Global effects of kinase inhibitors on signaling networks revealed by quantitative phosphoproteomics.
Aberrant signaling causes many diseases, and manipulating signaling pathways with kinase inhibitors has emerged as a promising area of drug research. Most kinase inhibitors target the conserved ATP-binding pocket; therefore specificity is a major concern. Proteomics has previously been used to identify the direct targets of kinase inhibitors upon affinity purification from cellular extracts. Here we introduce a complementary approach to evaluate the effects of kinase inhibitors on the entire cell signaling network. We used triple labeling SILAC (stable isotope labeling by amino acids in cell culture) to compare cellular phosphorylation levels for control, epidermal growth factor stimulus, and growth factor combined with kinase inhibitors. Of thousands of phosphopeptides, less than 10% had a response pattern indicative of targets of U0126 and SB202190, two widely used MAPK inhibitors. Interestingly, 83% of the growth factor-induced phosphorylation events were affected by either or both inhibitors, showing quantitatively that early signaling processes are predominantly transmitted through the MAPK cascades. In contrast to MAPK inhibitors, dasatinib, a clinical drug directed against BCR-ABL, which is the cause of chronic myelogenous leukemia, affected nearly 1,000 phosphopeptides. In addition to the proximal effects on ABL and its immediate targets, dasatinib broadly affected the downstream MAPK pathways. Pathway mapping of regulated sites implicated a variety of cellular functions, such as chromosome remodeling, RNA splicing, and cytoskeletal organization, some of which have been described in the literature before. Our assay is streamlined and generic and could become a useful tool in kinase drug development.
Pan
Cuiping
C
Department of Proteomics and Signal Transduction, Max Planck Institute for Biochemistry, Am Klopferspitz 18, D-82152 Martinsried near Munich, Germany.
Olsen
Jesper V
JV
Daub
Henrik
H
Mann
Matthias
M
D002070:Butadienes / Q000494:pharmacology; D000069439:Dasatinib; D030562:Databases, Protein; D004815:Epidermal Growth Factor / Q000494:pharmacology; D016044:Fusion Proteins, bcr-abl / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000201:enzymology / Q000473:pathology; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors; D008954:Models, Biological; D009570:Nitriles / Q000494:pharmacology; D010750:Phosphoproteins / Q000032:analysis*; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D020543:Proteome / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D013844:Thiazoles / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors
D002070:Butadienes; D007093:Imidazoles; D009570:Nitriles; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D020543:Proteome; D011725:Pyridines; D011743:Pyrimidines; D013844:Thiazoles; C113580:U 0126; D004815:Epidermal Growth Factor; D016044:Fusion Proteins, bcr-abl; D048051:p38 Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; C090942:4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)imidazole; D000069439:Dasatinib