A new expression cloning strategy for isolation of substrate-specific kinases by using phosphorylation site-specific antibody

Signal transduction from cell surface receptors to the nucleus is regulated in most part by protein phosphorylation. For the purpose of identification of kinases which play an important role at a particular phosphorylation st ep in a series of signal transduction pathways, we have developed a new exp ression-screening method using a phosphorylation site specific antibody and a vector encoding substrate polypeptide. We have applied this method for s creening kinases which phosphorylate STAT3 at serine(727). In this screenin g, antibody (PS727 antibody) specifically recognizing STAT3 in which serine (727) is phosphorylated was first prepared. Escherichia coli, bacteria expr essing a serine(727)-containing fragment of STAT3 which was fused to glutat hione-S-transferase (GST) (GST-STAT3-WT) were infected by lambda phage cDNA expression libraries. Phosphorylation of GST-STAT3-WT was effectively perf ormed in E. coli as expected, and clones positive for PS727 antibody immuno reactivity were selected. Isolated 53 clones encode four serine/threonine k inases; extracellular signal regulated kinase 1 (ERK1/p44-MAPK), dual speci ficity Yak1 related kinase (DYRK), dual specificity Yak1 related kinase 2 ( DYRK2) and homeodomain interacting protein kinase 2 (HIPK2), These kinases have a potential to phosphorylate serine(727) in STAT3 protein also in mamm alian cells. The present method is considered to be applicable in general t o isolate kinases. (C) 2001 Elsevier Science B.V. All rights reserved.