The role of the cyclic AMP-dependent protein kinase in the glucagon-stimulated phosphorylation of ATP-citrate lyase.
We have examined the mechanism whereby glucagon stimulates the phosphorylation of ATP-citrate lyase in intact rat hepatocytes. Purified ATP-citrate lyase is phosphorylated in vitro by the catalytic subunit of the cyclic AMP-dependent protein kinase, in a reaction wherein 2-3 mol phosphate/mol lyase are incorporated, at an initial rate that approaches that observed for mixed histone. This reaction is completely abolished by the protein kinase inhibitor protein. Limited tryptic digestion of ATP-citrate lyase phosphorylated in vitro by the cyclic AMP-dependent protein kinase yields a pattern of 32P-labeled peptides, indistinguishable from those observed in parallel digests of lyase isolated from 32P-labeled, glucagon-stimulated hepatocytes. Phosphorylase b kinase catalyzes the incorporation of 1 mol phosphate/mol lyase, albeit at less than 1/160 the rate observed for phosphorylase b. The phosphorylation of purified ATP-citrate lyase is also catalyzed by homogenates of hepatocytes. This reaction is stimulated by cyclic AMP. At 30 degrees C, in the presence of maximally stimulating concentrations of cyclic AMP, the addition of excess protein kinase inhibitor protein inhibits the phosphorylation of ATP-citrate lyase by 67%. Thus, hepatocytes contain both cyclic AMP-dependent and cyclic AMP-independent ATP-citrate lyase kinase activities. Pretreatment of hepatocytes with glucagon (10(-8) M for 2 min) prior to homogenization results in activation of an endogenous hepatocyte ATP-citrate lyase kinase, as well as histone kinase and phosphorylase b kinase; the glucagon-stimulated increment in lyase kinase (and histone kinase) is observed only when homogenates are assayed in the absence of added cyclic AMP, and is completely abolished by an excess of the protein kinase inhibitor protein. We conclude that the glucagon-stimulated phosphorylation of ATP-citrate lyase in intact hepatocytes is catalyzed directly by the cyclic AMP-dependent protein kinase.
D001275:ATP Citrate (pro-S)-Lyase / Q000378:metabolism*; D000818:Animals; D000242:Cyclic AMP / Q000494:pharmacology*; D005934:Glucagon / Q000494:pharmacology*; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008297:Male; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats
D005934:Glucagon; D000242:Cyclic AMP; D001275:ATP Citrate (pro-S)-Lyase; D011494:Protein Kinases