IQGAP1 protein regulates nuclear localization of β-catenin via importin-β5 protein in Wnt signaling

Abstract

In the canonical Wnt signaling pathway, the translocation of β-catenin is important for the activation of target genes in the nucleus. However, the molecular mechanisms underlying its nuclear localization remain unclear. In the present study, we found IQGAP1 to be a regulator of β-catenin function via importin-β5. In Xenopus embryos, depletion of IQGAP1 reduced Wnt-induced nuclear accumulation of β-catenin and expression of Wnt target genes during early embryogenesis. Depletion of endogenous importin-β5 associated with IQGAP1 also reduced expression of Wnt target genes and the nuclear localization of IQGAP1 and β-catenin. Moreover, a small GTPase, Ran1, contributes to the nuclear translocation of β-catenin and the activation of Wnt target genes. These results suggest that IQGAP1 functions as a regulator of translocation of β-catenin in the canonical Wnt signaling pathway.

Keywords: Beta-Catenin; Dvl; IQGAP; Importin; Nuclear Translocation; Nuclear Transport; Ran; Signal Transduction; Wnt Pathway; Wnt Signaling.

FIGURE 1.

Interaction between β-catenin, xDVL2, and xIQGAP1. A, interaction between ectopically expressed xDVL2 and β-catenin in HEK 293T cells. WB, Western blotting; IP, immunoprecipitation. B, interaction between ectopically expressed xIQGAP1 and β-catenin in HEK 293T cells. C, interaction among endogenous β-catenin, hDVL1, and hIQGAP1 in SW480 cells. D, interaction among ectopically expressed β-catenin, xDVL2, and xIQGAP1 in HEK 293T cells. Anti-FLAG antibody was used for the first immunoprecipitation, and the precipitate was subjected to a second immunoprecipitation with IgG or MYC antibodies. E, the second immunoprecipitation/input ratios for xDVL2, xIQGAP1, and β-catenin in panel D. F, nuclear localization of β-catenin-GFP in stage 10 Xenopus animal cap cells overexpressing Xwnt-8. Control-MO (15 ng), xIQGAP1-MO (15 ng), or xDVL1-MO, xDVL2-MO, and xDVL3-MO (each 5 ng) was co-injected with β-catenin-GFP mRNA (10 pg). Left, GFP signals. Center, DAPI staining of animal cap cells. Right, merge. G–J, the ratio of cells that had nuclear fluorescence signals of GFP. The average of ratio was taken with six explants in three independent experiments. Error bars represent S.D. with six explants. G, the ratio of nucleus-localized β-catenin-GFP in cells injected with xIQGAP1-MO and xDVL2-MO. Lane 1, n = 1770, 20.6%; lane 2, n = 1437, 10.9%; lane 3, n = 1662, 19.6%; lane 4, n = 1655, 35.8%; lane 5, n = 1763, 15.2%; lane 6, n = 1874, 33.9%. H, the ratio of nucleus-localized β-catenin-GFP in cells injected with xDVL1-MO, xDVL2-MO, and xDVL3-MO. Lane 1, n = 1858, 19.9%; lane 2, n = 4689, 6.4%; lane 3, n = 5382, 36.8%; lane 4, n = 3650, 18.0%. I, the ratio of nucleus-localized xDVL2-GFP in cells injected with β-catenin-MO. Lane 1, n = 971, 24.6%; lane 2, n = 989, 14.2%; lane 3, n = 347, 45.5%; lane 4, n = 326, 24.5%. J, the ratio of nucleus-localized xIQGAP1-GFP in cells injected with β-catenin-MO. Lane 1, n = 784, 21.9%; lane 2, n = 616, 20.9%; lane 3, n = 544, 39.0%; lane 4, n = 885, 27.9%.

FIGURE 2.

Cytoplasmic and nuclear distribution of xDVL2, xIQGAP1, and β-catenin in animal cap cells. Each MYC-tagged mRNA (100 pg) was co-injected into the animal poles of four-cell stage embryos with the indicated morpholino oligonucleotides, and the injected animal caps were dissected at stage 10. Lysates from the animal caps were fractionated and subjected to Western blotting (WB) with the indicated antibodies. Upper panels, β-catenin-MYC. Lower panels, left, xDVL2-MYC. Lower panels, right, xIQGAP1-MYC. In the case of β-catenin-MYC, cytoplasmic fraction was refractionated to cytosolic and membrane fractions.

FIGURE 3.

The role of xIQGAP1 in canonical Wnt signaling. A, interaction between ectopically expressed β-catenin and xIQGAP1 or xIQGAP1-ΔC in HEK 293T cells. B, the ratio of β-catenin-GFP localized in the nucleus in cells injected with xIQGAP1-ΔC mRNA. Lane 1, n = 635, 24.3%; lane 2, n = 1517, 21.2%; lane 3, n = 459, 44.7%; lane 4, n = 1271, 24.3%. C, quantitative RT-PCR analysis of early dorsal Wnt target genes (n = 3). Control-MO (15 ng) or xIQGAP1-MO (15 ng) was co-injected with xIQGAP1 (400 pg) or xIQGAP1-ΔC (400 pg) mRNA into two ventral blastomeres of four-cell embryos. RNAs from dissected ventral sectors of injected embryos were extracted at stage 10. RNAs from dissected dorsal and ventral sectors of uninjected embryos were used as controls. The value obtained for each gene was normalized to the level of ODC (ornithine decarboxylase). The value of dorsal sectors was set to 100, and other values were computed. Error bars represent S.D. in three experiments. D, quantitative RT-PCR analysis of early dorsal Wnt target genes (n = 3). Control-MO (15 ng) or xIQGAP1-MO (15 ng) was co-injected with β-catenin (20 pg) or NLS-β-catenin (20 pg) mRNA into two ventral blastomeres of four-cell embryos. The following procedure is indicated in panel C. E, the ratio of injected embryos exhibiting a partial secondary axis. The numbered lanes indicate the injected mRNAs and MOs consistent with the numbering in panel D. F, nuclear localization of NLS-β-catenin-GFP in stage 10 Xenopus animal cap cells overexpressing Xwnt-8. Left panels, GFP signals. Center panels, DAPI staining. Right panels, merge. G, the ratio of nucleus-localized NLS-β-catenin-GFP in cells injected with xIQGAP1-MO and xDVL1-, 2-, 3-MO in Xenopus animal cap cells at stage 10. Lane 1, n = 352, 92.0%; lane 2, n = 347, 92.5%; lane 3, n = 396, 89.9%; lane 4, n = 907, 94.7%; lane 5, n = 512, 93.6%; lane 6, n = 359, 92.4%. H, Western blotting analysis using β-catenin antibody. Control-MO (15 ng), xIQGAP1-MO (15 ng), xDVL2-MO (15 ng), xDVLs-MO (xDVL1-MO (5 ng), xDVL2-MO (5 ng), xDVL3-MO (5 ng), or β-catenin-MO (15 ng) was co-injected with β-globin-FLAG mRNA (100 pg) into two dorsal blastomeres of four-cell embryos. Lysates from whole embryos or cytoplasmic fractions were obtained dissected dorsal sectors at stage 10 and were subjected to Western blotting with the β-catenin and FLAG antibodies. I, ChIP assay for Siamois and Xnr3 promoter regions. Each indicated mRNA (200 pg) was injected into two dorsal blastomeres of four-cell embryos. Injected dorsal sectors were dissected and cross-linked at stage 10. Immunoprecipitates using anti-MYC antibody were examined by PCR using specific primers of Siamois and Xnr3 promoter regions.

FIGURE 4.

Interaction between xIQGAP1 and xIpo-β5 in canonical Wnt signaling. A, interaction between ectopically expressed xIpo-β5 and xDVL2, xIQGAP1, and β-catenin in HEK 293T cells. WB, Western blotting; IP, immunoprecipitation. B, interaction between endogenous human IQGAP1 and human importin-β5 in HEK 293T cells. C, interaction between xIQGAP1 and xIpo-β5 with ectopically expressed xDVL2 and β-catenin in HEK 293T cells. Immunoprecipitates obtained using anti-MYC antibody were subjected to Western blotting with the indicated antibodies. +, present; −, absent. D, the immunoprecipitation/input ratios for xIpo-β5 in panel C. Error bars represent S.D. in three experiments. E, quantitative RT-PCR analysis of early dorsal Wnt target genes (n = 3). Control-MO (80 ng) or xIpo-β5-MO (low dose: 40 ng, high dose: 80 ng) was injected into two dorsal blastomeres of four-cell embryos. The following procedure was described in Fig. 3C. F, quantitative RT-PCR analysis of early dorsal Wnt target genes (n = 3). Control-MO (40 ng), xIpo-β5-MO (40 ng), and Xwnt-8 (0.5 pg) mRNA were ventrally co-injected with xIpo-β5 mRNA (500 pg). G–J, the ratio of GFP-fused constructs localized in the nucleus in cells injected with xIpo-β5-MO or xIpo-β5 mRNA. G, xIQGAP1-GFP. Lane 1, n = 1859, 14.0%; lane 2, n = 1216, 9.7%; lane 3, n = 909, 35.1%; lane 4, n = 887, 8.3%. H, xDVL2-GFP. Lane 1, n = 932, 18.9%; lane 2, n = 540, 16.9%; lane 3, n = 669, 37.2%; lane 4, n = 402, 22.4%. I, β-catenin-GFP. Lane 1, n = 1125, 18.3%; lane 2, n = 636, 18.5%; lane 3, n = 1317, 40.0%; lane 4, n = 752, 23.7%. J, β-catenin-GFP. Lane 1, n = 495, 19.4%; lane 2, n = 966, 22.8%; lane 3, n = 796, 39.2%; lane 4, n = 399, 51.4%. Error bars represent S.D. with six explants.

FIGURE 5.

The role of xRan1 in canonical Wnt signaling. A, interaction between ectopically expressed xIQGAP1 and xRan1 in HEK 293T cells. B, interaction between ectopically expressed xIpo-β5 and xRan1 in HEK 293T cells. C, interaction between endogenous human IQGAP1 and human Ran in HEK 293T cells. D, interaction between endogenous human importin-β5 and human Ran in HEK 293T cells. E–G, quantitative RT-PCR analysis of early dorsal Wnt target genes (n = 3). E, control-MO (80 ng) or xRan1-MO (low dose: 40 ng, high dose: 80 ng) was injected into two dorsal blastomeres of four-cell embryos. RNAs from dissected dorsal sectors of injected embryos were extracted at stage 10. The following procedure was described in Fig. 3C. F, control-MO (40 ng), xRan1-MO (40 ng), and Xwnt-8 (0.5 pg) mRNA were ventrally co-injected with xRan1 mRNA (500 pg). G, the indicated amounts of control-MO, xIpo-β5-MO, and xRan1-MO were ventrally co-injected with Xwnt-8 mRNA (0.5 pg). H, the ratio of β-catenin-GFP localized in the nucleus in cells injected with xRan1-MO. Lane 1, n = 914, 21.8%; lane 2, n = 1006, 13.6%; lane 3, n = 2155, 31.8%; lane 4, n = 2245, 18.8%. I, the ratio of β-catenin-GFP localized in the nucleus in cells injected with xRan1 mRNA. Lane 1, n = 466, 19.1%; lane 2, n = 718, 22.3%; lane 3, n = 1582, 40.8%; lane 4, n = 448, 56.7%. J, Western blotting analysis using β-catenin antibody. Control-MO (40 ng), xIpo-β5-MO (40 ng), xRan1-MO (15 ng), or β-catenin-MO (15 ng) was co-injected into two dorsal blastomeres of four-cell embryos. The following procedure was described in Fig. 3H.

FIGURE 6.

Interaction between xIQGAP1 and xRan1 in canonical Wnt signaling. A, interaction between ectopically expressed xRan1 and xIQGAP1 or xIQGAP1-ΔRGD in HEK 293T cells. WB, Western blotting; IP, immunoprecipitation. B, interaction between ectopically expressed xIpo-β5 and xIQGAP1 or xIQGAP1-ΔRGD in HEK 293T cells. C, quantitative RT-PCR analysis of early dorsal Wnt target genes (n = 3). Control-MO (15 ng), xIQGAP1-MO (15 ng), and Xwnt-8 (0.5 pg) mRNA were ventrally co-injected with xIQGAP1 (400 pg) or xIQGAP1-ΔRGD mRNA (400 pg). D, the ratio of β-catenin-GFP localized in the nucleus in cells injected with xIQGAP1 constructs mRNA. Lane 1, n = 771, 21.4%; lane 2, n = 2165, 20.5%; lane 3, n = 856, 42.5%; lane 4, n = 549, 22.6%. E, Ran activation assay. xRan1 and xIQGAP1, xRanGAP, or xRanGEF were ectopically expressed in HEK 293T cells. Immunoprecipitates obtained using anti-active-Ran antibody were subjected to Western blotting with the indicated antibodies. F, interaction between ectopically expressed xIQGAP1 and xRanGAP or xRanGEF in HEK 293T cells. No interaction was observed. G, GAP assay. GTP-loaded GST-xRan1-FLAG was incubated with cell lysates from HEK 293T cells transfected with pCS2+ (control), xRanGAP-MYC, or xIQGAP1-MYC and immunoprecipitated with anti-active Ran antibody. H, interaction between ectopically expressed xRanGAP and active form of xRan1 (G19V, Q69L) with xIQGAP1 in HEK 293T cells. Their interactions were inhibited by expression of xIQGAP1 in a dose-dependent manner. I, GEF assay. GST-xRan1-FLAG was incubated with cell lysates from HEK 293T cells transfected with pCS2+ (control), xRanGEF-MYC, or xIQGAP1-MYC and immunoprecipitated with anti-active Ran antibody. J, interaction between ectopically expressed xRanGEF and the inactive form of xRan1 (T24N) with xIQGAP1 in HEK 293T cells. K, interaction between ectopically expressed xIQGAP1 and active or inactive form of xRan1 in HEK 293T cells. WT, wild type; GTP, active form of xRan1; GDP, inactive form of xRan1.