Journal Article

Regulation of a Plant SNF1-Related Protein Kinase by Glucose-6-Phosphate

Huber, Steven C., Zvi Plaut, Dikran Toroser

2000

Overview

One of the major protein kinases ($\\text{PK}_{\\text{III}}$) 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 $\\text{PK}_{\\text{III}}$ activity. Using the continuous assay, along with the fixed-time-point 32P-incorporation assay, we demonstrate that $\\text{PK}_{\\text{III}}$ 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 $\\text{PK}_{\\text{III}}$ in the following order: Glc-6-P > mannose-6-P, fructose-1,$6\\text{P}_{2}$ > ribose-5-P, 3-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 $\\text{PK}_{\\text{III}}$ activity potentially provides a mechanism for metabolic regulation of the reactions catalyzed by these important protein kinases.

• Publisher: American Society of Plant Physiologists,American Society of Plant Biologists
• Subject: Agriculture / Agronomy. Soil science and plant productions / amino acid sequences / antagonists & inhibitors / assays / Barium / Barium - metabolism / BASIC BIOLOGICAL SCIENCES / Biological and medical sciences / Cell Biology and Signal Transduction / Chromatography / Commercial regulation / Contaminants / Economic plant physiology / Enzymatic activity / ENZYME ACTIVITY / Enzymes / enzymology / Fluorescence / fructose / fructose 6-phosphate / Fundamental and applied biological sciences. Psychology / GENE REGULATION / GLUCOSE / Glucose-6-Phosphate / Glucose-6-Phosphate - pharmacology / hexosyltransferases / Hydrogen-Ion Concentration / INHIBITION / isolation & purification / isotope labeling / Magnesium / Magnesium - metabolism / mannose / Metabolism / Metabolites / Nutrition. Photosynthesis. Respiration. Metabolism / pharmacology / Phosphatases / PHOSPHATES / PHOSPHORYLATION / PHOSPHOTRANSFERASES / Plant physiology and development / Plants / Plants - enzymology / Protein metabolism / Protein Serine-Threonine Kinases / Protein-Serine-Threonine Kinases - antagonists & inhibitors / Protein-Serine-Threonine Kinases - isolation & purification / Protein-Serine-Threonine Kinases - metabolism / ribose / Spinach / Spinacia oleracea / Substrate Specificity