PP4 regulates Smo phosphorylation. (A) PP4RNAi elevates Smo phosphorylation. S2 cells were co-transfected with UAS-Myc-Smo and UAS-GFP, and treated with Hh-conditioned or control medium, or treated with OA, PP4 dsRNA, PP4R dsRNA, Mts dsRNA, Wdb dsRNA or GFP dsRNA. Cell extracts were immunoprecipitated and blotted with anti-Myc to detect Smo phosphorylation. Arrow indicates hyperphosphorylated forms of Smo and arrowhead indicates hypophosphorylated and unphosphorylated forms. The efficiency of knockdown of individual phosphatase is shown in Fig. S2A in the supplementary material. (B) PP4 downregulates the Hh-induced Smo phosphorylation. S2 cells were transfected with indicated constructs and treated with or without Hh. The levels of Smo phosphorylation were examined by immunoprecipitation. The expression levels of HA-PP4 or HA-Mts were shown by probing cell lysates with HA antibody. (C) PP4 interacts with Smo C-tail. Extracts from S2 cells expressing indicated constructs were immunoprecipitated with Myc antibody followed by western blot with HA antibody. The expressed proteins were shown by probing immunoprecipitates with anti-Myc, or probing the cell lysates with anti-HA. The asterisk indicates the slow mobility of the Myc-SmoΔCT (Jia et al., 2003). (D) SmoCT truncations interacting with PP4. See immunoprecipitation results in Fig. S3A in the supplementary material. (E) Deletion of the PP4-binding domain in Smo elevates its phosphorylation. Extracts from S2 cells expressing Myc-Smo or Myc-SmoΔ626-678 and treated with or without Hh were immunoprecipitated and blotted with the anti-Myc antibody. (F-G′) Wing discs expressing UAS-Myc-Smo or UAS-Myc-SmoΔ626-678 by ap-Gal4 were immunostained to show Myc expression in F′ and G′, and ectopic dpp-lacZ expression in F and G. Expressing Myc-SmoΔ626-678 induced higher level of ectopic dpp-lacZ expression (arrowhead in G), compared with expressing Myc-Smo (arrowhead in F). (H) Hh downregulates Smo-PP4 interaction. Extracts from S2 cells expressing Flag-PP4 with or without Hh treatment were incubated with the bacterially expressed GST or GST-Smo557-686 fusion proteins. The bound PP4 proteins were analyzed by western blot with Flag antibody. The middle panel shows the GST and GST-Smo fusion proteins. The lower panel indicates the equal amount of input PP4. (I) Smo-PP4 interaction is attenuated by Cos2 RNAi. S2 cells were transfected with HA-PP4 and Flag-SmoCT and treated with or without Cos2 dsRNA. The SmoCT-bound PP4 was examined by immunoprecipitation with anti-Flag and western blot with anti-HA. Equally expressed PP4 proteins were ensured by probing the lysates with anti-HA. The efficiency of Cos2 RNAi is shown in Fig. S2B in the supplementary material. (J) PP4 directly interacts with Cos2MB and Cos2CT. Bacterially expressed and purified GST, GST-PP4, His-Cos2MB and His-Cos2CT were used for pull-down assay. Antibodies used for western blots are indicated.

PP2A is essential for Hh signaling. (A,A′) A wild-type wing disc was stained to show the expression of Ci^FL and dpp-lacZ. The Ci antibody only recognizes Ci^FL in wing discs. (B,B′) A wing disc co-expressing UAS-MtsRNAi with UAS-P35 by MS1096 Gal4 was immunostained with Ci and β-gal antibodies. Arrowhead in B indicates the decreased Ci^FL levels. Arrowhead in B′ indicates the downregulated dpp-lacZ expression. MS1096 Gal4 is expressed at lower level in the ventral region than in the dorsal region of the wing disc (Jia et al., 2003). (C-C′) A wing disc expressing UAS-WdbRNAi by MS1096 Gal4. Arrowhead in C indicates the decreased Ci^FL and arrowhead in C′ indicates the downregulated dpp-lacZ. (D-E′) Wing discs expressing UAS-Mts or UAS-Wdb by ap-Gal4. Arrowheads in D and E show the elevation of Ci^FL and arrowheads in D′ and E′ show the induced ectopic dpp-LacZ expression. (F-F″) A wing disc bearing wdb^IP homozygous clones that were marked by the lack of GFP expression was immunostained with anti-Ci and anti-SmoN antibodies. Arrowhead in F shows the reduction of Ci^FL. Arrowhead in F′ shows the unaffected Smo accumulation in wdb mutant cells. (G,H) Wing discs expressing UAS-HA-Ci^FL alone, or along with UAS-Wdb by C765 Gal4. Arrowhead in H indicates the ectopic Ptc-lacZ expression in A-compartment cells.

PP2A downregulates Ci phosphorylation and blocks Ci proteolytic processing. (A) Ci^FL phosphorylation is upregulated by PP2A RNAi. S2 cells were transfected with Flag-Ci and treated with OA or indicated dsRNA. Cell extracts were subjected to direct western blot with anti-Flag antibody. Arrow indicates hyperphosphorylated forms of Ci and arrowhead indicates the hypophosphorylated or unphosphorylated forms. β-Tubulin serves as loading control. The knockdown efficiency of individual phosphatase was estimated by the method used for Fig. 2A. (B) PP2A downregulates Ci^FL phosphorylation. S2 cells were transfected with Flag-Ci alone or along with indicated HA-tagged phosphatase and treated with or without OA. Cell lysates were probed with anti-Flag or anti-HA antibodies. (C) A disc shows the wild-type Ci^FL staining. (D) A wing disc shows the Ci^FL stabilization by the treatment of proteasome inhibitor MG132. (E,F) Wing discs expressing UAS-WdbRNAi by ap-Gal4 were treated with or without MG132 and stained to show Ci^FL. Arrowhead in E indicates the destabilized Ci^FL by Wdb RNAi. Arrowhead in F indicates that the destabilized Ci^FL by Wdb RNAi was restored by MG132 treatment. (G-H′) Wing discs bearing smo³ clones and expressing UAS-HA-Ci^FL alone or along with UAS-Wdb by MS1096 Gal4 were stained to show the expression of GFP (green) and hh-lacZ (red). Arrowheads in G and H indicate smo³ clones that are marked by the lack of GFP expression. Arrowheads in G′ and H′ indicate the hh-lacZ expression in smo³ cells. (I) Western blot analysis of protein extracts from wing discs expressing UAS-HA-Ci^FL or co-expressing UAS-HA-Ci^FL with UAS-Wdb using the MS1096 Gal4. Protein extracts were prepared from 400 wing discs, immunoprecipitated and blotted with HA antibody. (J) A model for the involvement of PP4 and PP2A in Hh signaling. PP4 negatively regulates Hh signal transduction by antagonizing the phosphorylation of Smo. PP2A positively regulates Hh pathway by counteracting kinases to downregulate Ci^FL phosphorylation and attenuate its proteasome-mediated processing.

Knockdown of PP4 elevates Smo accumulation in wing discs. (A-A″) A wing disc expressing GFP by ap-Gal4 was stained with anti-SmoN, β-gal (for ptc-lacZ expression). GFP indicates the ap-Gal4 expression domain in wing disc. (B-B″) UAS-PP4RNAi was expressed by ap-Gal4 and immunostained for Smo and ptc-lacZ expression. Arrowhead in B indicates the accumulation of Smo and arrowhead in B′ indicates the anterior expansion of ptc-lacZ. (C-C″) As expressing UAS-MtsRNAi by the ap-Gal4 caused cell lethality, the weak act>CD2>Gal4 was used and larvae were grown at 20°C to reduce the Gal4 levels, thus limiting the strength of Mts RNAi. (D,D′) A wing disc expressing UAS-HA-PP4 by ap-Gal4 was immunostained for Smo and HA. (E) A wing disc co-expressing UAS-PP4RNAi and UAS-HA-PP4 was immunostained with anti-SmoN antibody. Arrowhead indicates that HA-PP4 reduces the levels of Smo accumulation that are caused by PP4 RNAi. (F,F′) A wing disc expressing UAS-Mts by ap-Gal4 was immunostained with anti-SmoN antibody. (G) A wing disc co-expressing UAS-Mts with UAS-PP4RNAi by ap-Gal4 was stained with anti-SmoN antibody. All wing discs shown in this study were oriented with anterior towards the left and ventral towards the top.

PP4 RNAi elevates the Hh-induced Smo cell-surface accumulation. (A-P) CFP-tagged Smo constructs were transfected into S2 cells and treated with phosphatase inhibitor (OA), phosphatase dsRNA or Hh-conditioned medium. Cell-surface-localized Smo was visualized by immunostaining with anti-SmoN antibody. The total amount of expressed Smo was indicated by the CFP signal. (Q) Ratio of cell surface level to total level of Smo. Each data set was based on 15 individual cells. WT, CFP-Smo; SD123, CFP-SmoSD123; Δ626-678, CFP-Smo Δ626-678.

PP2A counteracts kinases to regulate Ci and Hh target gene expression. (A,A′) A wild-type wing disc was immunostained to show Ci^FL and ptc-lacZ expression. (B,B′) A wing disc overexpressing UAS-mC^* by MS1096 Gal4. Arrowhead in B shows the downregulated Ci^FL level and arrowhead in B′ shows the attenuated ptc-lacZ expression. (C,C′) A wing disc overexpressing UAS-Wdb by MS1096 Gal4. Arrowhead in C shows the upregulated Ci^FL. (D,D′) A wing disc co-expressing UAS-mC^* with UAS-Wdb by MS1096 Gal4. Arrowhead in D shows reinstated Ci^FL and arrowhead in D′ shows the partially restored ptc-lacZ expression. (E,F) Wing discs expressing UAS-CRL or co-expressing UAS-CRL with UAS-MtsRNAi by MS1096 Gal4 and stained to show the levels of Ci^FL.