TY - JOUR
T1 - Phosphoprotein analysis using antibodies broadly reactive against phosphorylated motifs
AU - Zhang, Hui
AU - Zha, Xiangming
AU - Tan, Yi
AU - Hornbeck, Peter V.
AU - Mastrangelo, Allison J.
AU - Alessi, Dario R.
AU - Polakiewicz, Roberto D.
AU - Comb, Michael J.
PY - 2002/10/18
Y1 - 2002/10/18
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.M206399200
DO - 10.1074/jbc.M206399200
M3 - Article
C2 - 12151408
AN - SCOPUS:0037131411
SN - 0021-9258
VL - 277
SP - 39379
EP - 39387
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 42
ER -