Identifying D-Group Mitogen-Activated Protein Kinases as Substrates of Arabidopsis Tyrosine Phosphatase RLPH2 Using...

Identifying substrates of protein phosphatases has been technically challenging and has hampered progress in the field of plant sciences. Small molecule inhibitors of protein phosphatases have aided in uncovering classes of phosphatases that target substrates, but that too has severe limitations. Here, we describe a method that enriches phosphorylated substrates using TiO₂ and phospho-tyrosine antibodies in phosphatase knockout lines of Arabidopsis thaliana. When compared to wild-type plants, this approach permits identification of putative substrates and specific phosphorylation sites by mass spectrometry, allowing for further in vitro or functional validation. The key to the approach described here is the use of phosphatase knockout lines to maintain substrates in a phosphorylated state and using phospho-tyrosine antibodies to enrich for tyrosine phosphorylated peptides. Key features • Requires genetic knockout lines for the protein phosphatase of interest to uncover increased phosphorylation of putative substrates compared to wild type. • Phospho-peptides are (quantitatively) compared using mass spectrometry. • Tyrosine phosphorylated peptides are immunoprecipitated after TiO₂ phospho-peptide enrichment. • Putative substrates identified with this protocol can later be validated with in vitro assays using synthetic phospho-peptides and/or phospho-proteins.