Transduction of the Hedgehog signal through the dimerization of Fused and the nuclear translocation of Cubitus interruptus

Abstract

The Hedgehog (Hh) family of secreted proteins is essential for development in both vertebrates and invertebrates. As one of main morphogens during metazoan development, the graded Hh signal is transduced across the plasma membrane by Smoothened (Smo) through the differential phosphorylation of its cytoplasmic tail, leading to pathway activation and the differential expression of target genes. However, how Smo transduces the graded Hh signal via the Costal2 (Cos2)/Fused (Fu) complex remains poorly understood. Here we present a model of the cell response to a Hh gradient by translating Smo phosphorylation information to Fu dimerization and Cubitus interruptus (Ci) nuclear localization information. Our findings suggest that the phosphorylated C-terminus of Smo recruits the Cos2/Fu complex to the membrane through the interaction between Smo and Cos2, which further induces Fu dimerization. Dimerized Fu is phosphorylated and transduces the Hh signal by phosphorylating Cos2 and Suppressor of Fu (Su(fu)). We further show that this process promotes the dissociation of the full-length Ci (Ci155) and Cos2 or Su(fu), and results in the translocation of Ci155 into the nucleus, activating the expression of target genes.

Figure 1

Hh induces the recruitment of Cos2/Fu to the membrane through the interaction between Smo and the Cos2/Fu complex. (A) S2 cells were co-transfected with Smo-CFP^C, SmoSA-CFP^C, SmoSD-CFP^C(green) and Cos2-YFP^C (red), and Myc-tagged Fu, treated with or without Hh, and immunostained with anti-Myc antibody (blue). In the absence of Hh, only a few Cos2/Fu complexes co-localized with wild-type Smo. In the presence of Hh, the majority of Co2/Fu complexes co-localized with wild-type Smo and was recruited to the cell plasma membrane. When SmoSA was co-transfected, Cos2/Fu complexes were still localized in the cytoplasm following Hh treatment. When SmoSD was co-transfected, Cos2/Fu complexes were localized on the cell plasma membrane even without Hh treatment. (B) Quantification of Smo co-localized with Cos2 by using Smo mutants to mimic Hh gradient. (C, E) FRET efficiency from the indicated constructs in S2 cells treated with or without Hh-condition medium (mean ± sd, n ≥ 15). (D, F) FRET efficiency between Smo-CFP^C/Cos2-YFP^C(D), or Cos2-CFP^C/Cos2-YFP^C(F), expressed in wing discs (mean ± sd, n ≥ 8). A, A-compartment cells away from the A/P boundary; P, P-compartment cells; A/P, A-compartment cells adjacent to the A/P boundary.

Figure 2

The binding regions of Smo with Cos2 and Fu in the absence or presence of Hh. (A) S2 cells were transfected with Smo-CFP^C and Cos2-YFP^C with control RNAi and Fu, or the endogenous Fu knocked down by dsRNA in the absence or presence of Hh. (B) S2 cells were transfected with Smo-CFP^C and Fu-YFP^N with control RNAi and Cos2, or the endogenous Cos2 was knocked down by dsRNAi in the absence or presence of Hh. Then, the co-localization of Smo with Cos2 (A) or Fu (B) was examined. (A′) Quantification of Smo co-localized with Cos2 by using dsRNA of Fu to knockdown endogenous Fu. (B′) Quantification of Smo co-localized with Fu by using dsRNA of Cos2 to knockdown endogenous Cos2. (C, D) S2 cells were transfected with indicated Smo mutant constructs and Fu construct with endogenous Cos2 knocked down (C), or Cos2 construct with endogenous Fu knocked down (D), treated with or without Hh, respectively. (E) FRET efficiency from the indicated constructs in S2 cells treated with or without Hh (mean ± sd, n ≥ 15).

Figure 3

The dimerization of Fu is induced by Hh and is Smo and Cos2 dependent. (A, B) FRET efficiency of Fu-CFP^N and Fu-YFP^N in S2 cells (mean ± sd, n ≥ 15) (A), or in wing discs (mean ± sd, n ≥ 10) (B). (C, D) In the absence/presence of Hh, FRET efficiency of Fu-CFP^N and Fu-YFP^N co-expressing Smo (C) or Cos2 (D) with control RNAi, Cos2 RNAi (C) or Smo RNAi (D), to knockdown the endogenous protein. (E) Smo-CFP^C, Cos2-YFP^C or Cos2ΔMD-YFP^C and Myc-Fu were co-transfected in S2 cells, with or without Hh treatment, to detect the effects of the Cos2 motor domain on the co-localization of Smo and the Cos/Fu complex. Myc-Fu was immunostained (blue). (E′) Quantification of Smo that co-localized with Cos2 or Cos2ΔMD in the absence or presence of Hh. (F) FRET efficiency of Smo-CFP^C and Cos2-YFP^C or Cos2ΔMD-YFP^C with Myc-Fu co-transfected in S2 cells with or without Hh treatment. (G) FRET efficiency of Smo-CFP^C and Cos2-YFP^C with control RNAi, or the endogenous Fu knocked down by Fu RNAi in the absence/presence of Hh, respectively.

Figure 4

Induced Fu dimerization triggers the Hh pathway, which is independent of Smo and Cos2. (A-E) The ptc-luc reporter assay shows the pathway activity changes in S2 cells transfected with Ci155 and Su(fu). Error bars, 1 sd (triplicate wells). S2 cells were co-transfected with the indicated Div-based Fu expression constructs (A), or FKBP-based Fu constructs and treated with or without AP20187 (B). (C) S2 cells were co-transfected with Fv2-FuG13V or Fv2-Fu, and treated with the graded AP20187. Along with the concentration change of the synthetic agent (AP20187), the ptc-luc reporter activity increased from low to high when the cells were transfected with Fv2-Fu (red line, Fv2-Fu; blue line, Fv2-FuG13V). (D, E) With Smo or Cos2 RNAi to knockdown endogenous protein expression, in both Fu dimerization system based on FKBP or Div, dimerized Fu could trigger ptc-luciferase reporter activity bypassing Smo and Cos2.

Figure 5

The dimerization of Fu promotes Fu auto-phosphorylation and Cos2 and Su(fu) phosphorylation. (A) Phosphorylation mobility shift of dimerized Fv2-Fu as induced by a synthetic agent (AP20187) in vitro with ATP. Fv2-FuG13V served as a control. Phosphatase was used to dephosphorylate the phosphorylated protein. (B) Phosphorylation of Cos2 by dimerized Fu. In Div-based dimerization system, dimerized Fu can phosphorylate Cos2 in vitro. S2 cells were transfected with Flag-Cos2, Div-FuG13V, Div-Fu, Div-FuKG13V, Div-FuK^*G13V or Div-FuK^*, respectively. Then, Cos2 or different Div-Fu fusion proteins were concentrated by immunoprecipitation, respectively. The in vitro kinase assays were followed. Phosphatase was used to dephosphorylate the phosphorylated proteins. Div-Fu (B1), Div-FuK (B2) and Div-FuK^* (B3) can phosphorylate Cos2 but not the G13V mutants. (C) Phosphorylation of Flag-Su(fu) by activated Fu in an in vitro Div-Fu system. Su(fu) can be phosphorylated by Div-Fu and Div-FuK^* but not Div-FuK. In (A-C), the mobility shift band of target proteins were labeled by stars. (D) Smo-CFP^C(green), Cos2WT/SA-YFP^C (red) and Myc-FuWT/G13V (blue) were co-transfected in S2 cells in the absence/presence of Hh, to detect the effect of Cos2 phosphorylation or Fu kinase activity on co-localization between Smo and Cos2/Fu complex.

Figure 6

Phosphorylated Cos2 and Su(fu) by dimerized Fu promote the dissociation of Ci155 from the Cos2/Fu complex or Su(fu) and its nuclear translocation. (A, B-B′, C, D-D″) S2 cells were transfected with Myc-Ci155, Flag-Cos2 (A-B′) or Flag-Su(fu) (C-D′), and the indicated constructs. Cell extracts were immunoprecipitated with anti-Flag antibodies, followed by immunoblotting with indicated antibodies. Western blot for Flag-Cos2 or Flag-Su(fu) is serving as loading control, respectively. The Ci155 expression in the lysate is detected with anti-Myc antibody. (A) S2 cells were transfected with Flag-Cos2, Myc-Ci155 and Smo phosphor-mimetic mutants, which mimicked Hh gradient. (B, B′) FKBP- or Div-based Fu constructs were transfected into S2 cells with Flag-Cos2 and Myc-Ci155. (C) S2 cells were transfected with Flag-Su(fu), Myc-Ci155 and Smo phosphor-mimetic mutants. SmoSD, which mimics the high Hh concentration, but not SmoSD¹ and SmoSD¹², could induce the dissociation between Su(fu) and Ci155. (D, D′) The dimerized Fu (Fv2-Fu with AP20187 treatment or Div-Fu) decreased the association between Su(fu) and Ci155, compared with Fv2-FuG13V with or without AP20187 treatment, Fv2-Fu without AP20187 treatment, or Div-FuG13V. (D′′) Div-FuK^* enhanced dissociation of Su(fu) with Ci155, compared with Div-FuK, Div-FuKG13V and Div-FuK^*G13V. (E) S2 cells were transfected with HA-Ci155, or HA-Ci155 and Flag-Su(fu), and treated with Hh or LMB, respectively, or with Hh and LMB treatment. The antibody detecting full-length Ci was used to stain for Ci155 (red), antibody recognizing Flag was used for immunostaining Flag-Su(fu) (green), and the nuclei were stained with DAPI (blue). The nuclei were circled by white line. (F, G) The percentage of S2 cells with Ci155 nuclear localization after co-transfected with Smo phosphor-mimetic mutants to mimic Hh gradient (F), or with dimerized Fu. FKBP- or Div-based dimerized Fu could antagonize Su(fu) and promote Ci155 nuclear translocation (G). In F and G, pUAST and Hh treatment served as negative and positive control, respectively.

Figure 7

In vivo activities of dimerized Fu variants. (A-H) Wing discs expressing the indicated dimerized Fu variants by act>CD2>Gal4. The immunostainings show the expression of Fu variants, dpp-lacZ (dppZ) (A-D) and endogenous Ptc (E-H). In the Div-Fu, Div-FuK and Div-FuK^* clones, which were marked by green, dppZ or Ptc (blue) expression was turned on in A-compartment (B, F, Div-Fu; C and G, Div-FuK; D, H, Div-FuK^*). However, as the control, DivIVA protein failed to upregulate dppZ or Ptc expression (A, E).