Promoter binding by the Adr1 transcriptional activator may be regulated by phosphorylation in the DNA-binding region

PLoS One. 2008 Sep 15;3(9):e3213. doi: 10.1371/journal.pone.0003213.

Abstract

Background: Post-translational modification regulates promoter-binding by Adr1, a Zn-finger transcriptional activator of glucose-regulated genes. Support for this model includes the activation of an Adr1-dependent gene in the absence of Adr1 protein synthesis, and a requirement for the kinase Snf1 for Adr1 DNA-binding. A fusion protein with the Adr1 DNA-binding domain and a heterologous activation domain is glucose-regulated, suggesting that the DNA binding region is the target of regulation.

Methodology/principal findings: Peptide mapping identified serine 98 adjacent to the Zn-fingers as a phosphorylation site. An antibody specific for phosphorylated serine 98 on Adr1 showed that the level of phosphorylated Adr1 relative to the level of total Adr1 decreased with glucose derepression, in a Snf1-dependent manner. Relative phosphorylation decreased in a PHO85 mutant, and this mutant constitutively expressed an Adr1-dependent reporter. Pho85 did not phosphorylate Adr1 in vitro, suggesting that it affects Adr1 indirectly. Mutation of serine 98 to the phosphomimetic amino acid aspartate reduced in vitro DNA-binding of the recombinant Adr1 DNA-binding domain. Mutation to aspartate or alanine affected activation of a reporter by full-length Adr1, and in vivo promoter binding.

Conclusions/significance: Mutation of Adr1 serine 98 affects in vitro and in vivo DNA binding, and phosphorylation of serine 98 in vivo correlates with glucose availability, suggesting that Adr1 promoter-binding is regulated in part by serine 98 phosphorylation.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • DNA / chemistry*
  • DNA Mutational Analysis
  • DNA-Binding Proteins / genetics*
  • DNA-Binding Proteins / physiology*
  • Glucose / metabolism
  • Models, Molecular
  • Mutation
  • Peptide Mapping
  • Phosphorylation
  • Promoter Regions, Genetic
  • Protein Binding
  • Protein Processing, Post-Translational
  • Protein Structure, Tertiary
  • Saccharomyces cerevisiae Proteins / genetics*
  • Saccharomyces cerevisiae Proteins / physiology*
  • Serine / chemistry
  • Transcription Factors / genetics*
  • Transcription Factors / physiology*
  • Transcriptional Activation*
  • Zinc Fingers

Substances

  • ADR1 protein, S cerevisiae
  • DNA-Binding Proteins
  • Saccharomyces cerevisiae Proteins
  • Transcription Factors
  • Serine
  • DNA
  • Glucose