Phosphoprotein analysis using antibodies broadly reactive against phosphorylated motifs

被引:215
作者
Zhang, H
Zha, XM
Tan, Y
Hornbeck, PV
Mastrangelo, AJ
Alessi, DR
Polakiewicz, RD
Comb, MJ
机构
[1] Cell Signaling Technol, Beverly, MA 01915 USA
[2] Univ Dundee, Med Res Council Prot Phosphorylat Unit, Dept Biochem, Dundee DD1 5EH, Scotland
关键词
D O I
10.1074/jbc.M206399200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The substrates of most protein kinases remain unknown because of the difficulty tracing signaling pathways and identifying sites of protein phosphorylation. Here we describe a method useful in detecting subclasses of protein kinase substrates. Although the method is broadly applicable to any protein kinase for which a substrate consensus motif has been identified, we illustrate here the use of antibodies broadly reactive against phosphorylated Ser/Thr-motifs typical of AGC kinase substrates. Phosphopeptide libraries with fixed residues corresponding to consensus motifs RXRXXT-*/S* (Akt motif) and S*XR (protein kinase C motif) were used as antigens to generate antibodies that recognize many different phosphoproteins containing the fixed motif. Because most AGC kinase members are phosphorylated and activated by phosphoinositide-dependent protein kinase-1 (PDK1), we used PDK1-/- ES cells to profile potential AGC kinase substrates downstream of PDK1. To identify phosphoproteins detected using the Akt substrate antibody, we characterized the antibody binding specificity to generate a specificity matrix useful in predicting antibody reactivity. Using this approach we predicted and then identified a 30-kDa phosphoprotein detected by both Akt and protein kinase C substrate antibodies as S6 ribosomal protein. Phosphospecific motif antibodies offer a new approach to protein kinase substrate identification that combines immunoreactivity data with protein data base searches based upon antibody specificity.
引用
收藏
页码:39379 / 39387
页数:9
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