Because MAPK signaling cascades are used by all eukaryotic cells to control growth and differentiation, the timing and duration of MAPK responses must be tightly regulated. For MAPKs that participate in multiple pathways, cross talk must be controlled as well. Although MAPK pathways have been subjected to intense investigation, the ways in which they are regulated are not fully understood. A priori, regulatory mechanisms could involve expression, enzymatic activity, sequestration and/or localization, and stability of MAPKs. Induction of MAPK responses is thought to depend on the activation of MAPKs by their MEKs, and their subsequent accumulation in the nucleus, where they phosphorylate transcription factors and other regulatory targets. Such signal-induced localization has been demonstrated in a number of systems (26
). Inactivation of MAPKs partially depends on the action of specific protein phosphatases and, in some cases, the regulated export of MAPKs from the nucleus is thought to play a role in downregulation (15
). The yeast mating signal is mediated by a heterotrimeric G protein that communicates with a MAPK cascade composed of Ste11 (MEKK), Ste7 (MEK), and Fus3 (MAPK). The free Gβγ
subunit activates the MAPK module, and both the inactive and the active forms of the Gα
subunit, Gpa1, negatively regulate the response. Prior to this work, genetic evidence suggested that Gpa1 stimulates adaptation by indirectly inactivating Ste4 (Gβ
) and through a mechanism acting at the level of Fus3 (13
; A. T. Ellicott and D. E. Stone, unpublished results). In a proteomic screen for gene products that associate with Gpa1 in cells responding to pheromone, we identified Fus3 (44
). Additional experiments showed that the activated form of Gpa1 binds to the activated form of Fus3 in cells responding to pheromone. This discovery led us to study the relationship between the localization of Fus3 and adaptation to pheromone. We found that the relative level of nuclear Fus3 decreases under conditions in which cells are recovering from pheromone treatment or have adapted to chronic pheromone stimulation and that both Gpa1 and the dual-specific phosphatase Msg5 negatively affect the nuclear localization of Fus3. The adaptive functions of Gpa1 and Msg5 are partially dependent on one another and on the Kap104 importin. To our knowledge, this is the first time that the Gα
subunit of a heterotrimeric G protein has been shown to affect the subcellular localization of a MAPK.
Gpa1 and Msg5 downregulate the mating signal by affecting the localization of Fus3.
Because the expression of Fus3 is induced ~5-fold by pheromone (53
), it is difficult to detect signal-induced nuclear localization of Fus3 comparable to that documented for other MAPKs. Using a myc-tagged form of Fus3, Elion et al. found that the level of the kinase in the nucleus is slightly enhanced by pheromone treatment (6
), and van Drogen et al. came to the same conclusion using a Fus3-GFP reporter (65
). To determine whether the nuclear concentration of Fus3 rises and falls as cells respond to pheromone and adapt to it, we developed a quantitative assay that measures the relative proportion of Fus3 in the nucleus and cytoplasm. Although the RNCF values varied considerably within a given culture, four observations indicate that this measurement is sensitive to physiologically relevant changes in Fus3 localization. First, pheromone treatment of wild-type cells causes a 25 to 50% increase in the mean RNCF (Fig. to ). Statistical analysis showed this difference to be highly significant (P
< 0.0001). We speculate that the variability of the RNCF values within a given culture is due to the lack of cell cycle synchrony. Second, the increase in nuclear Fus3 is easily detectable after only 20 min of pheromone treatment, suggesting that the buildup of Fus3 in the nucleus is relevant to the induction of mating response. Third, the mean RNCF of wild-type cultures decreases as the cells recover and begin to bud (Fig. ). Fourth, Fus3 does not concentrate in the nucleus when cells that have adapted to chronic stimulation are rechallenged (Fig. ). This observation correlates with the insensitivity of such cells to pheromone-induced cell cycle arrest and may be relevant to the ability of recovered cells to “remember” their exposure to pheromone and remain unresponsive to further stimulation for multiple generations (46
The availability of a quantitative assay for the relative level of nuclear Fus3 enabled us to ask whether downregulation of the mating signal by Gpa1 and Msg5 is associated with changes in Fus3 localization. The data shown in Fig. , , and clearly indicate that both of these proteins, when manipulated to promote hyperadaptation, inhibit the pheromone-induced accumulation of Fus3 in the nucleus, and both are dependent on the wild-type function of Kap104 to accomplish this. It is noteworthy that both Gpa1 and Msg5 can inhibit the nuclear localization of Fus3 without affecting Fus3 expression. Is the decreased concentration of nuclear Fus3 in stimulated cells the cause or the result of the mating pathway downregulation? Although this question cannot be definitively answered on the basis of correlative results, Gpa1 and Msg5 are both known to interact directly with Fus3. Moreover, mutational disruption of the Gpa1-Fus3 interaction increased pheromone sensitivity and localization of Fus3 to the nuclei of stimulated cells (Fig. ), implying that Gpa1 regulates the mating signal by directly binding to the MAPK. Therefore, we favor the former possibility: Gpa1 and Msg5 inhibition of Fus3 accumulation in the nucleus is an adaptive mechanism.
It is important to note that the Gpa1 and Msg5 adaptive functions are partially interdependent. Deletion of MSG5
-induced recovery from pheromone arrest (Fig. ) and, when combined with the loss of Ptp3, another cytoplasmic phosphatase that targets Fus3, slightly lessens the effect of Gpa1E364K
on Fus3 localization (Fig. ). Furthermore, the msg5Δ ptp3Δ
double deletion is completely epistatic to the effect of excess wild-type Gpa1 on Fus3 localization in stimulated cells (Fig. ). Conversely, the effect of Msg5 overexpression on Fus3 localization is weakened by inactivation of Gpa1 (Table ). Together, these results suggest that Msg5 acts with Gpa1 to downregulate the mating signal by inhibiting the nuclear localization of Fus3. Because Gpa1 is found primarily at the plasma membrane (19
), its effect on Fus3 localization is likely due to sequestration. Msg5 might also act as a cytoplasmic tether for Fus3, in the same way that Ptp3 serves as a cytoplasmic anchor for the Hog1 MAPK of S. cerevisiae
). Alternatively, or in addition, Msg5 might inhibit nuclear accumulation of Fus3 by dephosphorylating it. Signal-induced nuclear localization of other MAPKs is known to be dependent on their phosphorylation state (for examples, see references 26
), and the same may be true for Fus3. Consistent with this possibility, recent evidence suggests that phosphorylation of Fus3 promotes its release from the Ste5 scaffolding protein (65
). Dephosphorylation of Fus3 might therefore promote its binding to Ste5.
Although the data do not yet allow us to formulate a detailed model of the Gpa1-Msg5 functional relationship, we can propose two general possibilities. Gpa1 and Msg5 may act in parallel, separately impinging on Fus3. When the kinase dissociates from one of the regulators, the other may capture it. In this scenario, Msg5 might sequester Fus3 and/or inactivate it. A distinct possibility is that Gpa1 and Msg5 act on Fus3 in a complex. Perhaps Gpa1 facilitates the docking of the phosphatase to the kinase. In this case, the primary role of Msg5 would be dephosphorylation of Fus3 rather than sequestration of it. Coimmunoprecipitation experiments might allow us to detect Gpa1-Fus3-Msg5 complexes if they exist. Whatever the mechanism, it is clear that the role of Msg5 is distinct from that of Ptp3. Whereas deletion of either phosphatase affects the basal RNCF, only overexpression of Msg5 represses the induced RNCF.
Based on photobleaching studies of cells expressing Fus3-GFP, van Drogen et al. concluded that Fus3 shuttles constitutively between the cytoplasm and nucleus and that its rate of transport is not affected by pheromone treatment (65
). According to their model, Fus3 is recruited to the plasma membrane in stimulated cells by Ste5, where it is activated by Ste7. Phosphorylation of Fus3 triggers its dissociation from Ste5. Some of the activated kinase molecules then move to the nucleus, whereas some concentrate at the shmoo tip. What controls this differential localization of activated Fus3? We have shown that the activated form of Gpa1 binds to the activated form of Fus3 in cells responding to pheromone and that the Gpa1-Fus3 interaction is important both in the chemotropic sensing of pheromone and in adaptation to pheromone (44
). Moreover, Gpa1 concentrates at the shmoo tips of responding cells (44
). Therefore, we propose that Gpa1 captures some of the activated Fus3 molecules as they are released from Ste5, thereby targeting the kinase to its substrates at the plasma membrane and simultaneously modulating the intensity of the mating signal.
Does this mean that Gpa1 inhibits the Fus3-dependent mating responses in the nucleus while at the same time promoting mating-specific events at the plasma membrane? It is interesting to suppose that Msg5, whose expression is strongly induced by pheromone, acts as a temporal switch that alters the consequences of the Gpa1-Fus3 interaction. Early in the mating reaction, when relatively little Msg5 is present, the activated Fus3 that dissociates from Gpa1 is free to move to the nucleus. When the expression of Msg5 is fully induced (near the time of zygote formation), however, any activated Fus3 that dissociates from Gpa1 is sequestered and/or inactivated by the phosphatase. Thus, toward the end of the mating reaction (perhaps as mating partners begin to fuse or in newly formed zygotes), Gpa1 and Msg5 work together to prevent the transport of activated Fus3 to the nucleus. This relieves the cell cycle block and downregulates mating-specific gene expression when these responses are no longer appropriate. Note that in this model, the Kd for the Gpa1-Fus3 interaction determines the relevance of Msg5 to the adaptive function of Gpa1. The model correctly predicts that, in the Fus3-GFP localization assay, Gpa1E364K, which binds Fus3 considerably better than does wild-type Gpa1, is less badly compromised by deletion of MSG5 and PTP3 than is wild-type Gpa1 (Fig. ).
An important point in the model proposed by van Drogen et al. (65
) is that pheromone treatment does not alter the rates of nuclear import or export of Fus3. Nevertheless, Fus3 clearly accumulates in the nuclei of cells responding to pheromone. Most likely, this is due to decreased cytoplasmic tethering and/or increased nuclear anchoring of phosphorylated Fus3. There are a number of examples in which transcription factors retain activated MAPKs in the nuclei of yeast and mammalian cells (3
). Although van Drogen et al. found that deletion of known Fus3 interactors had no effect on the nucleocytoplasmic distribution of Fus3 (65
), their assessment was not quantitative. Differences of 0.2 in the mean RNCF of a cell population are not easily discerned, despite their significance. Alternatively, the retention factors responsible for the pheromone-induced change in Fus3 distribution may not be known.
Involvement of Kap104 in the pheromone response.
It is clear from our data that Kap104 is required for proper regulation of the pheromone response. Deletion of the carboxy-terminal 91 amino acids of Kap104, which make up about one-quarter of the presumed cargo-binding domain, confers supersensitivity to pheromone in cells expressing wild-type Gpa1 and compromises the hyperadaptive activity of Gpa1E364K and excess Msg5. These effects of the kap104Δ827-918 mutation were apparent both in pheromone-induced growth inhibition assays (Fig. ) and in pheromone-induced transcriptional assays (data not shown) and could not be attributed to abnormally low expression of either Gpa1 or kap104Δ827-918 (Fig. ). Furthermore, the kap104Δ827-918 mutation suppressed the effects of excess Gpa1E364K and excess Msg5 on Fus3 localization (Fig. ). Thus, the epistatic effects of kap104Δ827-918 on Msg5- and Gpa1-mediated adaptation to pheromone correlate with restoration of normal Fus3 levels in the nuclei of stimulated cells.
A second indication that Gpa1-mediated adaptation depends on Kap104 is that the hyperadaptive activity of Gpa1E364K is potentiated by Kap104 overexpression. Approximately twice as many cells expressing Gpa1E364K and excess Kap104 can overcome what are normally lethal concentrations of pheromone, as can control cells expressing Gpa1E364K and the wild-type level of Kap104 (Fig. ), despite the decreased growth rate and increased sensitivity to pheromone conferred by Kap104 overexpression. Immunoblot analysis demonstrated that this enhanced resistance to pheromone is not due to an increase in the steady-state level of Gpa1 (Fig. ). Thus, changes in both the structure and expression of Kap104 affect the adaptive signaling mechanism(s) stimulated by Gpa1: overexpression of Kap104 enhances recovery, whereas truncation of Kap104 confers an adaptive defect.
How does Kap104 affect Fus3 localization? One possibility is that Kap104 imports a protein that acts as a nuclear tether for Fus3, although this is purely speculative. Another possibility is that Kap104 directly exports Fus3. This is unlikely because there is no precedent for a Ran-dependent karyopherin protein acting as both an importer and an exporter. Based on the structural and biochemical evidence, interaction of a given karyopherin with Ran-GTP in the nucleus is thought to stimulate either cargo release (for an importer) or cargo binding (for an exporter) (see references 7
and references therein). Kap104 is an import receptor. It is concentrated on the cytosolic side of nuclear pore complexes, where it is thought to interact with the RNA-binding proteins Nab2 and Nab4 (1
). Thus far, Nab2 and Nab4 are the only known Kap104 cargo proteins. Moreover, Fus3 does not contain a region similar to the Kap104-binding domain found in Nab2 (56
) or a stretch of residues like the M9 sequence of hnRNP A1 (rich in glycine, serine, and asparagine) that mediates the interaction of human transportin with its cargo (67
). If Kap104 is not itself the Fus3 export receptor, then perhaps it recycles the unknown transporter. After cargo release, all transport receptors must move back or be moved back through the NPCs to their point of origin. This is true for both importers and exporters. In at least one case, recycling of one transporter depends on the action of another: karyopherin α (importin) is returned to the cytoplasm by the exporter, CAS1/Cse1 (30
). A deficiency in recycling the Fus3 exporter could lead to increased levels of Fus3 in the nucleus, just as we observed when kap104Δ827-918
cells overexpressing Gpa1E364K
or Msg5 were challenged with pheromone (Fig. ). Of course, this explanation assumes that Fus3 is escorted across the nuclear membrane by specific transport receptors, and this has not been established. Fus3 may bind directly to nuclear pore proteins, as does Erk2 (66
A third way in which Kap104 might affect the pheromone response pathway is by ensuring the proper expression of a key regulator. Because Kap104 is essential for the reimport of Nab2 and Nab4 and thus indirectly responsible for the export of mature mRNAs, loss of Kap104 function would be expected to adversely affect gene expression. Given that Nab2-GFP is mislocalized in kap104Δ827-918
cells (Fig. ), it would be surprising if the mutant cells expressed all genes normally. What genes might be required to limit the amount of nuclear Fus3? We have found that Gpa1 and Msg5 are likely to play a role in controlling the localization of Fus3. However, the steady-state level of Gpa1 is not reduced in mutant strain A11 (Fig. ), and the signal intensity of an Msg5-GFP reporter appears to be unaffected by the truncation of Kap104 (data not shown). The phenotypes conferred by kap104Δ827-918
could also be due to a decrease in the level of an unknown Fus3 exporter, an increase in the level of a Fus3 nuclear tether or, less directly, by loss of transcription factors such as Mot2 or Mot3, which are required for downregulation of the mating signal (5
). Although the mechanism is unclear, our data indicate that Kap104 is required for proper regulation of the pheromone response and in particular for the Gpa1/Msg5-mediated effect on Fus3 localization.