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Previous Article | Next Article 
Molecular and Cellular Biology, March 2005, p. 1793-1803, Vol. 25, No. 5
0270-7306/05/$08.00+0 doi:10.1128/MCB.25.5.1793-1803.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Department of Developmental and Cell Biology,1 Center for Complex Biological Systems, University of California, Irvine, California2
Received 16 July 2004/ Returned for modification 3 September 2004/ Accepted 29 November 2004
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Scaffold proteins are also believed to promote specificity by additional mechanisms which exploit their defining ability to bind simultaneously to multiple components of the same pathway. In particular, it has been proposed that scaffolds may sequester their bound components into pathway-dedicated signaling particles, thereby physically isolating them from other pathways. In so doing, scaffolds may both prevent their bound compounds from straying into other pathways when active (Fig. 1B) and protect them from intrusions from other pathways when inactive (Fig. 1C). For instance, Ste5 may prevent pheromone-activated Ste11 from straying into the invasive growth and osmostress pathways and insulate Fus3 from Ste11 activated by these pathways.
Ste5's first function during mating is not as a scaffold but as an adapter. Following pheromone stimulation, Ste5 binds to the released ß
subunits of the pheromone receptor-coupled G protein, towing bound Ste11MEKK to the vicinity of the plasma membrane (38), where Ste11 gets phosphorylated and activated by the Ste20 protein kinase (46; reviewed in reference 3). The scaffold function of Ste5 then facilitates signal transmission from Ste11MEKK to Ste7MEK to Fus3MAPK (Fig. 1B) (15). The crucial role of both the adapter and scaffold functions of Ste5 in pheromone signal transmission makes it difficult to test whether Ste5 actively prevents signals from leaking out of the mating pathway (Fig. 1B). It is possible, however, to test whether Ste5 actively prevents signals from leaking into the mating pathway (Fig. 1C). Here we have done so, and performed additional experiments that provide insight into the influence of the Ste5 scaffold protein and the distinct scaffolding requirements of the Fus3 and Kss1 MAPKs on signaling specificity.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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N (9) contains the STE11N allele driven by the GAL1 promoter.
Yeast strain construction.
The yeast strains used in this study are shown in Table 2. Strain LFY105 (MATa ste5, 
1278b lineage) was transformed with a low-copy-number plasmid expressing wild-type Ste5 from its own promoter, the corresponding empty vector (5), the adapter-defective mutant Ste5F514L (F514L), or the scaffolding-defective mutant Ste5V763A S861P (VASP).
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::LEU2 ste7::ura3 1278b lineage) was derived from JCY107 (ste7::ura3) with a 3.5-kb ste5::LEU2 allele described previously (41). The resulting strain was cotransformed with YEpU-FT1Z and either YCpT3-STE7 (ste5) or YCpT (57). The MATa 1278b lineage strains used in Fig. 3B have been described (12, 13); they were transformed with either YEpU-FUS1Z or YEpU-FT1Z. YLB257 (MATa ste5::LYS2 ste7::ADE2 pbs2::URA3), used in Fig. 3C, was derived from YLB105 (MATa ste5::LYS2 ste7::ADE2; S288C lineage) with a 4.1-kb pbs2::URA3 allele (7). The resulting strain was cotransformed with wild-type STE7 and YEpT-FUS1Z plus either YCpH-STE5 (STE5) or YCpH (ste5).
In Fig. 4A to C, LFY105 was transformed with either "empty" YCpU () or YCpU-STE5 (+) and cotransformed with YEpU-FT1Z. The resulting strains were then further transformed with either YCpH (+) or YCpH-STE11-4 (11-4). Transformation with plasmid YCpU-STE5-V763AS861P (22) was used to generate the indicated strains in Fig. 4C. Figure 4D strains were derived by transforming YDV500 (MATa ste11) with either YCpH (–) or YCpH-STE11-4 (+). Strain YPH499 (MATa STE+; S288C lineage) and the isogenic derivatives YDM600 (kss1::hisG) and YDM200 (fus3::LEU2) used in Fig. 4E were transformed with YCpH-STE11-4 (11-4) or empty vector (+), as described above.
In Fig. 5A, JCY100 was transformed with either YCpUG (–), YCpUG-STE5-GST (Ste5-GST), or YGU-STE11N (11N). In Fig. 5B and 5C, YPH499 was transformed with YEpL-FUS1Z, and either YCpUG (–) or YCpUG-STE5-GST (+). The resulting strains were then further transformed with either YCpH (–) or YCpH-STE11-4 (+).
In Fig. 6A, strains JCY100 and its isogenic derivative JCY120 (fus3::TRP1) (11) were transformed with YCpUG (–) or YCpUG-STE5-GST (+). The resulting strains were then further transformed with either YCpH (–) or YCpH-STE11-4 (+). In Fig. 6B, strain BYB87 (MATa ste4) was transformed with either YCpUG (EV) or YCpUG-STE5(C177A C180A)-GST (STE5*-GST) and then either YCpH (EV) or YCpH-STE11-4 (Ste11-4). Strain DC17 (MAT
his1) was used as the tester strain for the mating assay.
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0.8). Pheromone (Fig. 2A and 4E) or sorbitol (Fig. 3C) was added directly to growing cultures with no additional manipulation. Alternatively, 2 optical densities of yeast cells were plated as a lawn and grown for 24 h on plates selective for plasmid maintenance prior to harvesting (Fig. 3A, 4A, and 4C). To overexpress genes driven by the GAL1 promoter (Fig. 5A), S. cerevisiae cells were grown in standard media (with dextrose) to mid-exponential phase, washed with water, and resuspended in media containing 2% galactose and 0.2% sucrose. The cultures were harvested 3 h later. For low-level GAL1-driven expression in Fig. 5B and 5C, yeast cells were grown as a lawn on plates containing 2% sucrose and 0.007% to 0.2% galactose for 24 h before harvesting. In Fig. 6A, cells were spotted on 2% sucrose and 0.067% galactose plates or plates with 2.0% dextrose. For Fig. 6B, cells were grown on plates containing 2% sucrose and 0.067% galactose for 24 h before initiating the mating assay.
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Bioassays. Reporter gene assays were performed as described (4, 6), with either cell extracts (Fig. 3A and 3C) (4) or semipermeablized cells (Fig. 3B, 4B, and 5B) (6). Invasive growth (Fig. 3A) was scored as described (40). Cell cycle arrest (Fig. 6A) was assayed by spotting 10 µl of standard medium containing equal numbers of cells onto plates with the appropriate carbon source and then incubating for 6 days. The mating assay (Fig. 6B) was performed as described (22), except that after yeast cells were grown on plates containing 2% sucrose and 0.067% galactose for 24 h, they were replica plated onto a rich plate with the mating tester strain for 4 h and then replica plated onto 2% dextrose plates selective for diploids and grown overnight before being photographed.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To specifically inactivate the scaffold function of Ste5 while leaving its adapter function intact, we replaced Ste5 with the Ste5V763A S861P mutant, which cannot bind to Ste7MEK but still binds to other ligands (22). When this was done, Fus3 activation by pheromone was abolished, but Kss1 activation was only slightly reduced (Fig. 2A, lane 4). Thus, Fus3 requires Ste5 scaffolding to receive the mating signal, as expected based on other results (22). Surprisingly, however, Kss1 does not require Ste5 scaffolding to receive the mating signal.
A schematic interpretation of these results is shown in Fig. 2B. In cells containing the adapter-defective Ste5, Ste11 cannot be activated, so neither Fus3 nor Kss1 is activated. In cells containing the scaffolding-defective Ste5, Ste11 can be activated and can apparently activate non-Ste5-bound Ste7, which then activates Kss1 but is unable to activate Fus3. Hence, these results suggest that active Ste11 may dissociate from Ste5 with a half-time that is relatively fast compared to the duration of pheromone stimulation (and in this case the stimulus duration was only 15 min). Alternatively, scaffold-bound Ste11 may be able to phosphorylate free Ste7. In either case, the results do not support the model shown in Fig. 1B; rather, they suggest that Ste5-activated Ste11 is not restricted to acting on Ste5-bound Ste7, and may be able to leak out into other pathways.
In the absence of Ste5, leaking from the invasive growth pathway to Fus3MAPK decreases. A prediction of the sequestering barrier model of scaffold function (Fig. 1C) is that removing a scaffold might enhance signal crossover, or leaking, between pathways. That is, if a pathway-dedicated scaffold blocks leaking into its pathway, then removing the scaffold may increase such leaking. Specifically, if Ste5 protects Fus3MAPK from Ste7MEK or Ste11MEKK activated by the invasive growth pathway or from Ste11 activated by the osmostress pathway, then removing Ste5 would be predicted to increase Fus3 activation by these inappropriate stimuli.
The filamentation-invasion pathway signals via the Ste11-to- Ste7-to-Kss1 MAPK cascade to drive the expression of genes under the control of filamentation response elements (FREs) (11, 29, 31). Accordingly, a portion of the cellular pool of Kss1MAPK was phosphorylated in cells undergoing invasive growth (Fig. 3A, lane 1), as previously reported (41). Fus3MAPK activation presumably needs to be minimized in these circumstances, since Fus3 positively regulates mating-specific functions and inhibits invasive growth (11, 31, 40, 41). Consistent with this expectation, although some Fus3 activation was detected, it was minimal. Surprisingly, however, in invasively growing cells lacking Ste5, phosphorylated Fus3 was no longer detectable (Fig. 3A, lane 2). This result is the opposite of what would be expected if Ste5 were simply acting as a passive physical barrier to prevent invasive growth signals from activating Fus3. In contrast, when Ste5 was absent, Kss1 phosphorylation was still robust, and Kss1-dependent, FRE-driven gene expression was only slightly reduced (Fig. 3A). Consistent with this, such cells could grow invasively (Fig. 3A), as previously reported (40).
The source of the minimal Fus3 phosphorylation seen in Fig. 3A could have been leaking from the invasive growth pathway, or it could have been (Ste5-dependent) basal signaling from the mating pathway, or a combination of both. Similarly, some of the Kss1 phosphorylation seen in Fig. 3A could have come from basal signaling from the mating pathway (31, 41); this would explain why FRE-driven expression decreased slightly when Ste5 was removed (Fig. 3A). In order to clarify the source of the Fus3 and Kss1 phosphorylation in cells undergoing invasive growth, we examined strains lacking key, pathway-dedicated upstream components of the mating and invasive growth pathways.
First we examined a strain lacking Ste4, the ß subunit of the pheromone receptor-coupled G protein. In this strain, spontaneous activation of the G protein is impossible; thus, basal signal flux through the mating pathway should diminish (18, 44). Indeed, phosphorylation of both Kss1 and Fus3 was decreased in cells lacking Ste4Gß, though Kss1 phosphorylation was still clearly evident and Fus3 phosphorylation was still detectable (Fig. 3B, lane 2 versus lane 1). Furthermore, both FRE-driven expression and basal expression of the mating-specific gene FUS1 were decreased (Fig. 3B), consistent with the decrease in Kss1 and Fus3/Kss1 phosphorylation, respectively.
Next, we examined a strain lacking Msb2, a transmembrane protein that has recently been shown to be a component of the invasive growth pathway and to reside upstream of the MAPK cascade (12). In this strain, Kss1 phosphorylation but not Fus3 phosphorylation was noticeably reduced, and both FUS1 and (especially) FRE-driven expression were decreased (Fig. 3B, lane 3). Finally, in a strain lacking both Ste4Gb and Msb2, both Kss1 and Fus3 phosphorylation was absent and both FUS1 and FRE-driven expression was dramatically reduced (Fig. 3B, lane 4).
These observations lead to the following conclusions. Ste4-dependent signals appear to be preferentially channeled to Fus3, and Msb2-dependent signals are preferentially channeled to Kss1. However, in ste4 cells, some Fus3 phosphorylation is still detectable, and this is dependent upon Msb2. Thus, the invasive growth pathway does leak into the mating pathway to weakly activate Fus3. Conversely, in msb2 cells, some Kss1 phosphorylation is still detectable, and this is dependent upon Ste4. Thus, some of the Kss1 phosphorylation seen in invasively growing cells is attributable to basal signaling from the mating pathway. Removal of Ste4 or Ste5 apparently has two opposing effects on Kss1 phosphorylation and invasive growth that seem to essentially cancel each other out: a decrease in Kss1 phosphorylation due to reduced basal signaling through the mating pathway, and an increase in Kss1 phosphorylation and invasive growth due to reduced inhibition by Fus3-dependent mechanisms (41).
Despite these complications, it is evident that in invasively growing cells, the removal of Ste5 did not increase Fus3 activation (as would be predicted by the sequestering barrier model); instead, Ste5 removal decreased Fus3 activation.
In the absence of Ste5, leaking from the osmostress pathway to Fus3MAPK decreases. In wild-type cells, osmotic stress signals activate Hog1MAPK and do not leak into either the mating or invasive growth pathway (14, 37). However, a leak can be created by removing or inactivating the kinase activity of Hog1 or Pbs2MEK (14, 19, 34). In such cells, presumably due to the lack of a negative-feedback loop that normally limits the extent of Ste11MEKK activation, osmotic stress increases the expression of mating genes such as FUS1 (19, 34) as well as FRE-driven invasive growth genes (14) and stimulates Fus3MAPK (19) and Kss1MAPK (14) phosphorylation. As determined by monitoring gene expression, the osmostress-to-mating leak occurs in cells containing Ste5 and is not increased in the absence of Ste5 (34), suggesting that Ste5 does not play a strong role in preventing this leak.
To more precisely determine the role of Ste5 in modulating signal crossover from the osmostress pathway and to ascertain if the MAPKs were differentially affected by such crossover, strains lacking Pbs2 and either lacking or containing Ste5 were constructed and treated with sorbitol to induce osmotic stress. This treatment resulted in potent Kss1 phosphorylation in cells containing Ste5 (Fig. 3C, lane 1 versus lane 2). Fus3 was also phosphorylated in these circumstances, but not nearly to the same extent as Kss1 (Fig. 3C, lane 1 versus lane 2). Strikingly, removal of Ste5 almost completely eliminated this low-level signal crossover to Fus3 but had less of an effect on signal crossover to Kss1 (Fig. 3C, lane 3). Consistent with the phosphorylation data, mating gene expression (as reported by FUS1-lacZ) decreased in the absence of Ste5, while filamentation gene expression (FRE-lacZ) did not (Fig. 3C). These results indicate that Ste5 enhances rather than lessens misdirected osmotic stress signaling to Fus3.
Fus3MAPK activation by Ste11-4 is weak and dependent on Ste5 scaffolding. To bypass the adapter function of Ste5 and study its scaffold function in isolation, we used a constitutively active version of Ste11MEKK, Ste11-4, which has a point mutation in its kinase domain that renders it independent of Ste5 adapter function (45). The constitutive activity of this mutant (expressed at nearly endogenous levels under the control of its own promoter on a single-copy plasmid) mimics the sustained signaling presumed to occur during filamentation and invasion; indeed, in haploid cells containing Ste11-4, FRE-driven expression was increased (Fig. 4B), as previously reported for diploids (31). Moreover, both Kss1MAPK and Fus3MAPK phosphorylation levels were increased (Fig. 4A, lane 2 versus lane 1, also Fig. 4C, lane 2 versus lane 1), but not to the same degree: the increase in the level of Kss1 phosphorylation was consistently and substantially greater than that of Fus3.
Initially, the weak activation of the mating pathway seemed surprising, since the Ste11-4 allele was isolated in a screen for hyperactivators of FUS1, a mating gene (45). Ste11-4 did in fact drive FUS1 expression, but it did so inefficiently relative to pheromone stimulation (Fig. 4B). Strikingly, when Ste5 was absent from cells containing Ste11-4, phosphorylated Fus3 disappeared and FUS1 expression decreased, but Kss1 phosphorylation levels remained high and FRE-driven expression increased (Fig. 4A, lane 2 versus lane 3, and Fig. 4B). Activation of Fus3 required the scaffolding function of Ste5, because the scaffolding-defective mutant of Ste5 (Ste5V763A S861P) was unable to channel the signal from Ste11-4 to Fus3 (Fig. 4C).
A modest decrease in FUS1 expression levels when Ste5 was removed from cells containing Ste11-4 was observed previously (45). The above results indicate that this modest change in gene expression is the net result of dramatic and opposing changes in the phosphorylation levels of the two MAPKs.
The relative insensitivity of Fus3 to Ste11-4-promoted activation was not caused by a decrease in Fus3 protein levels (Fig. 4A and 4C), nor was it attributable to competition between the plasmid-expressed Ste11-4 and endogenous Ste11 protein, because it also occurred when Ste11-4 was expressed in an ste11 background (Fig. 4D). In addition, there was no restoration of Ste11-4-promoted Fus3 activation in cells lacking Kss1 (Fig. 4E, lane 7); thus, Kss1 was not blocking Fus3 activation. Yet another possibility was that exposure to chronic stimulation was somehow desensitizing Fus3. However, Fus3 was capable of being activated, because it was rapidly phosphorylated following the addition of mating pheromone (Fig. 4E, lanes 4 and 8). Finally, it was unlikely that Ste5 was sequestering Fus3 into a complex from which Ste11-4 was excluded because if this were true, Fus3 activation should have increased (and not decreased) when Ste5 was removed. Ste11-4-promoted activation of Kss1 still occurred in cells lacking Fus3 (Fig. 4E, lane 11).
Although the observation that Ste11-4 signaling to Kss1 increased in the absence of Ste5 is consistent with the idea that Ste5 sequesters limiting amounts of Ste11 and/or Ste7 away from the invasive growth pathway, this model does not explain why Fus3 phosphorylation decreased in the absence of Ste5. Hence, we favor an alternative explanation for the increase in Ste11-4 signaling to Kss1 in the absence of Ste5, that it is a result of the loss of the Fus3-dependent feedback circuit documented previously (41).
To summarize, as in the previous experiments, Kss1 phosphorylation did not require Ste5 scaffolding, whereas Fus3 phosphorylation was strongly dependent on Ste5 even when Fus3 was receiving a misdirected, leaking signal.
Active Ste5 signals preferentially to Fus3. Ste5 is thought to exist in an inactive conformation in resting cells and to adopt an active, open conformation by binding to the pheromone receptor-coupled G protein during mating (15, 23, 43, 48). However, a low level of active Ste5 is present even in the absence of pheromone due to a low level of spontaneous activation of the G protein (44). We speculated that this low level of active Ste5 was responsible for the small amount of signal crossover to Fus3MAPK that we observed (e.g., in Fig. 3A, 3C, 4A, and 4C). In other words, perhaps signal crossover was limited because a component required for crossover (active Ste5) was limiting.
The fusion of glutathione S-transferase (GST) to the C terminus of otherwise wild-type Ste5 creates a constitutively active Ste5 mutant by inducing Ste5 to adopt an active conformation (22, 48). This mutant signals in the absence of pheromone but still requires Ste4Gß, indicating that it spontaneously associates with Gß to form a membrane-associated signaling complex. When Ste5-GST was greatly overproduced (by expressing it from the inducible GAL1 promoter under fully inducing conditions) in otherwise normal, unstimulated cells, both Kss1 and Fus3 were hyperphosphorylated (Fig. 5A). Notably, the signal from active Ste5 was preferentially channeled into Fus3 (Fig. 5A), perhaps because Ste5 binds to Fus3 with a higher affinity than it does to Kss1 (25). These results suggest that Ste5 can channel signal to both Fus3 and Kss1 but shows a preference for Fus3.
In contrast, when another constitutively active allele of Ste11MEKK (Ste11N) (9) was greatly overproduced from the GAL1 promoter, Kss1 was preferentially hyperphosphorylated (Fig. 5A), recapitulating the results obtained with the Ste11-4 mutant or osmostress-activated Ste11 (Fig. 3 and 4). The Ste11N mutant lacks the Ste5-binding domain of Ste11 (24) and thus is unlikely to signal to Fus3 and Kss1 via Ste5. Accordingly, the absence of Ste5 did not diminish Fus3 activation by overproduced Ste11N (data not shown). Thus, the requirement for Ste5-mediated scaffolding in Fus3 phosphorylation can be partially bypassed by overexpressing active Ste11.
Active Ste5 is responsible for signal crossover to the mating pathway. If signal crossover was constrained because the amount of active Ste5 was limiting, then increasing the amount of active Ste5 should increase crossover. To test this proposition, we sought to express the Ste5-GST protein at a level that would not, by itself, substantially activate Fus3; this was achieved by growing cells containing the Ste5-GST expression plasmid in a low concentration (0.067%) of the inducer galactose. This treatment resulted in a modest induction of FUS1 expression (Fig. 5B) and Kss1 and Fus3 phosphorylation (Fig. 5C, compare lane 2 to lane 1). Cells expressing only the Ste11-4 allele also exhibited modest FUS1 expression (Fig. 5B) and preferential phosphorylation of Kss1 (Fig. 5C, lane 3), as seen above (Fig. 4). Strikingly, cells expressing Ste11-4 in conjunction with Ste5-GST displayed greatly increased FUS1 expression (Fig. 5B) and Fus3 phosphorylation (Fig. 5C, lane 4). Kss1 phosphorylation was also increased by this combination (Fig. 5C, lane 4), consistent with the ability of Ste5 to guide some signal to Kss1 (Fig. 3).
Our interpretation of these results is illustrated in Fig. 5D. Only when both Ste5 and Ste11 were active was signal effectively channeled to Fus3. Efficient channeling required active Ste5 protein, because wild-type Ste5, when expressed at a low level comparable to Ste5-GST, was unable to channel the Ste11-4-generated signal to Fus3 (Fig. S1). Hence, active Ste5 allowed the otherwise inefficient Ste11-4-generated signal to further activate Kss1 and to potently activate Fus3, leading to increased signal crossover.
Increased signal crossover changes the developmental option chosen by the cell. Presumably the reason why S. cerevisiae and other organisms have evolved multiple mechanisms to limit signal crossover is because such crossovers would have a negative impact on their fitness. To begin to explore this issue, we investigated some of the functional consequences of the increased leaking that we observed when the cellular concentration of active Ste5 was increased. Two dramatic consequences were readily apparent.
First, whereas otherwise wild-type cells containing the Ste11-4 mutant exhibited increased invasiveness (data not shown), cells containing both Ste11-4 and low levels of Ste5-GST went into cell cycle arrest, as indicated by a lack of growth under inducing conditions (Fig. 6A). This arrest was partially suppressed in the absence of Fus3MAPK, consistent with the hypothesis that the aberrant activation of Fus3 was (at least partially) responsible for the arrest.
Second, Ste5-promoted signal crossover was able to induce the mating program. To demonstrate this, Ste5-GSTC177A C180A, a mutant of Ste5-GST that is active even in the absence of Ste4 (23), was expressed or not in an ste4 strain, with or without coexpression of Ste11-4. Ste5-GSTC177A C180A expressed at a low levels did not stimulate mating on its own (Fig. 6B), although when expressed at higher levels it is capable of doing so (23). Similarly, cells containing only Ste11-4 did not mate efficiently. In contrast, cells containing both Ste11-4 and Ste5-GSTC177A C180Awere able to mate efficiently with a partner of the opposite mating type even though they lacked Ste4Gß and thus were unable to effectively receive the pheromone signal from that mating partner (Fig. 6B). Hence, aberrant signal crossover caused by Ste5's being active at an inappropriate time resulted in a miscued induction of cell cycle arrest and mating.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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First, we tested the following simple hypothesis: if the Ste5 scaffold protein acts as a passive physical barrier to protect the Fus3 MAPK from misdirected signals and thereby prevents leaking into the mating pathway, then removing Ste5 should increase such leaking. In contrast to this prediction, we found that signal crossover from the invasive growth and osmostress pathways to Fus3 was not increased in the absence of Ste5; in fact, it was diminished (Fig. 3 and 4). Therefore, we conclude that Ste5 does not act as a sequestering barrier that isolates the mating pathway from intrusions from other pathways (i.e., it does not act as shown in Fig. 1C).
Second, we found that rather than preventing leaking into the mating pathway, active Ste5 actually promotes such leaking. Increasing the cellular concentration of active Ste5 resulted in increased signal crossover into the mating pathway, as measured by Fus3MAPK and Kss1MAPK activation, mating gene expression, and the downstream endpoints of mating and cell cycle arrest (Fig. 5 and 6). This aberrant signal crossover caused the cells to undergo Fus3-promoted cell cycle arrest and to attempt to mate under conditions that would normally encourage invasive growth (Fig. 6). These results suggest that when Ste5 is active, it directs some of the signal from activated Ste11MEKK to Fus3 and does not distinguish which pathway the activated Ste11 came from. The idea that constitutively active scaffolds may promote enhanced signal crossover is potentially relevant to the pathogenesis of cancer and other diseases.
Distinct requirements of MAPKs for scaffolding. Our third main finding was a dramatic difference in the dependency of the MAPKs Fus3 and Kss1 on Ste5 scaffolding. Whereas both MAPKs required the adapter function of Ste5 during mating, Ste5 scaffolding was not required for Kss1 activation by legitimate signals from the mating or invasive growth pathways or for Kss1 activation via signal crossover from the osmostress pathway. In marked contrast, Fus3 phosphorylation was strongly dependent on Ste5 whether Fus3 was receiving a bona fide mating signal or a misdirected signal leaking from the invasive growth or osmostress pathway (Fig. 2, 3, and 4).
Kss1 activation during mating required Ste5's adapter function (i.e., its ability to bind Ste11MEKK) but not its scaffold function (i.e., its ability to bind Ste7MEK). The adapter function requirement is consistent with the generally accepted proposal that Ste11 is activated at the membrane while bound to Ste5. Apparently, however, Ste5 did not prevent bound, active Ste11 from leaking out and encountering nonscaffolded Ste7. Hence, this experiment (Fig. 2A, lane 4) provides some evidence against the model shown in Fig. 1B. A caveat to this interpretation is that it is based upon analysis of a mutant of Ste5 that cannot bind Ste7; perhaps Ste7 binding to Ste5 reduces Ste11 dissociation from Ste5.
The mechanism underlying the strikingly different Ste5 dependencies of Fus3 versus Kss1 remains to be determined. These two MAPKs show greater than 50% sequence identity, are activated by the same MEK (Ste7), and have some key substrates in common (e.g., the Ste12, Dig1 and Dig2 transcription factors) (8). In vitro, Fus3 activation clearly requires Ste7 but does not require Ste5 (16, 33), although a differential enhancement of Fus3 versus Kss1 activation by purified Ste5 protein has been reported (8). The requirement for Ste5 in Fus3 activation is evidently intimately associated with the scaffolding function of Ste5, since the Ste5V763A S861P mutant, which cannot bind to Ste7, did not support Fus3 activation. Perhaps Ste5 causes a conformational change in Fus3 that is required for its phosphorylation by cobound Ste7. Presumably, Kss1 is exempt from these requirements or has them provided by another protein.
Possibly, Kss1 requires a different scaffold for its activation. If so, then our results suggest that this putative scaffold must be active most of the time (including during mating and osmostress) and that it does not protect Kss1 from signal crossover from the osmostress pathway (this work) or the mating pathway (41). Thus, this putative scaffold apparently does not contribute to specificity. The simpler model, which we favor, is that Kss1 activation is largely scaffold independent.
Model of how leaking into the mating pathway is prevented. Apparently, the Ste5 scaffold acts not as an insulant or barrier but as a conduit or sluice that, when open, undiscerningly guides signals from shared upstream components towards Fus3MAPK. Despite this seeming handicap, signal crossover into the mating pathway is normally limited. A model for how this may be achieved is presented in Fig. 7. This model is based upon the idea that Ste5 is in an inactive, closed conformation in nonmating cells and converts to an active, open, membrane-localized configuration in response to pheromone stimulation (15, 23, 43, 48). We suggest that since Fus3 requires Ste5 scaffolding to receive potentially leaky signals and Ste5 is inactive in nonmating cells, Fus3 ignores such signals most of the time.
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In contrast to Fus3, Hog1MAPK and Kss1MAPK are independent of Ste5 scaffolding and so can receive signals when Ste5 is inactive, although Kss1 still requires the adapter function of Ste5 during mating. During invasive growth, Kss1 is activated to a lesser extent than it is during mating (L. J. Flatauer, unpublished observations); Kss1 is also less effective than Fus3 at driving mating gene expression (39) or causing cell cycle arrest (8). Hence, invasive growth signals do not significantly stimulate mating via Kss1.
Comparison to other results. Our experiments indicate that Ste5 does not act via sequestration to prevent signals from leaking into the mating pathway. Furthermore, they suggest that Ste5 may also not be very effective at preventing signals from leaking out of the mating pathway. That stable kinase-scaffold associations have the potential to limit leaking out of the scaffolded pathway was demonstrated by Harris et al. (20), who showed that Ste11MEKK, covalently attached to Ste5, stimulated mating but not stress responses whereas Ste11, covalently attached to Pbs2MEK, stimulated stress responses but not mating. Evidence consistent with the idea that selective recognition of Ste7MEK is one important factor in limiting leaking out of the mating pathway was provided by Park et al. (35), who found that covalently attaching Pbs2MEK to Ste5 created a mating-to-osmostress leak.
Pbs2MEK and Sho1 have been proposed to act together to scaffold the osmostress pathway (37, 50). However, leaking out of this pathway occurs in cells in which Sho1 and Pbs2 are both present (19, 34) and functional for signal transmission (42). Hence, the Sho1/Pbs2 scaffold apparently does not effectively sequester stress-activated Ste11 and restrain it from straying into other pathways, at least not under the conditions studied.
Other mechanisms act to restrict signal crossover in certain directions. MAPK-dependent mechanisms limit leaking from the osmostress pathway (14, 19, 34) and from mating into invasive growth (41). The mechanisms that prevent signals from leaking into the Hog1MAPK pathway are unknown at present. Possibly, in analogy to the model we have proposed here, Hog1 or Pbs2 requires activation by stress in order to receive signals from Ste11.
At first consideration, our finding that pheromone-activated Ste11 was apparently able to dissociate from Ste5 and phosphorylate Ste7 is inconsistent with the results of the diverter scaffold experiment of Park et al. (35): if pheromone-activated Ste11 readily dissociates from Ste5, then why did Park et al. find it necessary to attach Pbs2 to Ste5 in order to get pheromone-activated Ste11 to phosphorylate Pbs2? Perhaps membrane localization of Pbs2 activates it somehow, making it competent to be phosphorylated by Ste11. Further work will be required to clarify this issue.
Two other groups have also recently found evidence of differential MAPK activation. Maleri et al. found that constitutively active Ste7 selectively activates Kss1 (32); this finding is consistent with our proposal that Ste5 must be activated by upstream components of the mating pathway in order to transmit signals to Fus3. Andersson et al. (1) observed the selective, scaffold-independent activation of Kss1 in response to several manipulations, such as expression of Ste11-4. In contrast to our findings, however, these authors reported that Ste4 and Ste5 are needed to activate Kss1 during invasive growth. The difference between their results and ours seems to be attributable to their use of the noninvasive S228C lineage for most of their experiments. The S288C lineage has acquired multiple mutations during the process of repeated selection in the laboratory for nonclumpy cells that formed round colonies (27). Perhaps one or more of these mutations decreased the ability of Kss1 to be activated in a scaffold-independent fashion (1). Like Andersson et al., we too have observed that in S228C-derived strains, Kss1 activation during mating is more dependent on the scaffold function of Ste5 (data not shown). Furthermore, we found a strong requirement for Ste4 in Kss1 activation during invasive growth in a 1278b-derived strain that lacked the Msb2 protein (Fig. 3B). Hence, one possibility is that the S228C lineage may be defective in a component that signals (in an Ste5-independent fashion) from Msb2 to Kss1.
Conclusions. Here we have shown the Ste5 scaffold protein does not act as an insulant or barrier to block signal crossover into the S. cerevisiae mating pathway. Instead of preventing misdirected signals from activating Fus3MAPK, Ste5 actually helps misdirected signals to activate Fus3. Leaking into the mating pathway is limited under normal circumstances because Fus3 requires active Ste5 to receive any signals and Ste5 is activated only during mating. Kss1MAPK does not require Ste5 scaffolding to receive signals, even during mating, and so can function in two different pathways, one which uses Ste5 and one which does not. If generalized, these observations imply that scaffold proteins promote specificity by directing signal flow in certain directions at certain times but do not actively prevent signal crossover by sequestration.
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ACKNOWLEDGMENTS |
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This work was supported by Public Health Service Training Grant GM07311 (to L.J.F.) and by research grants GM60366 and GM69013 from the National Institute of General Medical Science.
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FOOTNOTES |
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Supplemental material for this article may be found at http://mcb.asm.org/.
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REFERENCES |
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