Snf1 protein kinase regulates phosphorylation of the Mig1 repressor in Saccharomyces cerevisiae.
In glucose-grown cells, the Mig1 DNA-binding protein recruits the Ssn6-Tup1 corepressor to glucose-repressed promoters in the yeast Saccharomyces cerevisiae. Previous work showed that Mig1 is differentially phosphorylated in response to glucose. Here we examine the role of Mig1 in regulating repression and the role of the Snf1 protein kinase in regulating Mig1 function. Immunoblot analysis of Mig1 protein from a snf1 mutant showed that Snf1 is required for the phosphorylation of Mig1; moreover, hxk2 and reg1 mutations, which relieve glucose inhibition of Snf1, correspondingly affect phosphorylation of Mig1. We show that Snf1 and Mig1 interact in the two-hybrid system and also coimmunoprecipitate from cell extracts, indicating that the two proteins interact in vivo. In immune complex assays of Snf1, coprecipitating Mig1 is phosphorylated in a Snf1-dependent reaction. Mutation of four putative Snf1 recognition sites in Mig1 eliminated most of the differential phosphorylation of Mig1 in response to glucose in vivo and improved the two-hybrid interaction with Snf1. These studies, together with previous genetic findings, indicate that the Snf1 protein kinase regulates phosphorylation of Mig1 in response to glucose.
Treitel
M A
MA
Departments of Genetics and Development, Columbia University, New York, New York 10032, USA.
Kuchin
S
S
Carlson
M
M
D001426:Bacterial Proteins / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D005656:Fungal Proteins / Q000378:metabolism / Q000502:physiology; D017930:Genes, Reporter; D005947:Glucose / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009687:Nuclear Proteins; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism* / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases / Q000235:genetics; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D005947:Glucose; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein; C065455:CYC8 protein, S cerevisiae; C085976:MIG1 protein, S cerevisiae; C071570:SNF1-related protein kinases