Unbiased Functional Proteomics Strategy for Protein Kinase Inhibitor Validation and Identification of <i>bona fide</i> Protein Kinase Substrates: Application to Identification of EEF1D as a Substrate for CK2

Protein kinases have emerged as attractive targets for treatment of several diseases prompting large-scale phosphoproteomics studies to elucidate their cellular actions and the design of novel inhibitory compounds. Current limitations include extensive reliance on consensus predictions to derive kinase–substrate relationships from phosphoproteomics data and incomplete experimental validation of inhibitors. To overcome these limitations in the case of protein kinase CK2, we employed functional proteomics and chemical genetics to enable identification of physiological CK2 substrates and validation of CK2 inhibitors including TBB and derivatives. By 2D electrophoresis and mass spectrometry, we identified the translational elongation factor EEF1D as a protein exhibiting CK2 inhibitor-dependent decreases in phosphorylation in ³²P-labeled HeLa cells. Direct phosphorylation of EEF1D by CK2 was shown by performing CK2 assays with EEF1D-FLAG from HeLa cells. Dramatic increases in EEF1D phosphorylation following λ–phosphatase treatment and phospho-EEF1D antibody recognizing EEF1D pS162 indicated phosphorylation at the CK2 site in cells. Furthermore, phosphorylation of EEF1D in the presence of TBB or TBBz is restored using CK2 inhibitor-resistant mutants. Collectively, our results demonstrate that EEF1D is a <i>bona fide</i> physiological CK2 substrate for CK2 phosphorylation. Furthermore, this validation strategy could be adaptable to other protein kinases and readily combined with other phosphoproteomic methods

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

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

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

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

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