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

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

Application of Multiplexed Kinase Inhibitor Beads to Study Kinome Adaptations in Drug-Resistant Leukemia

<div><p>Protein kinases play key roles in oncogenic signaling and are a major focus in the development of targeted cancer therapies. Imatinib, a BCR-Abl tyrosine kinase inhibitor, is a successful front-line treatment for chronic myelogenous leukemia (CML). However, resistance to imatinib may be acquired by BCR-Abl mutations or hyperactivation of Src family kinases such as Lyn. We have used <u>m</u>ultiplexed kinase <u>i</u>nhibitor <u>b</u>eads (MIBs) and quantitative mass spectrometry (MS) to compare kinase expression and activity in an imatinib-resistant (MYL-R) and -sensitive (MYL) cell model of CML. Using MIB/MS, expression and activity changes of over 150 kinases were quantitatively measured from various protein kinase families. Statistical analysis of experimental replicates assigned significance to 35 of these kinases, referred to as the MYL-R kinome profile. MIB/MS and immunoblotting confirmed the over-expression and activation of Lyn in MYL-R cells and identified additional kinases with increased (MEK, ERK, IKKα, PKCβ, NEK9) or decreased (Abl, Kit, JNK, ATM, Yes) abundance or activity. Inhibiting Lyn with dasatinib or by shRNA-mediated knockdown reduced the phosphorylation of MEK and IKKα. Because MYL-R cells showed elevated NF-κB signaling relative to MYL cells, as demonstrated by increased IκBα and IL-6 mRNA expression, we tested the effects of an IKK inhibitor (BAY 65-1942). MIB/MS and immunoblotting revealed that BAY 65-1942 increased MEK/ERK signaling and that this increase was prevented by co-treatment with a MEK inhibitor (AZD6244). Furthermore, the combined inhibition of MEK and IKKα resulted in reduced IL-6 mRNA expression, synergistic loss of cell viability and increased apoptosis. Thus, MIB/MS analysis identified MEK and IKKα as important downstream targets of Lyn, suggesting that co-targeting these kinases may provide a unique strategy to inhibit Lyn-dependent imatinib-resistant CML. These results demonstrate the utility of MIB/MS as a tool to identify dysregulated kinases and to interrogate kinome dynamics as cells respond to targeted kinase inhibition.</p></div

Validation of the MYL-R kinome profiles by immunoblotting.

<p>(<b>A and B</b>) MYL and MYL-R cell lysates were analyzed by immunoblot with the antibodies indicated. Representative data are shown. (<b>A</b>) ATM, BCR-Abl and c-Kit were decreased in MYL-R relative to MYL cells as predicted from the kinome profile above. (<b>B, left panel</b>) Both total and activated Lyn and PKCβ were increased in MYL-R cells as shown using antibodies to detect phosphorylation of the activation loop of Lyn (p-Y416) or the autophosphorylation site of PKCβ. (<b>B, right panel</b>) Total amounts of IKKα, MEK2 and ERK1/2 were similar in MYL and MYL-R cells, however, phospho-specific antibodies demonstrated these kinases were more active in MYL-R cells. (<b>C</b>) Kinases from MYL and MYL-R cell lysates were captured by MIBs pull-down and analyzed by immunoblot using antibodies directed against total protein. The relative amounts of kinases captured from each cell lysate correlated with the abundance ratios predicted by the MYL-R kinome profile. MIBs exposed to MYL-R cell lysate (<b>C, left panel</b>) captured less ASK1, Jak1, MLTK, Yes, GSK3α, CDK2 and dCK; and (<b>C, right panel</b>) captured more NEK9, IKKα, RIPK2, Lyn and ERK. See also, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066755#pone.0066755.s002" target="_blank">Figures S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066755#pone.0066755.s003" target="_blank">S3</a>.</p

Kinome response to targeted MEK and IKK inhibitor treatment.

<p>(<b>A</b>) MYL-R cells were treated for 24 hours with AZD6244 (AZD, 5 µM), BAY 65-1942 (BAY, 10 µM) or AZD (5 µM) plus BAY (10 µM), and kinases were isolated and quantified by MIB/MS in two independent experiments. The relative abundances (Drug/DMSO) of kinases in the MEK/ERK pathway are shown. <i>Error bars</i>, SE (N = 2). (<b>B</b>) MYL-R cells were treated for 24 hours with AZD (5 µM), BAY(10 µM) or AZD (5 µM) plus BAY (10 µM), and were analyzed by immunoblot using the antibodies indicated. Data are representative of three separate experiments. (<b>C</b>) MYL-R cells were treated for 12 hours with AZD (5 µM), BAY (10 µM), AZD (5 µM) plus BAY (10 µM), or dasatinib (1 nM). Total RNA was isolated and the expression of IκBα and IL-6 were evaluated by qRT-PCR. <i>Error bars</i>, SE (N = 2).</p

Lyn drives activation of MEK and increases activation of IKKα in MYL-R cells.

<p>(<b>A</b>) MYL-R cells were treated for 1 hour with dasatinib, as indicated, and lysates were analyzed by immunoblot with the antibodies indicated. Lyn phosphorylation was detected by p-SFK (Y416). For densitometry, the band densities of p-SFK, p-MEK and p-IKKα were normalized against the β-actin loading control and were plotted relative to the DMSO treatment. <i>Error bars</i>, SE (N = 3). (<b>B</b>) MYL-R cells were transduced with non-targeting shRNA (shCtrl) or shRNA directed against Lyn (shLyn) and lysates were analyzed by immunoblot with the antibodies indicated. Lyn phosphorylation was detected by p-SFK (Y416) antibody. For densitometry, the band densities of p-IKKα and p-MEK were plotted relative to total IKKα and MEK protein expression. <i>Error bars</i>, SE (N = 2).</p

Targeted inhibition of kinases detected by MIB/MS leads to induction of apoptosis.

<p>(<b>A</b>) MYL-R cells were treated for 48 hours with AZD6244 (AZD, 5 µM), BAY 65-1942 (BAY, 10 µM), AZD (5 µM) plus BAY (10 µM), or dasatinib (1 nM) and cell viability was assessed by MTS assay. <i>Error bars</i>, SE (N = 3). (<b>B</b>) MYL-R cells were treated for 24 hours with AZD (5 µM), BAY (10 µM), AZD (5 µM) plus BAY (10 µM), or dasatinib (1 nM) and caspase 3/7 activity was assessed by fluorometric assay. <i>Error bars</i>, SE (N = 2). (<b>C</b>) MYL-R cells were treated for 48 hours with AZD (5 µM), BAY (10 µM), AZD (5 µM) plus BAY (10 µM), or dasatinib (1 nM) and cell lysates were analyzed by immunoblot using the antibodies indicated. Data are representative of two separate experiments.</p

Application of MIB/MS to analyze the kinomes of drug-sensitive and -resistant leukemia cells.

<p>Kinases from MYL and MYL-R cells were affinity enriched with multiplexed inhibitor beads and quantified by LC-MALDI TOF/TOF mass spectrometry (MIB/MS). Results from three independent experiments were pooled as described in Materials and Methods. (<b>A</b>) Over 150 kinases were quantified from MYL-R relative to MYL cells across a broad spectrum of kinase families, as depicted in the phylogenetic tree of the human protein kinase superfamily. Yellow and blue dots signify kinases that were increased or decreased in abundance, respectively, in MYL-R relative to MYL cells while gray dots signify kinases that were unchanged. Kinome illustration reproduced courtesy of Cell Signaling Technology, Inc. (<a href="http://www.cellsignal.com" target="_blank">www.cellsignal.com</a>). (<b>B</b>) The trend of kinase abundance changes for all kinases quantified. <i>Dashed line</i>, ±1.5-fold change. (<b>C</b>) The kinome profile of MYL-R relative to MYL was derived from the kinases that were significantly changed after statistical analysis. The kinase abundance ratios and p-values from three independent experiments were combined and adjusted for multiple hypothesis testing and ratios with a Benjamini-Hochberg q-value <0.2 were considered significant. <i>Dashed lines</i>, ±1.5-fold change; <i>error bars</i>, SE (N = 3).</p

MYL-R cells exhibit increased NF-κB signaling.

<p>(<b>A</b>) Total RNA was isolated from MYL and MYL-R cells and the expression of NF-κB target genes was evaluated by qRT-PCR. <i>Error bars</i>, SE (N = 3). (<b>B</b>) MYL and MYL-R cells were treated for two hours with DMSO or the IKK inhibitor BAY 65-1942 (BAY, 10 µM) and then analyzed by immunoblot using the antibodies indicated. The entire blot is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066755#pone.0066755.s005" target="_blank">Figure S5</a>. <i>Error bars</i>, SE (N = 3). (<b>C</b>) MYL-R cells were treated for 12 hours with DMSO or BAY (10 µM) and total RNA was isolated and evaluated by qRT-PCR. <i>Error bars</i>, SE (N = 3).</p