Phosphoprotein analysis using antibodies broadly reactive against phosphorylated motifs

J Biol Chem. 2002 Oct 18;277(42):39379-87. doi: 10.1074/jbc.M206399200. Epub 2002 Jul 31.

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.

MeSH terms

  • 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

Substances

  • Antibodies
  • Peptides
  • Phosphoproteins
  • Proto-Oncogene Proteins
  • AKT1 protein, human
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt
  • Protein Kinase C