Regulation of a plant SNF1-related protein kinase by glucose-6-phosphate

One of the major protein kinases (PKIII) that phosphorylates serine-158 of spinach sucrose-phosphate synthase (SPS), which is responsible for light/dark modulation of activity, is known to be a member of the SNF1 related family of protein kinases. In the present study, we have developed a fluorescence-based continuous assay for measurement of PKIII activity. Using the continuous assay, along with the fixed-time-point P-32 incorporation assay, we demonstrate that PKIII activity is inhibited by glucose-6-phosphate (Glc-6-P). Relative inhibition by Glc-6-P was increased by decreasing pH from 8.5 to 5.5 and by reducing the concentration of Mg2+ in the assay from 10 to 2 mM. Under likely physiological conditions (pH 7.0 and 2 mM Mg2+), 10 mM Glc-6-P inhibited kinase activity approximately 70%. Inhibition by Glc-6-P could not be ascribed to contaminants in the commercial preparations. Other metabolites inhibited PKIII in the following order: Glc-6-P > mannose-6-P, fructose-1,6P(2) > ribose-5-P, 5-PGA, fructose-6-P. Inorganic phosphate, Glc, and AMP were not inhibitory, and free Glc did not reverse the inhibition by Glc-6-P. Because SNF1-related protein kinases are thought to function broadly in the regulation of enzyme activity and gene expression, Glc-6-P inhibition of PKIII activity potentially provides a mechanism for metabolic regulation of the reactions catalyzed by these important protein kinases.