Phosphoprotein analysis using antibodies broadly reactive against phosphorylated motifs.
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.
Zhang
Hui
H
Cell Signaling Technology, Beverly, Massachusetts 01915, USA.
Zha
Xiangming
X
Tan
Yi
Y
Hornbeck
Peter V
PV
Mastrangelo
Allison J
AJ
Alessi
Dario R
DR
Polakiewicz
Roberto D
RD
Comb
Michael J
MJ
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D015153:Blotting, Western; D019992:Databases as Topic; D004797:Enzyme-Linked Immunosorbent Assay; D018014:Gene Transfer Techniques; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry*; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D012190:Retroviridae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013379:Substrate Specificity
D000906:Antibodies; D010455:Peptides; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D011493:Protein Kinase C