Detecting the phosphorylation substrates of multiple kinases in a single experiment is a challenge, and new techniques are being developed to overcome this challenge. Here, we used a multiplexed assay for kinase specificity (
This content will become publicly available on April 3, 2025
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 of many kinases, i.e., their substrates or “clients”, 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 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 P2K phosphorylated peptides and novel predictions from a deep-learning phosphorylation site prediction model (MUsite) and a trained hidden Markov model (HMM) based tool, HMMER. Screening the library against purified P2K1 cytosolic domain (CD), 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 selection criteria for efficient further
- Award ID(s):
- 2048410
- PAR ID:
- 10509634
- Publisher / Repository:
- Frontiers
- Date Published:
- Journal Name:
- Frontiers in Plant Science
- Volume:
- 15
- ISSN:
- 1664-462X
- Page Range / eLocation ID:
- 1372361
- Subject(s) / Keyword(s):
- protein kinase, signaling, machine learning
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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