Phosphorylation of Adr1 Figure 1. Phosphorylation of Adr1 DBD by cell extracts. A. In vitro kinase assay with purified, recombinant Adr1 DBD (amino acids 17–160 of Adr1), [c-32P]-ATP and cell extracts from TYY201. R, cells were grown in repressing medium; DR, cells were derepressed in 0.05% glucose for 3 hours. Because of variability in the extracts, quantitative comparisons cannot be made between the repressed and derepressed lanes. B. Coomassie stain of the gel in A. C. In vitro kinase assay as in A, but with N-terminally truncated versions of the Adr1 DBD. Numbers indicate the amino acid in wild-type Adr1 that is the N-terminus of the truncated version. D. Coomassie stain of the gel in C. doi:10.1371/journal.pone.0003213.g001 pointing to regulation beyond the potential Ser230 modification [8,10]. Another possible site for post-translational control of Adr1 is its DNA binding domain (DBD, residues 76–165). Fusion of the Adr1 DBD to a heterologous activation domain confers glucose- regulated expression on target promoters. Conversely, a Gal4 DBD - Adr1 (amino acids 21–1323) fusion shows no glucose regulation of Gal4 targets [6]. The Adr1 DBD contains two canonical C2H2 zinc fingers (amino acids 99–160) and an amino terminal region of approximately 20 amino acids called PAR (proximal accessory region) that is essential for high affinity binding [14,15]. The structure of the DBD has been determined by NMR in the presence and absence of DNA. The accessory motif PAR is unstructured in the absence of DNA and assumes a more ordered conformation when bound to DNA [16]. Mutations in PAR affect DNA binding: changing Pro 87 or Pro 97 to Ala or Gly abrogates DNA binding and activity in vivo [17], while mutation of Arg 91 to Lys allows better in vitro binding and suppresses several loss-of-function mutations in the DBD [18,19]. Because phosphorylation is a common mechanism of post- translational modification, and the Adr1 DBD appears to be crucial to glucose repression, we looked for phosphorylation of the Adr1 DBD as a possible mode of regulation. Serine 98, which is in a region critical for PAR structural changes during DNA-binding [16], appeared to be phosphorylated in vitro and in vivo. Mutation of serine 98 affected DNA-binding and activation of Adr1-dependent genes, suggesting that it could play a role in glucose repression. A screen of yeast kinase deletion mutants identified the kinase complex Pho80/85 as affecting the phosphorylation state of Adr1 serine 98 and contributing to glucose repression of some Adr1- dependent genes. Results The Adr1 DBD is phosphorylated in vitro and in vivo Binding of DNA by Adr1 is a regulated step [7] and the DBD of Adr1 contains a number of serine and threonine residues that could be phosphorylated. To assess whether the Adr1 DBD might PLoS ONE | www.plosone.org 2 September 2008 | Volume 3 | Issue 9 | e3213 be a target for a yeast protein kinase, we incubated recombinant Adr1 DBD with yeast whole cell extracts and [c-32P]-ATP. SDS gel electrophoresis followed by autoradiography showed a prominent band at the position of the Adr1 DBD (18 kDa) that was absent if recombinant protein was omitted from the mixture (Figure 1A, B and Table 1 for yeast strains). N-terminal truncations of the Adr1 DBD showed that the site of phosphor- ylation was within amino acids 94–160, which includes the C- terminal portion of the proximal accessory region (PAR) and the Zn-fingers (Figure 1C, D). Two-dimensional peptide mapping and amino acid analysis of tryptic peptides from the phosphorylated protein (data not shown) suggested a single major site of Table 1. Yeast strains BY4741 and kinase MATa his3D1 leu2D0 met15D0 ura3D0 and deletions relevant gene knockout with kanMX strain CKY13 VBY20 NK85 NK87 NK135 NK136 NK139 TYY201 TYY497 doi:10.1371/journal.pone.0003213.t001 genotype MATa ade2-1 can1-100 ura3-1 leu2-3,112 trp1-1 his3-11,115 Dadr1::kanMX MATa adh3 ura3 his3 leu2::(pRS315-ADR1)X3 ADH2::YIpADH2/lacZ(TRP1) ADH2::YIpADH2/ GFP(URA3) ADR1-3HA::kanmx VBY20 ADR1-3HA::kanmx NK85 Dsnf1::kanmx NK85 Dpho85::NAT1 NK85 Dpho80::NAT1 NK85 Dpcl10::NAT MATa ade2 can1-100 his3-11,115 leu2-3,112 trp1-1 ura3-1 MATa ade2 can1-100 his3-11,115 leu2-3,112 trp1-1 ura3-1 Dadr1::LEU2 ADH2::YIpADH2/ lacZ(trp1::HIS3) source [43] this work [9] this work this work this work this work this work [32] [32]