First published online August 8, 2002; 10.1104/pp.004507
Plant Physiol, September 2002, Vol. 130, pp. 256-264
Biochemical Characterization of the Arabidopsis Protein Kinase
SOS2 That Functions in Salt Tolerance1
Deming
Gong,
Yan
Guo,
Andre T.
Jagendorf, and
Jian-Kang
Zhu*
Department of Plant Sciences, University of Arizona, Tucson,
Arizona 85721 (D.G., Y.G., J.-K.Z.); and Department of Plant Biology,
Cornell University, Ithaca, New York 14853 (A.T.J.)
The Arabidopsis Salt Overly Sensitive 2
(SOS2) gene encodes a serine/threonine (Thr) protein
kinase that has been shown to be a critical component of the salt
stress signaling pathway. SOS2 contains a sucrose-non-fermenting
protein kinase 1/AMP-activated protein kinase-like N-terminal catalytic
domain with an activation loop and a unique C-terminal regulatory
domain with an FISL motif that binds to the calcium sensor
Salt Overly Sensitive 3. In this study, we examined some
of the biochemical properties of the SOS2 in vitro. To determine its
biochemical properties, we expressed and isolated a number of active
and inactive SOS2 mutants as glutathione S-transferase
fusion proteins in Escherichia coli. Three
constitutively active mutants, SOS2T168D, SOS2T168D F, and
SOS2T168D308, were obtained previously, which contain either the
Thr-168 to aspartic acid (Asp) mutation in the activation loop or
combine the activation loop mutation with removal of the FISL motif or
the entire regulatory domain. These active mutants exhibited a
preference for Mn2+ relative to Mg2+ and could
not use GTP as phosphate donor for either substrate phosphorylation or
autophosphorylation. The three enzymes had similar peptide substrate
specificity and catalytic efficiency. Salt overly sensitive 3 had
little effect on the activity of the activation loop mutant SOS2T168D,
either in the presence or absence of calcium. The active mutant
SOS2T168D308 could not transphosphorylate an inactive protein
(SOS2K40N), which indicates an intramolecular reaction mechanism of
SOS2 autophosphorylation. Interestingly, SOS2 could be activated not
only by the Thr-168 to Asp mutation but also by a serine-156 or
tyrosine-175 to Asp mutation within the activation loop. Our results
provide insights into the regulation and biochemical properties of SOS2
and the SOS2 subfamily of protein kinases.
1
This work was supported by the National
Institutes of Health (grant no. R01GM59138 to J.-K.Z.).
*
Corresponding author; e-mail jkzhu{at}ag.arizona.edu; fax
520-621-7186.
© 2002 American Society of Plant Physiologists
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