Identification of Plant Kinase Substrates Based on Kinase Assay-Linked Phosphoproteomics

: Protein phosphorylation is one of the key events in the regulation of plant physiological responses to diverse environmental stimuli. As crucial regulators of phosphorylation, protein kinases have been linked to the control of seed germination, flowering, and stress responses. Identifying downstream substrates of kinases is important for dissecting kinase-substrate networks as well as delineating the underlying defense mechanisms in response to extracellular stimulation. Despite the fact that thousands of kinase-substrate networks have been identified in mammalian cells, the downstream substrates of important plant kinases are still elusive. Moreover, it remains challenging to identify bona fide kinase substrates from proteome-wide analyses. Thus, developing methodologies with high sensitivity and specificity is imperative for understanding plant kinase-substrate cascades. Here, we describe a proteomic strategy termed kinase assay-linked phosphoproteomics (KALIP) approach for large-scale identification of the direct substrates of plant kinases with high sensitivity and a low false-positive rate.