IQGAP1 Functions as a Modulator of Dishevelled Nuclear Localization in Wnt Signaling

<div><p>Dishevelled (DVL) is a central factor in the Wnt signaling pathway, which is highly conserved among various organisms. DVL plays important roles in transcriptional activation in the nucleus, but the molecular mechanisms underlying their nuclear localization remain unclear. In the present study, we identified IQGAP1 as a regulator of DVL function. In <i>Xenopus</i> embryos, depletion of IQGAP1 reduced Wnt-induced nuclear accumulation of DVL, and expression of Wnt target genes during early embryogenesis. The domains in DVL and IQGAP1 that mediated their interaction are also required for their nuclear localization. Endogenous expression of Wnt target genes was reduced by depletion of IQGAP1 during early embryogenesis, but notably not by depletion of other IQGAP family genes. Moreover, expression of Wnt target genes caused by depletion of endogenous IQGAP1 could be rescued by expression of wild-type IQGAP1, but not IQGAP1 deleting DVL binding region. These results provide the first evidence that IQGAP1 functions as a modulator in the canonical Wnt signaling pathway.</p></div

Localization of xIQGAP1-GFP.

<p>(<b>A</b>) Nuclear localization of xIQGAP1-GFP in stage 10 <i>Xenopus</i> animal cap cells over-expressing <i>Xwnt-8</i> and <i>xDVL1, 2, 3</i>-MO. GFP signals (left). DAPI staining of animal cap cells (center). Merge (right). (<b>B, C</b>)The ratio of cells that had nuclear fluorescence signals. The following procedure is indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060865#pone-0060865-g002" target="_blank">Figure 2C</a>. (<b>B</b>) The ratio of nuclear localized xIQGAP1-GFP in cells injected with <i>xDVL2</i>-MO. Lane 1: n = 388, 22.7%, lane 2: n = 361, 22.2%, lane 3: n = 616, 20.9%, lane 4: n = 534, 39.7%. P>0.1 [between lane 1 and lane 2], P<0.05 [between lane 3 and lane 4]. (<b>C</b>) The ratio of nuclear-localized xIQGAP1-GFP in cells injected with <i>xDVL1</i>-MO, <i>xDVL2</i>-MO and <i>xDVL3</i>-MO in <i>Xenopus</i> animal cap cells at stage 10. Lane 1: n = 1424, 23.0%, lane 2: n = 1306, 21.6%, lane 3: n = 1702, 37.6%, lane 4: n = 1409, 16.6%. P>0.1 [between lane 1 and lane 2], P<0.01 [between lane 3 and lane 4]. (<b>D</b>) Cytoplasmic and nuclear distribution of xIQGAP1 in animal cap cells. MYC-tagged <i>xIQGAP1</i> mRNA (100 pg) was injected into the animal poles of 4-cell stage embryos. The following procedure is indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060865#pone-0060865-g002" target="_blank">Figure 2C</a>. P<0.1 [between lane 5 and lane 6], P<0.1 [between lane 7 and lane 8]. (<b>E</b>) Interaction between ectopically-expressed xDVL2 and xIQGAP1 in HEK 293T cells. The transfected cultured cells were stimulated with recombinant human Wnt3A for 6 hours in lanes 3 and 4. The bars represent the IP/Input ratios of xDVL2-FLAG for each transfection. Error bars represent standard deviation of the mean in three experiments. Statistical significance was determined by Student's <i>t</i>-test. P<0.1 [between lane 2 and lane 4].</p

IQGAP associates with DVL.

<p>(<b>A</b>) Interaction between ectopically-expressed hIQGAP1 and hDVL1 in HEK 293 cells. Immunoprecipitates (IP) obtained using anti-FLAG antibody were subjected to Western blotting (WB) with the indicated antibodies. +, present; -, absent. (<b>B</b>) Interaction between endogenous hIQGAP1 and hDVL1 in HEK 293T cells. The cultured cells were stimulated with recombinant human Wnt3A for 6 hours (right panels). (<b>C–E</b>) Interaction between ectopically-expressed hIQGAP1, hIQGAP2, hIQGAP3 and hDVL isoforms in HEK 293T cells. (C) hIQGAP1. (D) hIQGAP2. (E) hIQGAP3. (<b>F</b>) A schematic of the domains of hIQGAP1 and truncated constructs. (<b>G</b>) Interactions between ectopically-expressed hDVL1 and truncated hIQGAP1 constructs. (<b>H</b>) Interactions among ectopically-expressed hIQGAP1 and truncated hDVL1 constructs. (<b>I</b>) A schematic of the domains of hDVL1 and truncated constructs.</p

Localization of xDVL2-GFP.

<p>(<b>a,b</b>) Localization of xDVL2-GFP in <i>Xenopus</i> animal cap cells at stage 10. (<b>A</b>) xDvl2-GFP localized in punctate structures in the cytoplasm (left). Over-expression of <i>Xfz7</i> recruited xDVL2-GFP to the plasma membrane (center). <i>xIQGAP1</i>-MO did not affect the membrane localization of xDVL2-GFP induced by <i>Xfz7</i> (right). (<b>B</b>) Nuclear localization of GFP and xDVL2-GFP caused by over-expression of <i>Xwnt-8</i> and <i>xIQGAP1</i>-MO. GFP signals (left). DAPI staining of animal cap cells (center). Merge (right). (<b>C, D</b>) The ratio of cells that had nuclear fluorescence signals. The average of ratio was taken with six explants in 3 independent experiments (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060865#s2" target="_blank">Materials and Methods</a>). Error bars represent standard deviation of the mean with six explants. Statistical significance was determined by Student's <i>t</i>-test. (<b>C</b>) The ratio of GFP localized in the nucleus in cells. <i>Xwnt-8</i> was co-injected in lanes 2. Lane 1: n = 291, 18.2%, lane 2: n = 320, 17.2%. P>0.1 [between lane 1 and lane 2]. (<b>D</b>) The ratio of xDVL2-GFP localized in the nucleus in cells injected with <i>xIQGAP1</i>-MO. <i>Xwnt-8</i> was co-injected in lanes 3 and 4. Lane 1: n = 918, 26.0%, lane 2: n = 1694, 11.0%, lane 3: n = 263, 46.4%, lane 4: n = 477, 29.4%. P<0.01 [between lane 1 and lane 2], P<0.01 [between lane 3 and lane 4]. (<b>E</b>) Cytoplasmic and nuclear distribution of xDVL2, xIQGAP1 and ß-catenin in animal cap cells. MYC-tagged <i>xDVL2</i> mRNA (100 pg) was injected into the animal poles of 4-cell stage embryos, and the injected animal caps were dissected at stage 10. Lysates from the animal caps were fractionated and subjected to Western blotting with indicated antibodies. Each relative intensity was measured by ImageJ, and its relative ratio was calculated against Input with beta-tubulin for cytoplasm or with Histone H3 for nuclear. Error bars represent standard deviation of the mean in three experiments. Statistical significance was determined by Student's <i>t</i>-test. P<0.1 [between lane 5 and lane 6], P<0.1 [between lane 7 and lane 8].</p

xIQGAP1 is required for the canonical Wnt pathway during early embryogenesis.

<p>(<b>A</b>) Quantitative RT-PCR analysis of early dorsal Wnt target genes (n = 3). Each indicated morpholino (15 ng) was injected into two dorsal blastomeres of 4-cell embryos. RNAs from dissected dorsal 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 standard deviation of the mean in three experiments. Statistical significance was determined by Student's <i>t</i>-test for each marker gene. The highest P values in three marker genes were chosen as a representative, as follows: P<0.05 [between lane 3 and lane 4], P<0.05 [between lane 3 and lane 5], P>0.1 [between lane 3 and lane 6]. (<b>B</b>) Phenotypes of injected embryos at stage 30. Control (upper panel). <i>Xwnt-8</i> mRNA (0.5 pg) was co-injected with <i>xIQGAP1</i>-, <i>xIQGAP2</i>- or <i>xIQGAP3</i>-MO (15 ng) into two ventral blastomeres of 4-cell embryos. (<b>C</b>) The ratio of injected embryos exhibiting a partial secondary axis. (<b>D</b>) Quantitative RT-PCR analysis of early dorsal Wnt target genes (n = 3). <i>xIQGAP1</i>-, <i>xIQGAP2</i>- or <i>xIQGAP3</i>-MO (15 ng) and <i>Xwnt-8</i> (0.5 pg) (20 pg) mRNA were ventrally co-injected. RNAs from dissected ventral sectors of injected embryos were extracted at stage 10. The following procedure is indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060865#pone-0060865-g004" target="_blank">Figure 4A</a>. P<0.01 [between lane 3 and lane 4], P<0.1 [between lane 3 and lane 5], P>0.1 [between lane 3 and lane 6]. (<b>E</b>) Quantitative RT-PCR analysis of early dorsal Wnt target genes (n = 3). <i>xIQGAP1</i>, <i>xIQGAP2</i> or <i>xIQGAP3</i> (400 pg) mRNA was dorsally injected. The following procedure is indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060865#pone-0060865-g004" target="_blank">Figure 4A</a>. P<0.05 [between lane 1 and lane 3], P<0.01 [between lane 1 and lane 4], P>0.1 [between lane 1 and lane 5]. (<b>F</b>) Quantitative RT-PCR analysis of early dorsal Wnt target genes (n = 3). <i>xIQGAP1</i>-MO (15 ng) and <i>Xwnt-8</i> (0.5 pg) (20 pg) mRNA were ventrally co-injected with <i>xIQGAP1</i> constructs: <i>xIQGAP1</i> (400 pg), <i>xIQGAP1-ΔDBR</i> (400 pg), <i>xIQGAP1-DBR</i> (400 pg) mRNA. RNAs from dissected ventral sectors of injected embryos were extracted at stage 10. The following procedure is indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060865#pone-0060865-g004" target="_blank">Figure 4A</a>. P<0.05 [between lane 3 and lane 4], P<0.05 [between lane 4 and lane 5], P<0.05 [between lane 5 and lane 6], P<0.05 [between lane 5 and lane 7]. (<b>G</b>) The ratio of injected embryos exhibiting a partial secondary axis. The numbered lanes indicate the injected mRNAs and MOs consistent with the numbering in Figure F. (<b>H</b>) The ratio of injected embryos that exhibited a secondary axis. Control-MO (15 ng) or <i>xIQGAP1</i>-MO (15 ng) was co-injected with <i>Siamois</i> mRNA (indicated dose).</p

The role of the xIQGAP1-binding region of xDVL2 in canonical Wnt signaling during early embryogenesis.

<p>(<b>A</b>) Quantitative RT-PCR analysis of early dorsal Wnt target genes (n = 3). Control-MO (15 ng) or <i>xDVL2</i>-MO (15 ng) was ventrally co-injected with <i>Xwnt-8</i> (0.5 pg) mRNA. RNAs from dissected ventral sectors of injected embryos were extracted at stage 10. The following procedure is indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060865#pone-0060865-g004" target="_blank">Figure 4A</a>. Error bars represent standard deviation of the mean in three experiments. Statistical significance was determined by Student's <i>t</i>-test for each marker gene. The highest P values in three marker genes were chosen as a representative, as follows: P>0.1 [between lane 3 and lane 4]. (<b>B</b>) Quantitative RT-PCR analysis of early dorsal Wnt target genes (n = 3). <i>xDVL1</i>-MO (10 ng), <i>xDVL2</i>-MO (10 ng), <i>xDVL3</i>-MO (10 ng) and <i>Xwnt-8</i> (0.5 pg) mRNA were ventrally co-injected with <i>xDVL2</i> constructs: <i>xDVL2</i> (25 pg), <i>xDVL2-ΔIBR</i> (25 pg), <i>xDVL2-IBR</i> (25 pg) mRNA. RNAs from dissected ventral sectors of injected embryos were extracted at stage 10. The following procedure is indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060865#pone-0060865-g004" target="_blank">Figure 4A</a>. P<0.05 [between lane 3 and lane 4], P<0.05 [between lane 4 and lane 5], P<0.1 [between lane 5 and lane 6], P<0.05 [between lane 5 and lane 7]. (<b>C</b>) The ratio of injected embryos that exhibited a partial secondary axis. The numbered lanes indicate the injected mRNAs and MOs consistent with the numbering in Figure B. (<b>D</b>) Quantitative RT-PCR analysis of early dorsal Wnt target genes (n = 3). <i>Xwnt-8</i> (0.5 pg) mRNA was ventrally co-injected with <i>xIQGAP1</i> constructs: <i>xIQGAP1</i> (400 pg), <i>xIQGAP1-ΔDBR</i> (1 ng), <i>xIQGAP1-DBR</i> (1 ng) mRNA. The following procedure is indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060865#pone-0060865-g004" target="_blank">Figure 4A</a>. P<0.01 [between lane 3 and lane 4], P>0.1 [between lane 3 and lane 5], P<0.05 [between lane 3 and lane 6].</p