Regulatory Interactions between the Reg1-Glc7 Protein Phosphatase and the Snf1 Protein Kinase

Abstract

Protein phosphatase 1, comprising the regulatory subunit Reg1 and the catalytic subunit Glc7, has a role in glucose repression in Saccharomyces cerevisiae. Previous studies showed that Reg1 regulates the Snf1 protein kinase in response to glucose. Here, we explore the functional relationships between Reg1, Glc7, and Snf1. We show that different sequences of Reg1 interact with Glc7 and Snf1. We use a mutant Reg1 altered in the Glc7-binding motif to demonstrate that Reg1 facilitates the return of the activated Snf1 kinase complex to the autoinhibited state by targeting Glc7 to the complex. Genetic evidence indicated that the catalytic activity of Snf1 negatively regulates its interaction with Reg1. We show that Reg1 is phosphorylated in response to glucose limitation and that this phosphorylation requires Snf1; moreover, Reg1 is dephosphorylated by Glc7 when glucose is added. Finally, we show that hexokinase PII (Hxk2) has a role in regulating the phosphorylation state of Reg1, which may account for the effect of Hxk2 on Snf1 function. These findings suggest that the phosphorylation of Reg1 by Snf1 is required for the release of Reg1-Glc7 from the kinase complex and also stimulates the activity of Glc7 in promoting closure of the complex.

Life in the midst of scarcity: adaptations to nutrient availability in Saccharomyces cerevisiae

“…When exponentially growing yeast cells are transferred to medium containing glucose but missing an essential nutrient such as nitrogen or phosphate, they arrest growth and enter the G 0 state due to nutrient deprivation (light line) http://doc.rero.ch (Hedbacker et al 2004;Kim et al 2005;McCartney et al 2005). Dephosphorylation of Snf1 is mediated by the protein phosphatase complex Glc7-Reg1 (Ludin et al 1998;Sanz et al 2000;McCartney and Schmidt 2001). Glc7 is the catalytic subunit of the phosphatase complex, whereas Reg1 is the regulatory subunit that targets the phosphatase to Snf1 since it can bind to the catalytic domain of the protein kinase.…”

“…However, gluconeogenic genes remain repressed in a reg1D strain, while genes involved in the utilization of alternative carbon sources are derepressed (Schuller 2003). The interaction between Glc7-Reg1 and the Snf1 kinase complex is stimulated upon glucose resupplementation, which was proposed to facilitate the fast dephosphorylation and subsequent inactivation of Snf1 by the phosphatase complex (Sanz et al 2000). In support of such a crucial role for the phosphatase in the regulation of Snf1 activity, it was recently reported that the dephosphorylation rate of Snf1 is more subject to glucose regulation than the phosphorylation rate (Rubenstein et al 2008).…”

“…Two other hexokinases, Hxk1 and Glk1, can also catalyse this reaction, but Hxk2 is believed to be the crucial hexokinase during growth on glucose, since Hxk1 and Glk1 are themselves subject to glucose repression (De Winde et al 1996). It was reported that in a hxk2D mutant, the glucose repression of several genes was severely reduced (Zimmermann and Scheel 1977;Entian 1980;Entian and Zimmermann 1980), the interaction between Snf1 and Snf4 was increased (Jiang and Carlson 1996;Sanz et al 2000) and that Snf1 still phosphorylated Mig1 in the presence of glucose (Treitel et al 1998;Ahuatzi et al 2007). Early reports suggested that the role of Hxk2 in glucose repression was limited to its metabolic role (Ma et al 1989;Rose et al 1991).…”

“…More recently, however, mutants were isolated with a distinct effect on catalytic activity and glucose signalling (Hohmann et al 1999;Kraakman et al 1999b;Mayordomo and Sanz 2001), indicative for a more specific role of Hxk2 in glucose repression. Since it was shown that overexpression of REG1 rescued defects in glucose repression due to deletion of HXK2 (Sanz et al 2000), one hypothesis states that Hxk2 exerts its regulatory role in glucose signalling via stimulation of the Glc7-Reg1 phosphatase complex. However, no direct interaction between Reg1 and Hxk2 could be detected (Sanz et al 2000), and other findings indicate that Hxk2 interferes further downstream with the glucose repression pathway.…”

“…Since it was shown that overexpression of REG1 rescued defects in glucose repression due to deletion of HXK2 (Sanz et al 2000), one hypothesis states that Hxk2 exerts its regulatory role in glucose signalling via stimulation of the Glc7-Reg1 phosphatase complex. However, no direct interaction between Reg1 and Hxk2 could be detected (Sanz et al 2000), and other findings indicate that Hxk2 interferes further downstream with the glucose repression pathway. A small portion of Hxk2 is located within the nucleus and appears to interact with the transcriptional repressor Mig1 (Herrero et al 1998;Randez-Gil et al 1998;Ahuatzi et al 2004).…”