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.