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Convergence of Wnt, growth factor, and heterotrimeric G protein signals on the guanine nucleotide exchange factor Daple

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Science Signaling  27 Feb 2018:
Vol. 11, Issue 519, eaao4220
DOI: 10.1126/scisignal.aao4220
  • Fig. 1 The GBA motif and PBM in Daple allosterically inhibit each other’s functions.

    (A) Schematic summarizing how Daple enhances noncanonical Wnt signaling downstream of Frizzled (FZD receptors). (B and C) Schematic showing two key intermolecular interplays encountered during noncanonical Wnt signaling via Daple, as shown in (27); that is, (i) FZD7’s internal PDZ-binding motif (PBM) and Daple’s C-terminal PBM compete for binding to the PDZ-domain of Dvl (B), and (ii) Dvl and Gαi compete for two distinct modules on Daple (C). (D and E) Immunoprecipitation assay assessing the binding of Gαi (D) or Dvl (E) to full-length Daple wild-type (WT) or mutants [F1675A (GBA-deficient, FA), ΔPBM, or the double mutant (FA + ΔPBM; also called 2M)]. Bound complexes were analyzed for Daple (myc) and Gαi3 [hemagglutinin (HA)]. Gβ was monitored as a positive control for Gαi3-bound proteins. Quantification of blots (n = 3) is shown in fig. S1 (A and B). IB, immunoblotting.

  • Fig. 2 Multiple Tyr kinases phosphorylate the C-terminal PBM module of Daple.

    (A) (Top) Schematic showing the domain arrangement of Daple. (Bottom) Alignment of Daple’s C terminus in various species displaying an evolutionary conserved PBM (red box). The two phosphotyrosines defined in this work are highlighted in yellow. (B) In vitro kinase assays using several recombinant kinases and wild-type (WT) or nonphosphorylatable mutant [Y2025F (YF) and Y2023/2025F (Y2F)] His-Daple-CT proteins as substrate. Reactions were analyzed for phospho-Tyr-Daple with antibody to pTyr. (C) Schematic summarizing the findings in (B). (D) Immunoprecipitation assays assessing the phosphorylation of Daple after epidermal growth factor (EGF) stimulation in Cos7 cells co-expressing epidermal growth factor receptor (EGFR) and WT or YF-mutant myc-Daple. Immune complexes were analyzed for phosphorylated (pan-pTyr) and total (myc) Daple. (E) Immunoprecipitation assay assessing the phosphorylation of Daple by Src in Cos7 cells co-expressing a constitutively active (CA) mutant of Src (Y527F) and WT or Y2F-mutant myc-Daple. Immunoblotting was performed as in (D). (F) Tyr phosphorylation of endogenous Daple in HeLa cells in response to EGF. Whole-cell lysates were analyzed for pTyr-Daple with anti-pYDaple (pTyr2023 and pTyr2025; antibody validation in fig. S2) and phosphorylation of AKT (pSer473). Blots are representative of three to five experiments. t, total; p, phosphorylated.

  • Fig. 3 Daple is required for activation of Gαi3, Rac1, and AKT signals and in the antagonistic inhibition of β-catenin–dependent Wnt signals downstream of EGF/EGFR.

    (A) Immunoprecipitation assay assessing Gαi3 activation after EGF stimulation in control (shLuc) and Daple-depleted (shDaple) HeLa cells. Western blot for active Gαi3 (Gαi3-GTP) and total Gαi3. Quantification of blots is shown in fig. S3. (B) Whole-cell lysates of EGF-stimulated HeLa cells in (A) were analyzed for phosphorylated AKT (pSer473), Daple, β-catenin, and actin (loading control). (C) Bar graphs display quantification of phosphorylated/total AKT. Data are means ± SD; n = 3 experiments. (D) Rac1 activity assay in lysates of HeLa cells described in (A) upon EGF stimulation. Data are means ± SD; n = 3. (E and F) Whole-cell lysates of EGF-stimulated HeLa cells expressing Daple-WT or Daple-F1675A (FA) mutant were analyzed for AKT phosphorylation, as described in (B). (G) Bar graphs display quantification as a ratio of phosphorylated to total AKT. Data are means ± SD; n = 3. (H and I) Rac1 activity upon EGF stimulation, assessed in lysates from HeLa cells described in (F). Data are means ± SD; n = 3. (J and K) Quantitative polymerase chain reaction (qPCR) analysis assessing EPCAM (J) and other Wnt target genes (K) induced by EGF in HeLa cells described in (A). Data are means ± SD, fold change in RNA after EGF stimulation; n = 3 experiments. n.s., not significant.

  • Fig. 4 Tyr phosphorylation inhibits Daple binding to Dvl but favors its binding to and activation of Gαi3.

    (A and B) His-Daple-CT glutathione S-transferase (GST) pulldown assays to investigate the impact of such phosphorylation on binding to Dvl [GST-Dvl2-PDZ (A)] and Gαi3 [GST-Gαi3 (B)]. (Top) Bound total and Tyr-phosphorylated Daple (“pYDaple,” as determined using an antibody to pTyr2023/20–25 Daple) were analyzed by immunoblotting (quantification shown in fig. S4, A and B; n = 3 experiments). (Bottom) Confirmation of phosphorylation of His-Daple-CT by immunoblotting. (C to E) Lysates of Cos7 cells co-transfected with Src-HA and myc-Daple were used as source of Daple for GST pulldown assays to assess the impact of Src-dependent phosphorylation of myc-Daple on its ability to bind Dvl [GST-Dvl2-PDZ (C)] and Gαi3 [GST-Gαi3 (D)]. Bound total (myc) and phospho-Daple (pYDaple) were analyzed by immunoblotting as in (A) and (B) (quantification shown in fig. S4C; n = 3). KD, kinase-deficient. (F) GST pulldown assays investigating the impact of phosphomimicking (YE) and nonphosphorylatable (YF) mutants of Daple on binding to Dvl (GST-Dvl2-PDZ). Bound Daple (myc) was analyzed by immunoblotting (quantification shown in fig. S4E; n = 3). (G) Homology-based model of the PBM in Daple (black ribbon and sticks) bound to the PDZ domain of Dvl2 (gray surface), generated using solved crystal structures of Dvl2-PDZ bound to multiple peptides (see Materials and Methods). Each Tyr is predicted to form H-bonds (yellow dotted lines) with each other and with Dvl2 (Asp331 and Ser286). (H and I) Immunoprecipitation assays assessing the impact of EGF stimulation on the abundance of Daple/Dvl (H) or Daple/Gαi3 (I) complexes in Cos7 cells. Dvl- or Gαi3-bound immune complexes were analyzed for Daple (myc) (fold changes in binding are quantified in fig S4, F and G). (J) Immunoprecipitation assays investigating the impact of phosphomimicking (YE) and nonphosphorylatable (YF) mutants of Daple on binding to Gαi3. Gαi3-bound immune complexes were analyzed for Daple as in (H) (quantification shown in fig. S4H). (K) Immunoblotting for Daple, Gαi3, and actin in whole-cell lysates from control (shControl) or Daple-depleted (shDaple) HeLa cells stably expressing WT or mutant (YE) Daple. (L) HeLa cells in K were analyzed for activation of Gαi3 as in Fig. 3A (quantified in fig. S4K). (M) HeLa cells expressing Daple-WT or mutants were pretreated with forskolin and isobutylmethylxanthine (see Materials and Methods) and treated (+) or not (−) with 50 nM EGF for 30 min before analyzing adenosine 3′,5′-monophosphate (cAMP) levels by radioimmunoassay. Bar graphs display cAMP concentrations. Data are means ± SD; n = 3. (N) Schematic summarizing our conclusions from (A) to (M), that is, that phosphorylation of Daple’s PBM by protein tyrosine kinases (PTKs) triggers dissociation of Daple/Dvl complexes and enables Daple to bind and activate Gαi.

  • Fig. 5 Tyr-phosphorylated Daple enhances noncanonical Wnt signals and triggers EMT and cell migration.

    (A and B) Daple-depleted HeLa cells stably expressing Daple-WT or Daple-Y2025E (YE) were analyzed for expression of β-catenin by immunoblotting and quantified. Blot (A) is representative; data (B) are means ± SD; n = 3. (C) Canonical Wnt responsive β-catenin/TCF/LEF target genes were analyzed by qPCR in HeLa cells described in (A). Bar graphs display the fold change in each RNA (y axis). Error bars are means ± SD; n = 3. (D and E) Anchorage-independent (D) and anchorage-dependent (E) colony growth assays from HeLa cells described in (A) (additional representative data are shown in fig. S5, D to G). Bar graphs display the number of colonies (y axis) seen in each cell line. Data are means ± SD; n = 3. (F) Rac1 activity assay analyzed in HeLa cells described in (A). (G) Western blot analysis for phosphorylated (pSer473) AKT and phosphorylated (pS552) β-catenin from HeLa cell lines used in (A). (H and I) Expression of epithelial-mesenchymal transition (EMT) markers vimentin and LOXL3 was analyzed by qPCR in HeLa cell lines described in (A). Bar graphs display the fold change in each mRNA (y axis) normalized to the expression in cells expressing vector control. Data are means ± SD; n = 3. (J) Chemotaxis of HeLa cell lines described in (A) was analyzed by Transwell assays along a serum gradient (0 to 10% FBS). Graphs display the number of cells per high-power field (HPF). Data are means ± SD; n = 3 experiments; representative images are shown in fig. S5H.

  • Fig. 6 Concurrently high expression of Daple (CCDC88C) and EGFR in colon cancer is associated with reduced disease-free survival.

    (A) Graph displaying individual arrays according to the expression levels of EGFR and Daple (CCDC88C) in a data set containing 466 patients with colon cancer. (B and C) Survival analysis using Kaplan-Meier curves according to Daple expression in tumors with high (B) versus low (C) EGFR expression. (D and E) Survival analysis using Kaplan-Meier curves according to EGFR expression in tumors with high (D) versus low (E) Daple expression. (F) Schematic of working model. (Left) In the absence of growth factors or Src activation, Dvl remains complexed to Daple, and Gαi is largely inactive. Stimulation with growth factors like EGF triggers phosphorylation of Daple’s PBM, either directly by EGFR or indirectly via activation of the non-RTK Src, which triggers dissociation of Dvl/Daple complexes and favors the assembly of Daple/Gαi complexes and subsequent activation of Gαi. (Right) Schematic summarizing the key events triggered by the ligand Wnt5a, in the context of our previous report (27).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/519/eaao4220/DC1

    Fig. S1. The GBA and PBM in Daple allosterically inhibit each other.

    Fig. S2. RTKs and non-RTKs target distinct sets of tyrosines within the C terminus of Daple.

    Fig. S3. Daple mediates EGF-EGFR signaling–induced activation of Gαi3.

    Fig. S4. Phosphorylation or phosphomimic Y2025E mutation within the PBM of Daple inhibits its binding to Dvl, enhances its binding to Gαi, but has no impact on binding to FZD7R.

    Fig. S5. Tyr phosphorylation of Daple’s PBM inhibits colony growth and enhances cell migration.

    Fig. S6. Tyr phosphorylation of Daple affects the localization and transcriptional activity of β-catenin.

    Fig. S7. Concurrent high Daple and EGFR expression in colon cancers is associated with poor prognosis.

  • Supplementary Materials for:

    Convergence of Wnt, growth factor, and heterotrimeric G protein signals on the guanine nucleotide exchange factor Daple

    Nicolas Aznar,* Jason Ear, Ying Dunkel, Nina Sun, Kendall Satterfield, Fang He, Nicholas A. Kalogriopoulos, Inmaculada Lopez-Sanchez, Majid Ghassemian, Debashis Sahoo, Irina Kufareva, Pradipta Ghosh*

    *Corresponding author. Email: nicolas.aznar{at}inserm.fr (N.A.); prghosh{at}ucsd.edu (P.G.)

    This PDF file includes:

    • Fig. S1. The GBA and PBM in Daple allosterically inhibit each other.
    • Fig. S2. RTKs and non-RTKs target distinct sets of tyrosines within the C terminus of Daple.
    • Fig. S3. Daple mediates EGF-EGFR signaling–induced activation of Gαi3.
    • Fig. S4. Phosphorylation or phosphomimic Y2025E mutation within the PBM of Daple inhibits its binding to Dvl, enhances its binding to Gαi, but has no impact on binding to FZD7R.
    • Fig. S5. Tyr phosphorylation of Daple's PBM inhibits colony growth and enhances cell migration.
    • Fig. S6. Tyr phosphorylation of Daple affects the localization and transcriptional activity of β-catenin.
    • Fig. S7. Concurrent high Daple and EGFR expression in colon cancers is associated with poor prognosis.

    [Download PDF]


    © 2018 American Association for the Advancement of Science

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