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

BRASSINOSTEROID-INSENSITIVE 1 (BRI1) encodes a putative Leucine-rich repeat receptor kinase in Arabidopsis that has been shown by genetic and molecula r analysis to be a critical component of brassinosteroid signal transductio n. 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 i n vivo function. Recombinant BRI1-KD autophosphorylated on serine (Ser) and threonine (Thr) residues with p-Ser predominating. Matrix-assisted laser d esorption/ionization mass spectrometry identified a minimum of 12 sites of autophosphorylation in the cytoplasmic domain of BRI1, including five in th e juxtamembrane region (N-terminal to the catalytic KD), five in the KD ton e each in sub-domains I and VIa and three in sub-domain VIII), and two in t he 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 resi dues within these peptides. The inability of an active BRI1-KD to transphos phorylate an inactive mutant KD suggests that the mechanism of autophosphor ylation is intramolecular. It is interesting that recombinant BRI1-KD was a lso 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 ph osphorylation of the peptide required R or K residues at P - 3, P - 4, and P + 5 (relative to the phosphorylated Ser at P = 0).