bottom panel.

Figure 2

Evc/Evc2 are not required for Smo activation upon Shh stimulation. (A-F) NIH3T3^Smo-CFP cells infected with control (CT) or the indicated Evc/Evc2 shRNA viruses and treated with or without Shh-conditioned medium were immunostained to show the expression of acetylated (Ac)-tubulin (Red) that labels the primary cilium, GFP (green) that labels the CFP-tagged Smo proteins and PS1 (blue) that labels the phosphorylated Smo. More than 50 cells were analyzed for each experiment and representative images were shown. The insets show enlarged views of the selected regions with shifted overlays. (G) The percentage of Smo-CFP (GFP) or phosphorylated Smo (PS1) positive primary cilia in cells that were infected by different shRNA-expression viruses and treated with or without Shh. Over 100 ciliated cells were counted for each time point, n = 3. (H) NIH3T3^Smo-CFP cells treated as in A-F were collected for immunoprecipitation with GFP antibody, followed by representative western blot analysis with PS1 or GFP antibodies, respectively.

Figure 3

Evc/Evc2 function downstream of Smo but upstream of Gli. (A-D) Representative Gli-luciferase assay in NIH3T3 cells transfected with SmoSD, SmoA1, SmoA1SD, Gli1 and shRNAs against Evc/Evc2, respectively, and treated with or without Shh-conditioned medium. Individual Evc/Evc2 knockdown efficiency was verified by real-time PCR (data not shown).

Figure 4

Evc/Evc2 are not required for constitutive Gli activity in Sufu^−/− cells. Representative Gli-luciferase assay in WT and Sufu^−/− MEF cells transfected with the indicated constructs and treated with or without Shh-conditioned medium. Individual Evc/Evc2 shRNA knockdown efficiency was verified by real-time PCR (data not shown).

Figure 5

Evc/Evc2 modulate Gli^A ciliary accumulation and inhibit Gli^R formation in response to Hh. (A-L) Shh-LIGHT2^FLAG-Gli1 and Shh-EGFP^FLAG-Gli2 cells were infected with control (CT) or the indicated Evc/Evc2 shRNA viruses twice; 2 days later, the cells were split for additional treatment with or without Shh-conditioned medium or real-time PCR analysis for knockdown efficiency. For Gli cilia localization test, cells were immunostained to show the expression of acetylated (Ac)-tubulin (Red) that labels the primary cilium, Flag (green) that labels the Flag-tagged Gli1 (A-F) or Gli2 (G-L) proteins and DRAQ5 (blue) that labels the nucleus. More than 50 cells were analyzed for each experiment and representative images were shown. The insets show enlarged views of the selected regions with shifted overlays. (M, N) Shh-EGFP^FLAG-Gli2cells treated as in G-L were collected and probed with anti-Flag primary antibody. Representative western blot was shown in M, and the ratio for Flag-Gli2^FL/Flag-Gli2^R was quantified with three independent western blot results using Image J software (N).

Figure 6

Hh promotes Smo binding to Evc/Evc2 and recruitment of Kif7. (A) Coimmunoprecipitation assays to determine the interaction between HA-tagged EVC (HA-EVC)/Flag-tagged EVC2 (Flag-EVC2) and the indicated forms of Myc-tagged Smo. NIH3T3 cells were cotransfected with the indicated Smo and Evc/Evc2 constructs, followed by immunoprecipitation and western blot analysis with the indicated antibodies. Cell lysates were also directly immunoblotted by the indicated antibodies. (B) Hh promotes the interaction between activated forms of Smo with Kif7, depending on Evc/Evc2 coexpression. Coimmunoprecipitation assays to determine the interaction between GFP-tagged Kif7 (Kif7-GFP) and the indicated forms of Myc-tagged Smo. NIH3T3 cells were cotransfected with the indicated Smo, Kif7 and Evc/Evc2 constructs, followed by immunoprecipitation and western blot analysis with the indicated antibodies. Cell lysates were also directly immunoblotted by the indicated antibodies.

Figure 7

A working model for Evc/Evc2 involvement in Hh signaling. Hh promotes the activation of Smo and its association with Evc/Evc2 complex in the primary cilia to transduce the signal intracellularly, leading to Gli activation.