Snf1 controls the activity of adr1 through dephosphorylation of Ser230.
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(++) 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(++) 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.
Ratnakumar
Sooraj
S
Department of Biochemistry, University of Washington, Seattle, Washington 98195-7350, USA.
Kacherovsky
Nataly
N
Arms
Erin
E
Young
Elton T
ET
D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D005838:Genotype; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D054645:Protein Phosphatase 1 / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
C074682:ADR1 protein, S cerevisiae; C092600:CAT8 protein, S cerevisiae; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D012694:Serine; C071570:SNF1-related protein kinases; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D054645:Protein Phosphatase 1; C070829:REG1 protein, S cerevisiae; D005947:Glucose