Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates

Abstract

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.P rotein kinases and their substrates represent the largest signaling network that regulates protein-protein interactions, subcellular localization, and ultimately cellular functions (1, 2). Deregulation of the signaling network often leads to disease states such as human malignancies, diabetes, and immune disorders. Although many kinases are excellent therapeutic targets, the precise connection between protein kinases and their direct substrates has not been fully elucidated for a majority of protein kinases. Besides classical genetic and biochemical methods, there have been a number of high throughput approaches for the identification of potential kinase substrates. Common methods include in vitro kinase assays using libraries of synthetic peptides (3), phase expression libraries (4), protein/peptide arrays (5-7), or cell extracts (8, 9), but these methods can often be misleading and provide many false positive results. The discovery of physiological substrates for specific protein kinases has remained challenging, even with recent advances in mass spectrometry.Mass spectrometry-based proteomics has become a powerful tool and been applied to map protein interaction networks, including kinase/phosphatase-substrate networks (10). Large-scale phosphoproteomics, however, does not typically reveal precise connections between protein kinases and their direct substrates (11,12). In recent years, there have been increasing attempts to develop mass spectrometry-based proteomic strategies for the identification of elusive kinase substrates (7,13,1...

Identification of Direct Tyrosine Kinase Substrates Based on Protein Kinase Assay-Linked Phosphoproteomics

“…Phosphorylation of Y354 on STIP1 has been characterized by a large-scale tyrosine phosphorylation study (51). This phosphorylation was also shown to be dependent on SYK in phosphoproteomic studies of MDA-MB-231 (41) and DG75 cells (28). Our in vitro kinase assay confirmed that Y354 was indeed the direct phosphorylation site of SYK.…”

“…Because in vitro kinase reactions typically display a degree of promiscuity (43), we further incorporated endogenous phosphoproteomic analysis from the previous study (28) using cells whose kinase of interest was either active or inhibited (Fig. 1, right panel).…”

“…The reactions were quenched and then samples were pooled, trypsin digested, enriched using PolyMAC, and analyzed by mass spectrometry. The experimental conditions for phosphatase treatment, subsequent phosphatase removal, and kinase activity were optimized as described previously (28). In each step, changes in phosphotyrosine content were examined by Western blotting (supplemental Fig.…”

“…To identify bona fide substrates, we compared the in vitro results with the in vivo SYK dependent phosphotyrosine events (supplemental Table S7). The in vivo data set was retrieved from our previous study SYK ( 28), in which the phosphotyrosine proteomes of DG75 cells that were treated with or without the SYK inhibitor piceatannol. The differential phosphotyrosine proteins were identified by mass spectrometry (Technical details in experimental procedure).…”

“…To bridge the gap between in vitro phosphorylation and physiological phosphorylation events, we have recently introduced an integrated strategy termed Kinase Assay-Linked Phosphoproteomics (KALIP) (28). By combining in vitro kinase assays with in vivo phosphoproteomics, this method was demonstrated to have exceptional sensitivity for high confidence identification of direct kinase substrates.…”