Using phospho-motif antibodies to determine kinase substrates.

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.