Recombinant Brassinosteroid Insensitive 1 Receptor-Like Kinase Autophosphorylates on Serine and Threonine Residues and Phosphorylates a Conserved Peptide Motif in Vitro

Author:

Oh Man-Ho,Ray William K.1,Huber Steven C.2,Asara John M.3,Gage Douglas A.4,Clouse Steven D.1

Affiliation:

1. Department of Horticultural Science (M.H.O., W.K.R., S.D.C.) and

2. United States Department of Agriculture/Agricultural Research Service and Department of Crop Science (S.C.H.), North Carolina State University, Raleigh, North Carolina 27695; and Departments of

3. Chemistry (J.M.A.) and

4. Biochemistry (D.A.G.), Michigan State University, East Lansing, Michigan 48824

Abstract

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 atP − 3, P − 4, andP + 5 (relative to the phosphorylated Ser atP = 0).

Publisher

Oxford University Press (OUP)

Subject

Plant Science,Genetics,Physiology

Reference51 articles.

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