A Peptide-Based Biosensor Assay To Detect Intracellular Syk Kinase Activation and Inhibition

Abstract

Spleen Tyrosine Kinase (Syk) has been implicated in a number of pathologies including cancer and rheumatoid arthritis and thus has been pursued as a novel therapeutic target. Because of the complex relationship between Syk’s auto- and other internal phosphorylation sites, scaffolding proteins, enzymatic activation state and sites of phosphorylation on its known substrates, the role of Syk’s activity in these diseases has not been completely clear. To approach such analyses, we developed a Syk-specific artificial peptide biosensor (SAStide) to use in a cell-based assay for direct detection of intracellular Syk activity and inhibition in response to physiologically relevant stimuli in both laboratory cell lines and primary splenic B cells. This peptide contains a sequence derived from known Syk substrate preference motifs linked to a cell permeable peptide, resulting in a biosensor that is phosphorylated in live cells in a Syk-dependent manner, thus serving as a reporter of Syk catalytic activity in intact cells. Because the assay is compatible with live, primary cells and can report pharmacodynamics for drug action on an intended target, this methodology could be used to facilitate a better understanding of Syk’s function and the effect of its inhibition in disease.

Time-Resolved Luminescence Detection of Spleen Tyrosine Kinase Activity through Terbium Sensitization

“…An additional aliquot (1 μL) of the kinase reaction mixture was taken at each time point for validation of phosphorylation using an ELISA-based chemifluorescent assay as previously described. (27) Briefly, each aliquot was quenched with 0.5 M EDTA and incubated in a 96-well Neutravidin coated plate (15 pmol biotin binding capacity per well, Thermo Scientific) in Tris-buffer saline (TBS) containing 0.1% BSA and 0.05% Tween 20 for 1h. Following incubation, each well was washed with the TBS buffer and the incubated with mouse anti-phosphotyrosine monoclonal antibody 4G10 (Millipore, 1:10,000 dilution in TBS buffer) for 1h.…”

Spleen Tyrosine Kinase–Mediated Autophagy Is Required for Epithelial–Mesenchymal Plasticity and Metastasis in Breast Cancer

“…S1). Therefore, to verify enhanced SYK activity following TGFb-induced EMT, we conducted a substrate phosphorylation assay using SYK Phage-TIDE, a peptide sequence (EDPDYEQPSA) identified by phage display to be a highly specific and sensitive substrate for SYK as compared with the Y525/526 containing activation loop autophosphorylation peptide (19,26). This peptide was conjugated to a DNA oligonucleotide, allowing for highly sensitive quantification of phosphorylated peptide via PCR following its capture using the antityrosine antibody 4G10.…”

High-throughput identification of FLT3 wild-type and mutant kinase substrate preferences and application to design of sensitive in vitro kinase assay substrates

“…Phosphorylation was measured using the previously described ELISA-based assay 31,36 and results are shown in Figure 5.…”

“…The assay reaction was started by adding the peptide substrate to a 37.5 µM reaction concentration in a 30 µL volume. Sample aliquots (10 µL) were quenched by combining 1:1 with 30 mM EDTA at 2 and 60-minute timepoints (or 30minute timepoint for TKI dose response assays).Chemifluorescence Detection of Phosphorylation-Quenched aliquots were incubated in a96-well streptavidin coated plate (125 pmol binding capacity, ThermoScientific) in Tris buffered saline (25 mM Tris-HCL and 150 mM NaCl) with 0.05% Tween 20 (TBS-T) containing 5% w/v skim milk for 1 h 36. Subsequently, each well was washed with TBS-T and incubated with antiphosphotyrosine mouse monoclonal antibody 4G10 (MilliporeSigma, 1:10,000 dilution in TBS-T).…”