Carbon Source-Dependent Phosphorylation of Hexokinase PII and Its Role in the Glucose-Signaling Response in Yeast

Abstract

The HXK2 gene is required for a variety of regulatory effects leading to an adaptation for fermentative metabolism in Saccharomyces cerevisiae. However, the molecular basis of the specific role of Hxk2p in these effects is still unclear. One important feature in order to understand the physiological function of hexokinase PII is that it is a phosphoprotein, since protein phosphorylation is essential in most metabolic signal transductions in eukaryotic cells. Here we show that Hxk2p exists in vivo in a dimeric-monomeric equilibrium which is affected by phosphorylation. Only the monomeric form appears phosphorylated, whereas the dimer does not. The reversible phosphorylation of Hxk2p is carbon source dependent, being more extensive on poor carbon sources such as galactose, raffinose, and ethanol. In vivo dephosphorylation of Hxk2p is promoted after addition of glucose. This effect is absent in glucose repression mutants cat80/grr1, hex2/reg1, and cid1/glc7. Treatment of a glucose crude extract from cid1-226 (glc7-T152K) mutant cells with -phosphatase drastically reduces the presence of phosphoprotein, suggesting that CID1/GLC7 phosphatase together with its regulatory HEX2/REG1 subunit are involved in the dephosphorylation of the Hxk2p monomer. An HXK2 mutation encoding a serine-to-alanine change at position 15 [HXK2 (S15A)] was to clarify the in vivo function of the phosphorylation of hexokinase PII. In this mutant, where the Hxk2 protein is unable to undergo phosphorylation, the cells could not provide glucose repression of invertase. Glucose induction of HXT gene expression is also affected in cells expressing the mutated enzyme. Although we cannot rule out a defect in the metabolic state of the cell as the origin of these phenomena, our results suggest that the phosphorylation of hexokinase is essential in vivo for glucose signal transduction.

Very low amounts of glucose cause repression of the stress-responsive gene HSP12 in Saccharomyces cerevisiae

“…However, an alternative interpretation of our findings could be that binding of the substrate glucose to hexokinase may induce a regulatory function of this protein. Indeed, a regulatory role for Hxk2p has been postulated previously (De Winde et al, 1996 ;Herrero et al, 1998 ;Randezgil et al, 1998). Therefore, we aimed to confirm the importance of sugar phosphorylation for signalling, by using a strain in which both HXK1 and HXK2 are deleted.…”

Functional Diversity of Protein Phosphatase-1, a Cellular Economizer and Reset Button

“…The Snf1 kinase then phosphorylates Reg1. The phosphorylation of Reg1 is antagonized by Reg1-associated PP1, but at low glucose concentrations, the balance is tipped in favor of a net phosphorylation of Reg1 by the hexokinase Hxk2, which is itself phosphorylated on Ser-15 in these conditons (299). Hxk2 interacts (weakly) with both Snf1 and Reg1, but it is not clear yet whether Hxk2 promotes the phosphorylation of Reg1 by the stimulation of Snf1 and/or by the inhibition of the associated PP1 (313).…”

Glucose Signaling in Saccharomyces cerevisiae

“…The exact opposite result (intact glucose signaling and low catalytic activity), however, was obtained by others upon deletion of an overlapping and slightly larger Hxk2 segment (residues 1 through 15) (231). Likewise, the phosphorylatable Ser14 residue in Hxk2 (193) is reported either to mediate (305) or not to mediate (146,231) glucose repression. Further analysis is needed to resolve these discrepancies.…”