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

  

Authors:	Liang, X.; Hajivandi, M.; Veach, D.; Wisniewski, D.; Clarkson, B.; Resh, M. D.; Pope, R. M.
Article Title:	Quantification of change in phosphorylation of BCR-ABL kinase and its substrates in response to Imatinib treatment in human chronic myelogenous leukemia cells
Abstract:	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. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA.
Keywords:	controlled study; protein phosphorylation; human cell; antineoplastic agents; protein analysis; imatinib; proto oncogene; enzyme substrate; cell line, tumor; chronic myeloid leukemia; pyrimidines; tyrosine; phosphorylation; amino acid sequence; molecular sequence data; isotope labeling; nucleotide sequence; leukemia cell; adaptor proteins, signal transducing; phosphoproteins; protein-tyrosine kinases; bcr abl protein; piperazines; cbl protein; phosphoric monoester hydrolases; phosphoprotein; leukemia, myeloid, chronic; drug treatment; protein sh2; human chronic myelogenous leukemia; phosphorylation of bcr-abl kinase; inositol phosphate; oncogene protein v-cbl
Journal Title:	Proteomics
Volume:	6
Issue:	16
ISSN:	1615-9853
Publisher:	Wiley V C H Verlag Gmbh
Date Published:	2006-08-01
Start Page:	4554
End Page:	4564
Language:	English
DOI:	10.1002/pmic.200600109
PUBMED:	16858728
PROVIDER:	scopus
DOI/URL:	http://www.scopus.com/inward/record.url?eid=2-s2.0-33748344291&partnerID=40&md5=615c64b2a00c1928b60692a1f1420f8a
Notes:	--- - "Cited By (since 1996): 23" - "Export Date: 4 June 2012" - "CODEN: PROTC" - "Source: Scopus"

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