First published online March 22, 2002; 10.1104/pp.010776
Plant Physiol, May 2002, Vol. 129, pp. 225-234
Expression, Activation, and Biochemical Properties of a Novel
Arabidopsis Protein Kinase1
Deming
Gong,
Zhizhong
Gong,
Yan
Guo, and
Jian-Kang
Zhu*
Department of Plant Sciences, University of Arizona, Tucson,
Arizona 85721
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.
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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