Expression, activation, and biochemical properties of a novel Arabidopsis protein kinase.
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. PKS6deltaF) or a substitution of threonine-178 with aspartic acid within the putative activation loop. We found that PKS6deltaF exhibited a strong preference for Mn2+ over Mg2+ as a divalent cation cofactor for kinase activity. PKS6DeltaF displayed substrate specificity against three different peptide substrates and had an optimal pH of approximately 7.5 and temperature optimum of 30 degrees C. The apparent Km values for ATP and the preferred peptide substrate p3 of PKS6deltaF were determined to be 1.7 and 28.5 microM, 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.
Gong
Deming
D
Department of Plant Sciences, University of Arizona, Tucson, Arizona 85721, USA.
Gong
Zhizhong
Z
Guo
Yan
Y
Zhu
Jian-Kang
JK
D000040:Abscisic Acid / Q000494:pharmacology; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005638:Fruit / Q000201:enzymology / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics; D018547:Plant Stems / Q000201:enzymology / Q000235:genetics; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D029681:Arabidopsis Proteins; D011993:Recombinant Fusion Proteins; D000040:Abscisic Acid; D005982:Glutathione Transferase; D011494:Protein Kinases; C462023:PKS6 protein, Arabidopsis; C406738:SOS2 protein, Arabidopsis; D017346:Protein Serine-Threonine Kinases