Optionally, 2 mM free PI 51 was added to the lysate

Optionally, 2 mM free PI 51 was added to the lysate. 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 around 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. Protein kinases are key regulators of cellular signaling and therefore represent attractive targets for therapeutic intervention in a variety of human diseases (1). 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 (2, 3). 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 usually 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 (4, 5). 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 (6). 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 (7, 8). 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 (5). However, because of the power of affinity chromatography Rabbit Polyclonal to SLC5A6 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 (9, 10). 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 (4). In addition to p38, SB 203580 potently inhibits hepatic cytochrome P450 enzymes and was further shown to affect cyclooxygenase and thromboxane synthase activities association experiments. Twenty-five microliters of drained PI 51 matrix or control matrix was incubated with 250 l of high-salt lysate Dynorphin A (1-13) Acetate for 3 h at 4C. Optionally, 2 mM free PI 51 was added to the lysate. After washing with 500 l of 2 lysis buffer without additives made up of 1 M NaCl (high salt) and with 500 l of 1 1 lysis buffer without additives made up of 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 l of low salt lysis buffer supplemented with 1 mM PI 51 or 10 mM ATP/20 mM MgCl2 as indicated. For precipitation of strep-tagged proteins, 250 l of lysate made up of 150 mM NaCl was incubated with StrepTactin-MacroPrep beads (IBA, G?ttingen, Germany) for 3 h at 4C. Beads were then washed three Dynorphin A (1-13) Acetate times with the same buffer without additives. After SDS/PAGE, proteins were transferred to nitrocellulose membrane and immunoblotted with the indicated Dynorphin A (1-13) Acetate antibodies. Radioactively labeled RICK-KRdC was visualized by autoradiography before detection with StrepTactin-horseradish peroxidase (IBA). Affinity Chromatography and Preparative Gel Electrophoresis. Frozen HeLa cells (2.5 109, 4C Biotech, Seneffe, Belgium) were lysed in 30 ml of buffer made up of 20 mM Hepes (pH 7.5), 150 mM.