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Quantification of change in phosphorylation of BCR‐ABL kinase and its substrates in response to Imatinib treatment in human chronic myelogenous leukemia cells

Phosphorylation by the constitutively activated BCR‐ABL tyrosine kinase is associated with the pathogenesis of the human chronic myelogenous leukemia (CML). It is difficult to characterize kinase response to stimuli or drug treatment because regulatory phosphorylation events are largely transient changes affecting low abundance proteins. Stable isotope labeling with amino acids in cell culture (SILAC) has emerged as a pivotal technology for quantitative proteomics. By metabolically labeling proteins with light or heavy tyrosine, we are able to quantify the change in phosphorylation of BCR‐ABL kinase and its substrates in response to drug treatment in human CML cells. In this study, we observed that BCR‐ABL kinase is phosphorylated at tyrosines 393 and 644, and that SH2‐domain containing inositol phosphatase (SHIP)‐2 and downstream of kinase (Dok)‐2 are phosphorylated at tyrosine 1135 and 299, respectively. Based on the relative intensity of isotopic peptide pairs, we demonstrate that the level of phosphorylation of BCR‐ABL kinase as well as SHIP‐2 and Dok‐2 is reduced approximately 90% upon treatment with Imatinib, a specific inhibitor of BCR‐ABL kinase. Furthermore, proteins, such as SHIP‐1, SH2‐containing protein (SHC) and Casitas B‐lineage lymphoma proto‐oncogene (CBL), are also regulated by Imatinib. These results demonstrate the simplicity and utility of SILAC as a method to quantify dynamic changes in phosphorylation at specific sites in response to stimuli or drug treatment in cell culture.