title: Using phospho-motif antibodies to determine kinase substrates.
abstract: Phosphorylation of substrates by protein kinases regulates a myriad of cellular processes, ranging from proliferation and migration to autophagy, senescence, and apoptosis. Kinase substrate selectivity is largely dependent on the amino acid sequence surrounding the phosphorylation site; therefore, substrate-directed, phosphorylation-state-sensitive, motif-specific ("phospho-motif") antibodies represent powerful tools to identify novel kinase substrates and to investigate mechanisms of substrate phosphorylation in many signaling pathways typically associated with human malignancies. Phospho-motif antibodies are engineered to recognize proteins that contain a phosphorylated residue in the context of a specific motif. They are raised against a library of phospho-peptides comprising both the phosphorylated residue and the surrounding residues that determine kinase specificity, with degenerate residues taking up the remaining positions. Currently, several categories of phospho-motif antibody are commercially available, which may be used to specifically detect Ser, Thr, Ser/Thr, or Tyr residues phosphorylated by different protein kinase families. These antibodies are commonly used in immunoprecipitation and/or immunoblotting protocols to determine kinase-induced substrate phosphorylation. This unit describes the use of phospho-motif antibodies to elucidate the kinase(s) responsible for phosphorylating substrate proteins.
authors.affiliation: Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Colin D
authors.initials: CD
authors.lastname: White
authors.forename: Alex
authors.initials: A
authors.lastname: Toker
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D000906:Antibodies / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D047468:Immunoprecipitation / Q000379:methods; D010748:Phosphopeptides / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D000906:Antibodies; D010748:Phosphopeptides; D011494:Protein Kinases