skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


  • NSF Public Access
  • Search Results
  • Unveiling orphan receptor-like kinases in plants: novel client discovery using high-confidence library predictions in the Kinase–Client (KiC) assay
Title: Unveiling orphan receptor-like kinases in plants: novel client discovery using high-confidence library predictions in the Kinase–Client (KiC) assay
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 furtherin vivoexperiments 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 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.  more » « less
Award ID(s):
2048410
PAR ID:
10509634
Author(s) / Creator(s):
; ; ; ; ; ; ; ;
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
More Like this
  1. ; ; ; ; ; ; ; ; ; ; et al (, Molecular & Cellular Proteomics)
    In eukaryotic organisms, protein kinases regulate diverse protein activities and signaling pathways through phosphorylation of specific protein substrates. Isolating and characterizing kinase substrates is vital for defining downstream signaling pathways. The Kinase Client (KiC) assay is an in vitro synthetic peptide LC-MS/MS phosphorylation assay that has enabled identification of protein substrates (i.e., clients) for various protein kinases. For example, previous use of a 2,100-member (2k) peptide library identified substrates for the extracellular ATP receptor-like kinase, P2K1. Many P2K1 clients were confirmed by additional in vitro and in planta studies, including Integrin-Linked Kinase 4 (ILK4), for which we provide the evidence herein. In addition, we developed a new KiC peptide library containing 8,000 (8k) peptides based on phosphorylation sites primarily from Arabidopsis thaliana datasets. The 8k peptides are enriched for sites with conservation in other angiosperm plants, with the paired goals of representing functionally conserved sites and usefulness for screening kinases from diverse plants. Screening the 8k library with the active P2K1 kinase domain identified 177 phosphopeptides, including calcineurin B-like protein (CBL9) and G protein alpha subunit 1 (GPA1), which functions in cellular calcium signaling. We confirmed that P2K1 directly phosphorylates CBL9 and GPA1 through in vitro kinase assays. This expanded 8k KiC assay will be a useful tool for identifying novel substrates across diverse plant protein kinases, ultimately facilitating the exploration of previously undiscovered signaling pathways. 
    more » « less
  2. ; ; ; ; ; ; ; ; ; (, Nature Communications)
    Abstract The mevalonate pathway plays a critical role in multiple cellular processes in both animals and plants. In plants, the products of this pathway impact growth and development, as well as the response to environmental stress. A forward genetic screen of Arabidopsis thaliana using Ca 2+ -imaging identified mevalonate kinase (MVK) as a critical component of plant purinergic signaling. MVK interacts directly with the plant extracellular ATP (eATP) receptor P2K1 and is phosphorylated by P2K1 in response to eATP. Mutation of P2K1-mediated phosphorylation sites in MVK eliminates the ATP-induced cytoplasmic calcium response, MVK enzymatic activity, and suppresses pathogen defense. The data demonstrate that the plasma membrane associated P2K1 directly impacts plant cellular metabolism by phosphorylation of MVK, a key enzyme in the mevalonate pathway. The results underline the importance of purinergic signaling in plants and the ability of eATP to influence the activity of a key metabolite pathway with global effects on plant metabolism. 
    more » « less
  3. ; (, Journal of Experimental Botany)
    Abstract Heterotrimeric G-proteins regulate multiple aspects of plant growth, development, and response to biotic and abiotic stresses. While the core components of heterotrimeric G-proteins and their basic biochemistry are similar in plants and metazoans, key differences exist in their regulatory mechanisms. In particular, the activation mechanisms of plant G-proteins appear diverse and may include both canonical and novel modes. Classical G-protein-coupled receptor-like proteins exist in plants and interact with Gα proteins, but their ability to activate Gα by facilitating GDP to GTP exchange has not been demonstrated. Conversely, there is genetic and functional evidence that plant G-proteins interact with the highly prevalent receptor-like kinases (RLKs) and are phosphorylated by them. This suggests the exciting scenario that in plants the G-proteins integrate RLK-dependent signal perception at the plasma membrane with downstream effectors. Because RLKs are active kinases, it is also likely that the activity of plant G-proteins is regulated via phosphorylation/dephosphorylation rather than GTP–GDP exchange as in metazoans. This review discusses our current knowledge of the possible RLK-dependent regulatory mechanisms of plant G-protein signaling in the context of several biological systems and outlines the diversity that might exist in such regulation. 
    more » « less
  4. ; ; ; ; ; (, The Plant Cell)
    Abstract Mitogen-activated protein (MAP) kinase signaling cascades play important roles in eukaryotic defense against various pathogens. Activation of the extracellular ATP (eATP) receptor P2K1 triggers MAP kinase 3 and 6 (MPK3/6) phosphorylation, which leads to an elevated plant defense response. However, the mechanism by which P2K1 activates the MAPK cascade is unclear. In this study, we show that in Arabidopsis thaliana, P2K1 phosphorylates the Raf-like MAP kinase kinase kinase (MAPKKK) INTEGRIN-LINKED KINASE 5 (ILK5) on serine 192 in the presence of eATP. The interaction between P2K1 and ILK5 was confirmed both in vitro and in planta and their interaction was enhanced by ATP treatment. Similar to P2K1 expression, ILK5 expression levels were highly induced by treatment with ATP, flg22, Pseudomonas syringae pv. tomato DC3000, and various abiotic stresses. ILK5 interacts with and phosphorylates the MAP kinase MKK5. Moreover, phosphorylation of MPK3/6 was significantly reduced upon ATP treatment in ilk5 mutant plants, relative to wild-type (WT). The ilk5 mutant plants showed higher susceptibility to P. syringae pathogen infection relative to WT plants. Plants expressing only the mutant ILK5S192A protein, with decreased kinase activity, did not activate the MAPK cascade upon ATP addition. These results suggest that eATP activation of P2K1 results in transphosphorylation of the Raf-like MAPKKK ILK5, which subsequently triggers the MAPK cascade, culminating in activation of MPK3/6 associated with an elevated innate immune response. 
    more » « less
  5. ; (, Stress Biology)
    Abstract In plant immunity, a well-orchestrated cascade is initiated by the dimerization of receptor-like kinases (RLKs), followed by the phosphorylation of receptor-like cytoplasmic kinases (RLCKs) and subsequent activation of NADPH oxidases for ROS generation. Recent findings by Zhong et al. illustrated that a maize signaling module comprising ZmWAKL-ZmWIK-ZmBLK1-ZmRBOH4 governs quantitative disease resistance to grey leaf spot, a pervasive fungal disease in maize worldwide, unveiling the conservation of this signaling quartet in plant immunity. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email