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

Ritsuko Matsuo, Wataru Ochiai, Kinichi Nakashima, Tetsuya Taga

Research output: Contribution to journalArticlepeer-review

60 Citations (Scopus)

Abstract

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 step in a series of signal transduction pathways, we have developed a new expression-screening method using a phosphorylation site specific antibody and a vector encoding substrate polypeptide. We have applied this method for screening kinases which phosphorylate STAT3 at serine(727). In this screening, antibody (PS727 antibody) specifically recognizing STAT3 in which serine(727) is phosphorylated was first prepared. Escherichia coli, bacteria expressing a serine(727)-containing fragment of STAT3 which was fused to glutathione-S-transferase (GST) (GST-STAT3-WT) were infected by λphage cDNA expression libraries. Phosphorylation of GST-STAT3-WT was effectively performed in E. coli as expected, and clones positive for PS727 antibody immunoreactivity were selected. Isolated 53 clones encode four serine/threonine kinases; extracellular signal regulated kinase 1 (ERK1/p44-MAPK), dual specificity 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 mammalian cells. The present method is considered to be applicable in general to isolate kinases.

Original languageEnglish
Pages (from-to)141-151
Number of pages11
JournalJournal of Immunological Methods
Volume247
Issue number1-2
DOIs
Publication statusPublished - Jan 1 2001
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Immunology and Allergy
  • Immunology

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