Comparing Immobilized Kinase Inhibitors and Covalent ATP Probes for Proteomic Profiling of Kinase Expression and Drug Selectivity

Kinases are involved in the regulation of many cellular processes and aberrant kinase signaling has been implicated in human disease. As a consequence, kinases are attractive drug targets. Assessing kinase function and drug selectivity in a more physiological context is challenging and often hampered by the generally low expression level of kinases and the extensive post-translation modification <i>in vivo</i>. Kinase drug selectivity screens by chemical proteomics have gained attention because they allow the profiling of hundreds of kinases against one drug at the same time. Here, we directly compared two such methods, notably, immobilized broad spectrum kinase inhibitors (kinobeads) and active site labeling using desthiobiotin-ATP and -ADP probes. Affinity purification of ∼100 kinases by either kinobeads or ATP/ADP probes was readily achieved using 1 mg of cellular protein. Bioinformatic analysis revealed a high degree of complementarity of the two techniques. Kinobeads covered the Tyrosine Kinase family particularly well and ATP probes enriched higher numbers of STE family kinases. A consecutive combination of both enrichment strategies therefore allowed for the coverage of a larger part of the kinome than any one technique alone. While kinobeads are very selective for kinases, the ATP/ADP probes also enriched a large number of other nucleotide binding proteins. Both methods were applied to the selectivity profiling of the small molecular Aurora kinase inhibitor tozasertib in K562 cells. Our data confirmed Aurora A, B, and BCR-ABL as the main targets of tozasertib and identified TNK1, STK2, RPS6KA1, and RPS6KA3 as submicromolar off targets

Comparing Immobilized Kinase Inhibitors and Covalent ATP Probes for Proteomic Profiling of Kinase Expression and Drug Selectivity

Kinases are involved in the regulation of many cellular processes and aberrant kinase signaling has been implicated in human disease. As a consequence, kinases are attractive drug targets. Assessing kinase function and drug selectivity in a more physiological context is challenging and often hampered by the generally low expression level of kinases and the extensive post-translation modification <i>in vivo</i>. Kinase drug selectivity screens by chemical proteomics have gained attention because they allow the profiling of hundreds of kinases against one drug at the same time. Here, we directly compared two such methods, notably, immobilized broad spectrum kinase inhibitors (kinobeads) and active site labeling using desthiobiotin-ATP and -ADP probes. Affinity purification of ∼100 kinases by either kinobeads or ATP/ADP probes was readily achieved using 1 mg of cellular protein. Bioinformatic analysis revealed a high degree of complementarity of the two techniques. Kinobeads covered the Tyrosine Kinase family particularly well and ATP probes enriched higher numbers of STE family kinases. A consecutive combination of both enrichment strategies therefore allowed for the coverage of a larger part of the kinome than any one technique alone. While kinobeads are very selective for kinases, the ATP/ADP probes also enriched a large number of other nucleotide binding proteins. Both methods were applied to the selectivity profiling of the small molecular Aurora kinase inhibitor tozasertib in K562 cells. Our data confirmed Aurora A, B, and BCR-ABL as the main targets of tozasertib and identified TNK1, STK2, RPS6KA1, and RPS6KA3 as submicromolar off targets

The β₁-adrenoceptor is phosphorylated at multiple sites in the third intracellular loop and at the C-terminus.

<p>(<b>A</b>) Phosphorylation of the ADRB1 with and without stimulation with NE (100 μM) for 5 min. Cells stably expressing the human ADRB1 were cultured in medium containing 1% FCS and ³²P. After cell lysis and IP of the ADRB1, the amount of phosphorylated ADRB1 (pADRB1) was quantified by phosphor imager analysis. Total ADRB1 was subsequently visualized by western blotting. (<b>B</b>) Quantification of ADRB1 phosphorylation from (A), n = 3. (<b>C</b>) Preparation of samples for mass spectrometry. (<b>D</b>) Western blot after crosslink IP of the ADRB1. HEK: untransfected HEK293 cells (negative control); ADRB1: HEK293 cells stably expressing the β₁-adrenoceptor. (<b>E</b>) Silver staining of SDS gel showing the ADRB1 after IP. (<b>F</b>) Phosphorylation pattern of the ADRB1. Amino acids marked in light grey and black indicate phosphorylation under basal conditions and after stimulation with 100 μM NE for 5 min, respectively. Pool of five experiments. Annotated spectra of the detected phosphosites are presented in detail in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176450#pone.0176450.s001" target="_blank">S1 Fig</a>.</p

Two serines in the distal C-terminus of the human ß₁-adrenoceptor determine ß-arrestin2 recruitment

<div><p>G protein-coupled receptors (GPCRs) undergo phosphorylation at several intracellular residues by G protein-coupled receptor kinases. The resulting phosphorylation pattern triggers arrestin recruitment and receptor desensitization. The exact sites of phosphorylation and their function remained largely unknown for the human β₁-adrenoceptor (ADRB1), a key GPCR in adrenergic signal transduction and the target of widely used drugs such as β-blockers. The present study aimed to identify the intracellular phosphorylation sites in the ADRB1 and to delineate their function. The human ADRB1 was expressed in HEK293 cells and its phosphorylation pattern was determined by mass spectrometric analysis before and after stimulation with a receptor agonist. We identified a total of eight phosphorylation sites in the receptor’s third intracellular loop and C-terminus. Analyzing the functional relevance of individual sites using phosphosite-deficient receptor mutants we found phosphorylation of the ADRB1 at Ser461/Ser462 in the distal part of the C-terminus to determine β-arrestin2 recruitment and receptor internalization. Our data reveal the phosphorylation pattern of the human ADRB1 and the site that mediates recruitment of β-arrestin2.</p></div

Comparing Immobilized Kinase Inhibitors and Covalent ATP Probes for Proteomic Profiling of Kinase Expression and Drug Selectivity

Kinases are involved in the regulation of many cellular processes and aberrant kinase signaling has been implicated in human disease. As a consequence, kinases are attractive drug targets. Assessing kinase function and drug selectivity in a more physiological context is challenging and often hampered by the generally low expression level of kinases and the extensive post-translation modification <i>in vivo</i>. Kinase drug selectivity screens by chemical proteomics have gained attention because they allow the profiling of hundreds of kinases against one drug at the same time. Here, we directly compared two such methods, notably, immobilized broad spectrum kinase inhibitors (kinobeads) and active site labeling using desthiobiotin-ATP and -ADP probes. Affinity purification of ∼100 kinases by either kinobeads or ATP/ADP probes was readily achieved using 1 mg of cellular protein. Bioinformatic analysis revealed a high degree of complementarity of the two techniques. Kinobeads covered the Tyrosine Kinase family particularly well and ATP probes enriched higher numbers of STE family kinases. A consecutive combination of both enrichment strategies therefore allowed for the coverage of a larger part of the kinome than any one technique alone. While kinobeads are very selective for kinases, the ATP/ADP probes also enriched a large number of other nucleotide binding proteins. Both methods were applied to the selectivity profiling of the small molecular Aurora kinase inhibitor tozasertib in K562 cells. Our data confirmed Aurora A, B, and BCR-ABL as the main targets of tozasertib and identified TNK1, STK2, RPS6KA1, and RPS6KA3 as submicromolar off targets

Comparing Immobilized Kinase Inhibitors and Covalent ATP Probes for Proteomic Profiling of Kinase Expression and Drug Selectivity

Kinases are involved in the regulation of many cellular processes and aberrant kinase signaling has been implicated in human disease. As a consequence, kinases are attractive drug targets. Assessing kinase function and drug selectivity in a more physiological context is challenging and often hampered by the generally low expression level of kinases and the extensive post-translation modification <i>in vivo</i>. Kinase drug selectivity screens by chemical proteomics have gained attention because they allow the profiling of hundreds of kinases against one drug at the same time. Here, we directly compared two such methods, notably, immobilized broad spectrum kinase inhibitors (kinobeads) and active site labeling using desthiobiotin-ATP and -ADP probes. Affinity purification of ∼100 kinases by either kinobeads or ATP/ADP probes was readily achieved using 1 mg of cellular protein. Bioinformatic analysis revealed a high degree of complementarity of the two techniques. Kinobeads covered the Tyrosine Kinase family particularly well and ATP probes enriched higher numbers of STE family kinases. A consecutive combination of both enrichment strategies therefore allowed for the coverage of a larger part of the kinome than any one technique alone. While kinobeads are very selective for kinases, the ATP/ADP probes also enriched a large number of other nucleotide binding proteins. Both methods were applied to the selectivity profiling of the small molecular Aurora kinase inhibitor tozasertib in K562 cells. Our data confirmed Aurora A, B, and BCR-ABL as the main targets of tozasertib and identified TNK1, STK2, RPS6KA1, and RPS6KA3 as submicromolar off targets

Phosphorylation at the distal C-terminus of the ADRB1 determines receptor internalization, but does not alter stimulation-dependent MAP kinase activation.

<p>(<b>A</b>) Representative Western blots using antibodies directed against activated MAPK1/3 (pMAPK1/3), MAPK1/3 and ADRB1. HSP90 was used as a loading control. Lysates were prepared from HEK293 cells stably expressing the indicated ADRB1 mutants and stimulated with NE (100 μM for 5 min) or control. (<b>B</b>) Quantification of MAPK1/3 activity from (A). Data are means+SEM, n = 3–9. (<b>C</b>) Internalization of ADRB1 mutants expressed in HEK293 cells (together with ARRB2) as determined by loss of cell surface receptor labeling with ³H-CGP-12177. Time points indicate duration of treatment with NE prior to labeling with ³H-CGP-12177. Data are means+SEM of n = 5 independent experiments. ** p ≤ 0.01, *** p ≤ 0.001 as determined by two-way ANOVA.</p

Comparing Immobilized Kinase Inhibitors and Covalent ATP Probes for Proteomic Profiling of Kinase Expression and Drug Selectivity

Kinases are involved in the regulation of many cellular processes and aberrant kinase signaling has been implicated in human disease. As a consequence, kinases are attractive drug targets. Assessing kinase function and drug selectivity in a more physiological context is challenging and often hampered by the generally low expression level of kinases and the extensive post-translation modification <i>in vivo</i>. Kinase drug selectivity screens by chemical proteomics have gained attention because they allow the profiling of hundreds of kinases against one drug at the same time. Here, we directly compared two such methods, notably, immobilized broad spectrum kinase inhibitors (kinobeads) and active site labeling using desthiobiotin-ATP and -ADP probes. Affinity purification of ∼100 kinases by either kinobeads or ATP/ADP probes was readily achieved using 1 mg of cellular protein. Bioinformatic analysis revealed a high degree of complementarity of the two techniques. Kinobeads covered the Tyrosine Kinase family particularly well and ATP probes enriched higher numbers of STE family kinases. A consecutive combination of both enrichment strategies therefore allowed for the coverage of a larger part of the kinome than any one technique alone. While kinobeads are very selective for kinases, the ATP/ADP probes also enriched a large number of other nucleotide binding proteins. Both methods were applied to the selectivity profiling of the small molecular Aurora kinase inhibitor tozasertib in K562 cells. Our data confirmed Aurora A, B, and BCR-ABL as the main targets of tozasertib and identified TNK1, STK2, RPS6KA1, and RPS6KA3 as submicromolar off targets

Phosphorylation at Ser461/Ser462 in the distal C-terminus determines arrestin binding.

<p>(<b>A</b>) Schematic of the human ADRB1 highlighting the phosphosites mutated in the distal C-terminus: ADRB1(Ala459/461/462) (light blue), ADRB1(Ala461/462) (orange) and ADRB1(Ala473/475) (dark grey). (<b>B</b>) Averaged FRET tracings comparing ADRB1(Ala459/461/462), ADRB1(Ala461/462) and ADRB1 (Ala473/475). Data are means±SEM of n ≥ 6 representative tracings. (<b>C</b>) Quantification of NE-induced changes of receptor-arrestin FRET obtained for the ADRB1 variants depicted in (B). Data are means+SEM of n ≥ 9 cells. *** p ≤ 0.001 vs. wild-type and n.s. = not significant. (<b>D</b>) Conservation of serine 459 (light blue), serine 461 and serine 462 (orange) among twelve species.</p

Phosphorylation sites in the C-terminus are crucial for β-arrestin2 recruitment.

<p>(<b>A</b>) Schematic of the FRET assay for β-arrestin2 recruitment (left) and confocal microscopy of representative cells expressing Cerulean-tagged ADRB1 and YFP-tagged β-arrestin2 (right, scale = 10 μm). (<b>B</b>) Representative tracings (left) and their ratiometric representation (right) of simultaneous recording of emissions from YFP (535 nm) and Cerulean (480 nm) upon stimulation of cells with norepinephrine (NE, 10 μM). (<b>C, D</b>) Schematics of the human ADRB1 highlighting the phosphosites mutated in the third intracellular loop and the C-terminus. (<b>E</b>) Averaged FRET tracings comparing wild-type ADRB1, ADRB1Δ3rdloop^a (= ADRB1(Ala260), light green) and a mutant devoid of all phosphosites in the third intracellular loop and C-terminus (ADRB1Δphos). (<b>F</b>) Quantification of NE-induced changes of receptor-arrestin FRET obtained for the ADRB1 variants depicted in (C). (<b>G, H</b>) Analogous experiments as in (E, F) for C-terminal mutants. Data are means±SEM from ≥10 tracings (E, G) and ≥ 30 cells (F, H). Kruskal-Wallis-Test with Dunn‘s post test. *** p ≤ 0.001 vs. wild-type and n.s. = not significant.</p