A common phosphotyrosine signature for the Bcr-Abl kinase

Abstract

The Bcr-Abl fusion kinase drives oncogenesis in chronic myeloid leukemia (CML). CML patients are currently treated with the Abl tyrosine kinase inhibitor imatinib, which is effective in early stages of the disease. However, resistance to imatinib arises in later disease stages primarily because of a Bcr-Abl mutation. To gain deeper insight into Bcr-Abl signaling pathways, we generated phosphotyrosine profiles for 6 cell lines that represent 3 Bcr-Abl fusion types by using immunoaffinity purification of tyrosine phosphopeptides followed by tandem mass spectrometry. We identified 188 nonredundant tyrosine-phosphorylated sites, 77 of which are novel. By comparing the profiles, we found a number of phosphotyrosine sites common to the 6 cell lines regardless of cellular background and fusion type, several of which are decreased by imatinib treatment. Comparison of this Bcr-Abl signature with the profile of cells expressing an alternative imatinib-sensitive fusion kinase, FIP1L1-PDGFRalpha, revealed that these kinases signal through different pathways. This phosphoproteomic study of the Bcr-Abl fusion kinase highlights novel disease markers and potential drug-responsive biomarkers and adds novel insight into the oncogenic signals driven by the Bcr-Abl kinase.

Figure 1.

Cell lines analyzed to identify the Bcr-Abl signature. The 7 cell lines analyzed to identify the Bcr-Abl signature (SUP-B15, SD1, BV173, KCL22, K562, KU812, and the control cell line U937) are listed along with fusion type, cell lineage, and number of known and novel tyrosine phosphorylation sites identified in each cell type.

Figure 2.

Identification of a Bcr-Abl kinase signature. (A) A pathway diagram of the 16 common proteins from Table 2 with cellular localization is shown. The proteins without highlighting were found in the Bcr-Abl–containing cells and U937 cells and were therefore not considered part of a signature for Bcr-Abl. The 6 signature proteins with a previous link to Bcr-Abl were highlighted in green (Abl, Bcr, Cbl, SHIP-2, Shc, and VASP). The signature proteins not previously identified in Bcr-Abl signaling are highlighted in blue (CD2AP and GRF1). (B) Expression of 2 novel human signature proteins in Bcr-Abl–containing (BV173 and K562) and control (EoL-1) cells was confirmed by Western blotting using antibodies to total CD2AP and VASP. (C) Phosphorylation in Bcr-Abl–containing cells was validated using antiphosphotyrosine immunoprecipitation followed by Western blotting using total CD2AP or VASP antibodies. Immunoblotting for p-cdc2 (Y15) was used to control for the efficiency of the phosphotyrosine immunoprecipitation because this site is ubiquitously phosphorylated in cell lines.

Figure 3.

Imatinib-responsive phosphoproteins. (A) The 4 cell lines that were used for targeted analysis (BV173, K562, KU812, and SUP-B15) were treated with diluent (CON) or imatinib (IM) to inhibit Bcr-Abl kinase activity. Samples were immunoblotted using antiphosphotyrosine antibody. (B) Cells treated as described in panel A were immunoblotted for phosphoproteins that exhibited a change in phosphorylation level, as determined by SILAC analysis in K562 cells. Phospho-Gab1 (Y406) was identified by mass spectrometry, but a phosphospecific antibody that recognizes this site was not available; therefore, we used a p-Gab1 (Y627) antibody. Protein loading was normalized with the use of β-actin.