An autophosphorylation site database for leucine-rich repeat receptor-like kinases in Arabidopsis thaliana

Srijeet K. Mitra, Ruiqiang Chen, Murali Dhandaydham, Xiaofeng Wang, Robert Kevin Blackburn, Uma Kota, Michael B. Goshe, Daniel Schwartz, Steven C. Huber, Steven D. Clouse

Research output: Contribution to journalArticle

Abstract

Leucine-rich repeat receptor-like kinases (LRR RLKs) form a large family of plant signaling proteins consisting of an extracellular domain connected by a single-pass transmembrane sequence to a cytoplasmic kinase domain. Autophosphorylation on specific Ser and/or Thr residues in the cytoplasmic domain is often critical for the activation of several LRR RLK family members with proven functional roles in plant growth regulation, morphogenesis, disease resistance, and stress responses. While identification and functional characterization of in vivo phosphorylation sites is ultimately required for a full understanding of LRR RLK biology and function, bacterial expression of recombinant LRR RLK cytoplasmic catalytic domains for identification of in vitro autophosphorylation sites provides a useful resource for further targeted identification and functional analysis of in vivo sites. In this study we employed high-throughput cloning and a variety of mass spectrometry approaches to generate an autophosphorylation site database representative of more than 30% of the approximately 223 LRR RLKs in Arabidopsis thaliana. We used His-tagged constructs of complete cytoplasmic domains to identify a total of 592 phosphorylation events across 73 LRR RLKs, with 497 sites uniquely assigned to specific Ser (268 sites) or Thr (229 sites) residues in 68 LRR RLKs. Multiple autophosphorylation sites per LRR RLK were the norm, with an average of seven sites per cytoplasmic domain, while some proteins showed more than 20 unique autophosphorylation sites. The database was used to analyze trends in the localization of phosphorylation sites across cytoplasmic kinase subdomains and to derive a statistically significant sequence motif for phospho-Ser autophosphorylation. Significance Statement High-throughput cloning and mass spectrometry were used to generate an extensive autophosphorylation site database of 496 unique phophosSer and phosphoThr sites for 68 Leucine-Rich Repeat Receptor-Like kinases in Arabidopsis thaliana. This database will be a useful resource for designing further experiments to characterize the biological function of multiple members of this large multi-gene family of plant signaling proteins.

Original languageEnglish (US)
Pages (from-to)1042-1060
Number of pages19
JournalPlant Journal
Volume82
Issue number6
DOIs
StatePublished - Jun 1 2015

Fingerprint

protein phosphorylation
Arabidopsis
Leucine
leucine
phosphotransferases (kinases)
Phosphotransferases
Arabidopsis thaliana
Databases
receptors
phosphorylation
Plant Proteins
Phosphorylation
Organism Cloning
molecular cloning
Mass Spectrometry
mass spectrometry
Disease Resistance
proteins
Morphogenesis
multigene family

Keywords

  • Arabidopsis thaliana
  • mass spectrometry
  • motif analysis
  • phosphorylation
  • receptor kinase

ASJC Scopus subject areas

  • Genetics
  • Plant Science
  • Cell Biology

Cite this

Mitra, S. K., Chen, R., Dhandaydham, M., Wang, X., Blackburn, R. K., Kota, U., ... Clouse, S. D. (2015). An autophosphorylation site database for leucine-rich repeat receptor-like kinases in Arabidopsis thaliana. Plant Journal, 82(6), 1042-1060. https://doi.org/10.1111/tpj.12863

An autophosphorylation site database for leucine-rich repeat receptor-like kinases in Arabidopsis thaliana. / Mitra, Srijeet K.; Chen, Ruiqiang; Dhandaydham, Murali; Wang, Xiaofeng; Blackburn, Robert Kevin; Kota, Uma; Goshe, Michael B.; Schwartz, Daniel; Huber, Steven C.; Clouse, Steven D.

In: Plant Journal, Vol. 82, No. 6, 01.06.2015, p. 1042-1060.

Research output: Contribution to journalArticle

Mitra, SK, Chen, R, Dhandaydham, M, Wang, X, Blackburn, RK, Kota, U, Goshe, MB, Schwartz, D, Huber, SC & Clouse, SD 2015, 'An autophosphorylation site database for leucine-rich repeat receptor-like kinases in Arabidopsis thaliana', Plant Journal, vol. 82, no. 6, pp. 1042-1060. https://doi.org/10.1111/tpj.12863
Mitra, Srijeet K. ; Chen, Ruiqiang ; Dhandaydham, Murali ; Wang, Xiaofeng ; Blackburn, Robert Kevin ; Kota, Uma ; Goshe, Michael B. ; Schwartz, Daniel ; Huber, Steven C. ; Clouse, Steven D. / An autophosphorylation site database for leucine-rich repeat receptor-like kinases in Arabidopsis thaliana. In: Plant Journal. 2015 ; Vol. 82, No. 6. pp. 1042-1060.
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