Identification of the Direct Substrates of the ABL Kinase via Kinase Assay Linked Phosphoproteomics with Multiple Drug Treatments

Ableson tyrosine kinase (ABL) plays essential roles in cell differentiation, division, adhesion, and stress response. However, fusion of the breakpoint cluster region (BCR) to ABL produces constitutive kinase activity that causes chronic myelogenous leukemia (CML). Small molecule tyrosine kinase inhibitors (TKIs) such as imatinib revolutionized the treatment of CML and other cancers, but acquired resistance to these inhibitors is rising. Thus, careful dissection of ABL signaling pathways is needed to find novel drug targets. Here we present a refined proteomic approach for elucidation of direct kinase substrates called kinase assay linked phosphoproteomics (KALIP). Our strategy integrates in vitro kinase assays at both the peptide and protein levels with quantitative tyrosine phosphoproteomics in response to treatment by multiple TKIs. Utilizing multiple TKIs permits elimination of off-target effects of these drugs, and overlapping the in vivo and in vitro data sets allows us to define a list of the most probable kinase substrates. Applying our approach produced a list of 60 ABL substrates, including novel and known proteins. We demonstrate that spleen tyrosine kinase (SYK) is a novel direct substrate of ABL, and we predict our proteomic strategy may facilitate identification of substrates in other cancers that have disrupted kinase signaling.