title: Identification of the phosphorylation targets of symbiotic receptor-like kinases using a high-throughput multiplexed assay for kinase specificity.
abstract: 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 (MAKS) to identify the substrates directly and to map the phosphorylation site(s) of plant symbiotic receptor-like kinases. The symbiotic receptor-like kinases nodulation receptor-like kinase (NORK) and lysin motif domain-containing receptor-like kinase 3 (LYK3) are indispensable for the establishment of root nodule symbiosis. Although some interacting proteins have been identified for these symbiotic receptor-like kinases, very little is known about their phosphorylation substrates. Using this high-throughput approach, we identified several other potential phosphorylation targets for both these symbiotic receptor-like kinases. In particular, we also discovered the phosphorylation of LYK3 by NORK itself, which was also confirmed by pairwise kinase assays. Motif analysis of potential targets for these kinases revealed that the acidic motif xxxsDxxx was common to both of them. In summary, this high-throughput technique catalogs the potential phosphorylation substrates of multiple kinases in a single efficient experiment, the biological characterization of which should provide a better understanding of phosphorylation signaling cascade in symbiosis.
authors.affiliation: Department of Agronomy, University of Wisconsin, 1575 Linden Drive, Madison, WI, 53706, USA.
authors.forename: Dhileepkumar
authors.initials: D
authors.lastname: Jayaraman
authors.affiliation: Department of Chemistry, University of Wisconsin, 1101 University Avenue, Madison, WI, 53706, USA.|Genome Center of Wisconsin, University of Wisconsin, 425 Henry Mall, Madison, WI, 53706, USA.
authors.forename: Alicia L
authors.initials: AL
authors.lastname: Richards
authors.affiliation: Department of Chemistry, University of Wisconsin, 1101 University Avenue, Madison, WI, 53706, USA.|Genome Center of Wisconsin, University of Wisconsin, 425 Henry Mall, Madison, WI, 53706, USA.|Department of Biomolecular Chemistry, University of Wisconsin, 420 Henry Mall, Madison, WI, 53706, USA.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Westphall
authors.affiliation: Department of Chemistry, University of Wisconsin, 1101 University Avenue, Madison, WI, 53706, USA.|Genome Center of Wisconsin, University of Wisconsin, 425 Henry Mall, Madison, WI, 53706, USA.|Department of Biomolecular Chemistry, University of Wisconsin, 420 Henry Mall, Madison, WI, 53706, USA.
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Coon
authors.affiliation: Department of Agronomy, University of Wisconsin, 1575 Linden Drive, Madison, WI, 53706, USA.|Department of Bacteriology, University of Wisconsin, 1550 Linden Drive, Madison, WI, 53706, USA.
authors.forename: Jean-Michel
authors.identifier: http://orcid.org/0000-0002-3128-9439
authors.initials: JM
authors.lastname: Ané
mesh_terms: D046913:Medicago truncatula / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000235:genetics / Q000502:physiology; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D013559:Symbiosis / Q000235:genetics / Q000502:physiology; D053719:Tandem Mass Spectrometry
chemical_list: D010940:Plant Proteins; D011494:Protein Kinases
keywords: 
  IMAC
  ; 
  Medicago truncatula
  ; kinase assay; protein phosphorylation; signaling; symbiosis; tandem mass spectrometry; technical advance