title: Phosphoprotein analysis using antibodies broadly reactive against phosphorylated motifs.
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 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.
authors.affiliation: Cell Signaling Technology, Beverly, Massachusetts 01915, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.forename: Xiangming
authors.initials: X
authors.lastname: Zha
authors.forename: Yi
authors.initials: Y
authors.lastname: Tan
authors.forename: Peter V
authors.initials: PV
authors.lastname: Hornbeck
authors.forename: Allison J
authors.initials: AJ
authors.lastname: Mastrangelo
authors.forename: Dario R
authors.initials: DR
authors.lastname: Alessi
authors.forename: Roberto D
authors.initials: RD
authors.lastname: Polakiewicz
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Comb
mesh_terms: 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
chemical_list: 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