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Originally published In Press as doi:10.1074/jbc.M407900200 on November 15, 2004

J. Biol. Chem., Vol. 280, Issue 4, 2529-2535, January 28, 2005
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Rom2p, the Rho1 GTP/GDP Exchange Factor of Saccharomyces cerevisiae, Can Mediate Stress Responses via the Ras-cAMP Pathway*

Jong-In Park{ddagger}, Emma J. Collinson, Chris M. Grant§, and Ian W. Dawes

From the Ramaciotti Centre for Gene Function Analysis, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney 2052, Australia

Received for publication, July 13, 2004 , and in revised form, November 8, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Ras-cyclic AMP pathway is connected to other nutrient-regulated signaling pathways and mediates the global stress responses of Saccharomyces cerevisiae. Here, we show that Rom2p, the Rho1 GTP/GDP exchange factor, can mediate stress responses and cell growth via the Ras-cAMP pathways. ROM2 was isolated as a suppresser of heat and NaCl sensitivity caused by the lack of the Ras-GTPase activator Ira2p or of cAMP phosphodiesterases. Subsequent analysis of strains with a rom2 deletion showed that Rom2p is essential for resistance to a variety of stresses caused by freeze-thawing, oxidants, cycloheximide, NaCl, or cobalt ions. Stress sensitivity and the growth defect caused by the rom2 deletion could be suppressed by depleting Ras or protein kinase A (PKA) activity or by overexpressing the high affinity cAMP phosphodiesterase Pde2p. In addition, overexpression of ROM2 could not rescue cells lacking the regulatory subunit of PKA, indicating that the Ras-adenylate, cyclase-PKA cascade is essential for Rom2p-mediated stress responses and cell growth. Deletion of IRA2 exacerbated the freeze-thaw sensitivity and growth defect of the rom2 mutant, indicating that Rom2p signaling may control Ras independently of IRA2. Increases in cAMP levels were detected in the rom2 deletion mutants, and these were comparable with the effects of an ira2 mutation. The effects of the deletion of ROM2 on sensitivity to hydrogen peroxide, paraquat, and cobalt ions, but not to caffeine, were reduced when a constitutive allele of RHO1 was introduced on a single copy plasmid. However, the effects of the deletion of ROM2 on sensitivity to diamide and NaCl were exacerbated. Taken together, our data indicate that Rom2p can regulate PKA activity by controlling cAMP levels via the Ras-cAMP pathway and that for those stresses related to oxidative stress, this cross-talk is probably mediated via the Rho1p-activated MAPK pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The ability of cells to appropriately respond to alterations in environment is essential for the survival of stress situations, and processes for stress sensing and the subsequent modulation of cell physiology require the involvement of signal transduction pathways. The Ras-cyclic AMP pathway is an essential nutrient-sensing pathway in yeast (1, 2) and is also important for stress tolerance of Saccharomyces cerevisiae. This pathway is involved in cellular responses to stresses mediated via genes with stress response elements in their promoters (3–7), Pos9/Skn7-mediated oxidative stress (8), high-salt stress (9, 10), and freezing and thawing (11, 12).

Ras proteins in yeast (encoded by RAS1 and RAS2) are associated with adenylate cyclase (encoded by CYR1/CDC35) to control cAMP synthesis. cAMP functions exclusively through the cAMP-dependent protein kinase A (PKA)1 (13, 14), which can phosphorylate various target proteins involved in transcription, energy metabolism, cell cycle progression, and the accumulation of glycogen and trehalose (4, 15–22). PKA is a heterotetramer of two regulatory subunits encoded by BCY1 and two catalytic subunits redundantly encoded by TPK1, TPK2, and TPK3 (23). cAMP binding to the regulatory subunits induces dissociation and activation of the catalytic subunits of PKA. This Ras-cAMP signaling is controlled by diverse regulatory components. The highly homologous Ira1p or Ira2p inhibits Ras activity by stimulating the GTPase activity of Ras, whereas Cdc25, the guanine nucleotide exchange factor, activates Ras (24–27). In addition, the cAMP phosphodiesterases, encoded by PDE1 or PDE2, act as a separate branch of the pathway to play a negative regulatory role by hydrolyzing cAMP (28, 29).

The Ras-cAMP pathway may be connected to other nutrient or stress-sensing pathways for yeast cell survival. The Rho1-protein kinase C (Rho1-PKC) pathway, known as the cellular integrity pathway for its role in cell wall biosynthesis and mating (30–32), can also mediate stress responses to temperature shift (33–35) and changes in osmolarity (35–37). The Rho1-PKC pathway is connected to the TOR2 pathway via Rom2p (38, 39), which is the guanine nucleotide exchange factor for Rho1p (40). TOR2 is a phosphatidylinositol kinase homolog and has an essential role in integrating nutrient signals to regulate cell growth and cell cycle progression (41, 42). Recently, it has been shown that the Ras-cAMP pathway can suppress a TOR deficiency in yeast (43), suggesting that the Ras-cAMP pathway mediates parts of TOR signaling.

In this study, we isolated ROM2 as a suppresser of the sensitivity to heat shock or osmostress caused by lack of IRA2 or cAMP phosphodiesterases and subsequently investigated the cross-talk between Rom2p and the Ras-cAMP pathway. We show that the sensitivities to various stresses and the growth defect caused by rom2 deletion can be suppressed by modulating activity of the Ras-cAMP pathway. We also show that Rom2p can control cAMP levels in the wild-type strain. Our study demonstrates that Rom2p can mediate negative regulation of the Ras-cAMP signal transduction pathway.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains, Media, and Transformation—The yeast strains used are described in Table I. Strain Ras2(II) was made by backcrossing JC302-26B (44) to CY4 (11). The heterozygous diploid strains Jrom2/fsm, Jrom2/pde2, and Jrom2/ras2 were made by mating the strain Jrom2 with the strains Fsm4C, Jpde2, and Ras2(II) respectively. The strain Wrom2/MB12 was made by mating strain W303rom2 with strain MB12 (45). Strains, otherwise specified, were grown at 30 °C with shaking at 180 rpm in 3 ml of medium in a 16 x 100-mm culture tube. Yeast extract peptone dextrose (YEPD) medium contained 2% (w/v) glucose, 2% (w/v) bactopeptone, and 1% (w/v) yeast extract, and synthetic minimal dextrose (SD) medium contained 2% (w/v) glucose, 0.17% (w/v) yeast nitrogen base (Difco), 0.5% (w/v) ammonium sulfate (Oxoid), and auxotrophic requirements at 40 mg/liter where necessary. Media were solidified by adding 2% (w/v) agar. Transformation of yeast cells was carried out using lithium acetate (46).


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  		TABLE I
Yeast strains used in this study

 
Gene Deletion—Genes were deleted using a PCR-based direct gene deletion technique (47). Primers used to amplify the deletion DNAs are given in Table II. PCR was performed using the single copy plasmid pRS413 as the template to prepare PDE2 or IRA2 disruption DNA and plasmid pRS414 for PDE1 disruption. Successful disruption was confirmed by PCR using primers designed from the upstream region of target DNA and from the internal region of disruption markers. Primers used for confirmation are listed in Table II. DNA for ROM2 deletion was prepared by PCR amplification of genomic DNA prepared from the rom2 deletion strain AS138-1C (38) using the PCR primers for rom2 deletion listed in Table II. Successful deletion was confirmed by PCR amplification of the ROM2 region using the same primers and comparing the size of the PCR products. The pde1 pde2 double deletion mutant was made by crossing the pde1 and pde2 mutants, the subsequent sporulation of the diploid strain, and dissection. All deletions were verified as single mutations by backcrossing each mutant strain to its parental wild-type strain and measuring the segregation of phenotypes.


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  		TABLE II
PCR primers used in this study

 
Plasmids—YEp13PDE2 was obtained as a multi-copy suppressor of the ira2 mutation by screening a yeast genomic DNA library (American Type Culture Collection number 37323) in the ira2 mutant Fsm4C. It contains DNA from chromosome XV spanning 1,011,721–1,015,679 base pairs where PDE2 is the only intact open reading frame. Plasmid 37-3, containing DNA from chromosome XII spanning 860,720–872,193 base pairs, was cloned from a similar screening using the yeast genomic DNA library YHP1, which is constructed in the p366 vector (American Type Culture Collection number 77162). p366ROM2 was made by truncating plasmid 37–3 using BstBI.

cAMP Determination—An appropriate volume of cell culture was harvested and washed in 20 mM potassium phosphate buffer (pH 7). Cells were homogenized in 5% (w/v) trichloroacetic acid with glass beads. Supernatants were collected, washed with water-saturated diethyl ether, and freeze-dried. cAMP was measured using an enzyme immunoassay system (Amersham Biosciences) as indicated by the manufacturer.

Freezing and Thawing Conditions—Cells were harvested by centrifugation and washed in 0.1 M sodium phosphate buffer (pH 7) and suspended to an A600 of 3 in the same buffer. Aliquots (0.3 ml) of cells were transferred to thin walled 1.5-ml polycarbonate tubes (Greiner Labortechnik), frozen at –20 °C for 2 h, and thawed at room temperature for 15 min as described previously (11). Survival was determined by diluting cells into YEPD medium at room temperature and plating on YEPD plates. Cells were grown at 30 °C for 2 days before colony counting.

Assay for Stress Resistance on Plates—5 µl of cell culture, diluted to an A600 of 0.1 or 1 in fresh SD medium, was spotted onto SD plates containing 3 mM H2O2, 0.5 mM diamide, 2.5 mM paraquat, 0.25 µg/ml cycloheximide, 1 mM CoCl2, 7 mM caffeine, or 1.5 M NaCl and incubated at 30 °C for visualization of the phenotype. For heat shock, cells were spotted on YEPD plates and incubated at 37 °C.

Long Term Starvation Survival Test—The ability of cells to survive starvation was tested as described previously (48). Briefly, 2-day-cultured cells were maintained in sterile water for 12 days at 30 °C. Viability was measured by counting colonies on YEPD plates and expressed as a percentage of the initial cell count.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation of ROM2 as a Stress Defense Gene—IRA2 and PDE2 are important genes encoding components of the Ras-cAMP signaling pathway that are important in maintaining resistance to a wide range of stress conditions (49). Using a genomic DNA library in the single copy YHP1 vector, we isolated a plasmid that could suppress heat shock and NaCl sensitivity caused by lack of IRA2 (Fig. 1A). This plasmid, containing ARC18, ROM2, SUR4, YLR373C, and YLR374C, was subsequently truncated with BstBI to produce p366ROM2. This identified ROM2 as the gene responsible for rescue of the stress sensitivity. ROM2 overexpression could also suppress heat shock sensitivity caused by the lack of cAMP phosphodiesterase activity in the pde1 pde2 double mutant, but it could not effectively rescue yeast cells lacking BCY1, which encodes the regulatory subunit of PKA (Fig. 1B). This finding indicates that Rom2p probably acts by affecting the level of cAMP, because the loss of Bcy1p leads to constitutive activation of PKA unaffected by cAMP levels. This isolation of ROM2 was interesting, because Rom2p is a component of the TOR-Rho1-PKC pathway (38, 39), indicating that Rom2p may mediate some form of cross-talk between the TOR-Rho1-PKC pathway and the Ras-cAMP pathway.



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  		FIG. 1.
ROM2 suppresses stress sensitivity of mutants in the Ras-cAMP pathway. Stress tolerance of cells grown on SD medium for 1 day was determined. A, cells of strain Fsm4C (ira2), transformed with the plasmid 37-3, were spotted onto SD plates and cultured at 37 °C for 1 day for heat shock. Cells were also spotted on SD plates containing 1.5 M NaCl for salt stress and cultured at 30 °C for 2 days. B, cells of Jpde12 (pde1 pde2) and dbcy1 (bcy1), transformed with p366ROM2, were tested for heat shock resistance. Cultures were diluted to A600 of 1.0 or 0.1, and 5 µl of each diluted fraction was used for spotting. Experiments were repeated at least three times with similar results.

 
Deletion of ROM2 Leads to Sensitivity to a Broad Range of Stresses—To examine the involvement of Rom2p in a range of stress responses, ROM2 was deleted in the wild-type strain CY4. Deletion of ROM2 rendered cells sensitive to freezing and thawing (Fig. 2A) and to H2O2, the superoxide generator paraquat, the sulfhydryl oxidant diamide, cycloheximide, NaCl, caffeine, and cobalt (Fig. 2B). Identical results were obtained with two other rom2 deletion mutant strains, AS138-1c and W303rom2, which were constructed on the different genetic backgrounds of strains JK9-3d and W303-1A, respectively (Fig. 2A illustrates the freeze-thaw response of AS138-1c; data not shown for W303rom2). These findings indicated that Rom2p is involved in a global response of yeast to a wide range of stresses and that the effect is unlikely to be restricted to one strain. Because the deletion of ROM2 elicited similar effects on stress responses as those that were noted for deletion of the negative regulators of the Ras-cAMP pathway, IRA2 or PDE2, we investigated the possibility of a relationship between Rom2p and the Ras-cAMP pathway.



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  		FIG. 2.
ROM2 is required for a global stress response. Stress resistance of the rom2 mutants AS138-1C and Jrom2 was determined. The rom2 mutants were also transformed with p366ROM2 or YEp13PDE2. A, freeze-thaw tolerance of cells grown on minimal SD medium for 2 days was determined by measuring viability after exposure to –20 °C for 2 h. Percentage survival is expressed relative to the culture viability immediately prior to freezing (%). The data shown are means (± S.E.) for triplicate measurements from a representative experiment. B, stress resistance of cells grown for 1 day was determined by spotting 5 µl of each diluted fraction (A600 of 1.0 or 0.1) onto SD plates containing each of the agents indicated. Experiments were repeated at least three times with similar results.

 
Rom2p Shows an Epistatic Relationship with the Ras-cAMP Pathway—To determine whether there is an epistatic relationship between Rom2p and the Ras-cAMP pathway, the rom2 deletion mutants were crossed with isogenic yeast strains carrying ras2, ira2, pde2, or the tpk1 tpk2 double deletion mutation (Table I; see also "Experimental Procedures"). Asci of the resulting diploids were dissected to generate cells carrying combinations of rom2 and each mutation. These mutants were then examined for their freeze-thaw stress resistance.

The ras2 deletion was found to completely nullify the freeze-thaw sensitivity and growth defect caused by the rom2 deletion (Fig. 3A). Likewise, the tpk1 tpk2 double deletion also suppressed the freeze-thaw sensitivity and growth defect of the rom2 mutant (Fig. 3D), indicating that components of the Ras-adenylate cyclase-PKA cascade (including the PKA activity) are essential for the ROM2-mediated freeze-thaw stress response and cell growth. Meanwhile, the deletion of IRA2 or PDE2, which are the negative regulators of the Ras-cAMP pathway, exacerbated the freeze-thaw sensitivity and growth defect of the rom2 mutant (Fig. 3, B and C), indicating that ROM2 may affect PKA activity in parallel with IRA2 or PDE2.



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  		FIG. 3.
ROM2 has an epistatic relationship with the Ras-cAMP pathway. Freeze-thaw tolerance and the growth of haploid segregants of the heterozygous diploid strains Jrom2/ras2 (rom2/ras2) (A), Jrom2/pde2 (rom2/pde2) (B), Fsm/rom2 (ira2/rom2)(C), or Wrom2/MB12 (rom2/tpk1 tpk2) (D) were determined. wt, wild-type. Cells were grown on YEPD medium for 2 days prior to testing. Freeze-thaw tolerance was determined by measuring viability after exposure to –20 °C for 2 h. Percentage survival (column) is expressed relative to the culture viability immediately prior to freezing (%). A cross (x) indicates the A600 of cultures at the time points. The data shown are means (± S.E.) for triplicate measurements from a representative experiment. Experiments were repeated at least three times with similar results.

 
To further investigate this possibility, we examined the effect of overexpressing PDE2 in the rom2 mutant. If the role of Rom2p in some of the stress responses is mediated by negative regulation of the Ras-cAMP pathway, overexpression of PDE2 should compensate for the rom2 deletion. Indeed, overexpression of PDE2 did confer stress tolerance to freeze-thawing (Fig. 2A) and to peroxides, paraquat, and cobalt (Fig. 2B). It also increased starvation survival of the rom2 mutant about 25-fold. After a long-term (12-day) starvation of yeast cells in water, the rom2 mutant showed ~2% survival in the absence of PDE2 overexpression, but this increased to ~50% with PDE2 overexpression; the wild-type CY4 did not lose viability under these conditions. However, and interestingly, overexpression of PDE2 did not confer resistance to diamide, cycloheximide, NaCl, or caffeine-induced stress (Fig. 2B), indicating that Rom2p can also mediate stress responses that are independent of PKA. Taken together, these data indicate that Rom2p may play a substantial part of its role in some stress responses and cell growth by negatively regulating the Ras-cAMP pathway.

Rom2p Can Affect Intracellular cAMP Levels—As indicated above, if Rom2p affects the activity of the PKA pathway, one way would involve its acting to alter the intracellular level of cAMP. We therefore further characterized Rom2p-mediated regulation of the Ras-cAMP pathway by determining the effects of deleting ROM2 on cellular cAMP levels. The level of cAMP was determined in the wild-type and in the rom2 mutant strain, Jrom2, during both exponential growth (6–7 h of culture; A600 = 1) and the stationary phase (48 h of culture). In both growth phases, the rom2 mutant showed elevated levels of cAMP (~1.8-fold higher than wild-type levels), and this was only slightly lower than the level caused by deletion of IRA2, which is one of the main negative regulators of the Ras-cAMP pathway (Table III). Taken together, our data indicate that Rom2p can modulate cAMP levels through the Ras-adenylate cyclase cascade and thus mediate stress responses and cell growth.


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  		TABLE III
Determination of cAMP levels

 
Sensitivity of a rom2 Mutant Is Rescued by a Constitutively Activated Allele of RHO1—Rom2p is a GTP exchange factor whose main target in S. cerevisiae is Rho1p, which acts on protein kinase C (Pkc1p) to signal through the cell integrity pathway involving the MAP kinase Mpk1p (50, 51). However, Rom2p-mediated signaling can be independent of the Rho1-PKC pathway. GTP-bound Rho1p is a potential activator of Bni1p, a formin that nucleates the formation of linear actin filaments involved in budding and mitotic spindle orientation (52), and of the transcription factor Skn7p (53). Moreover, Pkc1p is involved in an "arrest-of-secretion response" that appears not to involve MAP kinase signaling via Mpk1p (54). If Rom2p acts via the Rho1p-activated Pkc1p pathway, expression of a constitutively active single copy RHO1 allele in a rom2 mutant should lead to an increase in resistance to a range of toxic compounds. This was achieved by transforming the rom2 mutant with a centromeric plasmid harboring the constitutive allele of RHO1, rho1Q68L (55).

From Fig. 4 it can be seen that expression of the rho1Q68L allele in a rom2 mutant led to an increase in resistance to hydrogen peroxide, paraquat, and cobalt ions. However, it had no effect on resistance/sensitivity to caffeine. When exposed to 7 mM caffeine, a rom2 mutant was no more sensitive than the wild-type strain, and no effect was observed on sensitivity in a rom2 mutant expressing the rho1Q68L allele. When challenged with a higher dose of caffeine (7.25 mM), however, the rom2 mutant was more sensitive than the wild-type as observed in Fig. 2; but, again, no effect was observed on the sensitivity of expressing the rho1Q68L allele in the rom2 mutant. Furthermore, expression of the constitutively active RHO1 allele actually led to an increase in sensitivity to the thiol-specific oxidant diamide. Likewise, expression of rho1Q68L also led to an increase in sensitivity to NaCl (at 1.5 mM). However, at this concentration a rom2 mutant appeared to be no more sensitive to the chronic NaCl stress than the wild-type strain. When the concentration of NaCl was increased to 1.75 mM, a rom2 mutant was completely sensitive and, therefore, the increased sensitivity of a rom2 mutant expressing rho1Q68L was masked. These results indicate that the cross-talk between ROM2 and the Ras-cAMP pathway is probably mediated via the Rho1p-activated MAPK pathway for the conditions related to oxidative stress.



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  		FIG. 4.
Expression of a constitutive allele of RHO1 suppresses the sensitivity of the rom2 mutant to reactive oxygen species, cobalt ions, and NaCl, but not caffeine. The rom2 mutant Jrom2 was transformed with a centromeric plasmid harboring the constitutive allele (rho1Q68L) of RHO1. Stress resistance of cells grown for 1 day was determined by spotting 5 µl of each diluted fraction (A600 of 1.0 or 0.1) onto SD plates containing each of the agents indicated, namely 3 mM H2O2, 2.5 mM paraquat, 0.5 mM diamide, and 1 mM CoCl2 (A) or 1.5 M NaCl, 1.75 M NaCl, 7 mM caffeine, and 7.25 mM caffeine (B). Quantification of data in the spot tests was carried out using ImageJ (image quantification software produced by National Institutes of Health) and expressed in the bar charts under each spot test panel. The data shown are means ± S.E. from a representative experiment performed in triplicate. Experiments were repeated at least three times with similar results. *, p < 0.005 compared with the vector control (pRS315) (two-tailed t test); **, p < 0.0007 compared with the vector control (pRS315) (two-tailed t test); ***, p < 0.00007 compared with the vector control (pRS315) (two-tailed t test).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This study shows that Rom2p can mediate stress responses and cell growth by negatively regulating the Ras-cAMP pathway. Rom2p is one of two pleckstrin homology domain proteins that act as the guanine nucleotide exchange factor to activate Rho1p (50, 51). Because Rom2p connects the Rho1-PKC pathway to the TOR signaling pathway (38, 39), our data indicate that the Ras-cAMP pathway may interact with the TOR-Rho1-PKC pathway via Rom2p. In addition, because Rom2p, a positive regulator of the Rho1-PKC pathway, negatively regulates the Ras-cAMP pathway, these two signal transduction pathways may act in opposite directions for stress resistance.

The data obtained from the rom2 deletion mutants indicate that Rom2p is involved in determining the intrinsic stress tolerance of yeast cells to a broad range of stresses including freezing and thawing, long term starvation, oxidative stress, hyperosmotic stress, and metabolic stress. The fact that the expression of a constitutively active allele of RHO1 in the rom2 mutant led to an increase in resistance to the oxidant hydrogen peroxide and the superoxide anion generator paraquat high-lighted the possibility that the cross-talk between Rom2p and the Ras-cAMP pathway in response to reactive oxygen species is probably mediated via the Rho1p-activated MAP kinase pathway. In addition to increasing the resistance of the rom2 mutant to reactive oxygen species, this pathway also increased resistance to CoCl2. This is not surprising, given that metal ions such as cobalt can catalyze the generation of reactive oxygen species via the Fenton reaction and indirectly cause oxidative stress via the depletion of GSH, which ultimately leads to lipid peroxidation (56, 57). Interestingly, cells with a constitutively active Rho1p are more sensitive to diamide. This is less unexpected, however, because mutants that are sensitive to diamide are often resistant to other oxidants, especially hydrogen peroxide (58). Moreover, the rom2 mutant expressing a constitutively active form of Rho1p was also more sensitive to NaCl, indicating that the responses to both diamide and NaCl are possibly mediated through a similar mechanism. Furthermore, the expression of a constitutively active allele of RHO1 had no effect on resistance to caffeine, indicating that the response to this compound is probably mediated through another pathway. This extends the potential involvement of the Rho1-PKC pathway in stress responses well beyond the previously observed heat shock (33–35) and hypo-osmotic stresses (35–37). Recently, it has been shown that the PKC-activated MAP kinase Mpk1p is rapidly and multiply phosphorylated following the exposure of cells to low concentrations of linoleate hydroperoxide (59); hence, under one condition at least the cell integrity pathway MAP kinase is activated by one form of oxidative stress. Rom2p-mediated signaling can be independent of the Rho1-PKC pathway. As indicated above, there are other ways that Rom2p activation appears to act independently of Mpk1p (52, 53, 54), and these ways may be involved in the response to caffeine and NaCl.

Our genetic and biochemical data indicate that ROM2 can control PKA activity by regulating cAMP levels in some way and that this effect occurs via the Ras-adenylate cyclase cascade. This mechanism of cAMP control by ROM2 appears to involve Ras2p, because deletion of RAS2 effectively suppressed the stress sensitivity and growth defect caused by the deletion of ROM2. Interestingly, ROM2-mediated negative regulation of Ras2p appears to be independent of IRA2, because deletion of this gene exacerbated the stress sensitivity of the rom2 mutant. In future studies, it will be important to identify whether ROM2 regulates the Ras pathway through other regulators, such as CDC25 or IRA1, or whether ROM2 mediates an independent signaling to control Ras2p activity.

Although ROM2 can control the Ras-cAMP pathway, the Ras-cAMP pathway is not always required for Rom2p-mediated stress responses. Overexpression of PDE2 in the rom2 mutant conferred stress resistance to freezing and thawing and to peroxides, paraquat, cobalt, and starvation, but not to diamide, cycloheximide, NaCl, and caffeine. Therefore, although ROM2 can mediate cell growth and stress signaling largely through the Ras-cAMP pathway, it also mediates stress response in a cAMP-independent manner. Recently, it has been shown that each component of the Rho1-PKC-Mpk1p pathway can be activated by lateral input of independent stimuli including actin stress, hypo-osmotic stress, and heat stress, indicating that this signal transduction pathway is not merely operating in a linear "top-down manner" (60). Similarly, each regulatory component of the Ras-cAMP pathway may respond to different stress signals. For example, Ssa1p, a chaperone of the HSP70 super family, has been shown to interact with Cdc25p to reduce PKA activity, indicating that Cdc25p connects heat shock signaling to the Ras-cAMP pathway (61). These findings indicate that cellular stress responses may involve numerous cross-talks among diverse signaling pathways and that these are mediated at various levels of signaling cascades depending on the type of stimulus. Thus, Rom2p may selectively employ the Ras-cAMP pathway and other signaling pathways for stress signaling in a manner dependent on the type of stress signal. Because the Ras-cAMP pathway is essential for determining the intrinsic stress tolerance of yeast for a variety of stresses (3–12), the pathway may be connected to various other signaling pathways to help mediate stress responses for cell survival. Our finding, that Rom2p can participate in the process for determining intrinsic stress tolerance of yeast by signaling through the Ras-cAMP pathway, further expands the repertoire of the Ras-cAMP signaling pathway.


   FOOTNOTES
 
* This work was supported in part by the Australian Research Council and the Cooperative Research Centre for Food Industry Innovation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Supported by an Australian Research Council overseas postgraduate research scholarship and the Cooperative Research Centre for Food Industry Innovation (Australia). Present address: The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231. Back

§ Present address: The Faculty of Life Sciences, The University of Manchester, Jackson's Mill, P. O. Box 88, Sackville St., Manchester M60 1QD, United Kingdom. Back

To whom correspondence should be addressed. Tel.: 61-2-9385-2089; Fax: 61-2-9385-1050; E-mail: I.Dawes{at}unsw.edu.au.

1 The abbreviations used are: PKA, protein kinase A; PKC, protein kinase C; MAP, mitogen-activated protein; SD, synthetic dextrose. Back


   ACKNOWLEDGMENTS
 
We thank M. Hall, K. Tatchell, M. Yamamoto, Y. Ohya, and M. Mazon for providing us with strains and plasmids.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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