Phosphoprotein Analysis Using Antibodies Broadly Reactive against Phosphorylated Motifs

• Published in: The Journal of biological chemistry Vol. 277; no. 42; pp. 39379 - 39387
• Main Authors: Zhang, Hui, Zha, Xiangming, Tan, Yi, Hornbeck, Peter V, Mastrangelo, Allison J, Alessi, Dario R, Polakiewicz, Roberto D, Comb, Michael J
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 18.10.2002
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Animals; Antibodies - metabolism; Blotting, Western; Databases as Topic; Enzyme-Linked Immunosorbent Assay; Gene Transfer Techniques; Humans; Immunoblotting; Molecular Sequence Data; Peptides - metabolism; Phosphoproteins - chemistry; Phosphorylation; Precipitin Tests; Protein Binding; Protein Kinase C - metabolism; Protein-Serine-Threonine Kinases; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-akt; Retroviridae - genetics; Sequence Homology, Amino Acid; Signal Transduction; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M206399200

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 R X R XX T*/S* (Akt motif) and S* X R (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.