Expression, activation, and biochemical properties of a
novel Arabidopsis protein kinase.
Gong D, Gong Z, Guo Y, Zhu JK.
Department of Plant Sciences, University of Arizona, Tucson, Arizona 85721, USA.
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.