Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific
protein kinases. Here we describe an integrated proteomic strategy, termed
kinase assay linked with phosphoproteomics, which combines a sensitive
kinase reaction with endogenous
kinase-dependent phosphoproteomics to identify direct substrates of
protein kinases. The unique in vitro
kinase reaction is carried out in a highly efficient manner using a pool of
peptides derived directly from cellular
kinase substrates and then dephosphorylated as substrate candidates. The resulting newly phosphorylated
peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated
peptides with
phosphopeptides derived from endogenous
proteins isolated from cells in which the
kinase is either active or inhibited reveals new candidate
protein substrates. The
kinase assay linked with phosphoproteomics strategy was applied to identify unique substrates of
spleen tyrosine kinase (Syk), a
protein-tyrosine kinase with duel properties of an oncogene and a
tumor suppressor in distinctive cell types. We identified 64 and 23 direct substrates of Syk specific to B cells and
breast cancer cells, respectively. Both known and unique substrates, including multiple centrosomal substrates for Syk, were identified, supporting a unique mechanism that Syk negatively affects cell division through its centrosomal
kinase activity.