PRL-3 is phosphoryated in vitro by the Src tyrosine kinase, and endogenous PRL-3 is tyrosine-phosphorylated in cells.

<p>A) Schematic diagram of PRL-3 showing the relative positions of the six tyrosines evaluated in these studies, as well as the C-terminal CaaX motif. The numbers shown reflect the probability of phosphorylation at each site based on predictions by NetPhos 2.0. B) PRL-3 (“WT”) or a mutant in which all six tyrosines were substituted by phenylalanine (“All_F”) was fused to GST, purified from bacteria, and subjected to <i>in vitro</i> phosphorylation with purified Src. WT PRL-3 was phosphorylated by Src while the “All_F” PRL-3 mutant was not. Src itself is visible on the phosphotyrosine blot because it becomes autophosphorylated on Y416 (pSrc). Coomassie staining demonstrates equal amounts of PRL-3 protein in each reaction. C) To determine whether endogenous PRL-3 is also tyrosine phosphorylated in cells, SW480 cells were treated with the tyrosine phosphatase inhibitor pervanadate (VO₄, 100 µM, 1 h) to enhance detection of transient tyrosine phosphorylation events. Endogenous PRL-3 was immunoprecipitated using anti-PRL-3 antibody, followed by SDS-PAGE and western blot analysis for either PRL-3 or phosphotyrosine.</p

Effects on P7.p polarity are <i>chw-1</i>-specific.

<p>^a compared to <i>lin-17(n671)</i></p><p>^b compared to <i>lin-17(n671); gfp(RNAi)</i></p><p>^c compared to <i>lin-18(e620)</i></p><p>^d compared to <i>lin-18(e620); gfp(RNAi)</i></p><p>Animals were grown at 23°C and scored by DIC at late L4 stage. <i>n</i> is number of animals scored. Data for strains marked with an asterisk (*) are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133226#pone.0133226.t001" target="_blank">Table 1</a> (<i>lin-17</i>) or <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133226#pone.0133226.t002" target="_blank">Table 2</a> (<i>lin-18</i>). Analyses of statistical significance were performed using Fisher’s exact test.</p

PRL-3 requires the activity of a Src family kinase to promote invasion and RhoC activation.

<p>A) SW480 cells expressing WT PRL-3 were subjected to Matrigel invasion analysis as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064309#pone-0064309-g005" target="_blank">Figure 5</a>. The Src family kinase inhibitor SU6656 was added to both the top and bottom chambers at the indicated concentrations. Cells expressing phosphatase-inactive PRL-3 (C104S) served as a negative control. Data are shown normalized to basal levels of invasion of vector-expressing cells. Results from four assays are shown +/− SD. B) SW480 cells expressing WT PRL-3 were treated with the Src inhibitor, SU6656, and analyzed for levels of active, GTP-bound RhoC using a Rhotekin-RBD pull down assay. Images from a representative assay are shown. Lanes containing samples not relevant to this work have been removed. All panels were taken from the same assay using the same photographic exposure time. Phosphatase-inactive PRL-3 (C104S) served as a negative control. C) Data quantitated from at least three pull down assays as in panel B are shown +/− SEM.</p

Two Wnt pathways regulate VPC polarization.

<p><b>A</b>. Schematic of vulval precursor cells in the whole animal. <b>B.</b> Schematic of refined (central) and ground (posterior) Wnt signals regulating VPC polarity. <b>C.</b> A schematic of polarized 2°-1°-2° VPC lineages (P5.p (left), P6.p (center), and P7.p (right)). P5.p and p7.p lineages are asymmetric and mirror one another around a central axis (dotted line). (<b>D-F</b>) Illustration and images of vulval lineages, model polarity signals and morphology in animals that are (<b>D</b>) wild type, (<b>E</b>) <i>egl-20(n585)</i> using only refined polarity, or (<b>F</b>) <i>lin-17(n671)</i>; <i>lin-18(e620)</i> using only ground polarity. Arrows represent putative Wnt polarity signals received by VPCs, grayed arrows are inactivated polarity signals, and dotted lines represent central vulval axis.</p

PRL-3 promotion of invasion and cell motility requires an intact Y53.

<p>A) SW480 cells stably expressing WT PRL-3 (WT) or a phosphorylation-deficient mutant lacking tyrosine 53 (Y53F) were subjected to Matrigel invasion assays. Data from three independent assays are shown. For each sample, 5x10⁴ cells were placed in the top chamber in the absence of serum; the bottom chamber contained medium supplemented with 10% serum. After 72 h, invaded cells were fixed, stained and counted. Equal expression of WT HA-tagged PRL-3 and the Y53F mutant was confirmed by HA blot. B) H1299 cells were transiently transfected with EGFP-tagged PRL-3, the Y53F mutant or vector only. The motility rate of individual cells was evaluated using a BioStation IM live cell recorder. Data shown are from three independent assays, and include a total of at least 35 cells for each sample. Images of representative cells expressing each EGFP-tagged protein are shown below the graph. All images were captured under identical microscopic and photographic settings. Data in both A and B are shown +/− SEM, and <i>p</i>-values were calculated using a two-tailed Student's <i>t</i>-test.</p

Loss of CHW-1 suppresses P7.p polarity defects caused by loss of LIN-17/Fz.

<p>^a compared to N2</p><p>^b compared to <i>lin-17(n671)</i></p><p>^c compared to <i>lin-17(sy277)</i></p><p>^d compared to <i>lin-17(n671); gfp(RNAi)</i></p><p>Animals were grown at 23°C and scored by DIC at late L4 stage. <i>n</i> is number of animals scored. Analyses of statistical significance were performed using Fisher’s exact test.</p

CHW-1 regulates relative LIN-17/Fz and LIN-18/Ryk signaling in refined (central) polarity, and LIN-17/Fz and LIN-18/Ryk execute a specific repressive program to exclude activity of the ground (posterior) polarity system.

<p>Shown is a schematic of P7.p with anterior, the direction of refined (central) polarization, to the left and posterior, the direction of ground (posterior) polarization to the right.</p

Effects on P7.p polarity are <i>chw-1</i>-specific.

<p>^a compared to <i>lin-17(n671)</i></p><p>^b compared to <i>lin-17(n671); gfp(RNAi)</i></p><p>^c compared to <i>lin-18(e620)</i></p><p>^d compared to <i>lin-18(e620); gfp(RNAi)</i></p><p>Animals were grown at 23°C and scored by DIC at late L4 stage. <i>n</i> is number of animals scored. Data for strains marked with an asterisk (*) are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133226#pone.0133226.t001" target="_blank">Table 1</a> (<i>lin-17</i>) or <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133226#pone.0133226.t002" target="_blank">Table 2</a> (<i>lin-18</i>). Analyses of statistical significance were performed using Fisher’s exact test.</p

Loss of CHW-1 enhances P7.p polarity defects caused by loss of LIN-18/Ryk.

<p>^a compared to N2</p><p>^b compared to <i>lin-18(e620)</i></p><p>^c compared to <i>lin-18(n1051)</i></p><p>^d compared to <i>lin-18(e620); gfp(RNAi)</i></p><p>Animals were grown at 23°C and scored by DIC at late L4 stage. <i>n</i> is number of animals scored. Analyses of statistical significance were performed using Fisher’s exact test.</p

Loss of CHW-1 does not disrupt ground polarity.

<p>^a compared to <i>lin-17(n671)</i></p><p>^b compared to <i>lin-18(e620)</i></p><p>^c compared to <i>lin-17(n671); egl-20(n585)</i></p><p>^d compared to <i>egl-20(n585); lin-18(e620)</i></p><p>Animals were maintained at 23°C and scored by DIC at late L4 stage. <i>n</i> is number of animals scored. Analyses of statistical significance were performed using Fisher’s exact test.</p