Research ArticleCell Biology

Hedgehog reciprocally controls trafficking of Smo and Ptc through the Smurf family of E3 ubiquitin ligases

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Science Signaling  06 Feb 2018:
Vol. 11, Issue 516, eaan8660
DOI: 10.1126/scisignal.aan8660
  • Fig 1 A cell-based RNAi screen identifies Smurf family members as Smo E3 ubiquitin ligases.

    (A) Family tree of the HECT domain E3 ubiquitin ligases that we targeted by RNAi. (B) Schematic drawings of Smurf, Nedd4, and Su(dx), with the C2, WW, and HECT domains indicated. (C) S2 cells stably expressing Myc-Smo were treated with control dsRNA or dsRNA targeting the indicated HECT domain E3 ligases. After treatment with the proteasome inhibitor MG132, Myc-Smo was immunoprecipitated (IP) with an anti-Myc antibody and immunoblotted (IB) with antibodies recognizing ubiquitin or Myc. Loading was normalized by the amount of Myc-Smo. (D) Cell-based ubiquitylation assay. Immunoblot (IB) showing ubiquitylation of Myc-Smo immunoprecipitated (IP) from extracts from S2 cells treated with the indicated dsRNAs. Luc dsRNA is a negative control. (E) Smo ubiquitylation in S2 cells overexpressing Flag (Fg)–tagged Smurf family members Smurf, Nedd4, Su(dx), CG3356, or CG6190. (F) Smo ubiquitylation in S2 cells overexpressing tagged wild type (WT) (Fg-Smurf) and catalytically inactive (Fg-SmurfC1029A). Immunoblots are representative of three independent experiments.

  • Fig 2 The Smurf family of E3s regulates Smo cell surface accumulation in cultured cells.

    Immunostaining (A) and quantification (B) of cell surface Smo in S2 cells stably expressing Myc-Smo and treated with control (Luc) or the indicated dsRNA. Data are means ± SD from three independent experiments. n = 10 cells for each experimental condition. **P < 0.01, ***P < 0.001 (Student’s t test). (C) Immunostaining showing a mutant form of Smo that mimics phosphorylation by PKA and CK1 (Myc-SmoSD) and Fg-Smurf, Fg-Nedd4, or Fg-Su(dx) (Flag) in S2 cells expressing the indicated tagged proteins. (D) Quantification of cell surface Myc-SmoSD signals in (C). Data are means ± SD from three independent experiments. n = 10 cells for each experimental condition. ***P < 0.001 (Student’s t test). (E) Immunoblot showing Myc-SmoSD and Fg-Smurf, Fg-Nedd4, or Fg-Su(dx) in S2 cells expressing the indicated combinations of tagged proteins. Myc-CFP (cyan fluorescent protein) is a loading control. The antibody that recognizes green fluorescent protein (GFP) also recognizes CFP. (F) Quantification of Myc-SmoSD abundance in (E). Data are means ± SD from three independent experiments. ***P < 0.001 (Student’s t test). (G) Immunoblot showing Smo protein in extracts from cl-8 cells treated with either Hh-conditioned medium (Hh) or dsRNAs targeting Smurf, Nedd4, Su(dx), or all three (3E3s). (H) Quantification of Smo abundance in (G). Data are means ± SD from three independent experiments. **P < 0.01, ***P < 0.001 (Student’s t test). (I and J) Quantification of smo (I) and ptc (J) mRNA by quantitative reverse transcription PCR (qRT-PCR) in cl-8 cells treated with either Hh-conditioned medium or the indicated dsRNAs. (K) Luciferase activity in cl-8 cells expressing the ptc-luc reporter gene and treated with either Hh-conditioned medium or the indicated dsRNAs. Data are means ± SD from three independent experiments (I to K). Immunofluorescence images are representative of 10 cells for each condition. Immunoblots are representative of three independent experiments. Scale bars, 5 μm.

  • Fig 3 Smurf reduces Smo cell surface accumulation and Hh signaling in developing wings.

    (A to D) Late third-instar control (Ctrl) wing discs (A and C) or wing discs expressing UAS-Smurf-RNAi under control of the MS1096 Gal4 driver (B and D) were immunostained for Ci (red), Smo (green in A and B), or Ptc (green in C and D). Wing discs in these and the following panels are oriented with anterior to the left and ventral up. Arrows and arrowheads indicate the dorsal and ventral compartments of the wing pouch region, respectively. Ci staining marks the anterior compartment cells. (E to I) Late third-instar control wing disc (E) or wing discs expressing UAS-Fg-Smurf under control of the MS1096 Gal4 driver either alone (F to H) or together with UAS-Smurf-RNAi (I) using MS1096 were immunostained for Ci (red), Ptc (green in F), En (green in E and G), Smo (green in H and I), or Flag (blue). Dashed lines demarcate the anteroposterior boundary determined by the Ci expression domain (E and G). Arrows indicate the anterodorsal cells near the anteroposterior boundary (E to G) or posterodorsal compartment cells (H to I) of the wing pouch region. Images are representative of five wing discs per genotype. Scale bars, 50 μm.

  • Fig 4 Hh-induced phosphorylation of Smo inhibits Smurf recruitment to Smo.

    (A) Schematic drawings of full-length Smurf and deletion mutants. (B and C) S2 cells were cotransfected with constructs encoding Fg-Smurf and WT Smo (Myc-SmoWT), a deletion mutant lacking the C-tail (Myc-SmoΔCT), a mutant form of Smo that cannot be phosphorylated by PKA (Myc-SmoSA), or a phosphomimetic form of Smo (Myc-SmoSD) as indicated and treated with control or Hh-conditioned medium (Hh), followed by immunoprecipitation (IP) and immunoblotting (IB) with the indicated antibodies. Cells were treated with the proteasome inhibitor MG132 for 4 hours before lysis. Asterisks indicate the monomeric form of full-length (Myc-SmoWT) and truncated (Myc-SmoΔCT) Smo. (D) S2 cells were cotransfected with Myc-Smo and the indicated deletion forms of Fg-Smurf and subjected to immunoprecipitation (IP) and immunoblotting (IB) with the indicated antibodies. Asterisks indicate the full-length and truncated forms of Smurf (D). (E) Cell-based ubiquitin assay showing ubiquitylation of Myc-Smo immunoprecipitated from S2 cells coexpressing Myc-Smo and a truncated form of Smurf consisting of only the C-terminal HECT domain (Fg-Smurf-HECT). (F) S2 cells were cotransfected with constructs encoding Fg-Smurf-HECT and various forms of Myc-tagged Fz: full-length Fz (Myc-Fz), Fz fused to SAID, the autoinhibitory domain of Smo (Myc-FS), and Fz fused to phosphorylation-deficient (Myc-FSSA) or phosphomimetic (Myc-FSSD) forms of the SAID. The Fz proteins were immunoprecipitated (IP) and then immunoblotted (IB) with antibodies recognizing Myc or Fg. (G) S2 cells were cotransfected with constructs encoding full-length Fg-Smurf and Myc-FS, Myc-FSSA, or Myc-FSSD. The Myc-tagged proteins were immunoprecipitated (IP) and immunoblotted (IB) with antibodies recognizing Myc or Fg. (H) S2 cells with endogenous Smo depleted by dsRNA targeting smo 5′ untranslated region (5′UTR) were cotransfected with constructs encoding Myc-Smo, Myc-SmoΔSAID, and Fg-Smurf. The Myc-tagged proteins were immunoprecipitated (IP) and immunoblotted (IB) with antibodies recognizing Myc or Fg. Blots are representative of three independent experiments.

  • Fig 5 Gprk2 promotes Smurf recruitment to Smo.

    (A and B) Cell-based ubiquitylation assay of Myc-Smo in S2 cells treated with Smurf and Gprk2 dsRNAs either alone or in combination (A), or coexpressed with Gprk2 (Fg-Gprk2) or the kinase-dead form Fg-Gprk2KM (B). (C) Immunostaining showing Smo and GFP in late third-instar wing discs expressing UAS-GFP under the control of the brk Gal4 driver. (D to F) Immunostaining showing Smo in late third-instar wing discs expressing UAS-Smurf-RNAi (D), UAS-Gprk2-RNAi (E), or UAS-Smurf-RNAi + UAS-Gprk2-RNAi (F) under control of the brk gal4 driver. Arrows indicate the anterior brk expression domain. (G to I) S2 cells were transfected with the indicated constructs and treated with either control (Luc) dsRNA or dsRNAs targeting the coding sequence (G and I) or 5′UTR (H) of Gprk2, followed by immunoprecipitation (IP) and immunoblotting (IB) with the indicated antibodies. Cells were treated with the proteasome inhibitor MG132 for 4 hours before lysis. Images are representative of five wing discs per genotype. Blots are representative of three independent experiments.

  • Fig 6 Gprk2 promotes Smo-Smurf association by phosphorylating Smurf.

    (A and B) Immunoblots (IB) of lysates from S2 cells expressing Fg-Smurf and Myc-Gprk2 or Myc-Grpk2KM as indicated and treated with control or Hh-conditioned medium (Hh) in the absence or presence of λ phosphatase (λ-PP). (C) Immunoblots of lysates from S2 cells expressing Fg-Smurf and Myc-Gprk2 as indicated in the presence or absence of control (Luc) or Smo dsRNA. (D) Immunoblot (IB) showing Fg-Smurf and Myc-Grpk2 in Myc immunoprecipitates (IP) from S2 cells expressing the Myc-Gprk2 and Fg-Smurf and subjected to treatment with Hh-conditioned medium or dsRNAs as indicated. (E and F) Western blot of immunoprecipitates from S2 cells expressing the indicated combinations of the N terminus of Smurf (Fg-SmurfΔHECT), a form of SmurfΔHECT in which the Gprk2 phosphorylation site was mutated (Fg-SmurfΔHECT-SA), or the HECT domain of Smurf (Fg-Smurf-HECT) with WT (Myc-Gprk2) and kinase-dead (Myc-Gprk2KM) forms of Gprk2. (G) Western blot of immunoprecipitates from S2 cells expressing full-length Smurf (Fg-Smurf) or a mutant form of Smurf in which the Gprk2 phosphorylation site was mutated (Fg-SmurfSA) with wild type (WT) Myc-Gprk2. WT sequence of the N-terminal Ser cluster and the SA or SD substitutions are shown. (H) Immunoblot showing Smurf and Smo in immunoprecipitates from cells expressing Myc-Smo and the indicated forms of Smurf. (I) Immunoblot showing ubiquitylation of Smo in S2 cells expressing Myc-Smo and the indicated forms of Smurf. (J) Immunoblot showing Smurf and Smo in immunoprecipitates from cells expressing Myc-Smo and the indicated forms of Smurf and treated with control (Luc) or Gprk2 dsRNAs as indicated. (K and L) Immunoblot analysis of immunoprecipitates (IP) from cells expressing the indicated proteins. Note that Fg-Gprk2 and Fg-SmurfΔHECT or Fg-SmurfΔHECT-SA overlapped on Western blot. Blots are representative of three independent experiments.

  • Fig 7 Hh promotes Ptc ubiquitylation by dissociating Smurf from Smo and facilitating its binding to Ptc.

    (A) Ptc ubiquitylation assay in S2 cells treated with control or Hh-conditioned medium (Hh) in the presence of Luc, Smo, Smurf, or both Smo and Smurf dsRNA. (B) Immunoblotting (IB) or immunoprecipitates (IP) from S2 cells coexpressing Myc-Ptc and Fg-Smurf and treated with Hh-conditioned medium in the presence or absence of the indicated dsRNAs. (C and D) L>Ptc-GFP expression in control wing discs (C) or wing discs containing clones expressing UAS-Smo-RNAi under the control of act-Gal4 (D). Smo depletion decreased Ptc-GFP in anterior compartment cells away from the anteroposterior boundary (arrows in D) but not in posterior compartment cells (arrowheads in D). Images are representative of five wing discs per genotype. (E and F) Ptc-Smurf association (E) or Ptc ubiquitylation (F) in S2 cells expressing the indicated constructs in the presence of Luc or Smo dsRNA. (G and H) Ubiquitylation of Myc-Ptc or Myc-PtcΔL2 in S2 cells treated with control or Hh-conditioned medium in the presence of Luc or Ptc dsRNA (G) or in the presence of both Smo and Ptc dsRNA (H). (I) Smurf-Ptc (or PtcΔL2) association in S2 cells transfected with the indicated dsRNA and treated with control or Hh-conditioned medium. Blots are representative of three independent experiments.

  • Fig. 8 Model for how Hh regulates the reciprocal trafficking of Smo and Ptc through Smurf.

    In the absence of Hh (left), Gprk2-mediated phosphorylation of Smurf promotes Smurf association with Smo, leading to Smo ubiquitylation, internalization, and degradation, and Ptc is modestly ubiquitylated by Smurf. In the presence of Hh (right), Smo recruits PKA, which phosphorylates the Smo C-tail, thus inhibiting Smurf binding to and ubiquitinating Smo. Hh promotes the binding of released Smurf to Ptc, leading to increased Ptc ubiquitylation and degradation. The different shapes of unphosphorylated and phosphorylated Smurf represent open (ovals) and closed (circles) conformations.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/516/eaan8660/DC1

    Fig. S1. Cul4 acts in parallel with Smurf to promote Smo ubiquitylation and cell surface clearance.

    Fig. S2. Effect of Smurf family knockdown on Smo cell surface accumulation and Hh signaling in wing discs.

    Fig. S3. Effect of overexpression of Smurf family members on Smo cell surface accumulation and Hh signaling in wing discs.

    Fig. S4. Hh-stimulated and PKA-mediated phosphorylation of Smo inhibit the recruitment of Su(dx) and Nedd4.

    Fig. S5. Hh regulates Ptc ubiquitylation through the Smurf family of E3s.

    Fig. S6. Regulation of Ptc-E3 interaction by Hh and Smo.

    Fig. S7. Ptc interacts with Smurf regardless of Smurf phosphorylation by Gprk2.

  • Supplementary Materials for:

    Hedgehog reciprocally controls trafficking of Smo and Ptc through the Smurf family of E3 ubiquitin ligases

    Shuang Li, Shuangxi Li, Bing Wang, Jin Jiang*

    *Corresponding author. Email: jin.jiang{at}utsouthwestern.edu

    This PDF file includes:

    • Fig. S1. Cul4 acts in parallel with Smurf to promote Smo ubiquitylation and cell surface clearance.
    • Fig. S2. Effect of Smurf family knockdown on Smo cell surface accumulation and Hh signaling in wing discs.
    • Fig. S3. Effect of overexpression of Smurf family members on Smo cell surface accumulation and Hh signaling in wing discs.
    • Fig. S4. Hh-stimulated and PKA-mediated phosphorylation of Smo inhibit the recruitment of Su(dx) and Nedd4.
    • Fig. S5. Hh regulates Ptc ubiquitylation through the Smurf family of E3s.
    • Fig. S6. Regulation of Ptc-E3 interaction by Hh and Smo.
    • Fig. S7. Ptc interacts with Smurf regardless of Smurf phosphorylation by Gprk2.

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    Citation: S. Li, S. Li, B. Wang, J. Jiang, Hedgehog reciprocally controls trafficking of Smo and Ptc through the Smurf family of E3 ubiquitin ligases. Sci. Signal. 11, eaan8660 (2018).

    © 2018 American Association for the Advancement of Science

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