An autophosphorylation site database for leucine-rich repeat receptor-like kinases in Arabidopsis thaliana.
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.
Department of Horticultural Science, North Carolina State University, Raleigh, NC, 27695, USA.
Srijeet K
SK
Mitra
Department of Horticultural Science, North Carolina State University, Raleigh, NC, 27695, USA.
Ruiqiang
R
Chen
Department of Horticultural Science, North Carolina State University, Raleigh, NC, 27695, USA.
Murali
M
Dhandaydham
Department of Horticultural Science, North Carolina State University, Raleigh, NC, 27695, USA.
Xiaofeng
X
Wang
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC, 27695, USA.
Robert Kevin
RK
Blackburn
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC, 27695, USA.
Uma
U
Kota
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC, 27695, USA.
Michael B
MB
Goshe
Department of Physiology and Neurobiology, University of Connecticut, Storrs, CT, 06269, USA.
Daniel
D
Schwartz
USDA/ARS, University of Illinois, Urbana, IL, 61801, USA.
Steven C
SC
Huber
Department of Horticultural Science, North Carolina State University, Raleigh, NC, 27695, USA.
Steven D
SD
Clouse
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D016208:Databases, Factual; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary
D029681:Arabidopsis Proteins; D011494:Protein Kinases; C463660:BAK1 protein, Arabidopsis; C440035:SERK1 protein, Arabidopsis; C108270:BRI1 protein, Arabidopsis; D017346:Protein Serine-Threonine Kinases
Arabidopsis thaliana; mass spectrometry; motif analysis; phosphorylation; receptor kinase