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 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).
Oh
M H
MH
Department of Horticultural Science, North Carolina State University, Raleigh, North Carolina 27695, USA.
Ray
W K
WK
Huber
S C
SC
Asara
J M
JM
Gage
D A
DA
Clouse
S D
SD
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010840:Phytosterols / Q000378:metabolism; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013912:Threonine / Q000737:chemistry
D029681:Arabidopsis Proteins; D010840:Phytosterols; D011994:Recombinant Proteins; D013912:Threonine; D012694:Serine; D011494:Protein Kinases; C108270:BRI1 protein, Arabidopsis