Promoter Binding by the Adr1 Transcriptional Activator May Be Regulated by Phosphorylation in the DNA- Binding Region Nataly Kacherovsky, Christine Tachibana, Emily Amos¤, David Fox, III, Elton T. Young* Department of Biochemistry, University of Washington, Seattle, Washington, United States of America 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. Citation: Kacherovsky N, Tachibana C, Amos E, Fox D, Young ET (2008) Promoter Binding by the Adr1 Transcriptional Activator May Be Regulated by Phosphorylation in the DNA-Binding Region. PLoS ONE 3(9): e3213. doi:10.1371/journal.pone.0003213 Editor: Baohong Zhang, East Carolina University, United States of America Received June 25, 2008; Accepted August 25, 2008; Published September 15, 2008 Copyright: 2008 Kacherovsky et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: NIGMS 26079 and NIDDK67276. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ety@u.washington.edu ¤ Current address: Seattle Biomedical Research Institute, Seattle, Washington, United States of America Introduction Glucose repression ensures that yeast cells use preferred carbon sources until available supplies are exhausted. Much of the regulation of glucose-repressed genes occurs at the level of transcription [1,2], and in yeast, Adr1 has a pivotal role in expressing these genes [3,4]. Control of Adr1 occurs at several levels: through transcription of its gene [5]; post-translational modification of the protein [6]; and access to, or ability to stably bind promoters [7,8]. The latter two mechanisms appear to be the most critical, because in glucose-repressed cells, raising Adr1 protein levels to be comparable to those in derepressed cells does not activate transcription of ADH2 or other Adr1-regulated genes [6,9]. Adr1 does not bind its cognate promoters in these conditions, so even at elevated protein levels, post-translational and promoter-binding regulation keep its activity in check [6,7]. Several lines of evidence suggest regulation of Adr1 binding and activity by post-translational modification. Derepression of the Adr1-regulated gene ADH2 can occur in the absence of protein synthesis [6]. Snf1, a central kinase in transcription of glucose- regulated genes, is required for Adr1 promoter-binding [7]. Snf1 is the yeast homolog of the AMP-activated protein kinase, and activation of Snf1 in repressing conditions relieves some glucose repression of ADH2 and other Adr1-dependent genes, allowing up to 10% of derepressed transcription levels [8,10]. Snf1 does not appear, however, to phosphorylate and activate Adr1 directly, as Adr1 lacks a consensus site for Snf1 phosphorylation and no Snf1- Adr1 in vitro phosphorylation or association has been detected (N. Kacherovsky, K. Dombek, unpublished). Since Snf1 is not required for ADR1 expression [5] but is required for DNA-binding by Adr1, one hypothesis is that Snf1 indirectly regulates Adr1 post- translationally. Protein kinase A (PKA) has been implicated in direct phosphorylation of Adr1. Both yeast and mammalian PKA phosphorylate Adr1 in vitro [11], and inactivation of BCY1, encoding the regulatory subunit of protein kinase A, inhibits Adr1 expression and activity [10,12]. Whether PKA phosphorylates Adr1 in vivo has not been resolved, however [13]. Adr1 has a PKA consensus site at Ser230, and mutations of this residue or in this region lead to alleles called ADR1c that allow constitutive ADH2 expression and hyper-derepression [11,12]. Nonetheless, the hyper-active Adr1c proteins are still sensitive to glucose repression and respond to activation of Snf1 in repressing conditions, PLoS ONE | www.plosone.org 1 September 2008 | Volume 3 | Issue 9 | e3213