Mitogen activated protein kinases (MAPKs) respond to a wide array of external signals, and are presumed to be present in all eukaryotes. Cellular proliferation, differentiation, and stress responses are all regulated by MAPKs [1–4]. The well characterized MAPKs function as part of a signaling module in which a MAPK kinase kinase (MEKK) phosphorylates and thereby activates a MAPK kinase (MEK), which then phosphorylates and activates the MAPK. Upon activation, MAPKs phosphorylate targets in the nucleus, in the cytoplasm, and at the cell cortex [5, 6].
Because MAPKs affect targets throughout the cell, their localization is a critical aspect of signal regulation [7–9]. In fact, mislocalization of mammalian ERK MAPKs has been associated with altered signaling [10–13]. Moreover, the duration of ERK nuclear localization has been reported to be very important in determining the outcome of ERK activation: proliferation vs. differentiation [14–17]. Immunofluorescence and GFP-tagging experiments have demonstrated that pathway stimulation causes some MAPKs to translocate from the cytoplasm to the nucleus [18–20]. Although nuclear accumulation of MAPKs has been studied in model systems, the mechanisms that regulate this phenomenon are not fully understood.
A priori, the nuclear localization of MAPKs can be regulated in two ways: 1) regulated import or export across the nuclear membrane; 2) release from cytoplasmic tethers (anchoring proteins) and/or sequestration by nuclear tethers. Thus far, there are four examples of MAPKs that are subjected to active transport. In mammals, Erk2 interacts directly with the nuclear pore complex and undergoes Ran-dependent nuclear import [21–23], whereas Erk3 and Erk5 are actively exported from the nucleus by the CRM1 exportin [24, 25]. In budding yeast, the Hog1 MAPK undergoes both Ran- and Nmd5-dependent nuclear import, as well as Crm1-dependent nuclear export . A number of reports also suggest that tethers influence the localization of mammalian MAPKs. MEK is thought to be a cytoplasmic tether for ERK1/2, for example . When ERK is activated, it releases from MEK, allowing ERK to localize to the nucleus. PEA-15, hSef, the MKP-3 dual-specific phosphatase, and the tyrosine phosphatases HePTP and PTP-SL have also been reported to be cytoplasmic tethers for ERKs, and β-arrestin is thought to anchor ERK to endosomal vesicles [28–34]. As yet, DUSP5 is the only protein reported to tether ERKs in the nucleus . In the budding yeast S.cerevisiae, the Ptp2 phosphatase and the Msn2, Msn4, and Hot1 transcription factors act as nuclear tethers for the Hog1 MAPK, whereas the Ptp3 phosphatase and the Pbs2 MEK tether Hog1 in the cytoplasm [36–38]. Also in budding yeast, Spa2 retains the Mpk1 MAPK at sites of polarized growth . Finally, the Atf1 transcription factor of S.pombe has been shown to regulate the nuclear localization of the Spc1/Sty1 MAPK .
To better understand the mechanisms controlling MAPK localization, we are using the mating reaction of S.cerevisiae as a model signaling system. When haploid yeast cells of opposite mating type are mixed, they undergo a complex mating reaction leading to the formation of zygotes. Each mating type constitutively secretes a peptide mating pheromone that triggers cells of the opposite type to arrest in the G1 phase of the cell cycle, form mating projections (shmoos), and induce mating-specific genes in preparation for cell and nuclear fusion. The signal to mate is transmitted across the plasma membrane by a seven transmembrane domain receptor and its associated heterotrimeric G protein. Upon release from Gα (Gpa1), the Gβγ dimer binds to the Ste5 scaffolding protein and stimulates a Pak kinase. These proteins, in turn, are required for activation of the MAP kinase cascade. The MAP kinase module consists of Ste11 (the MEKK), Ste7 (the MEK), and Fus3 (the MAPK). Upon activation by Ste7 on the Ste5 scaffold, the Fus3 MAPK accumulates at its sites of action [41–43]. In the nucleus, Fus3 phosphorylates the Ste12 mating-specific transcription factor [44, 45], its two negative regulators, Dig1 and Dig2 [44, 46], and a regulator of cell division, Far1 [44, 45, 47]. At the cortex, Fus3 is thought to phosphorylate Bni1  and Gβ .
In proliferating cells, Fus3 is found in both the cytoplasm and the nucleus, with a slightly greater concentration of Fus3 in the nucleus [50, 51]. Upon pheromone stimulation, the relative proportion of Fus3 in the nucleus increases. To quantify this pheromone-induced nuclear accumulation of Fus3, we developed an assay that measures the relative amount of Fus3 in each compartment . Digital images of cells expressing a Fus3-GFP reporter were used to determine the ratio of nuclear to cytoplasmic fluorescence (RNCF). Using this assay, we confirmed that pheromone induces a measurable accumulation of Fus3 in the nucleus, and found that this increase in nuclear Fus3 correlates with the responsiveness of cells to pheromone. Cells that are resistant to pheromone-induced cell cycle arrest have lower RNCF values; cells that are hypersensitive to pheromone arrest have higher RNCF values. Moreover, the relative amount of Fus3 in the nucleus decreases as cells adapt to pheromone stimulation and re-enter the mitotic cycle. These results demonstrate that, although the changes in Fus3 localization during the induction and downregulation of the pheromone response are small (RNCF values typically range from 1.6 to 2.0), they are of great consequence to the cell. It is therefore of interest to determine what regulates the partitioning of Fus3 between the cytoplasm and the nucleus.
Using fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) analyses, van Drogen and co-workers found no evidence that the rate of Fus3 transport either into or out of the nucleus is regulated by pheromone . Rather, they concluded that Fus3 rapidly shuttles between the nucleus and cytoplasm by passive diffusion. If the rate of Fus3 transport across the nuclear membrane is not regulated by pheromone, then what causes the accumulation of Fus3 in the nuclei of pheromone-treated cells? One possibility is that the activation of Fus3 alters its affinity for cytoplasmic and nuclear tethering proteins. Consistent with this, Fus3 dissociates from the Ste5 scaffolding protein in the cytoplasm when it is phosphorylated by Ste7 . Combining this mechanism with an increased tendency to bind tethers in the nucleus could account for the observed signal-induced change in Fus3 localization.
Here we report that three proteins known to interact with Fus3 in the nucleus (Dig1, Dig2, and Ste12) contribute to its pheromone-induced redistribution. Our data suggest that Dig1 and Dig2 are nuclear tethers for Fus3, and that Ste12 influences Fus3 localization either by directly interacting with it, or by transcribing genes whose protein products are Fus3 tethers. We also provide evidence that the phosphorylation state of Fus3 is a key determinant of its localization, and that the Msg5 phosphatase regulates Fus3 localization by dephosphorylating it in both the cytoplasm and in the nucleus. This work supports the idea that the activation of MAPKs changes their affinity for anchoring proteins in the cytoplasm and the nucleus, thereby effecting their signal-induced redistribution.