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. 2007 Aug 1;21(15):1949-63.
doi: 10.1101/gad.1557407.

Fused-Costal2 protein complex regulates Hedgehog-induced Smo phosphorylation and cell-surface accumulation

Yajuan Liu  1 , Xuesong Cao, Jin Jiang, Jianhang Jia
Affiliations

Fused-Costal2 protein complex regulates Hedgehog-induced Smo phosphorylation and cell-surface accumulation

Yajuan Liu et al. Genes Dev. .

Abstract

The seven-transmembrane protein Smoothened (Smo) acts as a signal transducer in the Hedgehog (Hh) pathway that mediates many key developmental processes. In Drosophila, Hh-induced phosphorylation promotes Smo cell-surface accumulation and signaling activity; however, the mechanisms controlling Smo phosphorylation and cell-surface accumulation are still unknown. The intracellular signaling complex containing Fused (Fu) and Costal2 (Cos2) is thought to transduce the Hh signal downstream from Smo. Here, we identify a novel feedback mechanism that regulates Smo through the Fu-Cos2 complex. We found that Hh-induced Smo accumulation is inhibited in fu mutant clones or by expressing a dominant-negative form of Fu, and such inhibition is alleviated by removal of Cos2. Conversely, overexpressing Cos2 blocks Smo accumulation, which is reversed by coexpressing Fu. Cos2 blocks Smo accumulation through its C-terminal Smo-interacting domain, and Fu antagonizes Cos2 by phosphorylating Cos2 at Ser572. Furthermore, we found that Ser572 phosphorylation attenuates the Cos2-Smo interaction and promotes Cos2 instability. Finally, we provided evidence that Fu and Cos2 control Smo cell-surface accumulation by regulating Smo phosphorylation. Our data suggest that Cos2-Smo interaction blocks Hh-induced Smo phosphorylation, and that Fu promotes Smo phosphorylation by antagonizing Cos2.

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Figures

Figure 1.
Figure 1.
FuG13V has a dominant-negative effect in Hh pathway. (A–C) Adult wings from wild-type (A), fuA mutant (B), or flies expressing HA-FuG13V by MS1096 Gal4 (C). Note the fusion between L3 and L4 in fuA- and FuG13V-expressing wings. (D,D′) A wing disc expressing HA-FuG13V with act > CD2 > Gal4 was immunostained for HA (green) and ptc-lacZ (red). FuG13V blocked ptc-lacZ expression (arrows). (E,E′) A wing disc expressing GFP (green) with ap-Gal4 was immunostained to show the expression of En (red). (F–F′″) A wing disc expressing GFP (green) and HA-FuG13V with ap-Gal4 was immunostained to show the expression of En (red) and HA (blue). Expressing HA-FuG13V inhibited the expression of en in anterior dorsal cells near the A/P boundary (arrow in F). (G–G′″) A wing disc coexpressing Myc-FuWT with HA-FuG13V restored anterior dorsal En expression (arrow in G) that was inhibited by FuG13V. All wing discs shown in this study were oriented with anterior to the left and ventral on the top.
Figure 2.
Figure 2.
Fu is required for Hh-induced Smo accumulation. (A) A wild-type wing disc was immunostained to show Hh-induced Smo accumulation in P-cells. (B–B″) Expression of FuG13V (red) with ap-Gal4 blocked Smo accumulation in the dorsal compartment cells (arrow in B). (C–F) S2 cells were transfected with CFP-tagged Smo (C,D) or SmoSD123 (E,F) and treated with Hh-conditioned medium (D,F) or control medium (C,E). (G–J) S2 cells were cotransfected with HA-FuG13V plus either CFP-Smo (G,H) or CFP-SmoSD123 (I,J), followed by treatment with Hh-conditioned medium (H,J) or control medium (G,I). FuG13V blocked Hh-induced CFP-Smo cell-surface accumulation (H) but did not block CFP-SmoSD123 accumulation (I,J). SmoN antibody staining indicates the cell-surface-localized Smo, and CFP signal indicates the total amount of expressed Smo.
Figure 3.
Figure 3.
Fu regulates Smo through Cos2. (A–B′″) cos2 mutant discs were immunostained with anti-SmoN (blue), anti-HA (green), and anti-Ci (red) antibodies to show the effect of FuG13V on Smo accumulation. A cos2 heterozygous (+/−) wing disc expressing HA-FuG13V under MS1096 Gal4 showed attenuated Smo accumulation indicated by Smo staining in P-cells (arrow in A), whereas Smo accumulation was unaffected in a cos2 homozygous (−/−) disc expressing HA-FuG13V (arrow in B). The elevated Ci staining in A″ and B″ was due to the expression of the FuG13V and cos2 mutations, respectively. Of note, MS1096 expresses higher levels of Gal4 in dorsal compartment cells than in ventral compartment cells. The A/P boundary was outlined by a red line according to the Ci expression domain. (C–C′″) Wing disc containing fu mutant clones was immunostained to show the decrease in Smo levels (arrows in C). fu mutant clones were recognized by two copies of GFP expression (arrow in C′). (D–D′″) Wing disc containing fu cos2 double-mutant clones was immunostained to show the restored Smo levels (arrow in D). fu and cos2 mutant clones were recognized by staining with anti-Fu antibody (arrow in D′). (E) S2 cells were cotransfected with CFP-Smo and HA-FuG13V, plus Cos2 dsRNA followed by treatment with Hh-conditioned medium. Hh-induced Smo cell-surface accumulation was recovered by knocking down endogenous Cos2. (F,G) S2 cells were cotransfected with HA-Cos2WT plus either CFP-SmoSD123 or CFP-Smo, followed by Hh-treatment. Cos2 blocked Hh-induced CFP-Smo cell-surface accumulation (F) but did not block CFP-SmoSD123 accumulation (G).
Figure 4.
Figure 4.
The ability of Cos2 deletion mutants to block Smo accumulation. (A–F) Wing discs expressing HA-tagged Cos2 variants by ap-Gal4 were immunostained with anti-HA (blue), anti-Ci (green), and anti-SmoN (red) antibodies to show the ability of Cos2 deletion mutants to inhibit Smo. Arrows indicate Smo levels in dorsal P-cells. (G) Schematic drawing of Cos2 constructs with their ability to down-regulate Smo accumulation indicated.
Figure 5.
Figure 5.
Cos2 blocks Smo phosphorylation. (A) S2 cells were transfected with the indicated constructs and treated with Hh-conditioned or control medium. (Top panel) Cell extracts were immunoprecipitated with anti-Myc antibody followed by Western blot with anti-Myc antibody to detect the phosphorylation status of Smo. The arrow indicates hyperphosphorylated forms of Smo and the arrowhead indicates hypophosphorylated and unphosphorylated forms. (Bottom panel) The expression of individual Cos2 constructs was detected by direct Western blot with anti-HA antibody. Of note, Cos2CT ran out of the gel due to its small molecular weight (27 kDa). (WB) Western blot. (B, lane 4) S2 cells were cotransfected with Myc-Smo, HA-FuG13V, and Cos2 dsRNA followed by treatment with Hh-conditioned medium to detect the effect of Cos2 RNAi (cf. lane 3 without RNAi). (Lanes 1,2) S2 cells transfected with Myc-Smo followed by treatment with Hh-conditioned or control medium served as control. The inhibitory efficiency of Cos2 RNAi is shown in the right panel. (C) A wing disc overexpressing HA-Cos2WT driven by ap-Gal4 was immunostained with anti-SmoN antibody to show the blockade of Smo accumulation in dorsal P-cells (arrow). (D,D′) A wing disc overexpressing a strong line of mC* was stained with anti-SmoN (blue) and anti-PKA (red) to show Smo expansion in A-cells near the A/P boundary (arrowhead). (E,E′) A wing disc coexpressing HA-Cos2WT, mC*, and Fg-CKIα was immunostained with anti-SmoN (E) and anti-PKA (E′) antibodies. (F) A control disc indicates the coexpression of PKA (red) and Fg-CKIα (green) for the experiment in E and E′.
Figure 6.
Figure 6.
Fu phosphorylation of Cos2 at Ser572 alleviates its inhibition on Smo accumulation. (A) S2 cells were transfected with the indicated constructs and treated with Hh-conditioned or control medium. Cell extracts were subjected to direct Western blot with anti-HA antibody to detect the phosphorylation status of Cos2. The arrow indicates hyperphosphorylated forms of Cos2 and the arrowhead indicates hypophosphorylated and unphosphorylated forms. (Lanes 9,10) Of note, Fu RNAi resulted in a reduction in Cos2 level. The efficiency of Fu RNAi is shown in the right panel. (B–D″) Wing discs expressing HA-Cos2WT (B–B″), HA-Cos2S572A (C–C″), or HA-Cos2S572D (D–D″) by ap-Gal4 were immunostained with anti-SmoN (blue) and anti-HA (red) antibodies to show the effects of Cos2 variants on Smo accumulation in dorsal P-cells (arrows). (E–E″) A wing disc coexpressing HA-FuG13V with HA-Cos2S572D was stained with anti-SmoN (blue) and anti-Fu (red) antibodies. (F) S2 cells were cotransfected with Myc-tagged Smo and HA-tagged Cos2 mutants and treated with Hh-conditioned or control medium. (Top panel) Cell extracts were immunoprecipitated with anti-Myc antibody and Western-blotted with anti-Myc antibody to show the effects of Cos2 on Smo phosphorylation. The arrow indicates hyperphosphorylated forms of Smo and the arrowhead indicates hypophosphorylated and unphosphorylated forms. (Bottom panel) Lysates were subjected to direct Western blot with anti-HA antibody to show the expression of Cos2. Double the amount of the Cos2S572D cDNA construct was used in cotransfection to normalize Cos2 protein levels. (G–J) S2 cells were cotransfected with different combinations of the indicated constructs and treated with Hh-conditioned medium, followed by the cell-based assay to detect cell-surface-localized Smo. The expression of HA-tagged Cos2 constructs was visualized by anti-HA staining (blue).
Figure 7.
Figure 7.
Cos2 phosphorylation at Ser572 attenuates Cos2–Smo interaction and promotes Cos2 degradation. (A) Extracts from S2 cells expressing the indicated HA-tagged Cos2 constructs were incubated with GST-Smo557–686 fusion protein. (Top panel) The bound Cos2 proteins were analyzed by Western blot with anti-HA antibody. (Bottom panel) Equal amounts of GST-Smo557–686 fusion protein and equal amounts of input Cos2 protein were used. (Right panel) The interaction between GST and Cos2CT served as a negative control. (B) Quantification of Cos2 variants associated with GST-Smo557–686. The pull-down efficiency was determined by the amount of bound Cos2 divided by the amount of total input. Three independent experiments were performed. (C, top panel) The stability of Cos2WT, Cos2S572A, and Cos2S572D in transfected S2 cells was analyzed by direct Western blot with anti-HA antibody after incubation with cycloheximide for the indicated times. (Bottom panel) The β-tubulin levels served as loading control. (D) Quantification of Cos2WT, Cos2S572A, and Cos2S572D by Western analysis performed in C. (E) S2 cells were transfected with the indicated constructs and treated with Hh-conditioned or control medium. Cell lysates were subjected to direct Western blot with anti-HA antibody to detect Cos2 protein levels. β-tubulin served as a loading control. (F–G″) Wing discs expressing HA-FuG13V (F–F″) or HA-FuWT (G–G″) by act > CD2 > Gal4 were immunostained for HA (green) and Cos2 (red). (H) A model for regulating Smo phosphorylation and accumulation by a positive feedback loop. Fu is required for Hh-induced Smo phosphorylation and accumulation through antagonizing the inhibitory role of Cos2. By phosphorylating Cos2 at Ser572, Fu kinase prevents Cos2 from inhibiting Smo, which attenuates Cos2–Smo interaction and promotes Cos2 degradation.
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