Laboratoire Information Génétique et Développement, Institut de Génétique et Microbiologie, UMR CNRS C8621, Université Paris-Sud, Bâtiment 400, 91405 Orsay cedex, France1
Dpto. Bioquímica y Biol. Molecular, Universitat de Valencia, and Dpto. Biotecnologia IATA, CSIC, Apdo Correos 73.46100 Burjassot, Valencia, Spain2
Author for correspondence: Hervé Garreau. Tel: +33 1 69 15 46 30. Fax: +33 1 69 15 72 96. e-mail: garreau{at}igmors.u-psud.fr
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ABSTRACT |
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Keywords: Saccharomyces cerevisiae, stress response, transcription factor, cAMP-dependent protein kinase
Abbreviations: PKA, cAMP-dependent protein kinase; STRE, stress-response element
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INTRODUCTION |
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METHODS |
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pGEX-3X-MSN4B contains the NciINruI fragment from the MSN4 gene (Estruch & Carlson, 1993 ) inserted into the EcoRI site of pGEX-3X, after filling the 5' protruding NciI and EcoRI ends. It contains codons 34 to 256 of the MSN4 ORF.
pEL-32/45 was derived from pEL32 by replacing the PvuIIPvuII fragment (not including the MSN2 gene) with the EcoRI fragment (containing the MSN4 gene) from pEL45 (Estruch & Carlson, 1993 ). It contains both MSN2 and MSN4 genes under the control of their own promoters inserted into the YEp24 vector (Carlson & Botstein, 1982
).
pAdh1-Msn2-GFP, encoding a Msn2GFP fusion under the control of the strong constitutive ADH1 promoter, was a generous gift from C. Schüller (Görner et al., 1998 ).
Yeast strains.
The following strains were used: W303-1A (MATa ade2 his3 leu2 trp1 ura3) (Estruch & Carlson, 1993 ) and the diploid OL556 (MATa/MAT
cdc25-5/cdc25-5 his3/his3 leu2/leu2 trp1/TRP1 rca1/rca1 ura3/ura3) (Boy-Marcotte et al., 1996
). The construction of the OL556-STRE strain containing the reporter gene lacZ under the control of four STRE motifs has been already described (Boy-Marcotte et al., 1998
).
Antibodies.
Rabbit anti-Msn2p and anti-Msn4p antibodies were raised against the N-terminal parts of Msn2p and Msn4p (expressed in E. coli from pGEX-3X-MSN2B and pGEX-3X-MSN4B, respectively) and showed no cross-reactivity. They were purified by immunoblotting (Harlow & Lane, 1988 ) with the GST-fused peptides expressed in E. coli from pGEX-3X-Msn2B and pGEX-3X-MSN4B. For immunoblotting, these antibodies were used at 1/20 to 1/50 dilutions.
Yeast growth, protein extracts, immunoblotting and alkaline phosphatase treatment.
For the diauxic transition, yeast cells were grown in YNBS medium with 2% glucose (Boy-Marcotte et al., 1996 ). Glucose concentration in the culture medium was monitored enzymically with glucose oxidase using Sigma Diagnostic Glucose reagent kit no 510-A. When present in the medium, cAMP was at 3 mM. For heat shock, cultures grown at 26 °C in mid-exponential phase (OD710
1) were transferred into a prewarmed water bath at 38 °C. Protein extracts were prepared essentially as described by Volland et al. (1992)
, except extraction was performed directly on the cell culture without centrifugation. The precipitated proteins from 4x107 cells were dissolved in 500 µl modified Laemmli sample buffer. After gel electrophoresis (1030 µl per well) in the Laemmli system (Laemmli, 1970
), separated polypeptides were transferred onto PVDF sheets by the semi-dry transfer method (Kyhse-Andersen, 1984
). Immunoreactive bands were identified by anti-Msn2p or anti-Msn4p antibodies, alkaline phosphatase-conjugated anti-rabbit IgG antibody (Promega) and 5-bromo-4-chloro-3-indoxyl-1-phosphate (BCIP)/nitro blue tetrazolium colour development. For dephosphorylation by alkaline phosphatase, 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 µl-1; Roche Molecular Biochemicals) for 2 h at 37 °C. The control was incubated under the same conditions without alkaline phosphatase.
RNA preparation and analysis.
Total RNA extraction, gel electrophoresis of glyoxylated RNA and hybridization were performed as described earlier (Boy-Marcotte et al., 1996 ). Fifteen micrograms of total RNA per sample were fractionated on a 1% agarose gel. [
-32P]dCTP-labelled probes [3000 Ci (111 TBq) mmol-1] were synthesized by random priming with the Ready To Go DNA labelling kit (Pharmacia Biotech) according to the manufacturers instructions. The HSP12 PCR-generated fragment (310 bp) used as template was amplified from genomic DNA with the following primers specific to the HSP12 ORF sequence: upper primer 5'-CAATGTCTGACGCAGGTA-3' and lower primer 5'-CTTCTTGGTTGGGTCTTC-3'. Hybridized membranes were exposed to phosphor screens, which were scanned in a Molecular Dynamics PhosphorImager.
GFP fluorescence microscopy.
The Msn2pGFP fusion was detected without fixation essentially as described by Görner et al. (1998) .
ß-Galactosidase assay.
Determination of ß-galactosidase activity was performed as previously described (Boy-Marcotte et al., 1998 ).
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RESULTS |
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As a reporter gene of the trancriptional activity of Msn2/4p (Martinez-Pastor et al., 1996 ), the accumulation of HSP12 mRNA was followed under the same conditions in strain OL556. The HSP12 mRNA accumulation, which occurred when glucose becomes limiting, was impaired in the presence of cAMP (Fig. 1d
).
Msn2p and Msn4p are hyperphosphorylated upon heat shock; cAMP prevents and is able to reverse this effect
For the heat-shock experiments described in this section, we used strain OL556 transformed with the pEL32-45 plasmid, which allows overexpression of both Msn2p and Msn4p, to get strong enough immunostaining. Experiments without overexpression gave the same results although the immunostaining was weak (data not shown).
The phosphorylation state of Msn2p and Msn4p was examined when a heat shock from 26 °C to 38 °C was applied in the absence or in the presence of cAMP (Fig. 3a). At 26 °C, Msn2p was detected as two immunoreactive bands (Fig. 3b
, left, lane 1). After heat shock in the absence of cAMP, the faster-mobility band disappeared (Fig. 3b
, left, lanes 2 and 3), indicating hyperphosphorylation of Msn2p. In the presence of cAMP at 26 °C, the faster-mobility form of Msn2p was predominant (Fig. 3b
, left, lane 4) and the Msn2p pattern was not modified after heat shock (Fig. 3b
, left, lanes 5 and 6). Msn4p at 26 °C was detected as one predominant band (Fig. 3b
, right, lanes 1 and 4). After the heat shock in the absence of cAMP, slower-migrating bands became apparent and the proportion of the fast-migrating band present at 26 °C decreased (Fig. 3b
, right, lanes 2 and 3). It can be noticed that the heat-shock-induced modification in the phosphorylation patterns was different for Msn2p and Msn4p. In the presence of cAMP, no mobility shift was observed (Fig. 3b
, right, lanes 5 and 6).
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DISCUSSION |
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In both the diauxic transition and heat shock, hyperphosphorylation of Msn2/4p was correlated with induced transcriptional activity of Msn2/4p. Moreover, the correlation between activation and hyperphosphorylation could be extended to the subcellular localization of these factors. We have confirmed the results previously obtained (Görner et al., 1998 ): the nuclear translocation of Msn2p induced by heat shock was inhibited and reversed by cAMP (data not shown). Therefore, it appears that hyperphosphorylation could be required for activation of Msn2p and Msn4p. The question therefore arises whether hyperphosphorylation is required for stress-induced nuclear localization or if it occurs only after migration to the nucleus.
cAMPPKA pathway antagonizes both hyperphosphorylation and activation of Msn2/4p
We observed that high cAMP levels prevent stress-induced hyperphosphorylation of Msn2/4p and induction of HSP12 transcription. Thus, dephosphorylation of PKA sites could be a prerequisite for stress-promoted hyperphosphorylation and activation. In addition, the fact that added cAMP reversed hyperphosphorylation suggests that the PKA-phosphorylated sites have been dephosphorylated during stress-induced activation in order to restore responsiveness to cAMP. As illustrated in this report, Msn2p and Msn4p were already phosphorylated during exponential growth on glucose, when the intracellular cAMP level remains higher than in the absence of glucose (Russell et al., 1993 ). This basal phosphorylation could be due to PKA activity. The decrease in cAMP that occurs upon glucose exhaustion could lead to dephosphorylation of the PKA sites, allowing phosphorylation by stress-activated protein kinases. In this respect, it was recently reported how the TOR pathway leads to Msn2/4p sequestration in the cytoplasm by association with Bmh2p, a member of the 14-3-3 protein family. Inhibition of the TOR signalling pathway by rapamycin led to release of Msn2p from Bmh2p and to nuclear translocation of Msn2p (Beck & Hall, 1999
). The fact that these 14-3-3 proteins are anchoring partners for serine-phosphorylated proteins (Muslin et al., 1996
) strongly argues for such phosphorylation of Msn2/4p during exponential growth, to keep them present in the cytoplasm in the absence of stress.
These results suggest the existence of a switch between active and inactive forms controlled by two types of antagonistic phosphorylation. A similar phosphorylation switch has been observed at the diauxic transition on Not5p, a subunit of the Not complex, a negative regulator of TATA-less promoters. Not5p is phosphorylated upon glucose starvation and this phosphorylation is inhibited by high PKA activity (M. Collart, personal communication). Multiple phosphorylations with different effects on the proteins appear to be used by the cell for the fine tuning of regulation such as in the case of Pho4p, which is phosphorylated at several sites, each of them being involved at a different level of control (Komeili & OShea, 1999 ). Msn2p and Msn4p are other examples of such complex regulation of transcription factors by multiple phosphorylations.
Different stress signals are transduced by different pathways
In this report we show that different conditions (the diauxic transition, heat shock and growth on ethanol) give different patterns of hyperphosphorylation of Msn2p and Msn4p. That could be due to differences in the antagonistic activity of PKA, higher in glucose medium, even after heat shock, than in ethanol. Indeed, the intracellular cAMP level has been shown to be lower in ethanol-grown cells than in glucose-grown cells (François et al., 1987 ). Another explanation could be the involvement of different protein kinases, according to different stresses. Several stress-signalling pathways have been identified that involve specific kinases (Gustin et al., 1998
). Among them, the HOG pathway has been shown to activate Msn2/4p/STRE-driven transcription in response to osmotic shock (Inoue et al., 1998
; Martinez-Pastor et al., 1996
; Rep et al., 1999
). Another candidate could be the Snf1p protein kinase, which is activated during the diauxic transition (Wilson et al., 1996
). Although protein kinase C mediates a response to heat-shock-induced cell-wall damage, this pathway does not seem to be involved in Msn2/4p activating phosphorylation, since STRE/Msn2/4p-driven transcription is independent of this pathway (Kamada et al., 1995
).
Msn2p and Msn4p seem to be differentially regulated
We have observed some differences between Msn2p and Msn4p. Their cellular content appeared to be differentially regulated during diauxic transition: the amount of Msn4p transiently increased at the end of the exponential-growth phase, whereas Msn2p remained constant. This result is consistent with the data obtained by micro-array analysis (De Risi et al., 1997 ): MSN4 mRNA increases at the diauxic transition and MSN4 expression is repressed by Tup1p, whereas MSN2 mRNA decreases at the diauxic transition. The rapid decrease of Msn4p when growth is arrested due to glucose exhaustion suggests an instability of this protein, which was not observed for Msn2p. The ratios of these two factors could vary between growth conditions. Some subtle differences in the phosphorylation patterns between Msn2p and Msn4p after different stresses were also observed. This suggests differential susceptibility to stress-induced phosphorylation and to PKA. As with many other partially redundant genes, MSN2 and MSN4 could play partially overlapping functions with some biological specificity. The amount of the active forms induced by different stresses could be different for each of the two genes. That could lead to a differential induction of two partially overlapping subsets of genes among the Msn2/4p regulon, each depending on its own transcription factor. Indeed, as reported by Treger et al. (1998)
, the relative contribution of each factor varies among different Msn2/4p-regulated genes, indicating some specificity for their functions.
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ACKNOWLEDGEMENTS |
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Received 22 March 2000;
revised 12 June 2000;
accepted 15 June 2000.