Using Phospho‐Motif Antibodies to Determine Kinase Substrates
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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. Curr. Protoc. Mol. Biol. 101:18.20.1?18.20.11. © 2013 by John Wiley & Sons, Inc.
Keywords: phospho?motif antibody; phosphorylation; protein kinase
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PDF or HTML at Wiley Online LibraryTable of Contents
- Introduction
- Basic Protocol 1: In Silico Analysis to Identify the Kinase Responsible for Phosphorylating a Known Substrate
- Basic Protocol 2: Biochemical Validation of Protein Kinase–Induced Substrate Phosphorylation
- Reagents and Solutions
- Commentary
- Literature Cited
- Figures
- Tables
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PDF or HTML at Wiley Online LibraryMaterials
Basic Protocol 1: In Silico Analysis to Identify the Kinase Responsible for Phosphorylating a Known Substrate
Materials
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Figure 18.20.1 The specificity of phospho‐motif antibodies. HeLa cells were serum‐starved for 24 hr prior to being stimulated with 50 ng/ml IGF‐1 or 100 nM PMA for 10 min. Lysates were immunoblotted with anti‐Akt/PKB phospho‐motif (left panel) and anti‐PKD phospho‐motif (right panel) antibodies. Open arrows indicate putative Akt/PKB substrates and solid arrows indicate putative PKD substrates. Figure reproduced with permission from Doppler et al. () View Image
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Literature Cited
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