The Ras/PKA Signaling Pathway Directly Targets the Srb9 Protein, a Component of the General RNA Polymerase II Transcription Apparatus  Ya-Wen Chang, Susie C. Howard, Paul K. Herman  Molecular Cell  Volume 15, Issue 1, Pages 107-116 (July 2004) DOI: 10.1016/j.molcel.2004.05.021

Figure 1 The Presence of Srb9p Was Required for the RAS2val19 Suppression of srb Mutant Phenotypes (A) Elevated levels of Ras signaling activity suppressed the Rye− growth phenotype associated with srb9, srb10, and srb11 mutants. Strains carrying either a RAS2val19 plasmid (pJR1052) or a control vector were plated to YM-glucose or BCP-sucrose media and incubated for 3 days at 30°C. The strains examined were wild-type (PHY1184), srb9 (PHY1382), srb10 (PHY1397), and srb11 (PHY1378). (B) The presence of RAS2val19 suppressed the YGP1-SUC2 expression defect associated with srb9-11 mutants. The steady-state levels of YGP1-SUC2 and ACT1 mRNA were assessed in mid-log cultures of wild-type (PHY1184) and srb9-11 (PHY1382) mutants with a Northern RNA blot analysis. The strains contained either the RAS2val19 plasmid, pJR1052 (+), or a control vector (−). (C) The Ras effects on the Srb complex were mediated by the cAMP-PKA effector pathway. Wild-type (PHY1184) and srb9-11 (PHY1382) yeast strains carrying either RAS2val19 (pJR1052), PDE2 (pPHY2299), or the appropriate control plasmids were plated to YM-glucose or BCP-sucrose media and incubated for 2 days at 30°C. (D) The Srb9 protein was required for the RAS2val19 suppression of the Rye− growth phenotype associated with the srb mutants. Strains carrying either a RAS2val19 plasmid (pJR1052), an SRB9 plasmid (pPHY1070), or the appropriate control vector were plated to YM-glucose or BCP-sucrose media and incubated for 3 days at 30°C. For the srb11Δ experiments, the RAS2val19 plasmid was pPHY827. The strains examined were wild-type (PHY1184), srb9 (PHY1382), srb9Δ (PHY1621), srb10Δ (PHY1627), and srb11Δ (PHY1616). Molecular Cell 2004 15, 107-116DOI: (10.1016/j.molcel.2004.05.021)

Figure 2 The Two PKA Sites in Srb9p Were Required for the RAS2val19 Suppression of srb9-11 Phenotypes and for the Normal Function of the Wild-Type Srb9p (A) A schematic indicating the position of the two PKA sites in Srb9p and the amino acid substitutions being analyzed in the experiments described in (B) and (C). (B) At least one of the two PKA sites in Srb9p was required for the RAS2val19 suppression of the Rye− growth defect associated with srb9-11 mutants. The srb9Δ mutant strain, PHY1621, was transformed with plasmids encoding the indicated mutagenized versions of the srb9-11 allele. These strains also carried either the RAS2val19 plasmid, pJR1052, or a control vector, pRS414. Equal cell numbers of each strain were plated to YM-glucose or BCP-sucrose media and incubated for 3 days at 30°C. (C) The RAS2val19 suppression of the YGP1-SUC2 expression defect associated with srb9-11 mutants required the presence of at least one of the two PKA sites in Srb9p. The steady-state levels of YGP1-SUC2 and ACT1 mRNA were assessed with a Northern RNA blot analysis. The strains contained either the RAS2val19 plasmid, pJR1052 (+), or a control vector (−). The strains analyzed were those described in (B). (D) Alteration of the two PKA sites in the full-length Srb9p resulted in a specific set of gene expression defects. The steady-state levels of HSP12, HSP26, ACT1, FLO11, and FLO1 mRNA were assessed with a Northern RNA blot analysis. The ACT1 levels served as the loading control for this experiment. The relative degree of flocculation observed in mid-log phase cultures grown in YM-glucose medium is also shown. The strains analyzed were wild-type (PHY1184), srb10 (PHY1397), srb9-11 (PHY1382), srb9Δ (PHY1621), and srb9-AA (PHY1621 carrying pPHY1089). All of the strains except the latter carried the control vector, pRS413. Molecular Cell 2004 15, 107-116DOI: (10.1016/j.molcel.2004.05.021)

Figure 3 Substitution of an Acidic Amino Acid for the Serine Residue in Either Srb9p PKA Site Was Sufficient to Suppress the Rye− Growth Defect Associated with srb9-11 Mutants (A) A schematic indicating the position of the two PKA sites in Srb9p and the amino acid substitutions being analyzed in the described experiments. (B) The srb9Δ strain, PHY1621, was transformed with either a control vector (srb9Δ), the SRB9 plasmid (pPHY1070; SRB9), or a plasmid containing the indicated mutagenized version of the srb9-11 allele. Equal cell numbers of each strain were plated to YM-glucose or BCP-sucrose media and incubated for 3 days at 30°C. Molecular Cell 2004 15, 107-116DOI: (10.1016/j.molcel.2004.05.021)

Figure 4 Ser608 of Srb9p Was Phosphorylated In Vitro in a PKA-Dependent Manner (A) Srb9p was a phosphoprotein in vivo. The wild-type yeast strain, PHY1220, carrying either a control vector or the pWS121 plasmid encoding the HA-tagged Srb9p, was labeled with [32P] orthophosphate as described in Experimental Procedures. The labeled HA-Srb9p was immunoprecipitated with an antibody specific for the HA epitope, and the bound proteins were separated on an SDS-polyacrylamide gel. The amount of label incorporated into Srb9p was then assessed by autoradiography. A Western immunoblot with the anti-HA antibody was performed to assess the levels of HA-Srb9p present in mock-labeled cell extracts. (B) A schematic indicating the fragments of Srb9p that were present in the Srb9.1 and Srb9.2 Protein A fusion proteins. The Srb9.1 fusion contained amino acids 502–617 of Srb9p and Srb9.2 contained residues 1088–1387. (C) The Srb9.1 fusion protein was phosphorylated by bovine PKA in a Ser608-dependent manner. Srb9.1 fusion proteins containing either a serine or alanine residue at position 608 were incubated with bovine PKA and [γ-32P] ATP for 30 min at 25°C. The fusion proteins were eluted from the Sepharose beads and run out on an SDS-polyacrylamide gel. The amount of label incorporated into the different fusion proteins was then assessed by autoradiography. PA refers to the Protein A control sample. The position of the Srb9.1 fusion protein is shown. The presence (+) or absence (−) of the bovine PKA in the reaction mixes is indicated at the bottom of the panel. The results for two different Srb9.1 isolates are shown. (D) The Srb9.1 fusion protein was phosphorylated by the yeast PKA catalytic subunit, Tpk1p. The Srb9.1 and Srb9.2 fusion proteins containing either a serine or alanine residue in their respective PKA sites were incubated with a GST-Tpk1p fusion protein and [γ-32P] ATP for 30 min at 25°C. The amount of label incorporated into the different fusion proteins was then assessed as described in (C). The relative positions of the Srb9.1 and Srb9.2 fusion proteins are shown. All reactions shown contained the GST-Tpk1p enzyme. (E) Western immunoblot control. The levels of the indicated Srb9p fusion proteins in the precipitated fraction were assessed by a Western immunoblot analysis. The relative positions of the Srb9.1 and Srb9.2 fusion proteins are shown. The band corresponding to the Protein A control (PA) was run off this particular gel. The asterisk indicates an IgG band that is recognized by the secondary antibody. Molecular Cell 2004 15, 107-116DOI: (10.1016/j.molcel.2004.05.021)

Figure 5 Srb9p Was an In Vitro and In Vivo Substrate for PKA (A) The full-length Srb9p was phosphorylated by PKA in vitro in a Ser608-dependent manner. Top: Full-length Srb9 proteins with the indicated alterations of the two PKA sites were incubated with bovine PKA and [γ-32P] ATP for 30 min at 25°C. The proteins were eluted from the anti-HA affinity matrix and run out on an SDS-polyacrylamide gel. The amount of label incorporated into the different proteins was then assessed by autoradiography. The position of the full-length Srb9p is shown. Bottom: A Western immunoblot control performed with the anti-HA antibody. The strains examined carried plasmids encoding the indicated mutagenized versions of the full-length HA-tagged Srb9p. (B) Srb9p was recognized by an anti-PKA substrate antibody in a Ser608-dependent manner. The indicated HA-tagged Srb9 proteins were immunoprecipitated and run out on a 7.5% SDS-polyacrylamide gel. These proteins were transferred to a nitrocellulose membrane that was then probed with the anti-PKA substrate antibody (α-substrate) or the anti-HA (α-HA) antibody as described in the Experimental Procedures. The position of the full-length Srb9p is shown. (C) Elevated levels of Ras/PKA signaling activity resulted in an increase in the in vivo phosphorylation of Srb9p. The wild-type HA-tagged Srb9p was immunoprecipitated from the yeast strain, TVY614, carrying either a MET3-RAS2val19 plasmid (Howard et al., 2001) or a control vector. The phosphorylation state of the precipitated Srb9p was then analyzed with the anti-PKA substrate antibody as described above in (B). To shut down expression from the MET3-RAS2val19 allele, the strain was grown in a medium that contained 500 μM methionine; otherwise, the strains were grown in a minimal medium that lacked methionine. Molecular Cell 2004 15, 107-116DOI: (10.1016/j.molcel.2004.05.021)

Figure 6 Models Explaining the Specific and More General Ramifications of the Data Presented in This Report for Transcriptional Control (A) A schematic depicting how the Ras/PKA pathway in S. cerevisiae may be controlling gene expression via the Srb complex. In this model, PKA directly phosphorylates Srb9p and thus enhances the activity of the Srb complex. One consequence of this regulation would be the inhibition of expression of specific genes that are normally associated with the stationary phase of growth. The dotted line indicates that the Ras/PKA pathway might also regulate the expression of some of these genes by a second, Srb-independent mechanism. (B) A model outlining an alternative, or bypass, mode of transcriptional control whereby signaling pathways directly target components of the RNA pol II holoenzyme. This figure describes two different ways that signal transduction pathways could control gene expression by the RNA pol II enzyme. Signaling pathways, in general, are represented by a particular protein kinase (PK) in these examples. The “Indirect Model” refers to those instances where signaling pathways directly target the activator proteins (ACT) present at the enhancer, or UAS, elements of eukaryotic gene promoters. These activator proteins, in turn, convey this regulatory information to the RNA pol II machinery. In the “Bypass Model,” the signaling pathways directly target proteins that are part of the RNA pol II holoenzyme or other components of the general RNA pol II transcription machinery. See text for further details. Molecular Cell 2004 15, 107-116DOI: (10.1016/j.molcel.2004.05.021)