Phosphorylation and inactivation of yeast 6-phosphofructo-2-kinase contribute to the regulation of glycolysis under hypotonic stress

Phosphorylation of yeast 6-phosphofructo-2-kinase and its role for the regu lation of glycolysis under hypoosmotic conditions were investigated. 6-Phos phofructo-2-kinase was found to be phosphorylated in vitro by protein kinas e C at serine 652 and thereby inactivated. Protein phosphatase 2A reversed the phosphorylative inhibition of the enzyme. When yeast cells were shifted to hypotonic media, 6-phosphofructo-2-kinase was found to be phosphorylate d and inactivated. Under in vivo conditions, two phosphate residues were in corporated into the enzyme. One of them is bound to serine 652, indicating that this modification was probably caused by yeast protein kinase Cl. The second phosphate is bound to Ser8 within the N-terminal peptide T1-41 which contains several serine residues but no protein kinase C recognition seque nce. Site-directed mutagenesis confirmed that the phosphorylation of serine 652 but not the N-terminal modification is responsible for the in vivo ina ctivation of 6-phosphofructo-2-kinase. The obtained results suggest that th e phosphorylation of 6-phosphofructo-2-kinase mediates a response of the ce lls to an activation of the hypoosmolarity MAP kinase pathway. Via a suppre ssion of glycolysis, the inactivation of 6-phosphofructo-2-kinase is expect ed to be responsible for the observed accumulation of glucose 6-phosphate, an essential precursor of the cell wall glucans, and the decrease of glycer ol, an important osmolyte.