Development of a high-throughput fluoroimmunoassay for Syk kinase and Syk kinase inhibitors

Anal Biochem. 2003 Apr 15;315(2):256-61. doi: 10.1016/s0003-2697(03)00026-5.

Abstract

Syk is a tyrosine kinase which is indispensable in immunoglobulin Fc receptor- and B cell receptor-mediated signal transduction in various immune cells. This pathway is important in the pathophysiology of allergy. In this study we established a quantitative nonradioactive kinase assay to identify inhibitors of Syk. We used recombinant GST-tagged Syk purified from baculovirus-infected insect cells. As a substrate, biotinylated peptide corresponding to the activation loop domain of Syk, whose tyrosine residues are autophosphorylated upon activation, was employed to screen both ATP- and substrate-competitive inhibitors. After the kinase reaction in solution phase, substrate was trapped on a streptavidin-coated plate, followed by detection of the phosphorylated tyrosine with europium-labeled anti-phosphotyrosine antibody. The kinase reaction in solution phase greatly enhanced phosphorylation of substrate compared to that of plate-coated substrate. High signal-to-background ratio and low data scattering were obtained in the optimized high-throughput screening (HTS) format. Further, several kinase inhibitors showed concentration-dependent inhibition of recombinant Syk kinase activity with almost the same efficacy for immunoprecipitated Syk from a human cell line. These data suggest that this assay is useful to screen Syk kinase inhibitors in HTS.

MeSH terms

  • Buffers
  • Enzyme Inhibitors / analysis*
  • Enzyme Precursors / antagonists & inhibitors*
  • Enzyme Precursors / metabolism*
  • Fluoroimmunoassay / methods*
  • Humans
  • Hydrogen-Ion Concentration
  • Intracellular Signaling Peptides and Proteins
  • Kinetics
  • Protein-Tyrosine Kinases / antagonists & inhibitors*
  • Protein-Tyrosine Kinases / metabolism*
  • Syk Kinase
  • Thermodynamics

Substances

  • Buffers
  • Enzyme Inhibitors
  • Enzyme Precursors
  • Intracellular Signaling Peptides and Proteins
  • Protein-Tyrosine Kinases
  • SYK protein, human
  • Syk Kinase