Klaus Godl, Josef Wissing, Alexander Kurtenbach, +7, Peter Habenberger, Stephanie Blencke, Heidrun Gutbrod, Kostadinos Salassidis, Matthias Stein-Gerlach, Andrea Missio, Matt Cotten, and Henrik Daub
December 10, 2003
100 (26) 15434-15439
Small molecule inhibitors of protein kinases are widely used in signal transduction research and are emerging as a major class of drugs. Although interpretation of biological results obtained with these reagents critically depends on their selectivity, efficient methods for proteome-wide assessment of kinase inhibitor selectivity have not yet been reported. Here, we address this important issue and describe a method for identifying targets of the widely used p38 kinase inhibitor SB 203580. Immobilization of a suitable SB 203580 analogue and thoroughly optimized biochemical conditions for affinity chromatography permitted the dramatic enrichment and identification of several previously unknown protein kinase targets of SB 203580. In vitro kinase assays showed that cyclin G-associated kinase (GAK) and CK1 were almost as potently inhibited as p38α whereas RICK [Rip-like interacting caspase-like apoptosis-regulatory protein (CLARP) kinase/Rip2/CARDIAK] was even more sensitive to inhibition by SB 203580. The cellular kinase activity of RICK, a known signal transducer of inflammatory responses, was already inhibited by submicromolar concentrations of SB 203580 in intact cells. Therefore, our results warrant a reevaluation of the vast amount of data obtained with SB 203580 and might have significant implications on the development of p38 inhibitors as antiinflammatory drugs. Based on the procedures described here, efficient affinity purification techniques can be developed for other protein kinase inhibitors, providing crucial information about their cellular modes of action.
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Protein kinases are key regulators of cellular signaling and therefore represent attractive targets for therapeutic intervention in a variety of human diseases. Various small molecule inhibitors for target-selective inhibition of diseaserelevant protein kinases are currently in different stages of clinical testing, and the first drugs of this class have already received FDA approval. Most of these inhibitors interact with the relatively conserved ATP binding site and are therefore likely to target several protein kinases, even when assumed to be highly specific based on currently available data. Because the knowledge about an inhibitor's true selectivity is a prerequisite for the correct interpretation of its biological effects, efficient methods to determine the cellular targets of protein kinase inhibitors are of central importance for both signal transduction research and drug development. Inhibitor selectivity is usually assessed in parallel enzymatic assays for a set of recombinant protein kinases. Because even the largest of these currently available selectivity panels comprise <100 members of the protein kinase family, the great majority of the >500 human protein kinases are not tested, and, moreover, alternative protein targets such as different types of enzymes are not analyzed. Thus, efficient methods for proteome-wide assessment of kinase inhibitor selectivity are needed. Classical affinity chromatography employing immobilized protein kinase inhibitors has been occasionally used to address this important issue. In terms of sensitivity and efficiency, limitations of these previously described affinity purifications become apparent when the results are seen in comparison with published data from selectivity panels comprising only ≈30 human protein kinases. However, because of the power of affinity chromatography combined with new advances in protein identification, a substantial optimization of the affinity approach is urgently required to gain new insights into the cellular modes of action of kinase inhibitors.
Antiinflammatory drugs, such as SB 203580, that belong to the pyridinyl imidazole class of compounds were originally recognized as protein kinase inhibitors when the mitogen-activated protein kinase p38 was identified as their major cellular target. SB 203580 is deemed to be relatively specific for p38 with respect to protein kinase inhibition because it did not significantly inhibit a variety of other kinases in vitro. In addition to p38, SB 203580 potently inhibits hepatic cytochrome P450 enzymes and was further shown to affect cyclooxygenase and thromboxane synthase activities in vitro, although at higher concentrations than p38 kinase. But these described side effects did not impair the use of this pharmacological inhibitor in studies resulting in >1,000 published articles to characterize and implicate p38 function in cellular signaling and a plethora of biological processes. Despite the widespread application of SB 203580, its true selectivity for p38 has not yet been assessed on a proteome-wide scale.
Here, we describe the immobilization of a suitable analogue of SB 203580 on chromatography beads. With this reagent, we establish an efficient proteomics method for the identification of cellular targets affected by a protein kinase inhibitor. To achieve this goal, various biochemical parameters had to be thoroughly optimized. Importantly, by using these conditions, we not only identify several previously unknown SB 203580 targets of high significance but also establish a robust procedure of general utility for the analysis of kinase inhibitor selectivity.
Cell culture media and Lipofectamine were purchased from Invitrogen. Radiochemicals and epoxy-activated Sepharose 6B were from Amersham Biosciences. SB 203580 and histone H1 were from Merck. GST-activating transcription factor 2 (ATF2) was obtained from Upstate Biotechnology (Lake Placid, NY). All other reagents were from Sigma.
Antibodies used were rabbit polyclonal anti-p38 antibody (Cell Signaling Technology, Beverly, MA), polyclonal anti-Rip-like interacting caspase-like apoptosis-regulatory protein (CLARP) kinase (RICK) antibody (ABR), mouse monoclonal anti-glycogen synthase kinase 3α/β (GSK3α/β) antibody, goat polyclonal anti-CK1α and anti-CK1ε antibodies, rabbit polyclonal anti-c-jun N-terminal kinase (JNK) antibody (all from Santa Cruz Biotechnology), and mouse monoclonal anti-vesicular stomatitis virus G protein (VSV-G) antibody (Roche). Recombinant protein kinases purchased were human p38α, human JNK1α1, human JNK2α2 (Upstate Biotechnology), and rat CK1δ (New England Biolabs).
A partial cDNA encoding amino acids 24–646 of cyclin G-associated kinase (GAK) was PCR-amplified from human lung cDNA and inserted into vector pcDNA3 (Invitrogen) modified to attach a C-terminal VSV-G epitope. The GAK sequence encoding amino acids 26 to 392 was cloned into pGEX-4T1 for expression of recombinant GST fusion protein in Escherichia coli.
The full-length RICK coding sequence fused to a C-terminal hemagglutinin epitope tag was cloned into pPM7 expression vector. Kinase-inactive K47R and inhibitor-insensitive T95M mutants were generated by using a mutagenesis kit (Stratagene). Plasmids pPM7-RICK-dCst and pPM7-RICK-KRdCst express the first 353-aa residues of wild-type or kinase-inactive RICK fused to a C-terminal streptag epitope. The expression cassette from pPM7-RICK-dCst was inserted into an adenovirus genome by recombination in bacteria. Recombinant RICK enzyme production using adenovirus-directed expression is described elsewhere.
Pyridinyl imidazole (PI) 51 and PI 51peg were prepared by and purchased from Evotec-OAI (Abingdon, Oxon, U.K.). PI 51 was synthesized as described. PI 51peg was prepared by dissolving 0.59 mM PI 51, 0.59 mM diisopropylamine, and 0.59 mM 1-bromo-2-[2-(2-ethoxy-ethoxy)-ethoxy]-ethane in 10 ml of 1,4-dioxane, followed by refluxing the mixture for 16 h until complete consumption of PI 51. The mixture was cooled to room temperature, and the solvent was removed under vacuum. The residue was purified by column chromatography [silica gel; gradient dichlorometane/methanol (95:5) to dichlorometane/methanol/25% aq. ammonia (90:10:1)].
For immobilization, drained epoxy-activated Sepharose 6B was resuspended in 2 vol of 20 mM PI 51 dissolved in 50% dimethylformamide (DMF)/0.1 M Na 2 CO 3. After adding of 10 mM NaOH, coupling was performed overnight at 30°C in the dark. After three washes with 50% DMF/0.1 M Na 2 CO 3, remaining reactive groups were blocked with 1 M ethanolamine. Subsequent washing steps were performed according to the manufacturer's instructions. To generate the control matrix, epoxy-activated Sepharose 6B was incubated with 1 M ethanol-amine and equally treated as described above. The beads were stored at 4°C in the dark.
COS-7 and HeLa cells were cultured in DMEM supplemented with 10% FBS. COS-7 cells were transiently transfected as described. On the second day after transfection, cells were either lysed or phosphate-starved for a further 2 h in phosphate-free medium containing 10% dialysed FBS. Cells were then treated with inhibitor for 15 min and subsequently metabolically labeled with 70 μCi (1 Ci = 37 GBq) [32 P]orthophosphate per ml for 30 min before cell lysis.
HeLa cells or transfected COS-7 cells were lysed in buffer containing 50 mM Hepes (pH 7.5), 150 mM NaCl, 0.5% Triton X-100, 10% glycerol, 1 mM EDTA, 10 mM sodium pyrophosphate plus additives (10 mM sodium fluoride/1 mM orthovanadate/10 μg/ml aprotinin/10 μg/ml leupeptin/1 mM phenylmethylsulfonyl fluoride/0.2 mM DTT). Lysates were precleared by centrifugation and equilibrated to 1 M NaCl for in vitro association experiments. Twenty-five microliters of drained PI 51 matrix or control matrix was incubated with 250 μl of high-salt lysate for 3 h at 4°C. Optionally, 2 mM free PI 51 was added to the lysate. After washing with 500 μl of 2× lysis buffer without additives containing 1 M NaCl (high salt) and with 500 μl of 1× lysis buffer without additives containing 150 mM NaCl (low salt), the beads were eluted with 1.5× SDS sample buffer. To test different elution conditions for bound p38, beads were incubated in 100 μl of low salt lysis buffer supplemented with 1 mM PI 51 or 10 mM ATP/20 mM MgCl 2 as indicated. For precipitation of strep-tagged proteins, 250 μl of lysate containing 150 mM NaCl was incubated with StrepTactin-MacroPrep beads (IBA, Göttingen, Germany) for 3 h at 4°C. Beads were then washed three times with the same buffer without additives. After SDS/PAGE, proteins were transferred to nitrocellulose membrane and immunoblotted with the indicated antibodies. Radioactively labeled RICK-KRdC was visualized by autoradiography before detection with StrepTactin-horseradish peroxidase (IBA).
Frozen HeLa cells (2.5 × 10 9, 4C Biotech, Seneffe, Belgium) were lysed in 30 ml of buffer containing 20 mM Hepes (pH 7.5), 150 mM NaCl, 0.25% Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM DTT plus additives (10 mM sodium fluoride/1 mM orthovanadate/10 μg/ml aprotinin/10 μg/ml leupeptin/1 mM phenylmethylsulfonylfluoride/10% glycerol), cleared by centrifugation, and adjusted to 1 M NaCl. The filtrated lysate was loaded with a flow rate of 100 μl/min on an HR 5/2 chromatography column (Amersham Biosciences) containing 600 μl of PI 51 matrix equilibrated to lysis buffer without
