Rst2p is a common transcription factor for the cAMP-repressible genes ste11 and fbp1.
Rst2p is a transcription factor for ste11+
, which functions downstream of the PKA pathway (20
). We suspected that Rst2p might also regulate expression of fbp1+
, another gene known to be controlled by PKA in fission yeast (3
). We thus examined growth of the rst2
Δ strain on nonfermentable carbon sources. Expression of fbp1+
, encoding FBPase, is required to assimilate nonfermentable carbon sources and is induced sharply under such nutritional conditions (39
). Without FBPase, cells cannot generate glucose from nonfermentable carbon sources via gluconeogenesis and hence fail to proliferate. The rst2
Δ strain grew very poorly compared to the wild-type strain on medium containing 3% glycerol and 0.1% glucose (data not shown). A trace amount of glucose was added in this assay because S. pombe
cells did not feed on glycerol alone for an unclear reason. A more obvious difference was seen on medium containing 3% gluconate, where the wild-type strain grew robustly but the rst2
Δ strain did not grow at all (data not shown).
To demonstrate the necessity of Rst2p for transcription of fbp1
more directly, we measured the level of fbp1
mRNA in rst2+
Δ cells by Northern blotting. As shown in Fig. , fbp1
mRNA accumulated significantly in wild-type cells 1 h after the shift from the repression medium (MMR; see Materials and Methods) to the derepression medium (MMD). In contrast, no fbp1
mRNA was detected in rst2
Δ cells, indicating that Rst2p was essential to express fbp1
. Scanning of the promoter region of fbp1
revealed one STREP (CCCCTC), the consensus motif for Rst2p binding, at nucleotides −262 to −257 (Fig. ). A recent study has shown that this sequence is positioned in a cis
regulatory element of fbp1
designated UAS2 (27
). We prepared a double-stranded oligonucleotide harboring this sequence (nucleotides −273 to −249) and showed that Rst2p could bind to it in a gel shift assay (data not shown), as was demonstrated with the ste11
upstream sequence (20
). These observations strongly suggest that Rst2p is a transcriptional activator of both fbp1
and functions via binding to STREP in both cases.
FIG. 1. The rst2 gene function is required for transcription of the fbp1 gene. (A) Induction of fbp1 transcription by a medium shift. Cells, either rst2+ (JY333) or rst2Δ (JX233), were grown in MMR containing 8% glucose to a concentration (more ...)
Figure displays collectively the effects of the PKA-activated and the PKA-null mutations on expression of ste11
, as assayed by Northern blotting. Some of these observations have been reported previously (3
). In Fig. , induction of ste11
expression was triggered by nitrogen starvation, whereas the induction of fbp1
expression was triggered by a shift of carbon sources. Both genes were expressed very poorly in the cgs1
Δ strain, which lacked the regulatory subunit of PKA and hence retained high PKA activity (Fig. , lanes 5 and 6). In contrast, they were expressed constitutively in the pka1
Δ strain, which lacked the catalytic subunit of PKA (Fig. , lanes 7 and 8). Disruption of rst2
in the pka1
Δ strain abolished constitutive expression of both ste11
. Thus, it is obvious that PKA affects expression of ste11
through Rst2p. However, it is also notable that the regulation of ste11
expression by the PKA-Rst2p system is not so thorough as is the case for fbp1
. Expression of fbp1
was not detectable in cgs1
Δ and rst2
Δ cells, but a low level of residual ste11
expression was evident in them (Fig. , lane 4). This may imply an involvement of a PKA-independent regulatory pathway in ste11
expression (see Discussion).
FIG. 2. Effects of PKA-related mutations on expression of ste11 and fbp1. (A) To examine ste11 expression, cells of each genotype indicated (wild type [wt], JY333; rst2Δ, JX233; cgs1Δ, JX222; pka1Δ, JX384; pka1Δ (more ...) Rst2p takes different phosphorylation states according to the medium conditions.
To see whether Rst2p undergoes modification at the protein level, we probed Rst2p by using affinity-purified anti-Rst2p antibodies in Western blotting experiments (Fig. ). The antibodies recognized a band (doublet; see below) of ca. 80 kDa in an extract prepared from wild-type cells grown in MMR (lane 1). This band was not detected in the extract of rst2Δ cells (lanes 7 and 8), confirming its identity as Rst2p. The shift of wild-type cells from MMR to MMD resulted in a reduction of the mobility of Rst2p in SDS-PAGE, suggesting that the protein underwent certain modification by the medium shift (lanes 2 through 6). The amount of Rst2p in a cell did not appear to change significantly by this shift. These observations led us to speculate that Rst2p might be phosphorylated under the derepressing conditions. To test this possibility, we treated crude preparations of Rst2p with nonspecific alkaline phosphatase, as described in Materials and Methods. Rst2p derived from derepressed cells regained a faster mobility after the phosphatase treatment, and this was blocked by the addition of phosphatase inhibitors (Fig. , lanes 4 to 6), indicating that Rst2p was hyperphosphorylated under the derepressing conditions.
Rst2p derived from repressed cells was subjected to a similar phosphatase treatment (Fig. , lanes 1 to 3). Analysis by SDS-PAGE by using a soft gel (7.5%), as shown here, revealed that Rst2p under the repressing conditions existed in two forms, giving two close but separable bands in electrophoresis (lanes 1 and 3). Rst2p treated by phosphatase appeared to give only the lower band (lane 2). Because the intracellular PKA activity was supposed to be high in the repression medium, we suspected the possibility that the upper band represented Rst2p phosphorylated by PKA. Two lines of evidence, however, denied this possibility. One was that we could detect these two bands in cgs1Δ (PKA-active) cells just as in wild-type cells (Fig. , lane 2 versus lane 4). The other was that a mutant form of Rst2p that lacked the PKA target sites (M3; see below) still gave the two bands in both wild-type and cgs1Δ cells (lanes 3 and 5). Thus, we presume that the upper band represents a phosphorylated form of Rst2p but that PKA is not responsible for this phosphorylation.
PKA activity affects the phosphorylation state of Rst2p.
To examine a possible involvement of PKA in the hyperphosphorylation of Rst2p observed above, we analyzed Rst2p in cgs1
Δ (PKA-active) and pka1
Δ (PKA-null) cells grown under either the repressing or the derepressing conditions by SDS-PAGE (Fig. ). Rst2p from pka1
Δ cells under the repressing conditions assumed a ladder of slow-moving bands, the top of which reached the position of the band observed in wild-type cells under the derepressing conditions (lanes 6 and 9). Because pka1
is the single gene encoding PKA in fission yeast (24
), this suggested that the hyperphosphorylation of Rst2p in the derepression medium was brought about by a kinase(s) other than PKA. In contrast, Rst2p was not fully hyperphosphorylated in cgs1
Δ cells under the derepressing conditions (lane 11), suggesting that high activity of PKA is inhibitory for the hyperphosphorylation.
We then investigated effects of the M3 mutation on hyperphosphorylation of Rst2p. Whereas the wild-type Rst2p was not hyperphosphorylated in the repression medium (Fig. , lane 1, and Fig. , lane 2), the M3 mutant was partially hyperphosphorylated in the same medium (Fig. , lane 3). Notably, the pattern of slow-moving bands exhibited by the M3 mutant here was quite similar to those of the wild-type Rst2p and the M3 mutant in PKA-defective cells under the repressing conditions (lane 3 versus lanes 6 and 7). Furthermore, the M3 mutant was phosphorylated more extensively than the wild-type Rst2p in PKA-active cells under the derepressing conditions (lane 12 versus lane 11). These results suggest strongly that PKA suppresses hyperphosphorylation of Rst2p through phosphorylation of its target sites, which are mutated in M3. The data shown in Fig. also suggest that, besides the absence of this direct phosphorylation by PKA, two conditions are essential for the kinase(s) responsible for hyperphosphorylation of Rst2p to become fully active (or the phosphatase to become fully inactive). One is depletion of glucose from the medium (as typically deduced from lane 7), and the other is reduction of the cellular PKA activity below a certain threshold (as typically deduced from lane 12).
Mutations in the phosphorylation sites activate Rst2p ectopically.
We investigated biological consequences of mutations introduced into the phosphorylation sites on Rst2p. Cells moderately expressing the rst2-M3
allele showed markedly slow growth and flocculation in a synthetic medium, which contained a sufficient amount of nitrogen to suppress sexual development of wild-type cells. The rst2-M3
strain grew even more slowly than the pka1
Δ strain, which has been shown to grow poorly (24
). Inhibition of cell growth by the rst2-M3
mutation was less severe in the cgs1
Δ background, although the reason for this suppression is unclear.
To see the effects of the M1 and M3 mutations on sexual development, we transformed homothallic rst2Δ cells, whose genetic background was either cgs1+ or cgs1Δ, with a multicopy plasmid carrying each rst2 allele connected to the weak nmt1 promoter. The M2 mutation, which harbored a single T-to-A substitution in the central target site, was also examined. Each transformant was grown on SD medium, which represses the nmt1 promoter, and a portion was restreaked on SSA medium, which induced sexual development due to the scarcity of the nitrogen source and also derepresses the nmt1 promoter due to the lack of thiamine. The results are summarized in Fig. . When the host cells were cgs1+, the wild-type and the mutant rst2 alleles were equally active in the overall induction of sporulation, as assayed by iodine staining (Fig. ). A microscopic measurement of the conjugation efficiency of the same samples confirmed that they were nearly equivalent (Fig. ). However, when the host cells were cgs1Δ, differences arose among them. The wild-type allele could recover neither overall sporulation (Fig. ) nor conjugation (Fig. ) in cgs1Δ rst2Δ cells. However, each mutant allele could do so, although not very strongly (Fig. ). The double-mutant allele, M3, was the most active, and the M1 allele appeared to be more active than M2 (Fig. ).
FIG. 6. Unphosphorylatable mutations in the PKA target sites enhances the activity of Rst2p. (A) Iodine-staining assay of sporulation efficiency in various rst2 transformants. Cells of JX231 (h90 cgs1+ rst2Δ) and JW487 (h90 cgs1Δ rst2 (more ...)
These observations suggested that Rst2p not phosphorylatable by PKA was likely to stimulate unconditional expression of the target genes involved in sexual development. To examine this, we measured induction of ste11 expression in cgs1+ rst2Δ and cgs1Δ rst2Δ cells transformed with either the wild-type or the M3 mutant allele, driven by the weak nmt1 promoter. Because expression of the M3 allele in cgs1+ cells impaired their growth severely, all of the transformants were precultured in the presence of thiamine. They were transferred to thiamine-free medium, cultured for five rounds of cell division, and examined for expression of ste11. As shown in Fig. , induction of ste11 expression by nitrogen depletion occurred in cgs1+ cells transformed with either the wild-type or the M3 allele. Expression of ste11 was not inducible in cgs1Δ cells carrying the wild-type rst2 allele under the experimental conditions. However, cgs1Δ cells transformed with the M3 allele expressed ste11 intensively in response to nitrogen starvation. From these observations we conclude that a mutant form of Rst2p unphosphorylatable by PKA overcomes hyperactivation of PKA with respect to the induction of ste11 expression. It is also conclusive, however, that this mutation does not represent the total effects brought by nitrogen starvation.
PKA activity affects subcellular localization of Rst2p.
To examine whether localization of Rst2p could be affected by the PKA activity, we expressed Rst2p-GFP in the pka1
Δ, or cyr1
Δ strains. The cyr1
Δ strain lacks adenylyl cyclase and has no detectable level of cAMP (23
). As summarized in Fig. , Rst2p-GFP was almost exclusively nuclear in the pka1
Δ and cyr1
Δ strains and almost exclusively cytoplasmic in the cgs1
Δ strain, when they were grown in the MM medium with 2% glucose. The nuclear localization of Rst2p-GFP in the cyr1
Δ strains was canceled by the addition of 2 mM cAMP to the medium (Fig. ). Altogether, these results indicate that the PKA activity is a key determinant of the subcellular localization of Rst2p.
To see the effects of the M3 mutation on subcellular localization of Rst2p, we expressed HA-tagged Rst2-M3p from a multicopy plasmid in cgs1+ and cgs1Δ cells. HA-tagged Rst2p was expressed similarly as a control. When HA-tagged Rst2p was produced in cgs1+ cells, less than 20% of the cells exhibited nuclear accumulation of the protein under the repressing conditions (in MMR), whereas nearly 70% did so under the derepressing conditions (in MMD) (Fig. ). In contrast, more than 30% of the cells exhibited nuclear accumulation of HA-tagged Rst2-M3p under the repressing conditions, and ca. 50% did so under the derepressing conditions (Fig. ). These observations suggest that phosphorylation at the two PKA target sites on Rst2p, which are mutated in M3, may be pertinent with nuclear exclusion of the protein. However, the data also imply that PKA affects localization of Rst2p not only through phosphorylation of these sites but also in an additional way(s). Curiously, Rst2-M3p showed highly efficient nuclear accumulation in cgs1Δ cells placed under the repressing conditions (Fig. ). The wild-type Rst2p showed extensive cytoplasmic localization in cgs1Δ cells under the same conditions (Fig. ). These observations suggest that PKA regulates subcellular localization of Rst2p in a complex manner, probably involving as-yet-unidentified factors.
FIG. 8. Quantitative measurements of subcellular localization of the wild-type Rst2p and the M3 mutant under the repressing and derepressing conditions. Rst2p and Rst2-M3p, both tagged with HA, were expressed from the expression vector pREP81 in cells that were (more ...) DISCUSSION
In this study we have demonstrated that fission yeast Rst2p regulates expression of both ste11
through interaction with the cis
element STREP. Further, the obtained data suggest strongly that Rst2p is phosphorylated directly by PKA, thereby suppressing its transcription-activating activity. It is interesting that a single factor is used in order to regulate two mutually independent biological processes, namely, sexual development and gluconeogenesis. Induction of ste11
expression, a key step for the sexual development, can occur in response to starvation of a nitrogen source, whereas induction of fbp1
expression, which ensures growth on a nonfermentable carbon source, occurs in response to the consumption of glucose. Both types of nutritional starvation cause a decrease in the intracellular cAMP level (16
), and a role for PKA has been inferred in the induction of both ste11
). The present study establishes Rst2p as a pivotal mediator between the activity of PKA and transcription of the ste11
If fission yeast cells lose the PKA activity completely, which is an unnatural condition realized only in the pka1
Δ or cyr1
Δ strain, the expression of both ste11
becomes constitutive regardless of the nutritional conditions. On the contrary, their expression is always suppressed in the cgs1
Δ strain sustaining high PKA activity. Our analysis has shown that Rst2p lacking the PKA target sites (M3) can cause effects similar to those of the loss of PKA activity with respect to hyperphosphorylation of the protein and can promote ste11
transcription in cgs1
Δ cells starved for nitrogen. These observations strongly suggest that Rst2p is a substrate of PKA in vivo. However, the mutant form of Rst2p cannot induce ste11
transcription in the presence of ample nitrogen, although the complete loss of PKA activity can do so. In other words, the M3 mutant does not replace the loss of PKA completely in induction of ste11
expression. Furthermore, the results presented in Fig. , lanes 3 through 6, indicate that nitrogen can yet affect expression of ste11
to some extent independent of the PKA-Rst2 pathway, as has been inferred in previous studies (18
). Taking these results together, we propose the scheme shown in Fig. as the most rational based on current knowledge. In this scheme, phosphorylation of Rst2p by PKA serves as the major switch to turn off ste11
expression, but there are additional negative regulatory pathways that bypass Rst2p, one connecting the nitrogen source to ste11
expression and another connecting PKA to ste11
Diagram of the regulatory network involving the PKA-Rst2p pathway for transcriptional activation of ste11 and fbp1. The pathway established in this study is shown in thick lines. Other possible paths are shown in thin lines.
A similar scheme appears to apply to the relation of Rst2p to fbp1
expression. Our preliminary observations suggest that the M3 mutant form of Rst2p cannot activate fbp1
expression in the presence of glucose, nor can it do so under conditions of high PKA activity (unpublished results). This finding may parallel the observation that both depletion of glucose and reduction of cellular PKA activity are apparently required to hyperphosphorylate the M3 form of Rst2p completely (Fig. ). Furthermore, several factors other than Rst2p have been shown to be involved in the activation and repression of fbp1
transcription. Neely and Hoffman (27
) have shown that the PKA pathway regulates transcription of fbp1
in a dual manner, through the STREP motif (UAS2) and the CRE motif (UAS1), the latter of which is recognized by a complex of Atf1/Gad7p and Pcr1p. These authors have also shown that at least four forms of protein complexes bind to STREP and that the stress-responsive MAPK cascade regulates transcription of fbp1
at both STREP and CRE sites. Janoo et al. (17
) have reported that two homologs of the Saccharomyces
transcriptional repressor Tup1p are involved in repression of fbp1
expression. Thus, combined with these observations, our results lead to a simplified scheme illustrating the regulation of fbp1
expression as shown in Fig. .
A portion of Rst2p is phosphorylated in wild-type cells grown in MMR, but PKA, which is supposed to be active in these cells, does not appear to be responsible for this phosphorylation, as discussed in Results. We have thus far been unsuccessful in detecting in vivo phosphorylation of Rst2p by PKA as a shift of the mobility of the protein in gel electrophoresis. However, the results of mutational analysis suggest strongly that PKA affects the activity of Rst2p through both direct phosphorylation and indirect modification. Thus, biochemical evidence is awaited to show critically whether Rst2p is phosphorylated by PKA in living cells.
An unexpected observation is that cells expressing rst2-M3 are more impaired in vegetative growth than pka1Δ cells in synthetic medium, whereas they grow better than pka1Δ cells in rich medium. Furthermore, cgs1Δ suppresses the slow growth of rst2-M3 cells in synthetic medium to some extent. These observations suggest that emergence of Rst2p unphosphorylated by PKA, not precisely harmonized with the state of the PKA activity, can cause certain adverse effects on cell growth. Because both ste11 and fbp1 are unlikely to be fully expressed in rst2-M3 cells under growing conditions, these observations indicate that Rst2p is likely to have a target(s) other than these two genes, which is more closely related to growth control.
This study has revealed a clear correlation between the subcellular localization of Rst2p and the activity of PKA in the cell. When the PKA activity is high, Rst2p tends to be located in the cytoplasm. When it is low, Rst2p tends to be nuclear. Nuclear exclusion of Rst2p in the presence of glucose apparently explains why high PKA activity is inhibitory to the transcription-activating activity of Rst2p. It is currently not conclusive whether phosphorylation by PKA accelerates nuclear export or inhibits nuclear import of Rst2p. Because leptomycin B blocks nuclear export of Rst2p, it appears that Rst2p is exported from the nucleus by the exportin-dependent system. However, our attempts to identify possible nuclear localization signal or nuclear export signal on Rst2p have not been successful so far.
Although phosphorylation by PKA apparently affects nucleocytoplasmic localization of Rst2p, the shift between the cytoplasm and nucleus is never all-to-none under our experimental conditions, suggesting that the phosphorylation may also impair other aspects of Rst2p required for its transcription-activating activity. Judging from the proposed ternary structure of Zn finger domain, as reviewed by Wolfe et al. (42
), the N-terminal phosphorylation site, 10 residues apart from the DNA-binding domain (Fig. and Fig. ), does not seem to affect the ability of Rst2p to recognize either a base or a phosphate. Rather, phosphorylated Rst2p may be impaired in the ability to activate the transcription machinery, because there lies a glutamine stretch near the phosphorylation site, as is frequently found in the activating domain of transcription factors. Further studies are necessary to clarify these possibilities.
Msn2p and Msn4p, Zn finger proteins that recognize the STRE motif, have been shown to undergo hyperphosphorylation in response to a carbon source shift and heat shock, and high PKA activity blocks this hyperphosphorylation (11
). It is suspected, though not proven, that some stress-responsive kinases may be involved in this hyperphosphorylation in S. cerevisiae
. Garreau et al. hypothesized further that different stresses may cause different states of hyperphosphorylation (11
). It is very intriguing that transcription activators that recognize similar motifs (STRE and STREP) undergo analogous modifications in S. cerevisiae
and S. pombe
, which are only distantly related in phylogeny. Although the identity of the kinase(s) (and possible antagonizing phosphatase) involved in the hyperphosphorylation of Rst2p remains to be clarified, it appears likely that, in S. pombe
, Rst2p undergoes hyperphosphorylation differently under depletion of glucose and depletion of nitrogen. The full activation of fbp1
and that of ste11
may require different levels of hyperphosphorylation of Rst2p. These questions remain to be answered.