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 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 lambda phage 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.
Matsuo
R
R
Department of Molecular Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10, Kanda-surugadai, Chiyoda-ku, Tokyo 101-0062, Japan.
Ochiai
W
W
Nakashima
K
K
Taga
T
T
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D001665:Binding Sites; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D015850:Interleukin-6 / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010768:Phosphoserine / Q000276:immunology; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D050796:STAT3 Transcription Factor; D013379:Substrate Specificity; D015534:Trans-Activators / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000097603:Dyrk Kinases
D000906:Antibodies; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D015850:Interleukin-6; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D010768:Phosphoserine; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D050796:STAT3 Transcription Factor; D015534:Trans-Activators; D000097603:Dyrk Kinases