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

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

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

Identification of a Cryptic Bacterial Promoter in Mouse (<i>mdr1a</i>) P-Glycoprotein cDNA

<div><p>The efflux transporter P-glycoprotein (P-gp) is an important mediator of various pharmacokinetic parameters, being expressed at numerous physiological barriers and also in multidrug-resistant cancer cells. Molecular cloning of homologous cDNAs is an important tool for the characterization of functional differences in P-gp between species. However, plasmids containing mouse <i>mdr1a</i> cDNA display significant genetic instability during cloning in bacteria, indicating that <i>mdr1a</i> cDNA may be somehow toxic to bacteria, allowing only clones containing mutations that abrogate this toxicity to survive transformation. We demonstrate here the presence of a cryptic promoter in mouse <i>mdr1a</i> cDNA that causes mouse P-gp expression in bacteria. This expression may account for the observed toxicity of <i>mdr1a</i> DNA to bacteria. Sigma 70 binding site analysis and GFP reporter plasmids were used to identify sequences in the first 321 bps of <i>mdr1a</i> cDNA capable of initiating bacterial protein expression. An <i>mdr1a</i> M107L cDNA containing a single residue mutation at the proposed translational start site was shown to allow sub-cloning of <i>mdr1a</i> in <i>E</i>. <i>coli</i> while retaining transport properties similar to wild-type P-gp. This mutant <i>mdr1a</i> cDNA may prove useful for efficient cloning of <i>mdr1a</i> in <i>E</i>. <i>coli</i>.</p></div

Identification of putative transcriptional and translational start sites in <i>mdr1a</i> cDNA.

<p>An <i>in silico</i> sigma 70 binding site analysis was used to screen for a putative cryptic bacterial promoter in <i>mdr1a</i> cDNA. (A) Sequence of <i>mdr1a</i> cDNA shown with predicted -35 and extended -10 elements (red). The location in the cDNA for each element is noted above the sequence. (B) Predicted Shine-Dalgarno and ATG of a methionine that is in-frame with regards to the <i>mdr1a</i> ORF.</p

Mouse P-gp model in apo (open) conformation with methionine residue at position 107 highlighted.

<p>(A) Mouse P-gp is shown with the methionine to leucine mutation highlighted in TM1-ECL1 by the presence of a space filled leucine residue. The region of the drug-binding pocket (DBP) is indicated. (B) Enlarged view of M107L mutation. Figures were generated using PyMOL from PDB deposit 4M1M [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136396#pone.0136396.ref016" target="_blank">16</a>].</p

Mutations and their position in <i>mdr1a</i> cDNA after transformation into <i>E</i>. <i>coli</i>.

<p>The pCR-Blunt-II-TOPO vector containing <i>mdr1a</i> cDNA was used to transform <i>E</i>.<i>coli</i> and 20 colonies were selected for small-scale bacterial growth, plasmid purification, and analysis of plasmid DNA by agarose gel electrophoresis. Sixteen of the colonies selected for analysis contained large insertion or deletion mutations (not shown), and the remaining four colonies containing potentially correct <i>mdr1a</i> cDNA were sequenced. Each of the four sequenced plasmids contained mutated <i>mdr1a</i> cDNA, indicating a 100% mutational rate. Mutations observed were classified as point, insertion, or deletion mutations. Wild-type <i>mdr1a</i> cDNA (top) is compared to sequenced, “mutant” <i>mdr1a</i> cDNA (bottom). Insertion and point mutations are indicated in red in the mutant cDNA. Deleted base pairs are highlighted in red in wild-type cDNA, and the resulting mutated cDNA sequence is shown below. A) A point mutation (C→T) at location 1027 bp resulting in an introduced stop codon into <i>mdr1a</i> cDNA. B) A single adenine base pair insertion at location 1033 bp. C) Two examples of deletion mutations. All mutations resulted either directly (point mutations) or indirectly (insertion or deletion mutations) in the introduction of a stop codon into <i>mdr1a</i> cDNA. Locations of the introduced stop codons are indicated.</p

Schematic of GFP fusion constructs.

<p>GFP reporter constructs were generated to assess the ability of sequences of <i>mdr1a</i> cDNA to drive protein expression. Black lines represent DNA sequences fused upstream of GFP. GFP cDNA is represented in green and the presence of introduced RBSs are shown in blue where applicable. The p300-GFP plasmid contains the first 300 bps of <i>mdr1a</i> fused to GFP immediately preceded by a strong RBS. The p321-GFP plasmid contains the first 321 bps of <i>mdr1a</i> fused to GFP without the presence of an introduced RBS. pGFP and λpR-GFP were used as positive and negative control for GFP expression, respectively. p321-GFP and p-321-M107L-GFP are equivalent with the exception of the methionine to leucine mutation at position 107 in the amino acid sequence.</p

Fluorescence of <i>E</i>. <i>coli</i> transformed with GFP fusion plasmids.

<p>(A) Confocal images of <i>E</i>. <i>coli</i> transformed with GFP fusion plasmids. From left to right, columns represent fluorescence, bright field, and a merged image. Yellow bars indicate 20 μm. (B) Fluorescence of <i>E</i>. <i>coli</i> transformed with GFP fusion plasmids. Fluorescence spectroscopy was used to determine levels of GFP fluorescence in bacteria transformed with GFP plasmids. Confocal images and fluorescence spectroscopy values for bacteria transformed with λpR-GFP plasmids were omitted due to the signal saturation resulting from high GFP fluorescence. Data represent the mean and standard deviation of five individual colonies from one transformation event. Multiple groups were analyzed using one-way ANOVA where significance was defined as p < 0.05 in GraphPad Prism.</p