Interaction of the Srb10 kinase with Sip4, a transcriptional activator of gluconeogenic genes in Saccharomyces cerevisiae

Abstract

Sip4 is a Zn(2)Cys(6) transcriptional activator that binds to the carbon source-responsive elements of gluconeogenic genes in Saccharomyces cerevisiae. The Snf1 protein kinase interacts with Sip4 and regulates its phosphorylation and activator function in response to glucose limitation; however, evidence suggested that another kinase also regulates Sip4. Here we examine the role of the Srb10 kinase, a component of the RNA polymerase II holoenzyme that has been primarily implicated in transcriptional repression but also positively regulates Gal4. We show that Srb10 is required for phosphorylation of Sip4 during growth in nonfermentable carbon sources and that the catalytic activity of Srb10 stimulates the ability of LexA-Sip4 to activate transcription of a reporter. Srb10 and Sip4 coimmunoprecipitate from cell extracts and interact in two-hybrid assays, suggesting that Srb10 regulates Sip4 directly. We also present evidence that the Srb10 and Snf1 kinases interact with different regions of Sip4. These findings support the view that the Srb10 kinase not only plays negative roles in transcriptional control but also has broad positive roles during growth in carbon sources other than glucose.

FIG. 1

Srb10 is required for phosphorylation of Sip4 in vivo. (A) Strains used were MCY3605 (wild type [WT]) and MCY3634 (srb10Δ) expressing HA-Sip4 from its native promoter on pPL76 (the same as pHA-Sip4 [17]). Cultures were grown in 2% glycerol plus 3% ethanol. Extracts were prepared by the boiling method, and proteins were separated by SDS–6% PAGE, immunoblotted, and detected with anti-HA. (B) Strains were CTY10-5d (WT) and its derivatives, MCY3691 (srb10Δ) and MCY4024 (gal83Δ), expressing HA-Sip4 from pOV64, a LEU2-marked derivative of pPL76 (33). Cultures were grown to mid-log phase in 2% glucose (R, glucose-repressed) and shifted (S) to 2% glycerol plus 3% ethanol for 4 h, or cells were grown in 2% glycerol plus 3% ethanol (GE). For the cells grown in GE, only 40% as much protein was loaded for the WT and srb10Δ samples, whereas the full amount was loaded for the gal83Δ sample. Immunoblot analysis was done as described above. Similar results were obtained with a second set of srb10Δ and gal83Δ transformants.

FIG. 2

Srb10 affects transcriptional activation by LexA-Sip4. Strains were wild-type (WT) CTY10-5d and its srb10Δ derivative MCY3691 expressing LexA-Sip4 from pOV21. Strains also expressed HA-Srb10 or the kinase-dead mutant HA-Srb10D290A, as indicated, from pSK135 or pSK136 or carried the vector pSK134 so that values could be compared. Cells were grown in SC-Trp-Leu containing 2% glycerol plus 2% ethanol into early stationary phase and then diluted in fresh medium to an optical density at 600 nm of 0.2 and allowed to grow for 24 h. (A) Values for β-galactosidase activity are averages for three transformants. Error bars are shown. (B) Protein extracts were prepared from transformants expressing LexA-Sip4 and vector pSK134 by the boiling method. Proteins were separated by SDS–8% PAGE, immunoblotted, and probed with anti-LexA. Lines indicate phosphorylated (P) and unphosphorylated species. Immunoblot analysis of transformants expressing LexA-Sip4 and HA-Srb10 or HA-Srb10D290A with both anti-LexA and anti-HA confirmed the expression of both tagged proteins (data not shown). WT, wild type.

FIG. 3

Coimmunoprecipitation of Srb10 and Sip4. Strain W303-1A expressed HA-Srb10, Srb10, LexA₈₇-Sip4, and LexA₈₇-Mig1, as indicated, from pSK135, pSK45, pPL49 (the same as pLexA-Sip4 [17]), and pLexA-Mig1 (31). Protein extracts were prepared from cells grown to mid-log phase in 2% glucose, and proteins (11 μg) were immunoprecipitated (IP) with monoclonal anti (α)-HA antibody. Both the input proteins (1.2 μg) and the precipitates were separated by SDS-PAGE and immunoblotted with α-LexA. Immunoprecipitation of HA-Srb10 was confirmed by immunoblot analysis with α-HA.

FIG. 4

Two-hybrid interactions of Sip4, Srb10, and Cat8. Strain CTY10-5d was transformed with plasmids expressing the indicated GAD-Sip4 and LexA fusion proteins. LexA proteins were expressed from pSK33, pPL54, pOV48, pOV8, and pRJ55. The open bar represents Sip4 sequence (residues 1 to 829); the dark bar represents Zn₂Cys₆ zinc cluster (residues 45 to 76); LZ indicates a leucine zipper motif beginning at residue 503. Transformants were grown on medium containing 2% glucose, and two-hybrid interaction was monitored using the filter assay for blue color. Color is represented as follows: +++, strong blue; ++, moderate blue; +/−, very light blue; −, white. ND, not determined. LexA-Sip4(1–690) also interacted with GAD-Cat8(1–1203), and full-length LexA-Sip4 alone generated a light blue color but nonetheless clearly interacted with both GAD-Sip4 and GAD-Cat8 (residues 1 to 1433). The interaction of LexA-Snf1 with GAD-Sip4 and GAD-Sip4(402–829) has been reported previously (17, 33).