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.
Liang
Xiquan
X
Proteomics, R&D Department, Invitrogen Life Science, Carlsbad, CA 92008, USA. xiquan.l@invitrogen.com
Hajivandi
Mahbod
M
Veach
Darren
D
Wisniewski
David
D
Clarkson
Bayard
B
Resh
Marilyn D
MD
Pope
R Marshall
RM
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001549:Benzamides; D045744:Cell Line, Tumor; D016044:Fusion Proteins, bcr-abl; D006801:Humans; D000068877:Imatinib Mesylate; D007553:Isotope Labeling; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive; D008969:Molecular Sequence Data; D050722:Oncogene Protein v-cbl / Q000378:metabolism; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D010750:Phosphoproteins / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D000090063:Proto-Oncogene Mas; D011743:Pyrimidines / Q000494:pharmacology*; D014443:Tyrosine / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D000970:Antineoplastic Agents; D001549:Benzamides; C115089:DOK2 protein, human; C000715470:MAS1 protein, human; D050722:Oncogene Protein v-cbl; D010750:Phosphoproteins; D010879:Piperazines; D000090063:Proto-Oncogene Mas; D011743:Pyrimidines; D014443:Tyrosine; D000068877:Imatinib Mesylate; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl; D010744:Phosphoric Monoester Hydrolases; C098706:INPPL1 protein, human; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases