Kinase Activity Assay and Inhibitor Screening

Protein kinase activity assays and inhibitor screening were performed as previously described (19 (link), 20 (link), 42 , 43 (link)). Briefly, the kinase substrate GST-FLT3S was purified from E. coli cells by using a glutathione-Sepharose column. Recombinant proteins that contained the catalytic domains of wild type and D835H/D835Y mutant forms of human FLT3 (aa573-993), human JAK3 (aa806-1124), and human cKit (aa558-976) were expressed by using the baculovirus expression system (Invitrogen) and isolated from recombinant baculovirus-infected Sf9 insect cells by using the NTA-Ni resin. Phosphorylation of GST-FLT3S by isolated recombinant protein kinases was carried out in a reaction buffer that contained 25 mM Tris–HCl (pH 7.5), 10 mM MgCl₂, 0.2 mM ATP, and 2 mM dithiothreitol in the presence of various concentrations of protein kinase inhibitors. The level of GST-FLT3S tyrosine phosphorylation was determined by immunoblotting with antiphosphotyrosine antibody PY20 followed by horseradish peroxidase-conjugated secondary antibody. Detection and quantification of enhanced chemiluminescence signals were done by using FluorChem SP imaging system from Alpha Innotech (CA, USA).