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Mol Biol Cell
2010 Oct 15;2120:3590-600. doi: 10.1091/mbc.E10-02-0095.
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Involvement of p114-RhoGEF and Lfc in Wnt-3a- and dishevelled-induced RhoA activation and neurite retraction in N1E-115 mouse neuroblastoma cells.
Tsuji T
,
Ohta Y
,
Kanno Y
,
Hirose K
,
Ohashi K
,
Mizuno K
.
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The Wnt-induced planar cell polarity (PCP) signaling pathway is essential for polarized cell migration and morphogenesis. Dishevelled (Dvl) and its binding protein Daam1 mediate RhoA activation in this pathway. WGEF, a member of the Rho-guanine nucleotide exchange factor (Rho-GEF) family, was shown to play a role in Wnt-induced RhoA activation in Xenopus embryos. However, it has remained unknown which member(s) of a Rho-GEF family are involved in Wnt/Dvl-induced RhoA activation in mammalian cells. Here we identified p114-RhoGEF and Lfc (also called GEF-H1) as the Rho-GEFs responsible for Wnt-3a-induced RhoA activation in N1E-115 mouse neuroblastoma cells. We screened for Rho-GEF-silencing short-hairpin RNAs (shRNAs) that are capable of suppressing Dvl-induced neurite retraction in N1E-115 cells and found that p114-RhoGEF and Lfc shRNAs, but not WGEF shRNA, suppressed Dvl- and Wnt-3a-induced neurite retraction. p114-RhoGEF and Lfc shRNAs also inhibited Dvl- and Wnt-3a-induced RhoA activation, and p114-RhoGEF and Lfc proteins were capable of binding to Dvl and Daam1. Additionally, the Dvl-binding domains of p114-RhoGEF and Lfc inhibited Dvl-induced neurite retraction. Our results suggest that p114-RhoGEF and Lfc are critically involved in Wnt-3a- and Dvl-induced RhoA activation and neurite retraction in N1E-115 cells.
Figure 1. Screening of effective shRNA constructs targeting Rho-GEFs expressed in N1E-115 cells. (A) Expression of Rho-GEF mRNAs in N1E-115 cells. Total RNAs from N1E-115 cells were subjected to RT-PCR analysis using primers designed to amplify PCR products of ∼400 base pairs in length. Experiments were repeated twice, and similar results were obtained. (B) Schematic of the protocol for selecting effective shRNA constructs. The cDNA fragment (∼400 base pairs) of a target gene is fused to the 3′ terminus of luciferase cDNA and infected into Jurkat cells together with each individual shRNA construct from an RNAi library constructed by an EPRIL method. Effective shRNA constructs target the cognate sequences of the target gene on the luciferase-target chimeric mRNA, thus preventing luciferase expression. (C) Luciferase reporter analysis to select shRNA constructs targeting each of 13 Rho-GEFs. Jurkat cells were coinfected with retroviruses carrying a luciferase-target chimeric gene together with retroviruses carrying shRNA constructs targeting each Rho-GEF. The relative luminescent intensity for each type of shRNA-infected cells was compared with that for shRNA noninfected control cells. Data represent means ± SD of triplicate experiments. The target sequences for Rho-GEFs are listed in Supplemental Table S2. (D) Immunoblot analysis of Rho-GEF expression. N1E-115 cells were transfected with the indicated shRNA (#1) and cultured for 48 h. Expression of endogenous Rho-GEF proteins or cotransfected HA-WGEF was analyzed by immunoprecipitation (IP) followed by immunoblotting (IB) with the indicated antibodies. Actin in cell lysates was analyzed as a loading control.
Figure 2. Knockdown of LARG, p114-RhoGEF, or Lfc, but not WGEF, suppresses Dvl-induced neurite retraction. (A) Effects of Rho-GEF knockdown on Dvl-induced neurite retraction. N1E-115 cells were cotransfected with CFP-Dvl-1 (or control CFP) and the indicated shRNA plasmids at the ratio of 1:2, cultured for 24 h, serum-starved for 24 h, and then fixed. Merged images of phase-contrast and CFP fluorescence are shown. Scale bar, 50 μm. (B) Quantitative analysis of the percentage of neurite-bearing cells. Cells with neurites exceeding one cell body length were scored and expressed as a percentage of the total number of CFP-positive cells. Data represent means ± SD of three independent experiments (at least 50 cells in each experiment). *p < 0.01. (C) Effects of WGEF shRNA and the indicated shRNA (#1) on Dvl-induced neurite retraction. Quantitative analysis of the percentage of neurite-bearing cells was performed as in (B).
Figure 3. Effects of knockdown of LARG, p114-RhoGEF, or Lfc on neurite retraction induced by Wnt-3aCM or LPA. N1E-115 cells were cotransfected with CFP and LARG, p114-RhoGEF, or Lfc shRNA at the ratio of 1:5 and serum-starved for 24 h. Cells were treated for 10 min with control (L cell) CM, Wnt-3a–producing L cell CM, or LPA. The percentages of neurite-bearing cells in CFP-positive cells were counted. Data represent means ± SD of three independent experiments (at least 50 cells in each experiment). *p < 0.01.
Figure 4. Effects of single knockdown of LARG, p114-RhoGEF, or Lfc, or double knockdown of p114-RhoGEF and Lfc, on RhoA activation induced by Dvl-1 (A) or Wnt-3a (B). In A, N1E-115 cells were cotransfected with CFP or CFP-Dvl-1 and shRNA constructs, cultured for 24 h, and serum-starved for 24 h. In B, N1E-115 cells transfected with shRNAs were cultured for 24 h, serum-starved for 24 h, and treated with control or Wnt-3aCM for 10 min. The level of active RhoA was analyzed by a GST-RBD pull-down assay. Relative activity of RhoA was normalized by the immunoblot intensity of RhoA in the total cell lysates. Data represent means ± SD of three independent experiments, with the level in control cells taken as 1.0. *p < 0.01.
Figure 5. Binding of p114-RhoGEF, Lfc, and WGEF, but not LARG, to Dvl and Daam1. (A) Binding to Dvl. Flag-tagged Dvl-1 was coexpressed with CFP-tagged Rho-GEFs in HEK293 cells. Cell lysates were immunoprecipitated with an anti-Flag antibody and immunoblotted with anti-GFP and anti-Flag antibodies. (B) Binding to Daam1. Myc-tagged Daam1 was coexpressed with CFP-tagged Rho-GEFs in HEK293 cells. Cell lysates were immunoprecipitated with an anti-Myc antibody and immunoblotted with anti-GFP and anti-Myc antibodies. (C) Binding of Daam1 to Dvl. Myc-Daam1 and Flag-Dvl-1 were coexpressed in HEK293 cells. Cell lysates were immunoprecipitated with an anti-Flag antibody and analyzed by immunoblotting with anti-Myc and anti-Flag antibodies. (D) Binding of endogenous proteins. N1E-115 cells were serum-starved for 24 h and treated with Wnt-3aCM for 10 min or left untreated. Cell lysates were immunoprecipitated with an anti–p114-RhoGEF or an anti-Lfc antibody and immunoblotted with anti–Dvl-1, anti–p114-RhoGEF, and anti-Lfc antibodies. All experiments were repeated twice, and similar results were obtained.
Figure 6. Mapping of the binding regions of Dvl and Daam1. (A) Binding of p114-RhoGEF and Lfc to the middle region of Dvl. Flag-tagged Dvl-1 fragments and CFP-tagged p114-RhoGEF or Lfc were coexpressed in HEK293 cells. Cell lysates were immunoprecipitated with an anti-Flag antibody and analyzed by immunoblotting with anti-GFP and anti-Flag antibodies. (B) Binding of p114-RhoGEF and Lfc to the N-terminal region of Daam1. Myc- or HA-tagged Daam1 fragments and CFP-tagged Rho-GEFs were coexpressed in HEK293 cells and analyzed as in A. In A and B, schematic structures of Dvl-1, Daam1 and their fragments are shown.
Figure 7. Mapping of the binding regions of p114-RhoGEF and Lfc. (A) Binding of p114-RhoGEF fragments to Dvl-1 and Daam1. CFP-tagged p114-RhoGEF fragments were coexpressed with Flag-Dvl-1-(165-367) or HA-N-Daam1 in HEK293 cells. Cell lysates were immunoprecipitated with an anti-Flag or an anti-HA antibody and analyzed by immunoblotting with an anti-GFP antibody. (B) Binding of Lfc fragments to Dvl-1 and Daam1. CFP-tagged Lfc fragments were coexpressed with Flag-Dvl-1-(165-367) or HA-N-Daam1 in HEK293 cells. Cell lysates were analyzed as in A. Schematic structures of p114-RhoGEF, Lfc, and their fragments are shown.
Figure 8. The Dvl-binding fragment of p114-RhoGEF or Lfc suppresses Dvl-induced neurite retraction. (A) Effects of expression of p114-RhoGEF or Lfc fragments on Dvl-induced neurite retraction. N1E-115 cells were cotransfected with Flag-Dvl-1 and CFP or CFP-tagged p114-RhoGEF or Lfc fragments, cultured for 24 h, and serum-starved for 24 h. Cells were then fixed and immunostained with an anti-Flag antibody. Merged images of phase-contrast, anti-Flag immunostaining (red), and CFP fluorescence (cyan) are shown in the right. Scale bar, 50 μm. (B) Quantitative analysis of the percentage of neurite-bearing cells. Data represent means ± SD of three independent experiments (at least 50 cells in each experiment). *p < 0.01.
Figure 9. A model for the Wnt-3a– and Dvl-induced RhoA activation. Wnt-3a induces the formation of a ternary complex, composed of Dvl-1, Daam1, and p114-RhoGEF or Lfc, which in turn stimulates RhoA activation and neurite retraction. Details are described in text.
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