Phd1 is destabilized by Cdk8. (A) Flag-epitope-tagged Mga1 (lanes 2 and 3), Phd1 (lanes 4 and 5), and Tec1 (lanes 6 and 7) were expressed in wild-type (lanes 1, 2, 4, and 6) or cdk8 (lanes 3, 5, and 7) W303 strains, and steady-state levels of protein were detected by immunoblotting with anti-Flag (α-flag) (upper) or antitubulin (α-tubulin) antibodies (lower panel). Relative levels of the Flag fusion proteins were quantified and are indicated as normalized to tubulin below the lanes ([Fusion]). Lane 1 contains an extract from the strain bearing an empty vector. (B) Flag-tagged Phd1 (cloned from S288C) was expressed in wild-type (lanes 5 to 7) or cdk8 (lanes 2 to 4) W303 yeast grown to mid-log phase. Cycloheximide was added to the cultures, and protein extracts were prepared for immunoblotting at the times indicated above (minutes).

Localization of Phd1 phosphorylations. (A) Schematic representation of the Phd1 protein indicating the MluI-box DNA binding domain and serine 92 within a serine/threonine-rich region (SSSS). D1, D2, and D3 indicate the span of residues contained within deletion mutants used to localize N-terminal phosphorylations. (B) Full-length Phd1 (lanes 1 and 5) or the D1 (lanes 2 and 6), D2 (lanes 3 and 7), and D3 (lanes 4 and 8) N-terminal deletion mutants, fused to a C-terminal 3×Flag epitope, were expressed in wild-type (lanes 5 to 8) or cdk8 null (lanes 1 to 4) W303 haploid yeast strains. Steady-state levels of Flag-tagged protein were detected by immunoblotting. (C) W303 strains expressing full-length Phd1 (lane 2) or the D1 (lane 3), or D2 (lane 4) deletion-3×Flag fusions or bearing a vector control (lane 1) were labeled with ³²P_i. Phd-1–Flag fusion proteins were recovered by immunoprecipitation, resolved on SDS-PAGE, and visualized by autoradiography. (D) Comparison of the sequence surrounding Phd1 S92 (top line) with the S261 (middle) and S451 (bottom) sites on Ste12 phosphorylated by Cdk8. Conserved proline (purple) and glutamine (green) residues flanking the serine (pink) are indicated.

Serine 92 on Phd1 is necessary for Cdk8-dependent destabilization. (A) 3×Flag epitope-tagged S288C-derived S92 Phd1 (lanes 1 to 4 and 9 to 12) or Σ1278b-derived S92F Phd1 (lanes 5 to 8 and 13 to 16) were expressed in wild-type (lanes 1 to 8) or cdk8 (lanes 9 to 16) Σ1278b diploid strains and grown to mid-log phase. Cycloheximide was added to the cultures, and protein extracts were prepared for immunoblotting at the times indicated above (minutes). (B) Flag-tagged Phd1 proteins were quantified, and the relative abundance, normalized to tubulin, was plotted as a function of time post-cycloheximide addition.

Cdk8 phosphorylates serine 92 on Phd1 in vitro. (A) In vitro kinase reactions were performed with HA-tagged Cdk8 recovered from yeast by immunoprecipitation. Reaction mixtures contained equivalent amounts of recombinant S92 Phd1 (lane 2), S92F Phd1 (lane 3), Gal4 (lane 4), GST-CTD (lane 5), GST (lane 6), or no substrate (lane 1). Samples were resolved by SDS-PAGE and analyzed by autoradiography. RNA polymerase II coimmunoprecipitates with Cdk8 at low stoichiometry, and the largest subunit becomes phosphorylated on the CTD in these reactions (indicated by *, left). (B) Recombinant S92 Phd1 (lane 1), S92F Phd1 (lane 2), Gal4 (lane 3), and GST-CTD (lane 4) substrate proteins, detected by Coomassie blue stain. In vitro kinase reaction mixtures contained 3 (lanes 1, 2, and 4) or 2 (lane 3) μg protein. (C) Recombinant wild-type Cdk8 (lane 3) or Cdk8 bearing a mutation at the ATP-binding site (D290A; lane 2) complexes copurified with Flag-tagged cyclin C, produced by expression in insect cells using baculovirus, were added to in vitro kinase reaction mixtures containing recombinant S92 Phd1. Lane 1 contained no added kinase. (D) Total phosphate incorporation into recombinant S92 and S92F Phd1 substrates was determined from in vitro kinase assays, the results of which are shown in panel A. Results are from five independent assays, and differences between the values are statistically significant with P = 0.0011. (E) In vitro kinase reactions were performed with HA-Cdk8 and peptide substrates representing S92 and S92F Phd1. Total phosphate incorporation was determined from three independent assays, and the values were corrected relative to reaction mixtures containing no peptide.

Cdk8 phosphorylates multiple sites on Phd1 in vitro, including serine 92. Phd1 substrates from in vitro kinase reactions (A) were digested with trypsin (A) or trypsin and chymotrypsin (B and C); phosphopeptides were resolved in 2 dimensions by electrophoresis (horizontal) and then chromatography (vertical) and visualized by autoradiography. The substrate proteins were S92 Phd1 (A and B) and S92F Phd1 (C). In panel A, arrows indicate phosphopeptides produced by multiple phosphorylations of a single large peptide spanning serine 92. In panel B, the arrow indicates a phosphopeptide unique to S92 relative to S92F in panel C.

S92F Phd1 produced by Σ1278b causes enhanced pseudohyphal growth. (A) Σ1278b diploid yeast strains with phd1 (top) or both phd1 and cdk8 (bottom) null alleles were transformed with a vector control (vector, left) or plasmids expressing S92 Phd1 (center) or S92F Phd1 (right) from the TEF1 promoter. The strains were plated on limiting nitrogen agar plates (SLAD), and colonies were photographed after 3 days. (B) S288C produces pseudohyphae when expressing Σ1278b FLO8 and PHD1. Combinations of the Σ1278b and S288C FLO8 and PHD1 alleles were expressed in an S288C diploid. Yeast strains were cotransformed with a plasmid expressing Σ1278b FLO8 (top panels) or a vector control (bottom panels), plasmids expressing the Σ1278b S92F Phd1 (right panels) or S288C S92 Phd1 (center panels) from the TEF1 promoter or a vector control (left). Strains were plated on SLAD, and colonies were photographed after 3 days.

Phd1 becomes stabilized in nitrogen-starved cells. (A) A Σ1278b diploid strain transformed with a plasmid expressing S92 Phd1 from the TEF1 promoter was grown to mid-log phase in SD medium (Rich; lanes 1 to 4), or switched to SLAD for 2 h (lanes 5 to 8) or 4 h (lanes 9 to 12). Protein extracts were prepared from cultures treated with cycloheximide for the times indicated above (minutes) and immunoblotted with anti-Flag (top panels) or antitubulin (bottom panels) antibodies. (B) Flag-tagged Phd1 proteins were quantified, and the relative abundance, normalized to tubulin, was plotted as a function of time post-cycloheximide addition.

Ste12 contributes to induction of PHD1 in response to nitrogen limitation. (A) Schematic representation of the PHD1 promoter. Location of consensus binding sites for Ste12 (PRE), Tec1 (TCS), and Phd1 are indicated. The numbering indicates the location relative to the translational start site. Recombinant Ste12 binds to each of the PREs in vitro as measured by footprinting. Electrophoretic mobility shift assay (EMSA) with Phd1 also produces a complex with the consensus site at −348 (data not shown). Flo8, Mga1, and Sok2 also bind the PHD1 promoter in vivo (), but these factors either do not have defined consensus sequences, or we were unable to detect their cis-elements within the promoter sequence. (B) Recombinant Ste12 protein was used in footprinting reactions with PHD1 promoter template DNA. Reaction mixtures contained no protein (lane 1) or increasing amounts of recombinant Ste12 (lanes 2 to 7). Lane 8 contains an M&G G+A sequencing ladder. Shown is a region of the footprint spanning two PREs between −485 and −465 from the translational start site. (C) RNA was extracted from Σ1278b ste12 diploid yeast bearing plasmids expressing wild-type STE12 (lanes 3 and 6) or STE12 bearing a mutation of serine 261 to alanine (lanes 2 and 5), grown in rich medium (lanes 1 to 3) or switched to SLAD (lanes 4 to 6) for 2 h, and analyzed by Northern blotting with probes for PHD1 (top) or PDA1 (bottom). Levels of PHD1 expression proportional to cells growing in rich medium expressing WT Ste12 (lane 3) and normalized to PDA1 are indicated below the lanes. (D) Relative levels of PHD1 expression were quantified from Northern blots, as in panel A, from cells growing in rich medium (open bars) or from cells switched to SLAD for 2 h (hatched bars) or 4 h (solid bars).

S92 Phd1 protein accumulates in nitrogen-starved cells. (A) PHD1 was tagged with an in-frame 3×HA epitope in the Σ1278b (top panels) or W303 (bottom panels) backgrounds. Proteins from strains growing in rich medium (lanes 1) or switched to nitrogen-limiting conditions (SLAD) for the indicated times in hours (lanes 2 to 5) were immunoblotted with anti-HA antibodies (Phd1-3HA) or anti-histone H3 (H3). Levels of Phd1-3HA expression, relative to zero time (Σ1278b) or 4 h poststarvation (W303) and normalized to histone H3, are indicated. (B) PHD1 overexpression can induce pseudohyphae in an ste12 strain. Wild-type (top panels) and ste12 (bottom) Σ1278b yeast strains were transformed with the 2μm plasmid (2μ) expressing PHD1 (S92) (right panels) or a vector control (left). Colonies were grown on SLAD for 3 days.

STE12 and PHD1 are essential for haploid invasive growth. Haploid (MATa) Σ1278b-derived yeast strains bearing the indicated gene disruptions were spotted onto YPD plates and allowed to grow for 3 days at 30°C (Total; left panel). Yeast cells growing above the surface of the agar were gently washed away under a stream of water to reveal the extent of invasive growth (right panel).

Regulation of pseudohyphal differentiation by Cdk8. (A) PHD1 expression is regulated by multiple factors, including Ste12 (blue), Tec1 (yellow), Phd1 (green), and Sok2 (red). In rich medium, PHD1 expression is limited because Cdk8 inhibits the activity of Ste12, Phd1, and likely additional factors (purple) by direct phosphorylation and is repressed by Sok2. (B) Nitrogen limitation inhibits Cdk8 by signaling that may involve Snf1 and/or PKA, which allows accumulation of Ste12 (and Tec1) to cause upregulation of PHD1 expression. PKA signaling through Yak1 also inhibits the repressor Sok2. Phd1 accumulates in nitrogen-starved cells and autoactivates its own expression in a positive-feedback loop. Accumulation of combinations of these factors causes activation of downstream target filamentous response genes (FRG), in combination with Flo8, whose activity is positively regulated by PKA signaling.