Expression, Activation, and Biochemical Properties of a Novel Arabidopsis Protein Kinase1
AUTOR(ES)
Gong, Deming
FONTE
American Society of Plant Physiologists
RESUMO
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. PKS6ΔF) or a substitution of threonine-178 with aspartic acid within the putative activation loop. We found that PKS6ΔF exhibited a strong preference for Mn2+ over Mg2+ as a divalent cation cofactor for kinase activity. PKS6ΔF 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 PKS6ΔF 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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=155886Documentos Relacionados
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