Identification of L-type pyruvate kinase as a major phosphorylation site of endogenous cyclic AMP-dependent protein kinase in rat liver soluble fraction

Abstract

The mechanism by which hormones such as glucagon and catecholamines stimulate gluconeogenesis in liver has as yet not been elucidated. Injection of glucagon or epinephrine has been reported to lead to a lowered activity of the glycolytic enzymes phosphofructokinase and pyruvate kinase, whereas the gluconeogenic enzyme fructose-l ,6-diphosphatase shows an increased activity; all enzymes being measured under one particular condition [ 1,2]. The mechanism of these changes in enzyme activity has not been investigated. The most well-known action of glucagon on liver cells is the stimulation of cyclic AMP-dependent protein kinases. It is obvious to explain the action of glucagon on gluconeogensis by phosphorylation of the above-mentioned enzymes which results in a change of enzyme activity. This seems indeed to be the case for pyruvate kinase, as judged from in vitro studies (reviewed in [3]) and from in vivo experiments [4]. Phosphorylation of purified phosphofructokinase [S] and fructose-l ,6diphosphatase [6] with concurrent change in enzyme activity has been reported. Experimental evidence for in vivo regulation of these enzymes by phosphorylation-dephosphorylation reaction is up till now absent. Phosphorylation of rat liver cell sap [779] or rat hepatocyte soluble proteins [lo] has been studied in the presence of added exogenous protein kinase. The effect of glucagon and catecholamines on the phosphorylation of supernatant proteins has been studied [lo] in intact hepatocytes where endogenous protein kinase was used. Although it was not proven, pyruvate kinase was indicated as one of the proteins of which the phosphorylation is greatly stimulated by these hormones. No evidence for a stimulated phosphorylation of fructose-l ,6-diphosphatase was found. We investigated the cyclic AMP-dependent phosphorylation of rat liver soluble proteins by endogenous protein kinase(s). Phosphorylated proteins were analysed by SDS-polyacrylamide gel electrophoresis and subsequent autoradiography. With this system we found that L-type pyruvate kinase is the major protein which incorporates 32P from [y-32P]ATP in a cyclic AMP-stimulated reaction. The phosphorylation of pyruvate kinase is greatly diminished in the presence of the substrate phosphoenolpyruvate and not influenced by the allosteric activator fructose-l ,6-diphosphate. Phosphorylation of fructose-l ,6-diphosphatase was not detectable in this system.