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Plant Physiol, October 2000, Vol. 124, pp. 751-766
Recombinant Brassinosteroid Insensitive 1 Receptor-Like Kinase
Autophosphorylates on Serine and Threonine Residues and Phosphorylates
a Conserved Peptide Motif in Vitro1
Man-Ho
Oh,2
William K.
Ray,
Steven C.
Huber,
John M.
Asara,3
Douglas A.
Gage, and
Steven D.
Clouse*
Department of Horticultural Science (M.H.O., W.K.R., S.D.C.) and
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 Chemistry
(J.M.A.) and Biochemistry (D.A.G.), Michigan State University, East
Lansing, Michigan 48824
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).
1
This work was supported by the National Science
Foundation (Integrative Plant Biology Program), the U.S. Department of
Agriculture National Research Initiative Competitive Grants Program
(Plant Growth and Development), and the North Carolina Agricultural
Research Service.
2
Present Address: Kumho Life and Environment Science
Laboratory, 572 Ssangam-Dong, Kwangsan-Gu Kwangju 506-712, Korea.
3
Present Address: Harvard Microchemistry Facility,
Department of Molecular and Cellular Biology, Harvard University,
Cambridge, MA 02138.
*
Corresponding author; e-mail steve_clouse{at}ncsu.edu; fax
919-515-2505.
© 2000 American Society of Plant Physiologists
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