Phosphoprotein analysis using antibodies broadly reactive against phosphorylated motifs

Hui Zhang, Xiangming Zha, Yi Tan, Peter V. Hornbeck, Allison J. Mastrangelo, Dario R. Alessi, Roberto D. Polakiewicz, Michael J. Comb

Research output: Contribution to journalArticle

Abstract

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 sub-classes 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.

Original languageEnglish (US)
Pages (from-to)39379-39387
Number of pages9
JournalJournal of Biological Chemistry
Volume277
Issue number42
DOIs
StatePublished - Oct 18 2002
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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  • Cite this

    Zhang, H., Zha, X., Tan, Y., Hornbeck, P. V., Mastrangelo, A. J., Alessi, D. R., Polakiewicz, R. D., & Comb, M. J. (2002). Phosphoprotein analysis using antibodies broadly reactive against phosphorylated motifs. Journal of Biological Chemistry, 277(42), 39379-39387. https://doi.org/10.1074/jbc.M206399200