Plant Physiology Preview
Published on August 8, 2002; 10.1104/pp.004507
Received February 18, 2002
Returned for revision April 17, 2002
Accepted May 16, 2002
Biochemical Characterization of the Arabidopsis Protein Kinase SOS2 That Functions in Salt Tolerance
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.)
* Corresponding author; email: jkzhu{at}ag.arizona.edu.
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 an 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.
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