Recombinant brassinosteroid insensitive 1 receptor-like kinase autophosphorylates on serine and threonine residues and phosphorylates a conserved peptide motif in vitro

M. H. Oh, W. K. Ray, Steven C Huber, J. M. Asara, D. A. Gage, S. D. Clouse

Research output: Contribution to journalArticle

Abstract

BRASSINOSTEROID-INSENSITIVE 1 (BRI1) encodes a putative Leucine-rich repeat receptor kinase in Arabidopsis that has been shown by genetic and molecular analysis to be a critical component of brassinosteroid signal transduction. In this study we examined some of the biochemical properties of the BRI1 kinase domain (BRI1-KD) in vitro, which might be important predictors of in vivo function. Recombinant BRI1-KD autophosphorylated on serine (Ser) and threonine (Thr) residues with p-Ser predominating. Matrix-assisted laser desorption/ionization mass spectrometry identified a minimum of 12 sites of autophosphorylation in the cytoplasmic domain of BRI1, including five in the juxtamembrane region (N-terminal to the catalytic KD), five in the KD (one each in sub-domains I and Via and three in sub-domain VIII), and two in the carboxy terminal region. Five of the sites were uniquely identified (Ser-838, Thr-842, Thr-846, Ser-858, and Thr-872), whereas seven were localized on short peptides but remain ambiguous due to multiple Ser and/or Thr residues within these peptides. The inability of an active BRI1-KD to transphosphorylate an inactive mutant KD suggests that the mechanism of autophosphorylation is intramolecular. It is interesting that recombinant BRI1-KD was also found to phosphorylate certain synthetic peptides in vitro. To identify possible structural elements required for substrate recognition by BRI1-KD, a series of synthetic peptides were evaluated, indicating that optimum phosphorylation of the peptide required R or K residues at P - 3, P - 4, and P + 5 (relative to the phosphorylated ser at P = 0).

Original languageEnglish (US)
Pages (from-to)751-765
Number of pages15
JournalPlant physiology
Volume124
Issue number2
StatePublished - Jan 1 2000

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Brassinosteroids
brassinosteroids
Threonine
threonine
serine
Serine
phosphotransferases (kinases)
Phosphotransferases
peptides
Peptides
receptors
synthetic peptides
protein phosphorylation
In Vitro Techniques
matrix-assisted laser desorption-ionization mass spectrometry
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Arabidopsis
Leucine
leucine
signal transduction

ASJC Scopus subject areas

  • Physiology
  • Genetics
  • Plant Science

Cite this

Recombinant brassinosteroid insensitive 1 receptor-like kinase autophosphorylates on serine and threonine residues and phosphorylates a conserved peptide motif in vitro. / Oh, M. H.; Ray, W. K.; Huber, Steven C; Asara, J. M.; Gage, D. A.; Clouse, S. D.

In: Plant physiology, Vol. 124, No. 2, 01.01.2000, p. 751-765.

Research output: Contribution to journalArticle

Oh, M. H. ; Ray, W. K. ; Huber, Steven C ; Asara, J. M. ; Gage, D. A. ; Clouse, S. D. / Recombinant brassinosteroid insensitive 1 receptor-like kinase autophosphorylates on serine and threonine residues and phosphorylates a conserved peptide motif in vitro. In: Plant physiology. 2000 ; Vol. 124, No. 2. pp. 751-765.
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