Phosphorylation and Inactivation of Yeast 6-Phosphofructo-2-kinase Contribute to the Regulation of Glycolysis under Hypotonic Stress

Abstract

Phosphorylation of yeast 6-phosphofructo-2-kinase and its role for the regulation of glycolysis under hypoosmotic conditions were investigated. 6-Phosphofructo-2-kinase was found to be phosphorylated in vitro by protein kinase 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 phosphorylated and inactivated. Under in vivo conditions, two phosphate residues were incorporated into the enzyme. One of them is bound to serine 652, indicating that this modification was probably caused by yeast protein kinase C1. The second phosphate is bound to Ser8 within the N-terminal peptide T(1-41) which contains several serine residues but no protein kinase C recognition sequence. Site-directed mutagenesis confirmed that the phosphorylation of serine 652 but not the N-terminal modification is responsible for the in vivo inactivation of 6-phosphofructo-2-kinase. The obtained results suggest that the phosphorylation of 6-phosphofructo-2-kinase mediates a response of the cells to an activation of the hypoosmolarity MAP kinase pathway. Via a suppression of glycolysis, the inactivation of 6-phosphofructo-2-kinase is expected to be responsible for the observed accumulation of glucose 6-phosphate, an essential precursor of the cell wall glucans, and the decrease of glycerol, an important osmolyte.

High Osmolarity Glycerol (HOG) Pathway-induced Phosphorylation and Activation of 6-Phosphofructo-2-kinase Are Essential for Glycerol Accumulation and Yeast Cell Proliferation under Hyperosmotic Stress

“…The monofunctional PFK2 can be either activated or inactivated by in vivo phosphorylation (7,11). We have shown earlier that phosphorylation at Ser 652 by PKC under hyposmotic stress inactivates PFK2 (11).…”

“…The monofunctional PFK2 can be either activated or inactivated by in vivo phosphorylation (7,11). We have shown earlier that phosphorylation at Ser 652 by PKC under hyposmotic stress inactivates PFK2 (11). On the other hand, glucose induction of yeast cells activates PFK2 by a single phosphorylation at Ser 644 and a multiple phosphorylation within the peptide T 67-101 (10).…”

“…The phosphorylation and hence inactivation of PFK2 are under control of the PKC MAPK pathway and reduce the rate of glycolysis, leading to an accumulaton of glucose 6-phosphate (G-6-P) (11).…”

An integrated view on a eukaryotic osmoregulation system

“…Fru-2.6-bP is produced by a dedicated phosphofructo-2-kinase encoded by the genes PFK26 and PFK27 (Boles et al 1996). Pfk26 appears to be the target of a number of different protein kinase such as protein kinase A, the Slt2 MAPK and Hog1 (Dihazi et al 2001(Dihazi et al , 2003(Dihazi et al , 2004. Stimulation of Pfk26 by Hog1 has the potential to increase glycolytic flux.…”

Genome-wide Analysis of the Response to Cell Wall Mutations in the Yeast Saccharomyces cerevisiae

“…Another interesting example is the transcriptional activation of PFK26 and FBP26 encoding enzymes that are respectively responsible for the synthesis and the degradation of fructose 2,6-bisphosphate. This metabolite is an important biofactor in promoting glycolysis and inhibiting gluconeogenesis in mammals (59), whereas such a role in yeast is less apparent (60 Pkc1p-dependent phosphorylation (61). It was then proposed that this inactivation could lead to a reduction of glycolytic flux in favor of a redirection of glucose 6-phosphate to cell wall ␤-glucan synthesis.…”