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© 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 3189-3202 doi:10.1242/jcs.188854
RESEARCH ARTICLE
Skb5, an SH3 adaptor protein, regulates Pmk1 MAPK signaling by
controlling the intracellular localization of the MAPKKK Mkh1
ABSTRACT
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.
KEY WORDS: Schizosaccharomyces pombe, PKC, MAPKK kinase,
Skb5, SH3 adaptor protein
INTRODUCTION
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;
1
Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences,
Kindai University, 3-4-1 Kowakae, Higashiosaka City, Osaka 577-8502, Japan.
2
Division of Pharmaceutical Education, Faculty of Pharmacy, Kindai University,
3-4-1 Kowakae, Higashiosaka City, Osaka 577-8502, Japan.
*Author for correspondence ([email protected])
Y.K., 0000-0002-8539-5149; R.S., 0000-0002-7467-0096; S.M., 0000-00018675-365X; C.I., 0000-0002-1437-7824; N.I., 0000-0003-3222-5741; K.H., 00000002-9366-6172; S.M., 0000-0002-5069-8262; S.T., 0000-0002-9595-2076; A.K.,
0000-0002-1879-9493; R.S., 0000-0001-6946-0935
Received 4 March 2016; Accepted 6 July 2016
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
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Journal of Cell Science
Yuki Kanda1, Ryosuke Satoh1, Saki Matsumoto1, Chisato Ikeda1, Natsumi Inutsuka1, Kanako Hagihara1,
Sumio Matzno2, Sho Tsujimoto1, Ayako Kita1 and Reiko Sugiura1,*
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.
RESULTS
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
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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 coexpression 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 cellwall-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.
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Journal of Cell Science (2016) 129, 3189-3202 doi:10.1242/jcs.188854
Fig. 1. Skb5 overproduction negatively regulates the Pck2–Pmk1 MAPK signaling pathway. (A) Skb5 was identified as a regulator of the cytotoxicity induced
by Pck2 overproduction. Wild-type (WT) cells transformed with the control vectors (+vector) alone, pREP2-GST-Pck2 (+pck2 +) and the control vector, pREP2GST-Pck2 and the skb5+gene, or pREP2-GST-Pck2 and pmp1 + gene, were grown in EMM in the presence (Promoter OFF) or absence (Promoter OFF) of
thiamine at 27°C for 5 days. (B) Skb5 suppressed the cytotoxicity induced by Rho2 overproduction. WT cells transformed with the control vectors alone and with
Rho2 (+rho2 +) and the control vector, the skb5 + gene or the pmp1 + gene, were grown in EMM in the presence (Promoter OFF) or absence (Promoter ON) of
thiamine at 27°C for 3 days. (C) Skb5 failed to suppress the cytotoxicity induced by Pek1DD overproduction. WT cells transformed with the control vectors alone,
pREP2-GST-Pek1DD (+pek1DD) and the control vector, pREP2-GST-Pek1DD and the skb5 + gene, or pREP2-GST-Pek1DD and pmp1 + gene, were grown in EMM
in the presence (Promoter OFF) or absence (Promoter ON) of thiamine at 27°C for 6 days. (D) Skb5 deletion induced a vic-negative phenotype. WT, Skb5 deletion
(Δskb5) and Pmp1 deletion (Δpmp1) cells were grown in YPD or YPD in the presence of 0.06 M MgCl2+FK506, FK506, 0.06 M MgCl2, or 0.6 M MgCl2 at 27°C for
4 days. (E) Skb5 overproduction can suppress Pmk1 MAPK phosphorylation induced by Pck2 overproduction. The chromosome-borne nmt1-GFP-Pck2 cells
expressing endogenous Pmk1-GST and transformed with either the control vector or skb5+, were grown in EMM in the presence ( promoter OFF −) or absence
( promoter ON +) of thiamine at 27°C. Cell lysates and proteins bound to glutathione beads were analyzed by immunoblotting using anti-GFP antibodies (GFP–
Pck2), anti-GST antibodies (Pmk1–GST) and anti-phospho-Pmk1 antibodies ( phosphorylated Pmk1). Upper panel: blot representative of three independent
experiments. Lower panel: quantification of Pmk1 phosphorylation calculated by measuring intensities of the phosphorylated Pmk1 (detected by anti-phosphoPmk1 antibodies) versus total Pmk1, as loading control (detected by anti-GST antibodies), and presented relative to the value for the vector and promoter OFF
cells (set at 1) using Image J software. The mean of the three experiments is shown.
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
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Fig. 2. Skb5 suppresses Pmk1 MAPK signaling independently of the interaction with Ptc1. (A) Skb5 binds to Mkh1. GST pulldowns were carried out with
GST or GST–Skb5. Cells transformed with plasmids harboring GFP alone (vector), GFP–Pmk1, Pek1–GFP or GFP–Mkh1, were collected and the lysates were
incubated with purified GST or GST–Skb5. Proteins bound to glutathione beads were analyzed by SDS-PAGE and immunoblotted using anti-GFP or anti-GST
antibodies. (B) Skb5 binds to Ptc1. Cells transformed with plasmids harboring GFP alone (vector), GFP–Pmp1 or Ptc1–GFP, were collected and the lysates were
incubated with purified GST or GST–Skb5. Proteins bound to glutathione beads were analyzed by SDS-PAGE and immunoblotted using anti-GFP or anti-GST
antibodies. (C) Skb5 overproduction causes a vic (viable in the presence of immunosuppressant and chloride ion) phenotype in the absence of Ptc1. Upper panel:
Skb5 overexpression causes a vic phenotype in the WT cells. WT cells transformed with the control vector (+vector), the skb5+ gene or the pmp1+ gene were
grown in EMM and EMM containing 0.12 M MgCl2 plus FK506 at 27°C for 4 days. Lower panel: Skb5 overexpression causes a vic phenotype in ptc1 deletion cells.
Cells as indicated were grown in EMM and EMM containing 0.12 M MgCl2 plus FK506 at 27°C for 4 days. (D) Skb5 overproduction inhibits Pmk1 MAPK
phosphorylation both in the WT and ptc1 KO cells (Δptc1). Left panel: WT cells expressing endogenous Pmk1–GST transformed with the control vector or the
skb5 + gene, were grown in EMM at 27°C and incubated with 2 µg/ml micafungin for 1 h, and the phosphorylation of Pmk1 was analyzed as described in Fig. 1E
and presented relative to the value for the vector and with micafugin (set at 1). Right panel: Ptc1 deletion cells expressing endogenous Pmk1–GST transformed
with the control vector or the skb5 + gene were grown in EMM at 27°C and the phosphorylation of Pmk1 was analyzed as described in Fig. 1E and presented
relative to the value for the vector and with micafugin (set at 1).
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–
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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
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Fig. 3. The Skb5–Mkh1 interaction is important for the Skb5-mediated inhibition of Pmk1 MAPK signaling. (A) Amino acid sequence of Skb5 and its
Skb5YF2A mutant version. The underlined tyrosine (Y) 89 and phenylalanine (F) 135 of Skb5WT were mutated to alanine (A) residues to make Skb5YF2A.
(B) Skb5YF2A specifically lost its binding affinity for Mkh1. Cells transformed with plasmids harboring GFP, GFP–Mkh1 or Ptc1–GFP were collected and the lysates
were incubated with purified GST alone, GST–Skb5 or GST–Skb5YF2A. Cell lysates (lysate) and proteins bound to glutathione beads ( pulldown) were analyzed by
SDS-PAGE and immunoblotted using anti-GFP or anti-GST antibodies. (C) Skb5YF2A overexpression did not suppress the cytotoxicity induced by Pck2
overproduction. WT cells transformed with pREP2-GST-Pck2 (+pck2 +) and the control vector, pREP2-GST-Pck2 and the skb5 + gene, or pREP2-GST-Pck2 and
the skb5YF2A gene, were grown in EMM in the presence (Promoter OFF) or absence (Promoter ON) of thiamine at 27°C for 5 days. (D) Skb5YF2A overexpression
did not cause a vic phenotype. Cells overexpressing the skb5YF2A failed to grow in the presence of EMM plus 0.12 M MgCl2 and FK506 at 27°C. WT cells
transformed with the control vector, skb5+ gene and skb5YF2A gene were grown in EMM+0.12 M MgCl2 +FK506 at 27°C for 4 days. (E) Skb5YF2A overproduction
failed to inhibit Pmk1 MAPK signaling. The phosphorylation levels of Pmk1 were not inhibited upon Skb5YF2A overproduction. WT cells expressing endogenous
GST-tagged Pmk1, transformed with the control vector, the skb5+ gene or the skb5YF2A gene, were grown in EMM at 27°C and incubated with 2 µg/ml micafungin
for 1 h. Proteins bound to glutathione–Sepharose were analyzed as described in Fig. 1E. Lower panel: graph showing phosphorylation levels of Pmk1 analyzed
as described in Fig. 1E relative to the value for the vector and without micafungin (set at 1). The mean of the three experiments is shown.
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,
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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
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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
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Fig. 4. Skb5 overproduction affects Mkh1 localization to the growing ends. (A) Mkh1 localization to the growing ends is reduced by Skb5 overproduction, but
not by Skb5YF2A. Upper panel: WT cells expressing endogenous Mkh1–GFP transformed with the control vector (+vector), the skb5 + gene or the skb5YF2A gene,
were grown in EMM plus thiamine for 16 h. The fluorescence of Mkh1–GFP was observed as described in the Materials and Methods. Representative images
from n=3 experiments are shown. Arrows indicate cell tips. Scale bar: 10 μm. Middle panel: the number in each lane indicates the number of the cells with Mkh1
localization to the cell ends per 100 cells. Results are mean±s.d. (n=3). **P<0.01; n.s., not significant (Dunnett’s test; see Materials and Methods). Lower panel:
the number indicates the ratio of the number of the cells with Mkh1 localization to the growing ends to that with Mkh1 localization to the growing ends in cells
harboring the control vector with the promoter off (set at 1). Results are mean±s.d. (n=3). (B) Amino acid sequence of Mkh1 and its mutant version of Mkh13PA. The
underlined three prolines residues (544, 546 and 547) of Mkh1WT were mutated to alanine (A) residues to make Mkh13PA. (C) Mkh13PA specifically lost its binding
affinity to Skb5. Cells were transformed with plasmids harboring GFP alone, GFP–Mkh1 or GFP–Mkh13PA were collected and the lysates were incubated with
purified GST, GST–Skb5, GST–Pek1 or GST–Pck2. Cell lysates (lysate) and proteins bound to glutathione beads ( pulldown) were analyzed by immunoblotting
using anti-GFP and anti-GST antibodies. (D) Mkh13PA localization to growing ends does not change upon Skb5 overproduction. Upper panel: WT cells expressing
endogenous Mkh13PA transformed with the control vector or the skb5 + gene, were grown in EMM with thiamine for 16 h. Representative images from n=3
experiments are shown. Arrows indicate cell tips. Scale bar: 10 μm. Middle panel and lower panel: quantitative analysis of the results as described for panel
A. Results are mean±s.d. (n=3).
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
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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
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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
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Fig. 5. Skb5 localization to the growing ends needs
Skb5–Mkh1 interaction. (A) Skb5, but not Skb5YF2A,
accumulated in the growing ends upon its overproduction.
Upper panel: WT cell expressing endogenous Skb5–GFP
or Skb5YF2A–GFP and with the control vector, and the WT
cells transformed with the GFP–Skb5 or GFP–Skb5YF2A
(overproduction), were grown in EMM+thiamine for 16 h.
Representative images from n=3 experiments are shown.
Scale bar: 10 μm. Middle panel: the number indicates the
number of cells with Skb5 or Skb5YF2A cell-tip localization
in 100 cells in each sample. Lower panel: the graph shows
the ratio of the cells with Skb5 or Skb5YF2A cell-tiplocalization versus that in the cells expressing endogenous
Skb5. Results are mean±s.d. (n=3). *P<0.05, **P<0.01
(Tukey’s test; see Materials and Methods). (B) Skb5
localization to growing ends is decreased in an mkh1knockout (Δmkh1) cell. Upper panel: WT cells or Δmkh1
cells expressing endogenous Skb5 transformed with the
control vector were grown in EMM for 16 h. Representative
images from n=3 experiments are shown. Scale bar:
10 μm. Middle and lower panels: quantitative analysis of
the results as described in Fig. 4A. Results are mean±s.d.
(n=3). **P<0.01 (Student’s t-test; see Materials and
Methods).
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Fig. 6. Skb5 deletion increased Mkh1 cell-tip localization and Pmk1 phosphorylation. (A) Skb5 deletion (Δskb5), Skb5YF2A mutation and Mkh13PA mutation
induced a vic-negative phenotype. Upper panel: WT, skb5 YF2A mutant and Skb5 deletion cells were grown in YPD or YPD in the presence of 0.06 M
MgCl2+FK506 at 27°C for 4 days. Lower panel: WT, mkh13PA mutant and Skb5 deletion cells were grown in YPD or YPD in the presence of 0.06 M MgCl2+FK506
at 27°C for 4 days. (B) Skb5 deletion induced Pmk1 hyperphosphorylation. The WT and Skb5 deletion cells expressing endogenous Pmk1–GST, were grown in
EMM at 27°C and incubated with 2 µg/ml micafungin for 0, 20, 40 and 60 min, and the phosphorylation of Pmk1 was analyzed and quantified as described in
Fig. 1E. The data shown are representative of three independent experiments. (C) The skb5 YF2A mutation and the mkh13PA mutant did not increase Pmk1
hyperphosphorylation. Left panel: WT and skb5 YF2A mutant cells expressing endogenous Pmk1–GST, were grown in EMM at 27°C and incubated with 2 µg/ml
micafungin for 0, 20, 40 and 60 min, and the phosphorylation of Pmk1 was analyzed and quantified as described in Fig. 1E. Right panel: WT and mkh13PA cells
expressing endogenous Pmk1–GST were grown in EMM at 27°C and incubated with 2 µg/ml micafungin for 0, 20, 40 and 60 min, and the phosphorylation of
Pmk1 was analyzed and quantified as described in Fig. 1E. (D) Skb5 deletion increased Mkh1 cell-tip localization. Upper panel: WT and Skb5 deletion cells
expressing endogenous Mkh1 tagged with GFP or cells expressing the Mkh13PA mutant protein tagged with GFP under the native promoter were analyzed as
described in Fig. 4A. Middle and lower panels: quantitative analysis of the results as described in Fig. 4A. Results are mean±s.d. (n=3). **P<0.01 (Dunnett’s test;
see Materials and Methods).
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Fig. 7. Pck2 is required for efficient Skb5/Mkh1 localization at the cell tips. (A) Mkh1 localization to the growing ends was decreased in the absence of Pck2.
Upper panel: WT, ras1-knockout (Δras1) , rho2-knockout (Δrho2) or pck2-knockout (Δpck2) cells expressing endogenous Mkh1 tagged with GFP, were grown in
EMM for 16 h. Representative images from n=3 experiments are shown. Arrows indicate cell tips. Scale bar: 10 μm. Middle and lower panels: the number in
the graph indicates the number of cells with cell-tip-localization of GFP–Mkh1 per 100 cells as described in Fig. 4A. The ratio of cells with cell-tip localization to
those without, and the percentages of the cells with Mkh1 localization to the cell ends were analyzed as described in Fig. 4A. Results are mean±s.d. (n=3).
(B) Skb5 localization to the growing ends was decreased in the absence of Pck2. Upper panel: WT, Δras1, Δrho2 or Δpck2 cells expressing endogenous Skb5
tagged with GFP were grown in EMM for 16 h. Representative images from n=3 experiments are shown. Arrows indicate cell tips. Scale bar: 10 μm. Middle and
lower panels: The ratio of cells with cell-tip localization to those without, and the percentage of the cells with Skb5 localization to the cell ends were analyzed as
described in Fig. 4A. Results are mean±s.d. (n=3). (C) Pck2 influences the binding between Skb5 and Mkh1. Upper panel: WT or Pck2 deletion cells expressing
GFP–Mkh1 were transformed with plasmids harboring the control GST vector or GST–Skb5 and grown in EMM+thiamine at 27°C. Cells were collected and the
lysates were incubated with purified GST alone or GST–Skb5. Cell lysates (lysate) and proteins bound to glutathione beads ( pulldown) were analyzed by
immunoblotting using anti-GFP and anti-GST antibodies. Lower panel: quantification of the Mkh1–Skb5 binding in WT and Pck2 deletion cells. The intensities of
the bands in the pulldowns were analyzed using ImageJ software. Results are mean±s.d. (n=3). (D) Skb5 does not affect Pck2 localization in the growing ends.
Upper panel: WT cells expressing endogenous Pck2 tagged with GFP transformed with the control vector or the skb5+ gene, were grown in EMM+thiamine for
16 h. Representative images from n=3 experiments are shown. Arrows indicate cell tips. Scale bar: 10 μm. Middle and lower panels: quantification of the cell-tip
localization of Pck2–GFP was analyzed as described in Fig. 4A. Results are mean±s.d. (n=3).
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Fig. 8. Pck2 and Mkh1 localized to the cell tips in a cell-cycle-dependent manner. (A) cdc25-22 mutant cells expressing endogenous Mkh1–GFP from the
native promoter 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. Aliquots were
obtained at different time points, and the cell-tip Mkh1 localization ( ) was analyzed as described in Fig. 4A. The septation index (□) is also shown, which
indicates good cell cycle synchrony in the culture. (B) Cells from the cdc25-22 mutants expressing endogenous Pck2–GFP from the native promoter were grown
to the log phase at 25°C and analyzed as described in Fig. 8A. Aliquots were obtained at different time points, and the cell-tip Pck2 localization ( ) was analyzed
as described in Fig. 4A. The septation index (□) is also shown, which indicates good cell cycle synchrony in the culture.
▪
▪
DISCUSSION
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 Skb5mediated 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 Pck2induced 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
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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.
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
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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.
Protein detection
Anti-GFP (Ma et al., 2006a,b), anti-GST (Ma et al., 2006a,b) and antiphospho-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 midlog phase (optical density at 660 nm=0.5). Micafungin stock solution
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(200 mg/ml) was added to the culture medium to give the indicated
concentrations. After stress treatment, the culture medium was chilled in icecold 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).
Image quantification
The quantification of cell tip localization was performed for at least two
individual datasets, which analyzed up to 100 cells.
Statistical analysis
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.
Acknowledgements
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.
Competing interests
The authors declare no competing or financial interests.
Author contributions
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.
Funding
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].
Supplementary information
Supplementary information available online at
http://jcs.biologists.org/lookup/doi/10.1242/jcs.188854.supplemental
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