Laboratoire Information Génétique et Développement, Institut de Génétique et Microbiologie, UMR CNRS C8621, Université Paris-Sud, Bâtiment 400, 91405 Orsay Cedex, France
Correspondence
Michel Jacquet
Michel.Jacquet{at}igmors.u-psud.fr
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ABSTRACT |
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INTRODUCTION |
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The cAMP-PKA signalling pathway is tightly regulated. The synthesis of cAMP by adenylyl cyclase requires the activation of at least one of the RAS1 and RAS2 gene products (Toda et al., 1985). It is also positively modulated by Gpa2p and Gpr1p in response to glucose addition (Colombo et al., 1998
), and negatively regulated by Ira1p and Ira2p (Tanaka et al., 1990
). There are two phosphodiesterases of high- and low-affinity, Pde1p and Pde2p (Nikawa et al., 1987a
), able to reduce its level, but the most stringent regulation appears to be the feedback exerted by PKA activation upon cAMP accumulation (Nikawa et al., 1987b
). Different components of this pathway could be affected by stress and contribute to the control of the cellular response.
We have previously reported that Cdc25p, an upstream element of the cAMP-PKA pathway acting as a guanine nucleotide exchange factor (GEF) for Ras proteins, is able to interact with Ssa (Hsp70 family) and Hsp90 family chaperones (Geymonat et al., 1998). Since these chaperones are recruited to unfold proteins upon stress, their interaction with Cdc25 might be affected. Indeed, it was shown that the amount of Cdc25p was reduced in cells depleted for Ssa chaperones (Geymonat et al., 1998
). Therefore, we have chosen to examine if Cdc25p is modified upon stress. We have monitored its cellular content, subcellular distribution and phosphorylation state in response to heat shock and other stresses. The results indicated that stress triggers a reduction in the amount of Cdc25p without any change in either phosphorylation state or localization. We have measured the capacity of cells to produce cAMP using mutants in which adenylyl cyclase activity is fully deregulated and we found that this capacity decreases in parallel to the reduction of Cdc25p upon stress. Paradoxically, the low basal level of cAMP measured in wild-type cells increases slightly rather than being reduced. We discuss some hypotheses that could account for such a paradoxical result.
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METHODS |
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Cdc25p isolation, immunoblotting and alkaline phosphatase treatment.
Cdc25p was tagged at its own locus by three tandem HA epitopes as previously described (Geymonat et al. 1998). The tagged version of Cdc25p, Cdc25p-3HA, was fully functional. The Cdc25p protein was extracted as previously described (Garreau et al., 2000
). A solution of 1·85 M NaOH plus 1 % 2-mercaptoethanol were added to the cell culture at 1/10 the culture volume to break the cells. The proteins were precipitated by a 50 % TCA solution (5 % final concentration). The precipitated proteins from 4x107 cells were dissolved in 500 µl modified Laemmli sample buffer.
Membrane association of Cdc25p was determined as previously described (Garreau et al., 1996) with the following modifications: after centrifugation at 100 000 g for 30 min, the pellet was directly dissolved in Laemmli sample buffer instead of extracting Cdc25p from the membrane by a 2 mM EDTA solution (Gross et al., 1992a
, b
).
Proteins were separated by 7·5 % SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes by a semi-dry method. Cdc25p-3HA was detected by rat monoclonal anti-HA antibody 3F10 (Roche Molecular Biochemicals) at a dilution of 1 : 1000, and anti-rat IgG antibody (alkaline phosphatase conjugate). The band of Cdc25p-3HA was measured by densitometry using Gelscan software (Yvan Zivanovic, Université Paris-Sud).
Alkaline phosphatase treatment was performed as previously described (Garreau et al., 2000) Laemmli extracts (50 µl) were diluted 10-fold in alkaline phosphatase buffer (100 mM Tris/HCl, pH 8·5, 1 mM MgCl2, 0·1 mM ZnCl2), concentrated again to the initial volume by ultrafiltration, and incubated in the presence of 3 µl calf intestinal alkaline phosphatase (20 U µl1; Roche Molecular Biochemicals) for 2 h at 37 °C. The control was incubated under the same conditions without alkaline phosphatase.
RT-PCR measurement of CDC25 mRNA.
Total RNA was extracted as described previously (Geymonat et al., 1998), and 5 µg was used in reverse transcription performed with the Superscript II kit (Gibco-BRL). Specific primers for CDC25 and ACT1 were used for PCR amplification with 6 ng cDNA as the template. The PCR product was labelled by [32P]-dCTP in a 50 µl reaction volume. As an internal control, ACT1 primers were added to the CDC25 PCR reaction after the first 10 cycles and then PCR was continued for another 20 cycles. The [32P]-labelled PCR products were separated by 6 % PAGE. The signal was quantified by a Molecular Dynamics PhosphorImager equipped with ImageQuant software.
cAMP assay.
Cell extracts were prepared as previously described by Thevelein et al. (1987) with the following modifications: cells were harvested by filtration and cAMP was extracted with 1 ml 15 % perchloric acid with three freeze-thaw cycles in liquid nitrogen. After centrifugation, the supernatant was neutralized by 1·5 M potassium carbonate/1 M acetic acid buffer. Potassium perchloride was removed by centrifugation and the supernatant was subjected to the cAMP assay by using the correlate-EIA cyclic AMP kit (Assay Designs, non-acetylated method). The concentration of cAMP was calculated with the Origin 5.0 logistic curve-fitting program (Microsoft).
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RESULTS |
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When cells were transferred from 25 to 38 °C, the amount of Cdc25p, as measured by immunodetection with anti-HA antibodies, decreased significantly and reproducibly within 120 min (Fig. 1a, b). In contrast, the level of CDC25 mRNA, measured by quantitative RT-PCR, remained stable upon heat shock (Fig. 1c, d
). This is consistent with published microarray data that show the CDC25 mRNA level remains stable under various stress conditions including heat, oxidative, osmotic and acid shock (Causton et al., 2001
; Gasch et al., 2000
; Jelinsky & Samson, 1999
). Therefore, the reduction at the protein level observed in Fig. 1
is due to a post-transcriptional modulation resulting from either reduction of its synthesis or enhanced degradation.
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The level of Cdc25p also decreases under oxidative and ethanol stress conditions
To discover whether the reduction of the Cdc25p content of the cell is specific to heat shock or corresponds to a more general stress-related phenomenon we examined the fate of Cdc25p under other classical stress conditions. First, we examined the response to oxidative stress caused by 0·4 mM H2O2. In wild-type cells, oxidative stress induced a more rapid and transient arrest in cell division than that observed under heat-shock conditions. However, Cdc25p showed similar kinetics of decrease as observed in heat-shocked cells (Fig. 3). We also extended this test to a stress induced by 7 % ethanol, which causes cells to stop growing almost immediately. As shown in Fig. 3
, ethanol stress also caused the content of Cdc25p to decrease. These results show that the reduction of the cellular content of Cdc25p is a more general response of the cells to stress.
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We extended this observation to other stresses such as oxidative and ethanol shock in the tpk2w strain (OL618-12B). Oxidative stress induced a decrease in cAMP production in the tpk2w strain which was similar to that observed under heat shock (Fig. 5b). The addition of 7 % ethanol, which appears to have a stronger effect on the cell, also leads to a cAMP reduction. Similar to the cells in response to heat shock, the amount of cAMP in the isogenic wild-type strain was much lower and exhibited a small increase upon oxidative and ethanol stress (Fig. 5a
). Here again, when the feedback regulation was not operating, the reduction of cAMP accumulation was not limited to heat shock, but was also observed in different types of stress.
Cdc25p is a potential signal transducer in response to stress
To verify that the decrease of cAMP in tpk2w strains correlates with a decrease of Cdc25p in this genetic background, we constructed a tpkw strain in which CDC25 was tagged with the HA epitope. The Cdc25p amount was reduced in this strain after heat shock with similar kinetics to those of a wild-type strain (Fig. 6). The Cdc25p decrease parallels the reduction of the cAMP level found in a similar genetic background (compare Fig. 6
and Fig. 5b
).
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DISCUSSION |
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The decrease of Cdc25p after these stresses is not due to transcriptional repression, as can be deduced from many published microarray data (Causton et al., 2001; Gasch et al., 2000
; Jelinsky & Samson, 1999
) and confirmed by us, using PCR in the strain where Cdc25p decreases. The depletion of Cdc25p in S. cerevisiae could then be controlled either by arrest of its production, since it is an unstable protein that decays rapidly (Kaplon & Jacquet, 1995
), or by increased degradation. Regulation of the cellular content of the protein has also been reported for a mammalian homologue of Cdc25p, the Ras-GRF2 which contains a CDB motif, also present in Cdc25p, and is controlled by ubiquitination and degradation via the proteasome (de Hoog et al., 2001
).
Although we have not addressed the mechanism of depletion of Cdc25p in this report, we have reinforced our previous evidence for the involvement of chaperones in the maintenance of the cellular content of Cdc25p. Indeed, the interaction of Cdc25p with Ssa and Hsp90 chaperones could explain the change in cellular levels following stress, as previously proposed (Geymonat et al., 1998). The unfolding of a large number of proteins upon stress is commonly supposed to recruit these chaperones, which are no longer able to bind their usual partners. Cdc25p could then be destabilized or less accumulated when the availability of these chaperones is reduced by stress. Our finding that the rate of decay of Cdc25p is similar for protein found in both soluble and membrane fractions implies a similar sensitivity to stress or a fast shuttling between both fractions. Our analysis also demonstrated that heat shock did not change the cellular distribution of Cdc25p nor its phosphorylation state.
Cdc25p is present in a limiting amount in the cell. It is known that it could be fully inactivated by sequestration with the product of the dominant negative RAS2ala22 mutant, leading to growth arrest (Powers et al., 1984). Its average number has recently been estimated to be approximately 319 per cell compared to the 19 800 molecules of Ras2p (Ghaemmaghami et al., 2003
). Therefore, even a small reduction in its level is expected to affect the activity of Ras, and consequently adenylyl cyclase activity. The abrogation of the strong feedback applied on the production of cAMP allows us to show that the accumulation of cAMP is reduced upon stress and correlates with the reduction of Cdc25p. The reduction of the level of accumulated cAMP upon heat shock does not occur when CDC25 is deleted and adenylyl cyclase is activated by the product of RAS2ile152, an allele of Ras2p encoding a constitutively activated protein. This result implies that no other component of the cAMP pathway such as the adenylyl cyclase, Gpr1p, Gpa2p, Ira1p, Ira2p, Pde1p or Pde2p significantly contribute to the reduction of cAMP after heat shock. In contrast, the continuous increase of cAMP observed in this strain suggests that one or several of these components reacts in the opposite way, illustrating the complex dynamic of this system. These results led us to conclude that Cdc25p, which is sensitive to stress, has the potential to contribute to the mediation of stress signals within the cell.
The complexity of the regulation of the cAMP-PKA pathway was further emphasized by the paradoxical result that the level of cAMP measured in wild-type cells is much lower and increases upon stress. This appears to be in contradiction with the activation of the stress response transcription factors Msn2p and Msn4p, and leads us to propose the existence of more sophisticated regulation than a simple linear pathway. It could be due to an additional regulation acting directly on PKA, as suggested by Thevelein & de Winde (1999). However, it cannot be excluded that Cdc25p, activator of Ras proteins, also controls another Ras-dependent pathway. Supporting this hypothesis, several reports have shown an involvement of Ras in the control of the MAP kinase pathway leading to invasive growth (Cherkasova et al., 2003
; Cook et al., 1997
). This pathway could converge with the cAMP-PKA pathway upon Msn2p and Msn4p, which are also involved in the control of invasive and filamentous growth (Ho & Bretscher, 2001
). In this hypothesis, the reduction of adenylyl cyclase activation would be compensated by the feedback inhibition mechanism and thus remain unnoticed, whereas the other pathway could be more severely affected.
A second, more speculative, hypothesis deserves attention because it stresses the potential of a non-linear phenomenon occurring within cells. It is based upon the consideration that the level of cAMP measured within the cell population does not reflect the real concentration within individual cells but is the average of large fluctuations. The value of cAMP measured in wild-type cells corresponds to a cellular concentration of 0·52 µM. Meanwhile, the Kd for the PKA regulatory subunit is 10 µM. Thus if the cAMP content were homogeneous, PKA would never reach its full activity and the ability of adenylyl cyclase to produce several hundred-fold more cAMP would be useless for the cell. Therefore, we think that this value is the average of large variations. The strong feedback loop provides a good device for large amplitude fluctuations. Variations in cAMP level are known to occur upon glucose addition (Thevelein & Beullens, 1985
), and have been reported during metabolic oscillations of cellular populations (Satroutdinov et al., 1992
; Muller et al. 2003
). Oscillations in cAMP production might also be involved in the oscillatory shuttling of Msn2p in and out the nucleus (Jacquet et al., 2003
). Such oscillations in cAMP accumulation, especially if their amplitude is reduced while their frequency is increased, could explain how the average measured value increases whereas the maximal value decreases. In this case, if the downstream target needs a high threshold of cAMP to be inactivated, then it will respond to the diminution of the amplitude rather than to the average value.
Altogether, the results presented in this paper reveal a new aspect of the stress response in yeast. They show that the cellular level of Cdc25p and the maximal capacity to produce cAMP are reduced. They present a paradoxical result which points to more complex regulation of the signalling pathways in response to stress than previously thought.
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ACKNOWLEDGEMENTS |
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Received 12 March 2004;
revised 24 June 2004;
accepted 30 June 2004.
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