Snf1 Controls the Activity of Adr1 Through Dephosphorylation of Ser230

Abstract

The transcription factors Adr1 and Cat8 act in concert to regulate the expression of numerous yeast genes after the diauxic shift. Their activities are regulated by Snf1, the yeast homolog of the AMP-activated protein kinase of higher eukaryotes. Cat8 is regulated directly by Snf1, but how Snf1 regulates Adr1 is unknown. Mutations in Adr1 that alleviate glucose repression are clustered between amino acids 227 and 239. This region contains a consensus sequence for protein kinase A, RRAS 230 F, and Ser230 is phosphorylated in vitro by both protein kinase A and Ca 11 calmodulin-dependent protein kinase. Using an antiphosphopeptide antibody, we found that the level of Adr1 phosphorylated on Ser230 was highest in glucose-grown cells and decreased in a Snf1-dependent manner when glucose was depleted. A nonphosphorylatable Ser230Ala mutant was no longer Snf1 dependent for activation of Adr1-dependent genes and could suppress Cat8 dependence at genes coregulated by Adr1 and Cat8. Contrary to expectation, neither protein kinase A (PKA) nor Ca 11 calmodulin-dependent protein kinase appeared to have an important role in Ser230 phosphorylation in vivo, and a screen of 102 viable kinase deletion strains failed to identify a candidate kinase. We conclude that either Ser230 is phosphorylated by multiple protein kinases or its kinase is encoded by an essential gene. Using the Ser230Ala mutant, we explain a long-standing observation of synergy between Adr1 constitutive mutants and Snf1 activation and conclude that dephosphorylation of Ser230 via a Snf1-dependent pathway appears to be a major component of Adr1 regulation.

The AMP-activated Protein Kinase Snf1 Regulates Transcription Factor Binding, RNA Polymerase II Activity, and mRNA Stability of Glucose-repressed Genes in Saccharomyces cerevisiae

“…At the transcriptional level, promoter binding of Adr1 is regulated by Snf1-dependent histone hyperacetylation that occurs when glucose is depleted (20,32). Adr1 binding is also regulated by inhibitory phosphorylation of Ser-98 within its DNA binding domain, the reversal of which is Snf1-dependent (18). Together, these Snf1-dependent modifications allow Adr1 to bind the promoter and recruit RNA pol II when glucose is depleted.…”

“…Snf1 activates Cat8 by direct phosphorylation but activates Adr1 by promoting its dephosphorylation (6,18). Together they activate over 300 target genes involved in metabolic pathways that allow growth in the absence of a fermentable carbon source (25)(26)(27).…”

“…Activation by Adr1 c is due to the inability of 14-3-3 (known as Bmh in yeast) proteins to inhibit the constitutive activators, presumably because Bmh binding requires phosphorylation of Ser-230 (37). Snf1 has a role in overcoming Bmh-mediated inhibition because it promotes dephosphorylation of Ser-230 (18). However, Snf1 has a second role in activating the poised complex because the two effects, constitutively active Adr1 c and activated Snf1, are synergistic with regard to gene expression (38).…”

Transcriptional Regulation inSaccharomyces cerevisiae: Transcription Factor Regulation and Function, Mechanisms of Initiation, and Roles of Activators and Coactivators

“…The AMP-activated, protein kinase homolog Snf1 is essential for promoter binding of Adr1, Adr1-dependent chromatin remodeling, and transcription when glucose is exhausted (Di Mauro et al 2002;Young et al 2002;Agricola et al 2004Agricola et al , 2006Biddick et al 2008a,b). Glucose repression inhibits Adr1 activity by multiple mechanisms, including ADR1 expression (Blumberg et al 1988), DNA binding (Sloan et al 1999;Kacherovsky et al 2008), and transcription activation (Cherry et al 1989;Cook et al 1994;Tachibana et al 2007;Ratnakumar et al 2009). PKA phosphorylates S230 in the regulatory domain in vitro (Denis et al 1992), but its activity is not required for phosphorylation (Ratnakumar et al 2009) or glucose inhibition of Adr1 activity in vivo (Denis et al 1992;Dombek et al 1993).…”

“…As illustrated in Figure 2, Bmh has direct and indirect roles in Adr1 regulation, and both roles involve Snf1. Identifying the S230 kinase and the mechanism whereby Snf1 promotes its dephosphorylation (Ratnakumar et al 2009) might link Adr1 activity to other nutrient-signaling pathways.…”