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, sse1Δ sch9Δ 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 sse1Δ 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.
PROTEIN chaperones play intricate roles in normal growth processes and under conditions of environmental stress in the budding yeast Saccharomyces cerevisiae. Perhaps the most well-known role is the amelioration of the proteotoxic effects of elevated temperature by stabilizing and refolding denatured proteins. A concerted increase in chaperone capacity, commonly known as the heat-shock response, occurs rapidly following exposure to elevated temperature and other cytotoxic conditions and results in the transcriptional activation of hundreds of genes. Many of these heat-shock genes are required for the cell to recover, adapt, and resume growth at the elevated temperature. Transcriptional activation of heat-shock genes is attributed to the collective activation of two pathways governed by the transcription factors Msn2/4 and Hsf1, which bind to stress response elements (STREs) and heat-shock elements, respectively, in the promoters of their respective targets (reviewed in Trott and Morano 2003). Msn2 and Msn4 bind independently at the promoters of STRE-regulated genes and exhibit coordinate activation in response to a wide variety of cellular stresses, including changes in nutrient availability and osmolarity and growth conditions resulting in oxidative stress (Thevelein and de Winde 1999). Unlike Msn2/4, Hsf1 responds almost exclusively to heat shock, although induction of Hsf1 targets in the absence of heat shock has been observed in a subset of stress-induced genes (HSP12, HSP26) containing binding sites for both sets of transcription factors (Ferguson et al. 2004). Hsf1 is essential under normal and heat-shock conditions, a characteristic attributed to a cellular requirement for maintaining basal levels of heat shock and other transcripts in the absence of stress (Sorger et al. 1987).
Hsf1 is solely responsible for the expression of both heat-shock-inducible (HSP82) and constitutively expressed (HSC82) isoforms of the essential chaperone Hsp90 (Borkovich et al. 1989; Erkine et al. 1995). Hsp90 regulates the activity of multiple “client” proteins that play diverse cellular roles in signaling pathways and transcriptional activation, including the MAPKK Ste11, the respiratory gene transcription factor Hap1, and the amino acid starvation sensor kinase Gcn2 (Louvion et al. 1998; Zhang et al. 1998; Donze and Picard 1999; Trott and Morano 2003). Interestingly, Hsf1 has also been implicated as a client, as Hsp90 inhibition results in increased transcription from Hsf1 in yeast, and chaperone association with human Hsf1 blocks activation in vitro (Duina et al. 1998; Zou et al. 1998; Liu et al. 1999). Through its interactions with associated cochaperones, Hsp90 retains clients in an inactive state until the appropriate activation signal is received, which allows for folding and release of the active client (Pratt 1998). Hsp90 regulation is achieved via differential interaction with the cochaperones, some of which enhance or inhibit the Hsp90 ATPase cycle that drives client protein maturation (Pearl and Prodromou 2000). Hsp90 plays positive and negative regulatory roles with known substrates, highlighting the complexity of Hsp90 complex functions in yeast and mammals.
Sch9, a member of the Akt family of serine/threonine kinases, was previously identified as a nonchaperone regulator of Hsp90 activity, as its absence suppressed the temperature sensitivity of a strain containing low levels of Hsp90 and its cochaperones due to a deletion of the carboxy-terminal activation domain of Hsf1 [HSF(1-583)] (Morano and Thiele 1999). Deletion of SCH9 leads to increased activation of Hsp90-dependent clients, although the absence of a demonstrated biochemical interaction between Sch9 and Hsp90 suggests the involvement of additional yet-unidentified components in the regulatory circuit. The roles of Sch9 in yeast signaling remain elusive. Cells lacking SCH9 display extended longevity and defects in nutrient signaling, but it is not clear if these phenotypes derive from a common target (Crauwels et al. 1997; Fabrizio et al. 2001). Genetic studies have demonstrated that Sch9 functions in parallel with the protein kinase A (PKA) pathway in yeast and can suppress lethality caused by loss of PKA activity, suggesting some degree of functional overlap (Toda et al. 1988; Lorenz et al. 2000). However, Sch9 and PKA likely regulate unique targets as well, as mutations in both pathways have been demonstrated to have synergistic or opposing effects on gene expression (Roosen et al. 2005).
The activity of the three PKA catalytic subunits, Tpk1–3, is directly regulated by association with the Bcy1 regulatory subunit, which retains the catalytic subunits in an inactive state in the absence of cAMP (Toda et al. 1987). Synthesis of cAMP is dependent upon the enzymatic activity of adenylate cyclase (Cyr1) and requires association with and subsequent activation by the small G-proteins Ras1/2 or the heterotrimeric G-protein α-subunit Gpa2 (Kataoka et al. 1984; Lorenz and Heitman 1997; Colombo et al. 1998; Xue et al. 1998). Fermentative growth conditions (high glucose) favor the activated GTP-bound form of Ras1/2 and subsequently result in a high level of PKA-mediated signaling through the pathway. Msn2/4, whose activity is negatively regulated by PKA phosphorylation, is retained in the cytoplasm in the absence of cellular stress, but translocates into the nucleus in response to decreased signaling through the PKA pathway (Gorner et al. 1998). Negative regulation of the PKA pathway can occur at multiple nodes, including decreased activation of Ras1/2 and Gpa2 by the GTPase-activating proteins (GAPs) Ira1/2 and Rgs2, respectively, as well as hydrolysis of cAMP by the phosphodiesterases Pde1/2 (Sass et al. 1986; Tanaka et al. 1989; Versele et al. 1999).
Sse1 is a member of the Hsp110 group of protein chaperones. Represented in mammals by the Hsp105 and Apg gene families, the Hsp110s are divergent members of the Hsp70 chaperone class (Yasuda et al. 1995; Ishihara et al. 1999). Hsp110 and Hsp70 share a similar domain architecture composed of an amino-terminal ATPase domain and a substrate-binding domain containing an extended linker region in the carboxyl terminus. This linker contributes to the increased size of the chaperone relative to Hsp70 and may be responsible for the observed inability of Hsp110s to actively refold denatured proteins in vitro (Oh et al. 1999). However, both Chinese hamster ovary Hsp110 and yeast Sse1 possess “holdase” activity, the ability to stabilize unfolded proteins (Brodsky et al. 1999; Oh et al. 1999). While overexpression of Hsp110 confers thermotolerance to mammalian cells, no precise targets or functional roles have been identified (Oh et al. 1997). A better understanding of the roles of the Sse1 chaperone in yeast may therefore shed light on likely functions in more complex eukaryotes. Sse1 was originally identified as MSI3, a multi-copy suppressor of an ira1Δ mutation that causes constitutively high PKA activity (Shirayama et al. 1993). Overexpression also suppresses phenotypes associated with the high PKA activity of bcy1Δ cells, suggesting that Sse1 exerts a negative regulatory effect independent of Ras/cAMP signaling (Shirayama et al. 1993). Sse1 shares 76% identity with its homolog Sse2, although a biochemical role for either chaperone has not been clearly defined (Mukai et al. 1993). Sse1 weakly associates with Hsp90, and its absence results in decreased Hsp90 activity (Liu et al. 1999). In our efforts to further characterize the contributions of Sse1 to Hsp90 functions in vivo, we demonstrate that contrary to a previous report, Sse1/2 are an essential chaperone pair in S. cerevisiae. We have also uncovered a novel synthetic genetic interaction between Sse1 and Sch9. This interaction is observed specifically under growth conditions at elevated temperature and can be suppressed by decreased signaling through the PKA pathway. We demonstrate by both genetic and biochemical approaches that sse1Δ sch9Δ cells have abnormally high levels of unregulated PKA activity. Finally, we find that hyperactivation of the PKA pathway through expression of a constitutive RAS2 allele likewise results in temperature-sensitive growth. Together these results strongly link the poorly understood Sse1 chaperone with PKA signaling and suggest that modulation of PKA activity during thermal stress is required for adaptation and viability.
MATERIALS AND METHODS
Strains and growth conditions:
Previously described sch9Δ::HIS3, hsc82Δ::LEU2, and sse1Δ::G418 disruption strains were crossed to generate the appropriate double disruptants using standard yeast genetic techniques (Liu et al. 1999; Morano and Thiele 1999). The tpkw strain RS13-58A-1 (SP1 tpk1w tpk2::HIS3 tpk3::TRP1 bcy1::LEU2) was a kind gift of A. Vojtek (University of Michigan, Ann Arbor, MI) (Cameron et al. 1988). To demonstrate the synthetic lethality of an sse1Δ sse2Δ strain, sse1Δ cells expressing Yep24-SSE1 were transformed with an sse2::LEU2 deletion construct and screened by Southern blot analysis for insertion at the correct locus (Mukai et al. 1993). The construction of sse1Δ sch9Δ cdc25Δ was performed by PCR amplification of the cdc25Δ::hphMX4 cassette constructed in this study and subsequent transformation into sse1Δ sch9Δ cells. Strains were grown at 30° or 37° in nonselective (YP, 1% yeast extract, 2% peptone) or selective (synthetic complete, SC) media containing 2% glucose unless otherwise specified. Experiments involving the alternative carbon sources galactose (2%), glycerol (3%), and ethanol (2%) used YP-based media at the indicated concentrations. SC media containing 0.1% 5-fluoroorotic acid (5-FOA) was used to select for loss of URA3-marked plasmids. Cells spotted in serial dilution were taken from overnight cultures or directly from streak plates maintained at 4°. See Table 1 for yeast strains and plasmids used in this study.
pRS416-MET3-RASval19, YeplacPDE2, pSSE1, pCT31/32, and p416-Sch9 were previously described (Schmitt and McEntee 1996; Morano and Thiele 1999; Howard et al. 2001). The overexpression plasmid p416GPD BCY1 was constructed by PCR amplification and cloning of the BCY1 ORF into the BamHI-EcoRI sites of the yeast-Escherichia coli shuttle vector p416GPD (URA3, CEN) (Mumberg et al. 1995). P416CUP1MSN2 was constructed by PCR amplification and cloning of the MSN2 ORF with a 5′ FLAG-tag into p416CUP1 using BamHI-HindIII (Labbe and Thiele 1999). To construct a CDC25 deletion cassette with several hundred base pairs of homologous sequence flanking the ORF, template DNA containing a hygromycin B-resistance cassette (pAG32; Goldstein and McCusker 1999) was PCR amplified with primers homologous to the 5′ and 3′ ends of the CDC25 ORF. This product was transformed into wild-type cells containing a plasmid carrying the CDC25 gene, including endogenous promoter/terminator sequences (p415-CDC25; kind gift of S. Klein), where it integrated by homologous recombination, replacing the CDC25 coding sequence with the hygromycin resistance marker. This cassette was then used for deletion of the chromosomal CDC25 locus.
A Yep24-based genomic library (URA3, 2μ; Carlson and Botstein 1982) was transformed into sse1Δ sch9Δ cells (KMY67) and plated on selective media at 30° for several days. Ura+ cells representing ∼105 independent transformants were subsequently washed from the plates and replated at several different dilutions on selective media at 37°. Suppressor colonies obtained from these plates were retested for growth at 37° and their library plasmids recovered by plasmid rescue. Rescued plasmids with unique restriction enzyme patterns were retransformed to verify suppression and sequenced to determine the identity of the genomic insert. Deletion mapping was carried out to identify the suppressing locus.
PKA reporter assays:
For measurements of the activity of a STRE-lacZ reporter, W303, sse1Δ (KMY69), sch9Δ (KMY52), and sse1Δ sch9Δ (KMY67) cells containing pCT31/32 were grown to midlogarithmic phase in selective media at 30° (Schmitt and McEntee 1996). The culture was split, with half transferred to a 37° shaking water bath for 30 min and the other half remaining at 30° for the duration of the heat shock. Cells were harvested by centrifugation and β-galactosidase activity was assayed as previously described (Morano and Thiele 1999). Trehalose content was measured in YPD-grown stationary-phase cells by established extraction and quantitation methods using a glucose assay kit (Sigma Diagnostics, St. Louis) (Parrou and Francois 1997). Heat-shock sensitivity was assayed at 55° in the following manner: cells of a known concentration were spotted onto YPD plates in serial dilution, exposed to 55° for the indicated times, and subsequently grown at 30° for several days. Viability was assayed for each strain in reference to cells plated at the same dilution and not receiving heat-shock treatment.
Northern blot analysis:
RNA from midlog YPD cultures of W303, sse1Δ (KMY69), sch9Δ (KMY52), and sse1Δ sch9Δ (KMY67) cells was isolated by the hot phenol method and subsequently used for the analysis of FLO11 and ACT1 transcript levels by Northern blot analysis as described (Trott and Morano 2004). Probes to FLO11 and ACT1 were generated by T4 polynucleotide kinase end labeling of oligonucleotides generated from sequence within the open reading frame of each gene. Transcript levels following probe hybridization were quantitated using a Storm PhosphoImager (Amersham/Pharmacia, Piscataway, NJ).
Msn2 phosphorylation assays:
For single time-point glucose starvation experiments, cells were grown to midlogarithmic phase in 100 ml of YPD. One-half of the culture was harvested by centrifugation and frozen in dry ice (T0), while the remaining culture was harvested, washed in YP(-dextrose), and subsequently resuspended in 50 ml YP(-dextrose) at 30° with shaking for 10 min (T10). Cells were then collected and frozen in dry ice. For the glucose starvation time course, W303 and sse1Δ sch9Δ (KMY67) cells containing p416CUP1Msn2 were grown to midlog in 75 ml of selective media and subsequently induced with 100 μm CuSO4 for 1 hr at 30°. Following induction, 10 ml of culture was harvested for (T0), and the remaining culture was collected, washed with selective media (no dextrose), and added to a flask containing 60 ml of selective media (no dextrose) at 30° with continual shaking. Samples were harvested at intervals within a 1-hr time course and the resulting cell pellets were frozen for future use. Protein extracts were made by glass bead lysis in TEGN buffer (20 mm Tris-HCl, pH 7.0, 0.5 mm EDTA, 10% glycerol, 50 mm NaCl) with a protease inhibitor cocktail (2 μg/ml aprotinin, 2 μg/ml pepstatin A, 1 μg/ml leupeptin, 2 μg/ml chymostatin, and 2 mm phenylmethylsulfonyl fluoride). Crude cell extract was clarified by centrifugation at 0.8 × g at 4° and analyzed for Msn2 using antisera against Msn2 (kind gift of F. Estruch, University of Valencia, Valencia, Spain), phosphorylated Msn2 (S133, α-P-CREB; Cell Signaling, Beverly, MA), and Pgk1 (α-PGK1; Molecular Probes, Eugene, OR) protein levels using standard procedures for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting.
Simultaneous deletion of SSE1 and SCH9 results in a synergistic growth defect at elevated temperature:
Sch9 was shown in a previous genetic study to function as a negative regulator of Hsp90 and its associated cochaperones (Morano and Thiele 1999). However, no direct biochemical interactions between Sch9 and the Hsp90 complex have been detected, suggesting the involvement of additional intermediate proteins and/or pathways of regulation. To further explore the genetic relationship between Sch9 and Hsp90, strains were constructed with deletions of SCH9 and individual chaperone components of the Hsp90 complex and analyzed for growth defects at both normal (30°) and heat-shock temperatures (37°). As shown in Figure 1, the sch9Δ mutation resulted in a temperature-specific synthetic growth defect in combination with deletion of SSE1, as compared to either single deletion. Importantly, this phenotype was not observed in sch9Δ hsc82Δ cells, which have significantly reduced levels of Hsp90 due to the loss of the constitutively expressed Hsp90 isoform HSC82 (Borkovich et al. 1989). Furthermore, we failed to detect synthetic interactions between sch9Δ and sti1Δ, ydj1Δ, cpr6Δ, cpr7Δ, and sba1Δ (data not shown). These data suggest that the dramatic phenotype of sch9Δ sse1Δ cells was caused by a temperature-specific requirement for Sse1 in the absence of Sch9 activity.
Sse1 and Sse2 are an essential gene pair:
It was previously reported that sse1Δ sse2Δ cells constructed in the W303 strain background are viable and exhibit a growth rate comparable to the slow-growth phenotype of sse1Δ cells (Mukai et al. 1993). However, our attempts to reconstruct the sse1Δ sse2Δ strain in the same background using standard molecular genetic procedures resulted in the inability to obtain double deletions unless a complementing plasmid (Yep24-SSE1) was present. As shown in Figure 2, wild-type and sse1Δ or sse2Δ single deletion strains were able to grow on 5-FOA-containing medium, which requires loss of the complementing pSSE1 plasmid. In contrast, an sse1Δ sse2Δ strain carrying the same plasmid was unable to grow on 5-FOA, demonstrating that expression of either Sse1 or Sse2 is required for viability. The validity of this finding is further supported from several additional lines of evidence: (1) we were unable to obtain the double deletion by crossing an sse1Δ strain with an sse2Δ strain followed by tetrad dissection, (2) the same behavior was independently observed in both the W303 and the S288C strain backgrounds, (3) a high-copy screen to identify genes capable of suppressing the lethality of the sse1Δ sse2Δ strain identified only the SSE1 and SSE2 loci, and (4) SSE1 mutants incapable of complementing the sse1Δ mutation likewise failed to confer viability to the sse1Δ sse2Δ strain (Shaner et al. 2004; data not shown).
The temperature sensitivity exhibited by sse1Δ sch9Δ cells can be suppressed by reduction of PKA activity:
To investigate the nature of the synergistic severe temperature sensitivity of sse1Δ sch9Δ cells, we performed a high-copy genomic library screen to identify suppressors of the 37° growth phenotype. From ∼105 independent transformants, we isolated SSE1 and SSE2 multiple times, as expected. In addition, a single isolate of each of the homologous Ras-GAP genes IRA1 and IRA2 was recovered. The Ira1/2 proteins stimulate the inherent GTPase activity of Ras1/2, which subsequently lowers signaling through the PKA pathway through decreased activation of adenylate cyclase (Tanaka et al. 1989). As shown in Figure 3A, IRA1 overexpression strongly suppressed the temperature-sensitive growth defect, allowing colony formation at the highest cell dilution. Interestingly, the slow-growth phenotype of sse1Δ sch9Δ cells was not corrected by IRA1 overexpression, suggesting that these are separable phenotypes.
If the suppression observed by overexpression of IRA1 and IRA2 was due specifically to their roles as negative regulators of the PKA pathway, we surmise that overexpression of other downstream negative regulators of the PKA pathway would likewise allow growth of sse1Δ sch9Δ cells at 37°. As shown in Figure 3B, overexpression of BCY1, the regulatory subunit of PKA, and of PDE2, one of two phosphodiesterases responsible for attenuation of adenylate-cyclase-dependent PKA activation through hydrolysis of cAMP, also efficiently suppressed the temperature sensitivity of sse1Δ sch9Δ cells. Finally, we asked whether deletion of a positive regulator of PKA signaling would mimic overexpression of a negative regulator. The product of the CDC25 gene encodes a Ras guanine nucleotide exchange factor, previously shown to be required for Ras activation and subsequent stimulation of adenylate cyclase (Broek et al. 1987). An essential gene in the S288C strain background, deletion of CDC25 was recently shown to be viable in the W303 lineage (Folch-Mallol et al. 2004). This discrepancy is most likely due to the presence of the close homolog SDC25 in W303, which is not expressed in S288C due to the presence of an internal stop codon mutation (Damak et al. 1991; Purnelle and Goffeau 1997). Because simultaneous reduction of the PKA and Sch9 pathways results in extremely slow growth, an sse1Δ sch9Δ cdc25Δ strain was constructed in the presence of a plasmid overexpressing CDC25. After confirmation of the CDC25 disruption, the plasmid was lost through growth on 5-FOA-containing medium, and the resulting triple deletion strain was assayed for growth at 30° and 37° (Figure 3C). Loss of CDC25 resulted in slow-growing cells that nonetheless suppressed the temperature sensitivity caused by the sse1Δ sch9Δ deletions. Taken together, these genetic experiments demonstrate that decreased signaling through the PKA pathway can ameliorate the synthetic temperature sensitivity caused by loss of SCH9 and SSE1.
sse1Δ sch9Δ cells exhibit phenotypic characteristics typical of constitutively high PKA activity:
Mutations in the components of the PKA pathway that lead to activation of the downstream catalytic subunits (Tpk1–3) result in a common set phenotypic characteristics, including reduced activation of environmental stress response genes, decreased resistance to lethal heat shock, and decreased stationary-phase accumulation of the reserve carbohydrate, trehalose (Cameron et al. 1988; Boy-Marcotte et al. 1999). These effects can be attributed largely to decreased activity of the stress-responsive transcription factors Msn2/4, whose activity is negatively regulated by PKA-dependent phosphorylation via nuclear exclusion (Gorner et al. 1998). Because reduction of PKA signaling suppressed sse1Δ sch9Δ temperature sensitivity, we hypothesized that PKA might be hyperactivated in this strain. Wild-type, sse1Δ, sch9Δ, and sse1Δ sch9Δ cells were assayed for Msn2/4-dependent phenotypes using the following three reporter assays as an indicator of PKA status: (1) heat-shock-induced STRE-lacZ expression, (2) trehalose accumulation during stationary phase, and (3) resistance to severe 55° heat shock (Figure 4, A–C).
In contrast to wild type, sse1Δ sch9Δ cells displayed severely reduced expression of a STRE-lacZ reporter in response to a 1-hr heat treatment at 37°, consistent with the effects of “high PKA” on the environmental stress response (Figure 4A) (Marchler et al. 1993; Boy-Marcotte et al. 1999). The sse1Δ mutation alone only moderately decreased heat-shock induction, while loss of SCH9 resulted in derepression of basal STRE-lacZ activity, suggesting that SCH9 may contribute to negative regulation of Msn2/4 in parallel with PKA. Likewise, sse1Δ sch9Δ cells accumulated <25% of the trehalose of wild-type cells in stationary phase, whereas accumulation in sse1Δ cells was reduced by only ∼50% (Figure 4B). Again, sch9Δ cells exhibited the reverse trend, accumulating more than twice the trehalose of wild type and a 10-fold increase over the sse1Δ sch9Δ strain. This pattern was recapitulated when heat-shock resistance was examined over a 4-hr time course. Decreased viability was observed in sse1Δ and sse1Δ sch9Δ cells, whereas sch9Δ cells were hyperresistant to heat shock. Therefore, on the basis of three distinct assays of PKA-regulated, Msn2/4-dependent stress responses, sse1Δ sch9Δ cells exhibit high levels of PKA activity. It should be noted that Msn2/4 function is regulated by additional PKA-independent factors such as the Tor pathway, and thus low activity of Msn2/4 as observed in the reporter assays may not necessarily be exclusively attributed to elevated signaling through PKA (Pedruzzi et al. 2003). Because Msn2/4 function was nearly abolished in the sse1Δ sch9Δ strain, it was formally possible that loss of Msn2/4-dependent transcription was the cause of the 37° growth defect. However, overexpression of Msn2 failed to remediate the temperature sensitivity, consistent with previous reports that msn2Δ msn4Δ cells are not temperature sensitive (Martinez-Pastor et al. 1996; data not shown). Together, these data suggest a role for additional downstream target(s) of PKA that independently, or in coordination with Msn2/4, regulate a cellular process required for growth at elevated temperatures.
Although deletion of SCH9 has been shown to mimic mutations resulting in low-PKA phenotypes, an exception was demonstrated in which levels of the FLO11 transcript were elevated when SCH9 was disrupted in a flocculation-competent strain (Lorenz et al. 2000). Transcription at the FLO11 promoter is regulated through signals received from both the PKA and MAPK pathways and previously has been shown to require both PKA-dependent activation of the Flo8 transcription factor and inhibition of the repressor Sfl1 (Pan and Heitman 2002). Therefore, in at least one case, loss of SCH9 and reduction of PKA signaling results in opposite effects. We confirmed this finding, observing high levels of FLO11 transcripts in our sch9Δ strain (Figure 4D). Puzzlingly, wild-type S. cerevisiae laboratory strains (including W303 used in this study) are defective in flocculation due to a nonsense mutation in FLO8, although this mutation appeared not to affect FLO11 activation in our experiment (Liu et al. 1996). Similar to our findings with the Msn2/4-dependent reporter assays described above, the deletion of SSE1 in sch9Δ cells resulted in reversion of the sch9Δ phenotype (decreased levels of the FLO11 transcript), which adds genetic evidence for roles for Sch9 and Sse1 in PKA signaling to an Msn2/4-independent pathway.
sse1Δ sch9Δ cells are defective in glucose signaling through the PKA pathway:
Mutations in PKA pathway components that result in elevated catalytic activity lead to hyperphosphorylation of Msn2 at a cluster of serines located within the nuclear localization sequence, blocking nuclear translocation and causing cytoplasmic retention. Glucose starvation has been shown to dramatically decrease PKA-dependent phosphorylation with subsequent rapid translocation of Msn2 to the nucleus (Gorner et al. 2002). To gain additional direct insight into the nature of PKA pathway hyperactivation in sse1Δ sch9Δ cells, we examined wild type, sse1Δ sch9Δ cells, and the corresponding single deletion strains for the PKA-dependent phosphorylation status of Msn2 in response to glucose starvation. Gorner et al. (2002) previously demonstrated that a commercial antibody that specifically recognizes a PKA-phosphorylated motif in the mammalian transcription factor cAMP response element binding protein (CREB) also reacts with the yeast PKA-phosphorylated region of Msn2 and can thus be used as a biochemical assay of in vivo PKA activity. As previously reported, wild-type cells exhibited a significant decrease in PKA-dependent phosphorylation of Msn2 in response to a 10-min glucose starvation (Figure 5A). Consistent with our reporter assays, we observed that sse1Δ sch9Δ cells exhibited enhanced phosphorylation relative to wild type prior to glucose starvation and that this strain maintained a high level of phosphorylation despite glucose starvation. sch9Δ cells exhibited diminished phosphorylation before and after glucose starvation, consistent with the low-PKA phenotypes that we observed in the reporter assays. Interestingly, we found that while sse1Δ cells also displayed levels of PKA-phosphorylated Msn2 lower than those of wild type, we consistently observed lower levels of Msn2 protein in this strain in comparison to the other strains used in this experiment. As diminished stability or expression of Msn2 would mimic the high-PKA phenotypes observed in our reporter assays, it is possible that a portion of the observed phenotypes in sse1Δ cells may be due to compromised levels of the transcription factor.
The decrease in PKA phosphorylation of Msn2 is known to rapidly attenuate during glucose starvation, suggesting that PKA downregulation is a transient effect (Gorner et al. 2002). To determine if concomitant loss of Sch9 and Sse1 proteins rendered PKA signaling insensitive to glucose levels, we performed a more thorough time-course analysis of Msn2 phosphorylation, as shown in Figure 5B. Wild-type cells exhibited a significant decrease in phosphorylated Msn2 within 10 min of glucose withdrawal, followed by reestablishment of PKA-dependent phosphorylation by 30 min. In contrast, PKA-dependent phosphorylation of Msn2 in sse1Δ sch9Δ cells showed reduced attenuation during glucose starvation. The high level of phosphorylation exhibited by sse1Δ sch9Δ cells in glucose-replete conditions, as well as the absence of regulated dephosphorylation in response to glucose starvation, directly supports our hypothesis that this mutant combination results in hyperactivated, constitutively high PKA signaling.
The temperature sensitivity of sse1Δ cells can be suppressed by decreased signaling through the PKA pathway:
Cells bearing disruptions of SSE1 alone are moderately temperature sensitive for growth, whereas sse1Δ sch9Δ cells were unable to form colonies at 37°. To ask whether the latter phenotype represented enhancement of sse1Δ temperature sensitivity or a novel high temperature growth defect, we asked whether overexpression of the PKA pathway negative regulators BCY1 and PDE2 could likewise allow growth at 37°. Wild-type cells harboring empty vector and sse1Δ cells expressing empty vector or overexpressed PDE2 or BCY1 were spotted on selective medium and grown at 30° and 37° (Figure 6A). Overexpression of either of the two negative regulators resulted in suppression of sse1Δ temperature sensitivity. PKA signaling is maximal in the preferred carbon source glucose, and growth on nonfermentable carbon sources such as ethanol and glycerol is known to result in decreased signaling through the PKA pathway and subsequent activation of a subset of genes necessary for respiratory growth (Thevelein and de Winde 1999). If the temperature sensitivity of sse1Δ cells can be suppressed by decreased signaling through the PKA pathway, we expect that the temperature sensitivity of this strain would be suppressed by growth on alternate carbon sources. In support of our hypothesis, the growth defect of sse1Δ cells, including temperature sensitivity, was eliminated on all the nonfermentable carbon sources tested (Figure 6B). These data demonstrate that elimination of SCH9 significantly worsens a high temperature growth defect caused by loss of SSE1 and that this defect is intimately linked with PKA signaling.
Our data suggest that Sse1 functions as a negative regulator of the PKA pathway. If this hypothesis is correct, strains compromised for PKA function would be predicted to be hypersensitive to increased production of the chaperone. This idea was tested using temperature-sensitive mutants of the positive regulators RAS2 and CDC25 (Howard et al. 2001). The ras2-23 and cdc25-1 mutants exhibited a moderate reduction in growth rate at the semipermissive temperature of 35°. Both mutants displayed a pronounced growth defect in the presence of the SSE1 overexpression plasmid that was not observed in an isogenic wild-type control strain (Figure 7).
High constitutive signaling through the PKA pathway results in temperature-sensitive growth:
High constitutive signaling through the PKA pathway observed in sse1Δ sch9Δ cells correlated with temperature-sensitive growth at 37°. Moreover, reduction of PKA activity suppressed this phenotype, suggesting that unregulated PKA signaling interferes with cellular adaptation to high-temperature growth conditions. This effect cannot be attributed to decreased activity of Msn2/4, since loss of Msn2/4 has been documented to result in heat-shock sensitivity (inability to tolerate lethal temperatures), but not temperature-sensitive growth. A previous report demonstrated that mutant BCY1 alleles with distinct internal deletions exhibited differential temperature sensitivity (Cannon and Tatchell 1987). To ask directly whether constitutively high PKA signaling causes temperature sensitivity, we took advantage of a conditionally expressed, constitutively active allele of RAS2. RAS2val19 contains a valine substitution at position 19 that mimics the oncogenic activating mutation (Val17) found in mammalian Ras (Kataoka et al. 1984). This mutation further decreases the weak GTPase activity of Ras, locking it into the activating state for adenylate cyclase. In yeast, this results in constitutive cAMP production insensitive to changes in glucose concentration or growth phase and in subsequent high levels of PKA activity. This level of signaling is deleterious to growth, and strains bearing this allele are difficult to manipulate. Howard et al. (2001) placed the RAS2val19 allele under control of the methionine-repressible MET3 promoter, allowing precise control over expression. When this plasmid was transformed into wild-type cells and induced by growth on synthetic medium lacking methionine, growth at 30° was moderately slowed while growth at 37° was completely abolished (Figure 8). We verified that this effect required the catalytic activity of PKA by transforming the MET3-RAS2val19 plasmid into a strain with constitutively low, cAMP-independent PKA activity. This strain contains deletions in TPK2, TPK3, and BCY1, and an uncharacterized mutation in TPK1 (the so-called “wimp” allele) that greatly decreases kinase activity (Cameron et al. 1988). In this background, RAS2val19 induction slightly reduced the growth rate but failed to cause temperature sensitivity. Together, these data demonstrate that PKA signaling must be appropriately regulated to allow adaptation and growth at high, but sublethal, temperatures.
We initiated the current study with the goal of further exploring the role of the growth control kinase Sch9 in regulation of the Hsp90 chaperone in yeast. To date, investigations into regulation of Hsp90 activity have focused primarily on its cochaperones, which are required at different stages of client protein maturation and positively or negatively affect Hsp90's ATPase rate. The identification of Sch9, an Akt-like kinase with no known inherent chaperone activity, as a regulator of Hsp90 signal transduction activity established an intriguing link between Hsp90 and nutrient sensing/growth control signaling pathways in yeast. Sch9 has been implicated in several genetic studies to share functional overlap with the catalytic subunits of PKA. However, the lack of a direct demonstration of kinase activity in vitro or identification of bona fide binding partners makes it difficult to conclusively determine the role of Sch9 in the regulation of Hsp90 or the PKA pathway. An unexpected outcome of our efforts to elucidate Sch9's regulatory effects on Hsp90 is the discovery of a requirement for the Hsp90 cochaperone, Sse1, and Sch9 during growth at elevated temperature. We did not observe a similar genetic interaction between SCH9 and other Hsp90 cochaperones, including proteins known to inhibit Hsp90 ATPase activity such as Sti1, nor does the synthetic interaction between SCH9 and SSE1 shed light on the suppression of Hsp90-signaling defects in the HSF(1-583) truncation mutant. We therefore predict that Sch9 and Sse1 genetically interact in a pathway independently of their roles in Hsp90 complex regulation. In support of this possibility, although most Hps90 cochaperones appear to function exclusively in collaboration with Hsp90, a subset do not, including the Ssa family of Hsp70 chaperones related to Sse1.
Previous studies reported that deletion of SSE1 resulted in a moderate temperature sensitivity phenotype, while deletion of SSE2 had no phenotypic consequence. Additionally, disruption of both alleles simultaneously did not enhance the growth defects of the sse1Δ strain. After repeated attempts, we were unable to recreate an sse1Δ sse2Δ strain. Identical results were obtained in the W303 strain background, the same used by Mukai and coworkers, and the genome consortium strain BY4741, a derivative of the S288C strain background (Mukai et al. 1993; Winzeler et al. 1999). As described in the results, we utilized both classical and molecular genetic approaches to construct the sse1Δ sse2Δ double mutant, but were unable to introduce both mutations unless the deletion was covered by ectopic expression of SSE1. Extended incubation of deletion transformations failed to recover potential extremely slow-growing double disruptions. Moreover, in a high-copy suppressor screen designed to identify potential targets or modulators of SSE1/2 function we isolated only Sse1 and Sse2. We find that while SSE2 is capable of suppressing sse1Δ phenotypes, it can do so only when overexpressed. This is consistent with a recent genome-wide analysis of protein abundance that determined the number of Sse1 molecules/cell to be ∼72,000 vs. 6300 for Sse2. Sse2 can efficiently correct growth defects caused by loss of Sse1 when expression of both chaperones is equivalent, suggesting little functional specificity between the isoforms (Ghaemmaghami et al. 2003).
We have demonstrated that sse1Δ cells exhibit phenotypic characteristics typical of elevated PKA signaling, while sse1Δ sch9Δ cells display extremely high levels of PKA activity unresponsive to nutrient status. The temperature-sensitive growth phenotypes of both strains can be remediated by reduction of PKA pathway activity, suggesting that Sse1, with additional input from Sch9, negatively regulates PKA activity as depicted in Figure 9. These data are consistent with the previous isolation of SSE1 as MSI3, a high-copy suppressor of an ira1Δ strain with constitutively high PKA signaling (Shirayama et al. 1993). Moreover, in this study overexpression of SSE1 was also demonstrated to suppress the high-PKA phenotypes of a bcy1Δ strain. This latter finding is of critical importance, because all regulation of the PKA catalytic subunits Tpk1–3 through the Ras/cAMP pathway is dependent on the regulatory Bcy1 subunit. Therefore, Sse1 modulation of Tpk catalytic activity can be formally defined as a cAMP-independent mode of PKA regulation. This model is consistent with our observation that the hypomorphic mutations that reduce PKA activation by cAMP, ras2-23 and cdc25-1, render cells hypersensitive to SSE1 overexpression. On the basis of these criteria, the most likely point of action for Sse1 in PKA pathway function is with the kinases. We are currently exploring the possibility that Sse1 physically interacts with one or more of the catalytic subunits. Alternatively, the Sse1 chaperone may indirectly influence their subcellular localization, preventing access to key targets, or alter the activity of a yet-unidentified protein phosphatase that opposes phosphorylation by Tpk.
The relationship between Sch9 and the PKA pathway is complex. Sch9 overexpression suppresses the lethality of PKA-inactivating mutations, including loss of all three catalytic subunits (Toda et al. 1988). Consistent with PKA and Sch9 functioning in parallel, we observed a requirement for Sch9 in maintaining basal levels of phosphorylation of Msn2 in the absence of stress, a result that is supported by phenotypic characteristics of sch9Δ cells typical of strains with high levels of activated Msn2, including elevated STRE-lacZ expression, trehalose accumulation, and heat-shock resistance. In contrast, we observed dramatically increased transcript levels of FLO11 in the absence of SCH9, similar to that caused by overexpression of Tpk2, suggesting that at least one Tpk isoform may be activated in sch9Δ cells. Additionally, Sch9 has been implicated as a negative regulator of PKA, with moderate effects on catalytic activity as measured by phosphorylation of the model substrate kemptide, although the mechanism of this putative regulation is unknown (Crauwels et al. 1997). Because the PKA catalytic subunits and Sch9 clearly share structural and functional homology, it is possible that Sch9 may participate in a feedback inhibition mechanism to regulate cAMP levels and thereby PKA signaling (Figure 9, dashed line). Although the range of cellular cAMP fluctuation is likely not as dramatic as originally described, PKA status clearly affects steady-state cAMP accumulation (Nikawa et al. 1987). It is therefore possible that simultaneous activation of one or more Tpk subunits by increased cAMP levels due to elimination of Sch9 and cAMP-independent derepression by loss of Sse1 can synergistically result in the constitutively high PKA activity and subsequent phenotypes that we have observed. The ability of Sch9 to regulate both shared downstream targets and PKA pathway function itself may explain how opposite phenotypes are observed in sch9Δ vs. sse1Δ sch9Δ strains (Crauwels et al. 1997; Roosen et al. 2005).
Hyperactivation of the PKA pathway is classically associated with substantially decreased tolerance to lethal heat shock (temperatures >42°). Although heat-shock resistance can be increased by prior exposure to sublethal temperatures that induce the heat-shock and environmental stress responses, the heat-shock sensitivity assay has long been used as a convenient and accurate biomarker for PKA status in unstressed cells. This correlation is likely due to decreased activity of the general stress transcription factors Msn2/4, which regulate multiple factors contributing to thermal tolerance (Gasch et al. 2000). Most notable among these is production of the disaccharide trehalose and synthesis of the chaperone Hsp104. In fact, elevated levels of both cellular constituents are sufficient to confer nearly maximal thermotolerance (Lindquist and Kim 1996). We have demonstrated that unregulated high PKA activity caused by constitutively active Ras results in temperature sensitivity at 37°, consistent with the previous finding that bcy1 mutant alleles can result in differential temperature sensitivity at 37° or 39° (Cannon and Tatchell 1987). The stress of temperature sensitivity is distinct from heat-shock tolerance on the basis of multiple criteria. First, the former reflects an inability of the cell to modulate gene expression and physiology in response to chronic high but not deleterious temperature, while the latter assesses the cell's readiness to withstand acute lethal temperatures with no prior adaptation. Second, msn2Δ, msn2Δ msn4Δ, and hsp104Δ mutants all display reduced heat-shock tolerance, as do mutants unable to synthesize or accumulate trehalose, while none of these mutants display temperature-sensitive growth at 37° (De Virgilio et al. 1993; Lindquist and Kim 1996; Martinez-Pastor et al. 1996). Our findings suggest that cellular adaptation to high-temperature growth depends upon proper regulation of PKA signaling. It is therefore tempting to speculate that Sse1 and Sse2 may function together as a negative feedback loop to decrease PKA activity under heat-shock conditions, when chaperone levels are strongly induced. The relevant PKA target(s) responsible for this phenotype are at present unknown. An intriguing possibility is suggested by the recent identification of Spt5 and Srb9, components of the basal transcription apparatus and the Mediator complex, as PKA substrates. Deletion of multiple transcription proteins including SIN4, MED2, and SRB9-11 results in moderate-to-severe temperature sensitivity (Myers et al. 1998; Chang et al. 2001; Bourbon et al. 2004). However, whether unregulated phosphorylation of transcriptional components is the cause of the observed temperature sensitivity of strains with constitutively high PKA activity remains to be determined.
The authors thank Paul Herman, Johan Thevelein, Michel Jacquet, Francisco Estruch, Shoshana Klein, Anne Vojtek, and John McCusker for helpful advice and reagents and an anonymous reviewer for helpful comments on the manuscript. We also thank Brett Geissler for contributions in the initial phases of this study. This work was supported by Research Scholar Grant MBC-103134 from the American Cancer Society.
Communicating editor: A. P. Mitchell
- Received March 9, 2005.
- Accepted March 18, 2005.
- Genetics Society of America