In Vivo Phosphorylation of Arabidopsis thaliana 6-phosphofructo-2-kinase/fructose-2,6-bisphosphate 2-phosphatase

Abstract

Fructose 2,6-bisphosphate (F2,6BP) is a non-metabolic sugar-phosphate ubiquitously found in eukaryotes. This compound regulates the balance of glycolysis and gluconeogenesis. In plants, its physiological functions are thought to be an inhibition of sucrose synthesis in source leaves and to be an activation of glycolysis in sink organs. Both synthesis and degradation of F2,6BP are catalyzed by a bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphate 2-phosphatase (PFK 2/FBPase 2; EC 2.7.1.105/EC 3.1.3.46). In animals and yeast, it is well known that several isozymes are regulated by protein phosphorylation. In plant enzymes, on the other hand, it had been thought that the protein phosphorylation is not involved in the enzymatic regulation. An Arabidopsis cDNA for PFK 2/FBPase 2 was isolated and expressed in E. coli. Antisera prepared from the recombinant enzyme identified 96-kD and 92-kD protein bands of plant crude protein extracts. Results of in vitro calf intestine alkaline phosphatase treatment and an immunoprecipitation of the crude extracts from [32P]-labeled whole plants revealed that the 96-kD protein was phosphorylated in vivo. Immunoblot analysis showed that the 96-kD protein was enriched in younger leaves than in older leaves. These results indicate that Arabidopsis enzyme is phosphorylated. Now we try to identify the biochemical effects of this phosphorylation, especially whether this phosphorylation can change the enzymatic activities or not.