Proteomics analysis of cellular imatinib targets and their candidate downstream effectors.
Inhibition of deregulated protein kinases by small molecule drugs has evolved into a major therapeutic strategy for the treatment of human malignancies. Knowledge about direct cellular targets of kinase-selective drugs and the identification of druggable downstream mediators of oncogenic signaling are relevant for both initial therapy selection and the nomination of alternative targets in case molecular resistance emerges. To address these issues, we performed a proof-of-concept proteomics study designed to monitor drug effects on the pharmacologically tractable subproteome isolated by affinity purification with immobilized, nonselective kinase inhibitors. We applied this strategy to chronic myeloid leukemia cells that express the transforming Bcr-Abl fusion kinase. We used SILAC to measure how cellular treatment with the Bcr-Abl inhibitor imatinib affects protein binding to a generic kinase inhibitor resin and further quantified site-specific phosphorylations on resin-retained proteins. Our integrated approach indicated additional imatinib target candidates, such as flavine adenine dinucleotide synthetase, as well as repressed phosphorylation events on downstream effectors not yet implicated in imatinib-regulated signaling. These included activity-regulating phosphorylations on the kinases Btk, Fer, and focal adhesion kinase, which may qualify them as alternative target candidates in Bcr-Abl-driven oncogenesis. Our approach is rather generic and may have various applications in kinase drug discovery.
Breitkopf
Susanne B
SB
Department of Molecular Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.
Oppermann
Felix S
FS
Keri
Gyorgy
G
Grammel
Markus
M
Daub
Henrik
H
D001549:Benzamides; D016503:Drug Delivery Systems / Q000379:methods; D055808:Drug Discovery / Q000379:methods*; D016903:Drug Monitoring / Q000379:methods; D006801:Humans; D000068877:Imatinib Mesylate; D009713:Nucleotidyltransferases; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D011743:Pyrimidines / Q000494:pharmacology*; D015398:Signal Transduction
D001549:Benzamides; D010879:Piperazines; D011743:Pyrimidines; D000068877:Imatinib Mesylate; D011494:Protein Kinases; D009713:Nucleotidyltransferases; C020143:FMN adenylyltransferase