Regulation of 6-phosphofructo-2-kinase activity by cyclic AMP-dependent phosphorylation.

Abstract

Addition of glucagon to isolated rat hepatocytes resulted in inhibition of 6-phosphofructo-2-kinase (ATP:D-fructose-6-phosphate-2-phosphotransferase) activity in extracts of the cells and in a decrease in the intracellular level of fructose 2,6-bisphosphate. The effect on 6-phosphofructo-2-kinase was characterized by a decrease in the affinity of the enzyme for fructose 6-phosphate. To investigate the mechanism of action of glucagon, 6-phosphofructo-2-kinase from rat liver was partially purified by polyethylene glycol precipitation, DEAE-cellulose chromatography, (NH4)2SO4 fractionation, Sephacryl S-200 gel filtration, DEAE-Sephadex chromatography and Sephadex G-100 gel filtration. Incubation of the purified enzyme with the catalytic subunit of the cAMP-dependent protein kinase from rat liver and [.gamma.32P]ATP resulted in 32P incorporation into a protein with a subunit MW 49,000 as determined by NaDodSO4 disc gel electrophoresis. Associated with this phosphorylation was an inhibition of 6-phosphofructo-2-kinase activity that was also characterized by a decrease in the affinity of the enzyme for fructose 6-phosphate. Both the phosphorylation and the inhibition of the purified 6-phosphofructo-2-kinase were blocked by addition of the heat-stable protein kinase inhibitor. The glucagon-induced decrease in fructose 2,6-bisphosphate levels observed in isolated hepatocytes is due, at least in part, to cAMP-dependent phosphorylation and inhibition of 6-phosphofructo-2-kinase.