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


RESULTS
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
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.
Kacherovsky
Nataly
N
Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.
Tachibana
Christine
C
Amos
Emily
E
Fox
David
D
Young
Elton T
ET
D004247:DNA / Q000737:chemistry*; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000502:physiology*; D005947:Glucose / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010449:Peptide Mapping; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000502:physiology*; D012694:Serine / Q000737:chemistry; D014157:Transcription Factors / Q000235:genetics* / Q000502:physiology*; D015533:Transcriptional Activation; D016335:Zinc Fingers
C074682:ADR1 protein, S cerevisiae; D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D012694:Serine; D004247:DNA; D005947:Glucose
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