Regulation of a plant SNF1-related protein kinase by glucose-6-phosphate.
One of the major protein kinases (PK(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 PK(III) activity. Using the continuous assay, along with the fixed-time-point (32)P-incorporation assay, we demonstrate that PK(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 Mg(2+) in the assay from 10 to 2 mM. Under likely physiological conditions (pH 7.0 and 2 mM Mg(2+)), 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 PK(III) in the following order: Glc-6-P > mannose-6-P, fructose-1,6P(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 PK(III) activity potentially provides a mechanism for metabolic regulation of the reactions catalyzed by these important protein kinases.
Toroser
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United States Department of Agriculture, Agricultural Research Service, and Departments of Crop Science and Botany, North Carolina State University, Raleigh 27695-7631, USA.
Plaut
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Huber
S C
SC
D001464:Barium / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D001464:Barium; D019298:Glucose-6-Phosphate; C071570:SNF1-related protein kinases; D017346:Protein Serine-Threonine Kinases; D008274:Magnesium