Figure 6
Single His-repeats at 637,638 and 647,648 are essential for Dvl3 function in Wnt5a/cGMP/Ca 2+ non-canonical signaling to NF-AT-dependent transcription. A, sequence alignment of C-terminus of mouse Dvl1, 2, and 3 is displayed. Sequences that are common to at least two mDvls are shaded in gray. Unique sequences of mDvl3 are highlighted in yellow. His residues located within amino acid 631-650 of mDvl3 are displayed in red. B, Sequence alignment of mouse Dvl3 (631-650) with corresponding region of Dvl from fly Dsh, frog Dvl3 and mammalian Dvl3 is displayed. Common sequences are shaded in gray. HH637, 638 and HH647, 678 of mouse Dvl3 and corresponding single His-repeats of Dvl3 in other species are displayed in red. Other His residues in the region are displayed in blue. C, F9 cells stably expressing Fz2 were treated with siRNA targeting Dvl3 (siRNA). Dvl3-depleted cells were transfected with an empty vector (EV) or expression vector harboring either Myc-hDvl3, or one of hDvl3 mutants as indicated. Activity of NF-AT-reporter was measured in response to Wnt5a (upper panel). ***, p ≤ 0.001, compared with basal (-Wnt5a) in control (-siRNA and EV) group; ##, p ≤ 0.01, compared with (+Wnt5a) in control group. Expressions of Myc-hDvl3 or mutants were analyzed by immunoblotting by using anti-Dvl3 and anti-Myc antibodies (bottom panel). D, F9 cells stably expressing Fz1 were treated with siRNA targeting Dvl3 (siRNA). Rescue experiments were conducted by expression of either Myc-hDvl3, or Myc-hDvl3Δ(631-650) in Dvl3-depleted cells, followed by challenging cells with or without Wnt3a. Activity of Lef/Tcf-dependent transcription was measured by using a luciferase reporter (left panel). ***, p ≤ 0.001, compared with basal (-Wnt3a) in control (-siRNA and EV) group; ###, p ≤ 0.001, compared with (+Wnt3a) in control group. Expressions of Myc-hDvl3 or Myc-hDvl3Δ(631-650) were analyzed by immunoblotting (right panel). E, F9 cells expressing β2AR/Fz2 were treated with siRNA targeting Dvl3 (siRNA). Rescue experiments were conducted by expression of Myc-hDvl3 (FL), or one of Myc-hDvl3 mutants indicated, followed by activating the non-canonical pathway by treating cells with or without isoproterenol (Iso). Activity of NF-AT-reporter was measured and analyzed (upper panel). **, p ≤ 0.01, ***, p ≤ 0.001, compared with basal (-Iso) in control (-siRNA and EV) group; ##, p ≤ 0.01, ###, p ≤ 0.001, compared with (+Iso) in control group. The expression of each hDvl3 variant was evaluated by immunoblotting by using anti-Dvl3 and anti-Myc antibodies. The same blots were stained with anti-β-actin antibody (IB: actin) to establish loading equivalence. F, Dvl3-CT, Dvl3-CTΔ(631-650), Dvl3-CT HH637,8AA, or Dvl3-CT HH647,8AA was expressed in F9 clones stably expressing Fz2. The activity of NF-AT-dependent luciferase reporter was measured following stimulation of Wnt5a (upper panel). **p ≤ 0.01, compared with basal (-Wnt5a) in control (EV) group; ##, ≤ 0.01, compared with (+Wnt5a) in control group. Expression of Dvl3-CT or mutants at a similar level was shown by immunoblotting (bottom panel). The blots of β-actin are shown as loading controls.