Unveiling orphan receptor-like kinases in plants: novel client discovery using high-confidence library predictions in the Kinase–Client (KiC) assay

ABSTRACT: Plants are remarkable in their ability to adapt to changing environments, with receptor-like kinases (RLKs) playing a pivotal role in perceiving and transmitting environmental cues into cellular responses. Despite extensive research on RLKs from the plant kingdom, the function and activity (i.e., their substrates or “clients”) of many kinases remain uncharted. To validate a novel client prediction workflow and learn more about an important RLK, this study focuses on P2K1 (DORN1), which acts as a key receptor for extracellular ATP (eATP), playing a crucial role in plant stress resistance and immunity. We designed a Kinase-Client (KiC) assay library of 225 synthetic peptides, incorporating previously identified phosphorylation sites and novel predictions from a deep-learning phosphorylation site prediction model (MUsite) and a trained hidden Markov model (HMM) . Screening the library against purified P2K1, we identified 46 putative substrates, including 34 novel clients, 27 of which may be novel peptides, not previously identified experimentally. Gene Ontology (GO) analysis among phosphopeptide candidates revealed proteins associated with important biological processes in metabolism, structure development, and response to stress, as well as molecular functions of kinase activity, catalytic activity, and transferase activity. We offer efficient selection criteria for further in vivo experiments to confirm these discoveries. This approach not only expands our knowledge of P2K1's substrates and functions but also highlights effective prediction algorithms for identifying additional potential substrates. Overall, the results further support use of the KiC assay as a valuable tool in unraveling the complexities of plant phosphorylation and provide a foundation for predicting the phosphorylation landscape of plant species based on peptide library results.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

SUBMITTER: Gabriel Lemes Jorge  

LAB HEAD: Jay J. Thelen

PROVIDER: PXD047713 | Pride | 2024-03-25

REPOSITORIES: pride