Plant Physiology Preview
Published on March 22, 2002; 10.1104/pp.010776
Received August 23, 2001
Returned for revision December 11, 2001
Accepted January 15, 2002
Expression, Activation, and Biochemical Properties of a Novel
Arabidopsis Protein Kinase
Deming Gong , Zhizhong Gong , and Jian-Kang Zhu *
Department of Plant Sciences, University of Arizona, Tucson, Arizona 85721
* Corresponding author; email: jkzhu{at}ag.arizona.edu.
An Arabidopsis SOS2 (salt overly sensitive 2)-like protein kinase gene, PKS6, was expressed in leaves, stems, and siliques, but not detectable in roots of adult plants; its expression in young seedlings was up-regulated by abscisic acid. To determine the biochemical properties of the PKS6 protein, we expressed the PKS6 coding sequence as a glutathione S-transferase fusion protein in Escherichia coli. The bacterially expressed glutathione S-transferase-PKS6 fusion protein was inactive in substrate phosphorylation. We have constructed constitutively active forms of PKS6 by either a deletion of its putative auto-inhibitory FISL motif (i.e. PKS6 F) or a substitution of threonine-178 with aspartic acid within the putative activation loop. We found that PKS6F exhibited a strong preference for Mn2+ over Mg2+ as a divalent cation cofactor for kinase activity. PKS6F displayed substrate specificity against three different peptide substrates and had an optimal pH of approximately 7.5 and temperature optimum of 30°C. The apparent Km values for ATP and the preferred peptide substrate p3 of PKS6F were determined to be 1.7 and 28.5 µM, respectively. These results provide significant insights into the regulation and biochemical properties of the protein kinase PKS6. In addition, the constitutively active, gain-of-function kinase mutants will be invaluable for future determination of the in planta function of PKS6.
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