Membrane targeting of inhibitory Smads through palmitoylation controls TGF-β/BMP signaling

Abstract

TGF-β/BMP (bone morphogenetic protein) signaling pathways play conserved roles in controlling embryonic development, tissue homeostasis, and stem cell regulation. Inhibitory Smads (I-Smads) have been shown to negatively regulate TGF-β/BMP signaling by primarily targeting the type I receptors for ubiquitination and turnover. However, little is known about how I-Smads access the membrane to execute their functions. Here we show that Dad, the Drosophila I-Smad, associates with the cellular membrane via palmitoylation, thereby targeting the BMP type I receptor for ubiquitination. By performing systematic biochemistry assays, we characterized the specific cysteine (Cys556) essential for Dad palmitoylation and membrane association. Moreover, we demonstrate that dHIP14, a Drosophila palmitoyl acyl-transferase, catalyzes Dad palmitoylation, thereby inhibiting efficient BMP signaling. Thus, our findings uncover a modification of the inhibitory Smads that controls TGF-β/BMP signaling activity.

Keywords: Dad; Drosophila germ-line stem cell; inhibitory Smads; palmitoylation.

Fig. 1.

Dad associates with membrane proteins in a palmitoylation-dependent manner. (A and B) S2 cells were transfected with the indicated combinations of plasmids. At 48-h posttransfection, cell lysates were immunoprecipitated with anti-Flag (A) or anti-Myc (B) beads. Western blots were performed to analyze the presence of Myc or Flag-tagged proteins. (C) S2 cells were transfected with the indicated plasmids. At 48-h posttransfection, cell lysates were fractionated as described in Fig. S1. Western blots were performed to analyze the presence of Myc or Flag-tagged proteins. (D and E) S2 cells were transfected with plamids expressing Myc-Dad and dCalnexin-GFP. dCalnexin-GFP was used as an internal reference of membrane fraction. At 42-h posttransfection, the cells were treated with ethyl alcohol (EtOH, as a control) or 2-Brp (25 µM) for 6 h, and then lysed for fractionation. Western blots were performed to analyze the presence of Myc or GFP-tagged proteins (D). Densitometric analyses to quantify levels of Dad in membrane in D are shown in E, Error bars represent SD (n = 3). (F and G) S2 cells were transfected with the Myc-Dad or Flag-Rop expression construct. At 42-h posttransfection, the cells were further treated with ethyl alcohol (as a control) or 2-Brp (25 µM) for 6 h; cell lysates were immunoprecipitated with anti-Myc or anti-Flag beads, and then subjected to the S-palmitoylation assay to measure palmitoylation levels of Dad (F) or Rop (G). HAM and Palm are abbreviations for hydroxylamine and palmitoylation, respectively. All of the biochemical experiments were performed at least three times. In E, the Student’s t test was used to analyze statistical significance. ***P < 0.001 vs. the control groups. IB, immunoblotting; IP, immunoprecipitated; M, membrane; Rel., relative; T, total.

Fig. 2.

Dad forms a complex with Tkv to mediate its ubiquitination. (A) S2 cells were transfected with plasmids as indicated. At 48-h posttransfection, cell lysates were prepared and immunoprecipitated with anti-Flag beads, followed by Western blot analyses. (B) S2 cells were transfected with plasmids as indicated. At 48-h posttransfection, cells were treated with MG132 (50 µM) for 6 h, then lysed and immunoprecipitated with anti-Flag beads, followed by Western blot assays to detect the ubiquitination status of Tkv(ca). (C and D) S2 cells were treated with dsRNAs targeting gfp (as a control) or dad for 24 h, followed by transfections with the indicated plasmids. At 48-h posttransfection, cells were treated with MG132 (50 µM) for 6 h, then lysed for IP assays to detect the ubiquitination status of Tkv(ca) (C) or cells were harvested for quantitative real-time PCR (qRT-PCR) assays to examine relative mRNA levels of dad (D), Error bars represent SD (n = 3). (E and F) S2 cells were transfected with plasmids as indicated. At 48-h posttransfection, cells were treated with CHX (50 ng mL⁻¹) for various times, followed by immunoblotting to examine levels of Tkv protein. Densitometric analyses to quantify Tkv expression in E are shown in F. Error bars represent SD (n = 3). (G) A schematic diagram of the germarium with different cell types and organelles indicated as follows: cystoblast cells (CB), cap cells (CPC), germ-line stem cells (GSC), inner germarium sheath cells (IGC), somatic stem cells (SSC), terminal filament (TF), and cyst (differentiated germ cells with extended or branched fusomes). Among these, TFs, CPCs, and IGCs produce Dpp ligands. (H–L) Ovaries collected from indicated genotypes were stained with anti-Vasa (green) and anti-Hts (red) antibodies. Anti-Hts was used to outline the germarium and the morphology of the fusome, and the staining of anti-Vasa was used to visualize all germ cells in the germarium and egg chambers. (Scale bars, 10 µm.) (M) Quantification of the germarium phenotypes of ovaries in H–L. All of the biochemical experiments were performed at least three times. In D, the Student’s t test was used to analyze statistical significance. In F, the log-rank test was used to analyze statistical significance. *P < 0.05; ***P < 0.001 vs. the control groups. IB, immunoblotting; IP, immunoprecipitated; Rel., relative.

Fig. 3.

The Cys556 site is important for Dad palmitoylation and antagonizes Dpp signaling. (A) S2 cells were transfected with expression plasmids encoding wild-type Myc-Dad or its mutants as indicated. Forty-eight hours after transfection, cell lysates were prepared for S-palmitoylation assay. Western blots were performed to detect the presence of Myc-tagged proteins. (B) S2 cells were cotransfected with Myc-Dad or its mutations, dad-AE-luciferase and actinP-lacZ (used as an internal control). At 36-h posttransfection, cells were treated with BMP4 (10 ng mL⁻¹) for 12 h, the cells were lysed for luciferase assays and immunoblotting assays. The Student's t test was used to analyze statistical significance. **P < 0.01 vs. the control groups; N.S., not significant. (C) The S2 cells were cotransfected with Flag-Tkv(ca) and Myc-Dad or Myc-DadC556A mutant. At 48-h posttransfection, cell lysates were fractionated and the membrane fractions were immunoprecipitated with anti-Flag beads. Western blots were performed to analyze the presence of Flag- or Myc-tagged proteins. (D) S2 cells were transfected with the indicated plasmids. Forty-eight hours after transfection, cells were treated with MG132 (50 µM) for 6 h, then cell lysates were immunoprecipitated with anti-Flag beads, and subjected to immunoblotting analysis to detect the ubiquitination status of Tkv(ca). (E–I) Ovaries collected from indicated genotypes were stained with anti-Vasa (green) and anti-Hts (red) antibodies. (Scale bars, 10 µm.) (J) Quantification of the germarium phenotypes of ovaries in E–I. All of the biochemical experiments were performed at least three times. In B, the two-tailed Student’s t test was used to analyze statistical significance. **P < 0.01 vs. the control groups. IB, immunoblotting; IP, immunoprecipitated; M, membrane; Rel. Luc. Act., relative luciferase activity; T, total.

Fig. 4.

PAT dHIP14 regulates Dad palmitoylation. (A and B) S2 cells were transfected with Myc-Dad together with empty vector or PAT expression constructs as indicated. Forty-eight hours after transfection, cell lysates were prepared for S-palmitoylation assay, followed by immunoblotting analysis (A). Densitometric analyses to quantify levels of palmitoylation of Dad in A are shown in B, Error bars represent SD (n = 3). (C) S2 cells were treated with dsRNAs targeting gfp (as a control) or dhip14 for 24 h, followed by transfection with Myc-Dad. At 48-h posttransfection, cell lysates were prepared for S-palmitoylation assay to detect Dad palmitoylation levels, or qRT-PCR assays to examine relative levels of dhip14 mRNA, Error bars represent SD (n = 3). (D) S2 cells were transfected with plasmids as indicated. At 48-h posttransfection, cell lysates were prepared and immunoprecipitated with anti-Myc beads, followed by Western blot analyses. (E) S2 cells were cotransfected with the indicated expression vectors together with dad-AE-luciferase and actinP-lacZ (used as an internal control). At 36-h posttransfection, cells were treated with BMP4 (10 ng mL⁻¹) for 12 h; the cells were lysed for luciferase assays (Upper) and immunoblotting assays (Lower). (F–I) Ovaries collected from indicated genotype females, were stained with anti-Vasa (green) and anti-Hts (red) antibodies. The flies were fed at 18 °C and dissected at 1-d-old. (Scale bars, 10 µm.) (J) Quantification of the germarium phenotypes of ovaries in F–I. All of the biochemical experiments were performed at least three times. In B, C and E, the Student’s t test was used to analyze statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the control groups. IB, immunoblotting; IP, immunoprecipitated; Rel. Luc. Act., relative luciferase activity.