The molecular chaperone Sse1 and the growth control protein kinase Sch9 collaborate to regulate protein kinase A activity in Saccharomyces cerevisiae

Abstract

The Sch9 protein kinase regulates Hsp90-dependent signal transduction activity in the budding yeast Saccharomyces cerevisiae. Hsp90 functions in concert with a number of cochaperones, including the Hsp110 homolog Sse1. In this report, we demonstrate a novel synthetic genetic interaction between SSE1 and SCH9. This interaction was observed specifically during growth at elevated temperature and was suppressed by decreased signaling through the protein kinase A (PKA) signal transduction pathway. Correspondingly, sse1Delta sch9Delta cells were shown by both genetic and biochemical approaches to have abnormally high levels of PKA activity and were less sensitive to modulation of PKA by glucose availability. Growth defects of an sse1Delta mutant were corrected by reducing PKA signaling through overexpression of negative regulators or growth on nonoptimal carbon sources. Hyperactivation of the PKA pathway through expression of a constitutive RAS2 allele likewise resulted in temperature-sensitive growth, suggesting that modulation of PKA activity during thermal stress is required for adaptation and viability. Together these results demonstrate that the Sse1 chaperone and the growth control kinase Sch9 independently contribute to regulation of PKA signaling.

Figure 1.—

Simultaneous disruption of the molecular chaperone SSE1 and the growth control kinase SCH9 causes synthetic temperature sensitivity. The indicated strains were streaked onto YPD medium and incubated at 30° or 37° for 4 days. No sse1Δ sch9Δ colonies were visible even after extended incubation at 37°. Wild type (W303), sch9Δ (KMY52), hsc82Δ (KMY61), sse1Δ (KMY69), hsc82Δ sch9Δ (KMY59), sse1Δ sch9Δ (KMY67).

Figure 2.—

SSE1 and SSE2 are an essential gene pair. The indicated strains carrying pSSE1 (pYEp24-SSE1), a URA3-based vector, were streaked onto SC-URA and 5-FOA selection medium and incubated at 30° for 3 days. Wild type (W303) and sse1Δ (KMY67) or sse2Δ (ATY1) single deletion strains grew in the presence of 5-FOA, indicating the ability to lose the covering plasmid. The sse1Δ sse2Δ strain (ATY2) was unable to grow in the absence of pSSE1 (pYEp24-SSE1), demonstrating that expression of one of the two SSE genes is required for viability.

Figure 3.—

Reduction in PKA pathway activity suppresses temperature sensitivity of sse1Δ sch9Δ. (A) Tenfold serial dilutions of wild type (W303) and sse1Δ sch9Δ (KMY67) cells transformed with an empty vector or sse1Δ sch9Δ cells carrying the IRA1-containing library suppressor isolate were spotted onto SC-URA medium and incubated at 30° or 37° for 3 days. IRA1 overexpression is shown to correct the temperature sensitivity, but not the slow-growth phenotype, of sse1Δ sch9Δ cells. (B) Tenfold serial dilutions of the indicated strains transformed with empty vector (p416GPD) or plasmids overexpressing the BCY1 (o/e BCY1, p416GPDBCY1) or PDE2 (o/e PDE2, YEplacPDE2) open reading frames were spotted onto SC-URA medium and incubated at 30° or 37° for 3 days. BCY1 or PDE2 overexpression likewise corrects only the temperature-sensitivity phenotype of sse1Δ sch9Δ cells. (C) The indicated strains were streaked onto YPD medium and incubated at 30° and 37° for 5 days.

Figure 4.—

The PKA pathway is hyperactivated in sse1Δsch9Δ cells. (A) The indicated strains were transformed with a STRE-lacZ reporter plasmid (pCT31/32) whose expression is dependent upon the general stress response pathway. Cells were grown to an OD₆₀₀ of ∼0.5 and split into two equal aliquots and were either maintained at 30° or shifted to 37° for 1 hr. β-Galactosidase assays were performed using cell extracts as described in materials and methods. (B) Steady-state levels of the storage carbohydrate trehalose were measured enzymatically as described in materials and methods and normalized to total cell density (OD₆₀₀). (C) Heat-shock survival was assayed by incubating plates containing serial dilutions of the indicated strains at 55° for 0 to 240 min, followed by a 3-day incubation at 30° and determination of the resulting CFU. (D) Expression of the FLO11 gene was assayed by Northern blot analysis as described in materials and methods. ACT1 levels were determined as a load control. The strains for all experiments described in this figure were wild type (W303), sse1Δ (KMY69), sch9Δ (KMY52), and sse1Δ sch9Δ (KMY67).

Figure 5.—

sse1Δ sch9Δ cells are defective in glucose signaling through the PKA pathway. Western blot analysis was used to assay phosphorylation of Msn2 in the conserved PKA consensus site. (A) The indicated strains [wild type (W303), sse1Δ (KMY69), sch9Δ (KMY52), and sse1Δ sch9Δ (KMY67)] were grown in YPD medium to midlogarithmic phase at 30°. Cells were harvested and resuspended in either YPD medium or YP medium lacking glucose and incubated for an additional 10 min prior to harvesting. Protein extracts were resolved by SDS-PAGE and parallel Western blots were probed with antiphospho CREB (P-CREB), anti-Msn2 (Msn2), or anti-PGK (PGK) antibodies. Msn2 protein levels in the sse1Δ strain were reproducibly lower by two- to threefold for undetermined reasons. (B) Kinetic analysis of Msn2 phosphorylation in response to glucose starvation. Cultures of wild-type and sse1Δ sch9Δ cells were grown in YPD medium to midlogarithmic phase at 30° and shifted to YP medium lacking glucose. Equivalent aliquots were removed at the indicated time points and flash frozen after collection. Msn2 phosphorylation was assayed as described above.

Figure 6.—

Reduction in PKA pathway activity through negative regulators or alternate carbon sources partially suppresses sse1Δ temperature sensitivity. (A) Tenfold serial dilutions of wild-type (W303) and sse1Δ (KMY69) cells transformed with the indicated plasmids were plated onto SC-URA medium and grown at 30° and 37° for 3 days. (B) Tenfold serial dilutions of wild-type and sse1Δ cells were plated onto YP medium with the following sole carbon sources: 2% glucose, 2% galactose, 2% ethanol, or 3% glycerol. Plates were incubated at 37° for 3–6 days to allow for colony development. o/e, overexpressed.

Figure 7.—

PKA pathway mutants are hypersensitive to SSE1 overexpression. The plasmids pYEp24SSE1 (o/e SSE1) or pYEp24 alone (vector) were transformed into the indicated strains and 10-fold serial dilutions were plated on SC-URA medium for 4 days at 35°, the semipermissive growth temperature for the ras2-23 (PHY1340) and cdc25-1 (PHY1452) strains. o/e, overexpressed.

Figure 8.—

Hyperactive PKA activity inhibits high-temperature growth. Wild type (SP1) and low-PKA (tpk1^w tpk2 tpk3 bcy1; RS13-58A-1) strains transformed with pRS416 (vector) or pMET3-RAS2^val19, a plasmid expressing a methionine-regulatable activated RAS2 allele, were streaked onto SC-URA-MET plates supplemented with additional methionine or not. Plates were incubated at 37° for 3 days.

Figure 9.—

Model of interactions among Sse1, Sch9, and the PKA pathway. Adenylate cyclase (Cyr1) regulates PKA activity via the regulatory subunit Bcy1 through production of cAMP. The three catalytic subunits of PKA both positively and negatively regulate downstream targets by phosphorylation. The Sch9 kinase likely contributes additively to regulation of shared targets. Sch9 may also negatively regulate PKA pathway function by an unknown mechanism, as shown by the dashed line. In addition, Sse1 may act as a negative regulator of PKA catalytic activity in a Bcy1-independent manner. Simultaneous loss of Sse1 and Sch9 could therefore lead to hyperactivation of one or more Tpk subunits, while loss of Sse1 alone would result in only moderate PKA activation.