The mitogen-activated protein kinase (MAPK) cascade is a highly conserved signaling module composed of MAPK kinase kinases (MAPKKKs), MAPK kinases (MAPKK) and MAPKs. The MAPKKK Mkh1 is an initiating kinase in Pmk1 MAPK signaling, which regulates cell integrity in fission yeast (Schizosaccharomyces pombe). Our genetic screen for regulators of Pmk1 signaling identified Shk1 kinase binding protein 5 (Skb5), an SH3-domain-containing adaptor protein. Here, we show that Skb5 serves as an inhibitor of Pmk1 MAPK signaling activation by downregulating Mkh1 localization to cell tips through its interaction with the SH3 domain. Consistent with this, the Mkh13PA mutant protein, with impaired Skb5 binding, remained in the cell tips, even when Skb5 was overproduced. Intriguingly, Skb5 needs Mkh1 to localize to the growing ends as Mkh1 deletion and disruption of Mkh1 binding impairs Skb5 localization. Deletion of Pck2, an upstream activator of Mkh1, impaired the cell tip localization of Mkh1 and Skb5 as well as the Mkh1–Skb5 interaction. Interestingly, both Pck2 and Mkh1 localized to the cell tips at the G1/S phase, which coincided with Pmk1 MAPK activation. Taken together, Mkh1 localization to cell tips is important for transmitting upstream signaling to Pmk1, and Skb5 spatially regulates this process.
The mitogen-activated protein kinase (MAPK) signaling cascade is a highly conserved signaling module, which plays a central role in various physiological processes, including cell proliferation, gene expression, differentiation and cell survival (Nishida and Gotoh, 1993; Marshall, 1994; Herskowitz, 1995; Munshi and Ramesh, 2013). It is also conserved in lower eukaryotes such as yeasts and plays a key role in cell wall biosynthesis and stress responses (Levin, 2005; Park and Bi, 2007; Perez and Cansado, 2010). The abnormal activation of MAPK signaling leads to deregulated phosphorylation events that play a role in tumorigenesis (Dhillon et al., 2007; Santarpia et al., 2012). Therefore, understanding the mechanisms of negative regulation of MAPKs could lead to the discovery of drugs to target the Raf–MEK–ERK MAPK pathway and be important for cancer therapeutics.
The MAPK pathway transmits its signal through the sequential phosphorylation of MAPK kinase kinases (MAPKKKs) to MAPK kinases (MAPKKs) to MAPKs (Zheng and Guan, 1993; Gardner et al., 1994). Hence, protein phosphatases, such as those from the DUSP family and PP2C, which dephosphorylate MAPKs or upstream kinases play key roles in the negative regulation of these activation processes (Jeffrey et al., 2007; Pan et al., 2015). MAPKKKs lie at the apex of the MAPK pathway kinase module and play a crucial role in transmitting upstream signaling to MAPKKs and MAPKs. MAPKKKs have been known to be inactivated through dephosphorylation by PP5 (von Kriegsheim et al., 2006; Shah and Catt, 2006), and recent studies on RKIP (also known as PEBP1) (Yeung et al., 2000; Park et al., 2006) have revealed a new regulatory mechanism of MAPKKK regulation by an adaptor protein and its influence on MAPK activation. However, relatively little is known about the subcellular localization of MAPKKKs and their relevance to MAPK activation.
We have been studying the Pmk1 MAPK signaling module, composed of the MAPKKK Mkh1, the MAPKK Pek1 and the MAPK Pmk1, a key regulator of cell wall integrity in fission yeast (Toda et al., 1996; Sugiura et al., 1999; Sengar et al., 1997). Our previous genetic screen for negative regulators of Pmk1 MAPK signaling identified phosphatases (Sugiura et al., 1998) that inactivate MAPK signaling, including the Pmp1 dual-specificity phosphatase and the PP2C serine/threonine protein phosphatase (Takada et al., 2007) in addition to the Rnc1 RNA-binding protein (Sugiura et al., 2003) and the cell surface protein Ecm33 (Takada et al., 2010). Our genetic screen also identified components and activating regulators of Pmk1 MAPK, including the small GTPases Rho1, Rho2, Rho4 and Rho5, and the protein kinase C (PKC) protein Pck2, by isolating mutants of the farnesyl transferase Cpp1 and geranylgeranyl transferase Cwg2 (Ma et al., 2006a,b; Doi et al., 2015). Here, we have established a novel genetic screen for negative regulators of Pck2-mediated MAPK signaling activation by utilizing the cell growth defect induced by Pck2 overproduction and its recovery upon Pmk1 signaling inhibition (Takada et al., 2007). We identified Shk1 kinase binding protein 5 (Skb5), an SH3 adaptor protein that has been isolated as a binding partner for the p21-activated kinase (PAK) homolog Shk1 in fission yeast. Furthermore, Skb5 has been shown to directly activate Shk1 kinase activity (Yang et al., 1999). We showed that Skb5 inhibits Pck2-mediated MAPK signaling hyperactivation by interacting with Mkh1. Notably, Mkh1 was localized to the cell tips at the G1/S phase in addition to the previously described localization of the medial region (Madrid et al., 2006), and importantly, the cell-tip localization of the MAPKKK was regulated by the Skb5–Mkh1 interaction. Pck2 deletion impaired Mkh1 and Skb5 localization at cell tips as well as the Mkh1–Skb5 interaction. Possible roles of Skb5 as a spatial regulator of MAPKKKs and the physiological significance of MAPKKK localization to cell tips in terms of MAPK signaling activation will be discussed.
Skb5 overproduction negatively regulates Pck2–Pmk1 MAPK signaling
To identify new regulators of PKC–MAPK signaling in fission yeast, we established a genetic screen. This screen was based on previous findings from our laboratory (Takada et al., 2007) and others (Carnero et al., 2000) showing that the overexpression of Pck2 in wild-type (WT) cells results in severe growth defects, whereas the inhibition or deletion of the components of the Pmk1 MAPK pathway can reverse the growth defects. Consistent with this, the overproduction of protein from the pmp1+ gene, which we previously identified as a dual-specificity phosphatase that dephosphorylates and inactivates the Pmk1 MAPK (Sugiura et al., 1998), clearly suppressed the growth defect induced by Pck2 overproduction (Fig. 1A), indicating that this screen can reveal new genes involved in the negative regulation of PKC–MAPK signaling. We therefore screened for genes that when overexpressed can suppress the growth defect induced by Pck2 overproduction. Consequently, two classes of genes were identified, and sequence analysis revealed that skb5+, encoding an SH3-domain-containing adaptor protein, and pmp1+ were included. As shown in Fig. 1A, the overexpression of skb5+ and pmp1+ suppressed the growth defect induced by Pck2 overproduction in the absence of thiamine (Promoter ON), whereas cells harboring the control vector alone (+pck2+ +vector) failed to grow in the absence of thiamine.
Because Rho2 acts upstream of Pck2–Pmk1 MAPK signaling, and the overexpression of Rho2 is toxic to WT cells, but not to cells with deletions in components of the Pmk1 MAPK pathway (Ma et al., 2006a,b), the effects of Skb5 and Pmp1 overexpression on the growth of Rho2-overproducing cells were also examined. As shown in Fig. 1B, overexpression of Rho2 was toxic to the WT cells (Promoter ON; +rho2+ +vector), but the overexpression of the skb5+ and pmp1+ significantly reduced the toxicity of Rho2 overproduction, indicating that Skb5, similar to Pmp1, is involved in the negative regulation of Rho2- and Pck2-mediated MAPK signaling downstream of Pck2.
To further delineate the step at which Skb5 functions in Pmk1 MAPK signaling, we examined the effect of skb5+ on the growth defect induced by Pek1DD, which encodes a constitutively active MAPKK (Sugiura et al., 1999). As shown in Fig. 1C, the toxicity induced by Pek1DD overproduction was suppressed by the expression of Pmp1, consistent with the notion that Pmp1 dephosphorylates and inhibits Pmk1 MAPK (+pek1DD +pmp1+). In clear contrast, the overexpression of the skb5+ gene failed to suppress the toxicity induced by Pek1DD, indicating that Skb5, unlike Pmp1, could not reverse the hyperactivation induced by Pek1DD overproduction. Thus, Skb5 is likely to inhibit MAPK signaling downstream of Pck2 and upstream of Pek1.
If Skb5 serves as an inhibitor of Pmk1 MAPK signaling, then Skb5 deletion cells would be expected to exhibit phenotypes similar to those associated with Pmp1 deletion. As shown in Fig. 1D, Skb5 deletion induced hypersensitivity to 0.6 M MgCl2 as did Pmp1 deletion. However, cells with an Skb5 deletion (Δskb5) did not exhibit sensitivity to FK506, whereas the growth of cells with a Pmp1 deletion (Δpmp1) was significantly inhibited by this treatment (Fig. 1D). We then examined the combined effect of FK506 and MgCl2 on Δskb5 and Δpmp1 cells. Our previous findings established that the vic phenotype (for ‘viable in the presence of chloride ion’) is a strong indicator of MAPK signaling inhibition (Ma et al., 2006a,b; Doi et al., 2015). The results showed that Δskb5 and Δpmp1 cells failed to grow in medium containing 0.06 M MgCl2 and FK506, whereas the WT cells grew in this medium, indicating that Skb5 deletion induced a vic-negative phenotype (Fig. 1D). This finding is consistent with the notion that Skb5 inhibits Pmk1 MAPK signaling.
In order to confirm that the suppression of Pck2 overproduction by Skb5 was due to its effect on Pmk1 MAPK activation, the effect of Skb5 overexpression on the phosphorylation levels of Pmk1 MAPK was examined. For this, antibodies against phosphorylated Pmk1 (phospho-Pmk1), which recognize doubly phosphorylated Pmk1, were utilized (Sugiura et al., 1998). As shown in Fig. 1E, Pck2 overproduction driven under the nmt1 promoter stimulated Pmk1 phosphorylation without any environmental stimuli, whereas the co-expression of skb5+ significantly reduced the phosphorylation levels of Pmk1 as compared with the cells harboring the control vector alone, thus indicating that Skb5 is involved in the negative regulation of Pck2-mediated Pmk1 signaling. A negative control experiment with pek1-null mutant cells showed that Pmk1 was not phosphorylated at all even when Pck2 was overexpressed (Fig. S1).
Skb5 downregulates Pmk1 MAPK signaling independently of Ptc1
To obtain clues for the mechanisms underlying Pmk1 MAPK signaling suppression by Skb5, we focused on the ability of Skb5 to bind to several components of the MAPK signaling pathway. It has been reported that Skb5 binds to the Mkh1 MAPKKK and Ptc1 MAPK phosphatase (Stanger et al., 2012), and that both are involved in the regulation of Pmk1 MAPK signaling (Sugiura et al., 1999; Takada et al., 2007). To investigate whether this interaction is specific, the interaction between Skb5 and several components of the Pmk1 MAPK pathway, including Pmk1, Pek1 and Mkh1, was examined. Results clearly showed that Skb5 specifically interacted with Mkh1 (Fig. 2A, GST–Skb5). Co-precipitation experiments with the unfused GST protein did not lead to detection of Pmk1, Pek1 or Mkh1 in the pulldowns (Fig. 2A, GST).
As Ptc1 has also been shown to be involved in negative regulation of the MAPK Pmk1 (Takada et al., 2007), the Skb5 interaction with the dual-specificity MAPK phosphatase Pmp1 was also investigated. In this case, results showed that Skb5 interacted with Ptc1, but not with Pmp1 (Fig. 2B, GST–Skb5). Co-precipitation experiments with the unfused GST protein did not detect Pmp1 or Ptc1 (Fig. 2B, GST).
To determine whether the interactions between Skb5 and Mkh1 or Ptc1 are required for the suppression of MAPK signaling, the vic phenotype was utilized. WT cells fail to grow in the presence of the calcineurin inhibitor FK506 (0.2 μg/ml) and 0.12 M MgCl2, whereas cells deleted for the components of the Pmk1 MAPK pathway are viable in the same medium (Ma et al., 2006a,b). Consistent with this, cells overexpressing pmp1+ and skb5+ grew in the presence of FK506 and 0.12 M MgCl2, whereas cells harboring the control vector alone failed to grow (Fig. 2C, upper panel). Next, the effect of the overexpression of pmp1+ and skb5+ was examined in cells deleted for Ptc1 (Δptc1). Notably, the overproduction of Skb5 and Pmp1, fully suppressed the vic phenotype of the Δptc1 cells, indicating that Skb5 exerts its ability to suppress MAPK signaling even in the absence of Ptc1 (Fig. 2C, lower panel). Furthermore, Skb5 overproduction inhibited the hyper-phosphorylation of Pmk1 induced by the cell-wall-damaging agent micafungin, both in the WT and in the Δptc1 cells (Fig. 2D), thus indicating that the Skb5–Ptc1 interaction is not required for MAPK signaling suppression by Skb5.
Skb5 inhibits Pmk1 MAPK signaling through its binding to Mkh1
We next focused on the Skb5–Mkh1 interaction and its effect on Pmk1 MAPK, because the above results strongly suggested that Skb5 exerted its suppression through its interaction with Mkh1. Skb5 contains an SH3 domain and it has been reported that mutations in the SH3 domain impair its interaction with binding partners (Stanger et al., 2012). This prompted us to make a Skb5YF2A mutant wherein both the tyrosine (Y) 89 and phenylalanine (F) 135 in the SH3 domain of the Skb5 protein, were mutated to alanine (A) residues (Fig. 3A). As shown in Fig. 3B, the GST–Skb5YF2A mutant protein barely bound to GFP–Mkh1, whereas WT GST–Skb5 interacted with Mkh1 (Fig. 3B, middle panel). Importantly, Skb5YF2A maintained the ability to interact with Ptc1–GFP (Fig. 3B, right panel), indicating that the YF2A mutation in Skb5 specifically abolished the Mkh1–Skb5 interaction. It should be noted that the GST, GST-fused Skb5 and GST–Skb5YF2A proteins did not bind to the GFP control (Fig. 3B, left panel).
Next, the effect of the Skb5YF2A mutant protein on the suppression of Pmk1 MAPK signaling was examined. The overexpression of the mutant skb5YF2A could not rescue the lethality caused by overexpressing Pck2 (Fig. 3C). Similarly, the overexpression of skb5YF2A resulted in the failure to induce the vic phenotype whereas WT skb5+ did (Fig. 3D). As expected, the overexpression of skb5YF2A did not reduce the increase in Pmk1 phosphorylation levels induced by micafungin as did the WT skb5+ (Fig. 3E). Thus, the ability of Skb5 to interact with Mkh1 is required for Skb5 to inhibit Pmk1 signaling.
Skb5 affects Mkh1 localization at cell tips
To explore how Skb5 overproduction inhibits Pmk1 signaling by interacting with Mkh1, the endogenous Mkh1 protein tagged with GFP was visualized and the effect of Skb5 overproduction was examined. It has been reported that Mkh1 localizes to the cytoplasm and at the septum during cell division (Madrid et al., 2006). Here, we found that the endogenous Mkh1 protein fused to GFP (i.e. expressed from the native promoter) was localized to the cell tips in addition to the previously described localization (Fig. 4A, +vector, arrows). Notably, the overproduction of the skb5+ markedly reduced the Mkh1 fluorescence at the cell tips (Fig. 4A, +skb5+, arrows, promoter ON). A quantification of the proportion of cells where the endogenous Mkh1–GFP was localized at the cell tips showed that less than 10% of the Skb5-overproducing cells exhibited Mkh1 localization to the cell tips as compared with the cells harboring a control vector alone (Fig. 4A, lower panel, promoter ON). Next, the effect of the overproduction of the skb5YF2A mutant protein on the Mkh1 localization was investigated; skb5YF2A overproduction barely reduced the Mkh1 localization to the cell tips (Fig. 4A, +skb5YF2A, arrows, promoter ON), thus indicating that the Mkh1 localization change was induced largely upon Skb5 binding to Mkh1 through the SH3 domain.
Furthermore, the impact of the Skb5–Mkh1 interaction on Mkh1 localization was examined by investigating the effect of Mkh1 mutations that would disrupt Skb5 binding. It has been reported that the PxxP sequence is a preferred binding signature for the SH3 domains (Stanger et al., 2012), and that the mutation in the proline (P) residues in the budding yeast Bck1 MAPKKK, markedly impair its binding with the Nbp2 SH3 domain protein (Stanger et al., 2012). We then searched for the PxxP motif in Mkh1, and three proline residues, at positions 544, 546 and 547 were mutated into alanine (A) residues to make the Mkh13PA protein (Fig. 4B).
The resulting Mkh13PA protein showed a markedly reduced affinity for Skb5, whereas it maintained the ability to interact with Pek1 and Pck2 (Fig. 4C), indicating that the Mkh13PA mutation specifically impaired the binding between Mkh1 and Skb5. It should be noted that the GST, GST–Skb5, GST–Pek1 and GST–Pck2 protein did not bind to the GFP control and the unfused GST protein did not pulldown GFP–Mkh1 or GFP–Mkh13PA (Fig. 4C).
In order to evaluate the physiological significance of the Skb5–Mkh1 interaction, the Mkh13PA mutation was introduced into the chromosomal mkh1 locus. The resultant Mkh13PA–GFP protein also localized to the cytosol with intense fluorescence at the cell tips (Fig. 4D, +vector). Notably, however, skb5+ overexpression failed to reduce the Mkh13PA localization to the cell tips (Fig. 4D, +skb5+), which is clearly different from the observations with the endogenous Mkh1–GFP protein shown in Fig. 4A. The quantification also confirmed the above results (Fig. 4D, lower panel). Thus, the interaction between Skb5 and Mkh1 appears to play a key role in Mkh1 localization to cell tips.
Skb5 localization is also affected by Mkh1 interaction
Next, the subcellular distribution of endogenous Skb5 tagged with GFP was analyzed. The fluorescence of the Skb5–GFP protein expressed from its endogenous loci was not well defined, but diffusedly observed throughout the cytoplasm, and less than 20% of the cells exhibited localization to the cell tips (Fig. 5A, endogenous Skb5WT–GFP, arrows). The fluorescence of exogenously expressed GFP–Skb5 showed that there was the Skb5 localization around the cell periphery, and approximately half of the cells exhibited Skb5 localization to the cell tips (Fig. 5A, GFP–Skb5WT overproduction, arrows). The physiological significance of the Skb5–Mkh1 interaction was examined by introducing the YF2A mutation into the chromosomal skb5 gene. The endogenous Skb5YF2A–GFP protein also localized to the cell periphery (Fig. 5A, endogenous Skb5YF2A–GFP), although the frequency of cell tip localization was significantly reduced (to 33.3%) as compared with the WT endogenous Skb5 (Skb5WT). Quantification revealed that more than 40% of the cells exhibited Skb5 cell-tip localization when Skb5 was exogenously expressed as compared with less than 20% with the endogenous Skb5 (Fig. 5A, lower panel). Notably, however, when the GFP–Skb5YF2A mutant protein was exogenously expressed, it could barely be visualized at the peripheral and cell-tip localizations, and the frequency of the cells exhibiting cell-tip localization decreased markedly (Fig. 5A, Skb5YF2A overproduction).
In order to determine whether Mkh1 plays a role in Skb5 localization, the effect of Mkh1 deletion on the endogenous Skb5 localization was examined. Notably, Mkh1 deletion significantly reduced the Skb5 localization to the growing ends, and only ∼20% of the cells exhibited cell-tip localization (Fig. 5B). Thus, Skb5 can localize to cell tips at least partly through its interaction with Mkh1.
Skb5 deletion increases Mkh1 cell-tip localization and Pmk1 phosphorylation
To investigate whether the Mkh1–Skb5 interaction is important for downstream Pmk1 MAPK signaling activation, Skb5 deletion cells, Skb5YF2A mutant strains and Mkh13PA mutant strains were studied to see whether these strains exhibited altered Pmk1 phosphorylation levels. For this purpose, the Skb5YF2A mutant and Mkh13PA mutant strains were first examined for whether they displayed a vic phenotype. As shown in Fig. 1D and in Fig. 6A, the Δskb5 cells failed to grow in the presence of 0.06 M MgCl2 and FK506, whereas the WT cells grew well, indicating that the skb5-null cells display a vic-negative phenotype. Both the Skb5YF2A mutant and the Mkh13PA mutant cells also exhibited the vic-negative phenotype, similar to that observed in the Δskb5 cells (Fig. 6A), consistent with the hypothesis that the Skb5–Mkh1 interaction is important for Pmk1 MAPK signaling.
Next, Δskb5, Skb5YF2A mutant and Mkh13PA mutant cells were investigated for Pmk1 MAPK phosphorylation before and after the micafungin treatment. In the Δskb5 cells, Pmk1 MAPK phosphorylation levels were significantly higher as compared with the WT cells after the micafungin treatment (Fig. 6B). It should be noted that the difference in basal Pmk1 activation between the Skb5 deletion and WT type cells was indiscernible (Fig. 6B). In contrast, the Skb5YF2A mutant and Mkh13PA mutant cells exhibited similar Pmk1 phosphorylation levels both before and after micafungin treatment as compared with the WT cells (Fig. 6C).
In order to see whether the difference in Pmk1 phosphorylation levels in the mutant strains resulted from the difference in the amount of Mkh1 cell-tip localization, the endogenous Mkh1 protein was visualized in the WT and Skb5 deletion cells. The endogenous Mkh13PA mutant protein was also visualized and the fluorescence of the cell-tip-localized Mkh1 in these cells was quantified. Results showed that Skb5 deletion significantly increased Mkh1 cell-tip localization as compared with the WT cells (Fig. 6D). This is consistent with its role as a negative regulator of MAPK signaling through its interaction with Mkh1. In contrast, the Mkh13PA mutation did not significantly affect Mkh1 cell-tip localization (Fig. 6D). Thus, although the biochemical studies showed that Mkh13PA mutation impairs Mkh1–Skb5 binding (Fig. 4C), the Mkh13PA mutant protein might still maintain its biological ability to bind to Skb5, whereas Skb5 deletion totally abolishes the Skb5–Mkh1 interaction.
Pck2 influences Mkh1–Skb5 localization to the cell tips and their interaction
We next sought to assess possible upstream factors that could affect the Mkh1–Skb5 localization at the cell tips. As candidates, we investigated the effect of the deletion of the small G protein Rho2 and Pck2, both of which act upstream of Mkh1. The effects of the deletion of the Ras1 small G protein, which acts upstream of the Byr2–Byr1–Spk1 MAPK pathway, was also investigated. Notably, the localization of the endogenous Mkh1 protein at the cell ends was markedly abrogated upon Pck2 deletion (Δpck2), but not upon Ras1 (Δras1) or Rho2 (Δrho2) deletion (Fig. 7A). Similar effects were obtained with endogenous Skb5 localization, as deletion of Pck2 specifically abrogated Skb5 cell-tip localization (Fig. 7B). Quantification revealed that only half of the Δpck2 cells exhibited cell-tip localization of Mkh1 and only 20% of the cells exhibited cell-tip localization of Skb5 as compared with that in the WT cells (Fig. 7A,B).
This prompted us to further study the effect of Pck2 deletion on the Mkh1–Skb5 interaction. GST pulldown experiments showed that Pck2 deletion significantly impaired the Mkh1–Skb5 interaction, as the GFP–Mkh1 protein was barely detectable in the GST pulldown extracted from Δpck2 cells harboring GST–Skb5 (Fig. 7C). Quantification showed that the interaction between Mkh1 and Skb5 in Pck2 deletion cells was ∼50% of that in the WT cells (Fig. 7C).
Finally, the effect of Skb5 overproduction on the endogenous Pck2 localization was examined, and the results showed that Pck2 was localized to the cell tips irrespective of Skb5 overproduction (Fig. 7D). These data are consistent with the above findings showing that Skb5 does not interact with Pck2 and that Skb5 specifically impaired Mkh1 localization at the cell tips.
Pck2 and Mkh1 localized to the cell tips in the G1/S phase of the cell cycle
In order to reveal the role of Mkh1 at cell tips and the significance of Pck2–Mkh1–Skb5 localization at cell tips, cell cycle synchronization experiments were performed by using the cell cycle mutant cdc25-22 expressing the Mkh1–GFP protein from the native promoter. Cells from this mutant were grown to log phase at 25°C, shifted to 37°C for 4 h to synchronize the cells in the G2 phase, and then shifted back to 25°C. As shown in Fig. 8A, the proportion of cells with cell-tip localization of Mkh1 oscillates as a function of the cell cycle, reaching a maximum during the G1/S phase.
We also visualized the endogenous Pck2–GFP protein in the cdc25-22 mutant, and the Pck2 protein expressed from its native promoter was similarly observed to be localized at the cell tips during the G1/S phase of the cell cycle (Fig. 8B). It should be noted that in addition to our findings, other researchers have shown changes in Pmk1 phosphorylation during the cell cycle, with it reaching a maximum during the G1/S phase (Madrid et al., 2006; Satoh et al., 2009). Collectively, Pck2–Mkh1 localization at the cell tips closely coincided with Pmk1 activation at the G1/S phase, suggesting that the cell-tip localization of the upstream kinases (Pck2 and Mkh1) could stimulate Pmk1 MAPK, which leads to the oscillation of MAPK activation during the cell cycle.
In this study, we utilized a forward genetic screen in fission yeast to identify molecules involved in Pmk1 MAPK signaling inhibition. Mkh1 MAPKKK acts upstream of Pmk1 and plays a pivotal role in MAPK activation. However, to date, factors that influence Mkh1 localization remain poorly characterized. Here, we identified Skb5 as a modulator of Pmk1 signaling and showed that the interaction of Mkh1 with the Skb5 SH3 domain affects Mkh1 localization at the cell tips, thereby attenuating Pmk1 MAPK signaling.
Our genetic and biochemical experiments demonstrated that Skb5 inhibits Pmk1 MAPK signaling at the level of Mkh1. Importantly, this data clearly showed that Ptc1, a previously reported binding partner of Skb5 was not required for the Skb5-mediated inhibition of Pmk1 signaling. Moreover, an analysis of Pmk1 phosphorylation when expressed with the Skb5YF2A mutant protein, which has an impaired binding to Mkh1, indicated that Skb5 inhibits Pmk1 MAPK signaling by specifically interacting with Mkh1.
How can Skb5 overproduction affect intracellular localization of Mkh1 at the cell tips and inhibit MAPK signaling?
Two lines of evidence suggested that Skb5 affects Mkh1 localization at the cell tips by interacting with Mkh1 through its SH3 domain, which leads to Pmk1 signaling inhibition. First, the overproduction of Skb5, but not that of Skb5YF2A, specifically reduced Mkh1 localization at the cell tips. In addition, the overproduction of Skb5, but not that of Skb5YF2A, rescued Pck2-induced cytotoxicity and inhibited Pmk1 MAPK activation. Second, the Mkh13PA mutant, which specifically reduces its affinity for Skb5, was insensitive to Skb5 overproduction, and remained at the cell tips. This was further confirmed by analysis with the endogenous Skb5WT and Skb5YF2A mutant alleles, which were integrated into the chromosomal skb5 locus. More importantly, Skb5 deletion induced Mkh1 cell-tip localization and increased Pmk1 MAPK phosphorylation levels. It should be noted that although the Skb5YF2A mutant alleles and the Mkh13PA mutant alleles exhibited a vic-negative phenotype, these mutant alleles did not cause increased Pmk1 activation. Thus, although biochemical studies suggested that the Mkh13PA and Skb5YF2A mutations significantly impaired Skb5–Mkh1 binding, these proteins might still maintain the ability to interact with the binding partners. Alternatively, as-yet-unidentified factors that can determine Mkh1 cell-tip localization might exist.
What is the physiological significance of Mkh1 localization to the cell tips?
Previous studies in mammals have reported that the Raf kinase [also known as Raf1; the MAPKKK upstream of ERK1 and ERK2 proteins (ERK1/2, also known as MAPK3 and MAPK1, respectively)] is translocated from the cytoplasm to the plasma membrane through binding with the upstream GTP-bound Ras. This recruitment of Raf to the plasma membrane induces a conformational change of Raf, allowing its phosphorylation in the plasma membrane by several kinases such as Src, PKC and Akt, resulting in the activation of the Raf kinase and downstream MAPK signaling (Marais et al., 1995; Barnard et al., 1998; Hibino et al., 2011).
In our study, Pck2, a target of Rho small GTPases and an upstream activator of Mkh1, which binds to and activates Mkh1, also localizes to the cell tips. Moreover, Pck2 and Mkh1 localized to the cell tips at the G1/S phase of the cell cycle, coincident with Pmk1 MAPK activation. Interestingly, Pck2 deletion impairs Mkh1 localization to the cell tips as well as the Mkh1–Skb5 interaction. These findings support the hypothesis that Mkh1 localization to the cell tips is important for Mkh1 to efficiently receive and transmit the Pck2-mediated signaling to the downstream MAPKK Pek1 and MAPK Pmk1. In this regard, Pck2 might stimulate Mkh1 localization to the cell tips, thereby facilitating the signal transduction from the upstream Rho small GTPases to Pmk1 MAPK through Mkh1. It is noteworthy that Pck2 deletion also abrogated Skb5 localization to the cell tips. Thus, it is intriguing to speculate that Pck2-mediated Mkh1 phosphorylation might enhance Mkh1 localization to the cell tips as well as the Skb5–Mkh1 interaction. In line with this, the Skb5–Mkh1 interaction also seems to be important for Skb5 cell-tip localization, based on the findings that Skb5, but not Skb5YF2A, localized to the growing ends upon overproduction. Consistent with this, Mkh1 deletion reduced Skb5 localization to the growing ends, suggesting that Skb5 needs Mkh1 to localize to the growing ends and thereby affects Mkh1 localization at the cell tips. Thus, we hypothesize that Skb5 recognizes the cell-end-localized and presumably active form of Mkh1 through the Skb5 SH3 domain, and this interaction might recruit Skb5 to the cell tips.
Given that Skb5 plays a role as a negative regulator of MAPK signaling, Skb5 might have a higher affinity to the cell-tip-localized and/or active form of Mkh1, thus raising the possibility that activation of MAPKKK recruits its adaptor molecule, thus making a negative-feedback loop system. Our observations showing that the Skb5YF2A mutation with impaired Mkh1 binding, as well as Mkh1 deletion, markedly reduce Skb5 localization to the growing ends, further support this hypothesis.
The additional question that arises is: what is the in vivo role of Skb5 in inhibiting Mkh1 after its activation by Pck2? Alternatively, does Skb5 serve to keep Mkh1 inactive prior to recruitment to the tips? The evidence in favor of the latter possibility is the observation that Skb5 deletion induced Mkh1 cell-tip localization. Thus, although basal Pmk1 phosphorylation levels might not be affected by Skb5 deletion, these data suggest an in vivo role of Skb5 as a spatial regulator of Mkh1. However, as hypothesized above, the Pck2-mediated MAPK activation signal induced Mkh1 cell-tip localization and Mkh1–Skb5 interaction at the cell tips, thus indicating that Skb5 might also play a role in inhibiting Mkh1 after its activation by Pck2. In line with this view, Skb5 deletion significantly stimulated Pmk1 phosphorylation after micafungin treatment. Thus, Skb5 might be both required for spatial regulation before and after the upstream Pck2 activation. Future studies will be necessary to clarify the molecular mechanisms of Skb5-mediated inhibition of the Pck2–Mkh1–Pmk1 MAPK signaling pathway.
Based on a previous paper showing that Skb5 is as an activator of the p21-activated kinase (PAK) homolog Shk1 (Yang et al., 1999), which is a member of the Cdc42 signaling cascade, and a paper reporting a possible crosstalk between the Cdc42 pathway and the Pmk1 MAPK pathway (Merla and Johnson, 2001), it would be intriguing to investigate whether Skb5 is also involved in the suppression of the Cdc42-mediated signaling pathways (Merla and Johnson, 2001). These previous findings prompted us to examine the effect of the skb5+ overexpression, and the results showed that Skb5 overexpression modestly suppressed Cdc42-induced lethality (Fig. S2). However, in a previous report by Madrid et al. (2006), the overexpression of Cdc42G12V did not significantly stimulate Pmk1 MAPK activation, and the authors concluded that the Cdc42 GTPase and PAK kinases Pak1 and Pak2 do not regulate the basal and stress-induced activation of Pmk1. Therefore, the suppression of Cdc42-mediated lethality by Skb5 overexpression might simply reflect the Skb5 function as an activator and a binding partner for Shk1 and Pak1, an important kinase downstream of Cdc42. Future studies will be required to fully reveal the function of Skb5 as a crucial regulator of several kinases involved in polarity and morphogenesis.
Intriguingly, Src-like adaptor protein (SLAP) and SLAP2 (also known as SLA and SLA2), Skb5 orthologs in higher eukaryotes (http://www.pombase.org/spombe/result/SPCC24B10.13), have been reported to act as negative regulators of T cell receptor (TCR) signaling. SLAP interacts with a set of proteins relevant to TCR signal transduction, such as ZAP-70 and Vav through its SH2 domain (Tang et al., 1999; Holland et al., 2001). In addition, accumulating evidence has revealed an emerging role of SLAP as a key regulator in receptor tyrosine kinase (RTK) signaling. SLAP has been shown to interact with a subset of RTKs including Eph receptors and PDGFRs. (Wybenga-Groot and McGlade, 2015). Interestingly, SLAP function in the regulation of TCR and RTK signaling is closely coupled with its binding to the ubiquitin ligase Cbl through its C-terminal region, allowing for ubiquitylation of substrates such as EphA2 and its subsequent degradation (Wybenga-Groot and McGlade, 2015). This prompted us to investigate the protein amount of Mkh1 in the absence and presence of Skb5 overproduction, with a presumption that Skb5 might couple Mkh1 to the ubiquitin-mediated degradation. However, no Mkh1 protein degradation was observed upon Skb5 overproduction (data not shown), thus indicating that the effect of Skb5 on cell-tip Mkh1 localization could not be attributed to Mkh1 degradation, but reflects the dispersal of Mkh1 localization from the cell tips to the cytoplasm. Further investigations regarding the regulatory mechanism of Mkh1 localization to cell tips and factors involved in the process are necessary. However, given that Skb5 shows similarity with SLAP and SLAP2 in the SH3 domain, Skb5 might downregulate factors involved in Pmk1 MAPK signaling cooperatively with unidentified ubiquitin ligases.
MATERIALS AND METHODS
Strains, media, and genetic and molecular biology methods
Schizosaccharomyces pombe strains used in this study are listed in Table S1. The complete media (yeast extract with peptone-dextrose, YPD, or yeast extract with supplements, YES) and the minimal medium (Edinburgh minimal medium, EMM) are as described previously (Sabatinos and Forsburg, 2010; Toda et al., 1996). Standard genetic and recombinant DNA methods (Sabatinos and Forsburg, 2010) were used except where otherwise noted. PCR-based genomic epitope tagging was performed using standard methods (Bahler et al., 1998). Proteins were N-terminally or C-terminally tagged with GFP or GST expressed from the respective endogenous loci. The GFP- or GST-tagging did not alter the protein function of these molecules as evidenced by the observations that the phenotypes in terms of the lethality in the presence of the Cl− and FK506 as well as the sensitivity or tolerance to micafungin are indiscernible from those of the untagged WT cells (data not shown).
Isolation of the skb5+
The chromosome-borne nmt1-GFP-pck2+ cells (Pck2 overproducing) were constructed as described in Bahler et al. (1998). The thiamine-repressible nmt1-GFP-pck2+ integrated Pck2-overproducing cells were transformed using an S. pombe genomic DNA library constructed in the vector pDB248 (Beach et al., 1982). Leu+ transformants were replica-plated onto EMM plates at 27°C without the addition of thiamine, and plasmid DNA was recovered from transformants that showed plasmid-dependent growth in the absence of thiamine. The recovered plasmids suppressed the lethality induced by Pck2 overproduction. DNA sequencing showed that the suppressing plasmids contained SPCC24B10.13 (skb5+) and pmp1+.
Protein expression and site-directed mutagenesis
For protein expression in yeast, the thiamine-repressible nmt1 promoter was used (Maundrell, 1990). Expression was repressed by the addition of 4.0 mg/ml thiamine to EMM and was induced by washing and incubating the cells in EMM lacking thiamine. The GST- or the GFP-fused gene was subcloned into the pREP1 vectors. Skb5YF2A and Mkh13PA were generated using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA). The primers used are summarized in Table S2.
Anti-GFP (Ma et al., 2006a,b), anti-GST (Ma et al., 2006a,b) and anti-phospho-Pmk1 (Sugiura et al., 1999) were used as the primary antibodies (1:20,000 dilutions). Anti-rabbit-IgG (Cell Signaling) was used as the secondary antibody (1:4000 dilution). Membranes were developed with Chemi-Lumi One Super (Nacalai tesque). Protein levels were quantified using ImageJ software (http://rsb.info.nih.gov/ij/).
Growth conditions and stress treatment
Unless otherwise stated, cells were cultivated at 27°C in EMM (Sabatinos and Forsburg, 2010). Prior to stress treatment, the cells were grown to mid-log phase (optical density at 660 nm=0.5). Micafungin stock solution (200 mg/ml) was added to the culture medium to give the indicated concentrations. After stress treatment, the culture medium was chilled in ice-cold water for 1 min. The cells were harvested by a brief centrifugation at 4°C (700 g).
Microscopy and miscellaneous methods
Light microscopy methods (e.g. fluorescence microscopy) were carried out as described previously (Kita et al., 2004; Satoh et al., 2012). Photographs were taken using an AxioImager M1 microscope (Carl Zeiss, Germany) equipped with an LSM700 microscope (Carl Zeiss) and ZEN 2012 software (Carl Zeiss). Images were processed with ZEN 2012 software. Cell extract preparation and immunoblot analysis were performed as previously described (Sio et al., 2005).
The quantification of cell tip localization was performed for at least two individual datasets, which analyzed up to 100 cells.
All results are expressed as mean±s.d. of several independent experiments. Data were analyzed using a one-way ANOVA, followed by a post hoc test using Dunnett's multiple comparison (Fig. 4A; upper row of graphs, Fig. 6D; upper row of graphs), a one-way ANOVA, followed by a post hoc test using Tukey-Kramer's multiple comparison (Fig. 5A; upper row of graphs), or by Student's t-test (Fig. 5B; upper row of graphs). P-values less than 5% were regarded as significant. Asterisks indicate significant differences, and n.s. indicates not significant.
We thank T. Toda, K. Nakano and the Yeast Resource Center (YGRC, NBRP; http://yeast.lab.nig.ac.jp/nig) for providing strains and plasmids, and Professor William Figoni for critical reading of the manuscript. We are grateful to the members of the Laboratory of Molecular Pharmacogenomics for their support.
R.S. designed this project. Y.K., R.S., S.M., C.I., N.I., S.T. and A.K. performed experiments. Y.K., S.M., S.T., A.K., K.H., and R.S. analyzed the data. Y.K. and R.S. wrote the manuscript. All authors reviewed the manuscript.
This work was supported by research grants from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) (to R.S.). This work was also supported by the MEXT-Supported Program for the Strategic Research Foundation at Private Universities 2014–2018 [grant number S1411037].
The authors declare no competing or financial interests.