Journal of Cell Science Control of Sty1 MAPK activity through stabilisation of the Pyp2 MAPK phosphatase Katarzyna M. Kowalczyk*, Sonya Hartmuth*, David Perera ` , Peter Stansfield and Janni Petersen § University of Manchester, Faculty of Life Sciences, C.4255 Michael Smith Building, Oxford Road, Manchester M13 9PT, UK *These authors contributed equally to this work ` Present address: Department of Oncology, University of Cambridge, Hutchison/MRC Research Centre, Hills Road, Cambridge CB2 0XZ, UK § Author for correspondence ([email protected]) Accepted 29 April 2013 Journal of Cell Science 126, 3324–3332 ß 2013. Published by The Company of Biologists Ltd doi: 10.1242/jcs.122531 Summary In all eukaryotes tight control of mitogen-activated protein kinase (MAPK) activity plays an important role in modulating intracellular signalling in response to changing environments. The fission yeast MAPK Sty1 (also known as Spc1 or Phh1) is highly activated in response to a variety of external stresses. To avoid segregation of damaged organelles or chromosomes, strong Sty1 activation transiently blocks mitosis and cell division until such stresses have been dealt with. MAPK phosphatases dephosphorylate Sty1 to reduce kinase activity. Therefore, tight control of MAPK phosphatases is central for stress adaptation and for cell division to resume. In contrast to Pyp1, the fission yeast Pyp2 MAPK phosphatase is under environmental control. Pyp2 has a unique sequence (the linker region) between the catalytic domain and the N-terminal MAPK-binding site. Here we show that the Pyp2 linker region is a destabilisation domain. Furthermore, the linker region is highly phosphorylated to increase Pyp2 protein stability and this phosphorylation is Sty1 dependent. Our data suggests that Sty1 activation promotes Pyp2 phosphorylation to increase the stability of the phosphatase. This MAPK-dependent Pyp2 stabilisation allows cells to attenuate MAPK signalling and resume cell division, once stresses have been dealt with. Key words: Schizosaccharomyces pombe, Sty1, MAPK, Pyp2, Phosphatase, DUSP6 Introduction In all eukaryotes mitogen-activated protein (MAP) kinase cascades regulate cell growth and cell homeostasis. Following changes in the cell environment MAPKs play an important role in modulating intracellular signalling to instigate changes in a range of processes, including transcriptional control. Higher eukaryotes and budding yeast have several such MAPK pathways, each of which responds to a particular type of stress (Brewster et al., 1993; Schu ¨ller, et al., 1994; Han et al., 1994). In contrast, in fission yeast Schizosaccharomyces pombe one main MAPK, Sty1 (also known as Spc1 or Phh1), is activated in response to a variety of extracellular stimuli (Millar et al., 1995; Shiozaki and Russell, 1995a; Degols et al., 1996; Shiozaki and Russell, 1996). Strong Sty1 activation transiently blocks cell division to prevent segregation of damaged organelles or chromosomes (Degols et al., 1996; Hartmuth and Petersen, 2009). To allow for cells to adapt following stress and for cell division to resume, Sty1 is negatively regulated by deactivating phosphatases (Millar et al., 1995; Shiozaki and Russell, 1995a; Shiozaki and Russell, 1995b). The phosphatases Pyp1 and Pyp2 dephosphorylate tyrosine 173 in the activation site (Dal Santo et al., 1996; Millar et al., 1992). Levels of Pyp1 remain constant upon exposure to stress (Chen et al., 2003). In contrast, Pyp2 protein expression is enhanced by the Sty1-activated transcription factor Atf1 following cell exposure to most environmental stresses (Wilkinson et al., 1996). However, nutrient stress induces a very rapid decline in Pyp2 levels. This is regulated through proteasome mediated Pyp2 degradation (Petersen and Nurse, 2007). Thus, Pyp2 protein levels are under tight control. In this report we provide evidence that the stability of the Pyp2 MAPK phosphatase is regulated through a destabilisation domain that links the N-terminal MAPK-binding site with the C-terminal phosphatase domain. We designate this domain the ‘linker region’. The Pyp2 linker region is highly phosphorylated to enhance protein stability. The absence of Sty1 activity severely impaired Pyp2 phosphorylation and significantly reduced Pyp2 protein stability. Thus, Sty1-dependent Pyp2 phosphorylation stabilises the phosphatase, which in turn reduces Sty1 activity. Such a Sty1 self-regulatory mechanism allows the cell to efficiently attenuate MAPK signalling to promote stress adaptation and cell cycle progression. Results The Pyp2 linker region is a destabilisation domain In order to identify potential Pyp2-specific regulatory domains, the protein sequences of Pyp1 and Pyp2 were compared. A high degree of homology within the N-terminal MAPK-binding site (26% identity) and the C-terminal catalytic domains (40% identity) was observed. Interestingly, Pyp2 has an additional region of ,270 amino acids linking the N- and C-termini. This sequence is absent in Pyp1 (Fig. 1A). We named this Pyp2- specific domain the ‘linker region’. To investigate its role in Pyp2 regulation, the linker region (a.a. 130–313; deletion of XhoI fragment see Fig. 1A) was deleted from the genomic pyp2 locus (pyp2.linker-free or pyp2.LF). Importantly, the shorter linker-free Pyp2 retains phosphatase activity since we were able to generate a pyp2.LF pyp1D double mutant (data not shown). It has 3324 Research Article
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Control of Sty1 MAPK activity through stabilisation ofthe Pyp2 MAPK phosphatase
Katarzyna M. Kowalczyk*, Sonya Hartmuth*, David Perera`, Peter Stansfield and Janni Petersen§
University of Manchester, Faculty of Life Sciences, C.4255 Michael Smith Building, Oxford Road, Manchester M13 9PT, UK
*These authors contributed equally to this work`Present address: Department of Oncology, University of Cambridge, Hutchison/MRC Research Centre, Hills Road, Cambridge CB2 0XZ, UK§Author for correspondence ([email protected])
Accepted 29 April 2013Journal of Cell Science 126, 3324–3332� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.122531
SummaryIn all eukaryotes tight control of mitogen-activated protein kinase (MAPK) activity plays an important role in modulating intracellularsignalling in response to changing environments. The fission yeast MAPK Sty1 (also known as Spc1 or Phh1) is highly activated inresponse to a variety of external stresses. To avoid segregation of damaged organelles or chromosomes, strong Sty1 activation
transiently blocks mitosis and cell division until such stresses have been dealt with. MAPK phosphatases dephosphorylate Sty1 to reducekinase activity. Therefore, tight control of MAPK phosphatases is central for stress adaptation and for cell division to resume. In contrastto Pyp1, the fission yeast Pyp2 MAPK phosphatase is under environmental control. Pyp2 has a unique sequence (the linker region)
between the catalytic domain and the N-terminal MAPK-binding site. Here we show that the Pyp2 linker region is a destabilisationdomain. Furthermore, the linker region is highly phosphorylated to increase Pyp2 protein stability and this phosphorylation is Sty1dependent. Our data suggests that Sty1 activation promotes Pyp2 phosphorylation to increase the stability of the phosphatase. ThisMAPK-dependent Pyp2 stabilisation allows cells to attenuate MAPK signalling and resume cell division, once stresses have been dealt
and arrowheads indicate hypophosphorylated Pyp2. (B) Cultures
expressing Pyp2.myc from the nmt81 promoter were incubated at
42 C for 10 minutes. (C) Cultures expressing Pyp2.myc from the
nmt81 promoter were split in two. The control was kept at 28 C, while
the other half was incubated at 42 C for 10 minutes. Afterwards both
cultures were treated with cycloheximide. Samples were harvested at
the indicated time-points. (D) Quantification of relative protein levels
from C. *P50.015.
Journal of Cell Science 126 (15)3328
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Pyp1. We called this domain the ‘linker region’. Deletion of the
Pyp2 linker region (pyp2.LF) stabilises the phosphatase,
suggesting that sequences within this domain are responsible forregulating protein turnover. Pyp2 is phosphorylated within the
linker region and this promotes protein stability. There are a
number of possible reasons why phosphorylation may enhanceprotein stability here. First, as Pyp2 is degraded by the proteasome
(Petersen and Nurse, 2007), phosphorylation within the linker
region may block its ubiquitylation, which would normally targetthe protein for degradation. Preliminary data (supplementary
material Fig. S4) indicates that the unphosphorylated pyp2.AA
mutant has increased levels of ubiquitylation, which therefore may
contribute to the reduced protein stability. Second, Pyp2
phosphorylation is likely to change the protein charge andinfluence its conformation. Increased negative charge and/or
altered conformation may prevent Pyp2 from interacting with the
degradation machinery and therefore promote its stability.
Cells appear to be particularly reliant on the Pyp2 phosphatase
in response to heat stress, as the pyp2.LF mutant is only sensitiveto heat stress. This might be explained by the observation that
Pyp1 becomes insoluble and unable to inactivate Sty1 in responseto heat stress (Nguyen and Shiozaki, 1999), which may then
explain why cells are sensitive to altered Pyp2 regulation.
Furthermore, enhanced Atf1-dependent transcription caused by
increased Sty1 activity results in elevation of Pyp2 but not Pyp1
levels (Chen et al., 2003). Further characterisation of the pyp2.LF
mutant following heat stress showed that the cells arrest growth
at 37 C, however, they do not die (supplementary material Fig.
S2).
Our data presented here suggests that an additional,
transcription-independent control of the Pyp2 phosphatase ispresent in cells and that this relies upon Sty1 activity. Deletion of
sty1 results in a major reduction in Pyp2 protein levels. However,this is unlikely to be exclusively due to reduced transcription,
because pyp2.LF can be detected in cells deleted for sty1.
Importantly, when transcribed from the nmt81 promoter, whichis unlikely to be regulated by Sty1 signalling (Chen et al., 2003),
Pyp2 levels are reduced. Deletion of Sty1 abolished the majority of
Pyp2 phosphorylation. Because phosphorylation of the linkerregion stabilises Pyp2, the unphosphorylated phosphatase in sty1Dcells is likely to be unstable. We therefore propose a model
whereby a Sty1 self-regulatory mechanism functionsindependently of transcription (Fig. 7B). Hence, elevated Sty1
activity will promote Pyp2 phosphorylation and consequently, anincrease in phosphatase stability. This will return Sty1
phosphorylation/activity back to steady state levels when the
Fig. 5. Pyp2 stability is regulated through serine 234
and threonine 279 in the linker region.
(A–C) Western blot analysis of TCA-extracted total
protein from early exponential cell cultures. Arrows
indicate hyperphosphorylated Pyp2 and arrowheads
indicate hypophosphorylated Pyp2. (A) Western blot of
Pyp2.myc from pyp2.S234 and pyp2.T279
unphosphorylatable or phosphomimetic mutants.
(B,C) TCA-extracted protein samples from early
exponential cultures of wt, pyp2.S234A and pyp2.S234E
myc-tagged strains. (C) The cells were heat stressed at
promoter were treated as in Fig. 4C. Following western
blot analysis, relative protein levels were quantified; the
graphs represent two independent experiments.
*P,0.045.
MAPK control of MAPK phosphatase stability 3329
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activity of the MAPK kinase Wis1 subsides. In contrast, a sudden
reduction in Sty1 activity will destabilise Pyp2. Thus, elevated
Pyp2 degradation will in turn lead to increase Sty1
phosphorylation/activation. The latter can be observed following
mild nutrient stress, as under these conditions TOR-pathway-
dependent degradation of Pyp2 promotes Sty1 activity (Petersen
and Nurse, 2007). Interestingly, TOR control of Pyp2 is controlled
through the linker region as well (supplementary material Fig. S3),
because the pyp2.LF mutant is completely unable to respond to
nutrient stress. This suggests that the Pyp2 linker region is
modified in response to several environmental stresses. However,
because the pyp2.S234 and Pyp2.T279 mutants still advance
mitosis following nutrient stress, we propose that TOR control of
Pyp2 is regulated through sites other than the Sty1 controlled S234
and T279. Therefore, control of phosphatase stability by post-
translational modification gives cells the possibility to quickly and
efficiently fine-tune MAPK activity.
Because of their physiological role in the control of MAPK
activity, misregulation of MAPK phosphatases has been linked to
diseases such as cancer (reviewed in Haagenson and Wu, 2010;
Bermudez et al., 2010). For instance, in pancreatic cancer, levels
of the MAPK phosphatase DUSP6, which is responsible for
Fig. 6. Sty1 control of Pyp2.S234 phosphorylation.
(A,C) Western blot analysis of TCA-extracted total protein
from early exponential cell cultures. Arrows indicate
hyperphosphorylated Pyp2 and arrowheads indicate
hypophosphorylated Pyp2. (B) Sty1 in vitro kinase assay, using
Pyp2-GST or GST as substrates. (C) Extracts were probed with
anti-phospho-Sty1 or anti-Sty1 antibodies. Right: signal
quantification. (D) Left: cells exponentially grown in YES were
shifted from 28 C to 37 C. At the indicated time-points samples
were collected and fixed, and the number of dividing cells
assessed; 500 cells were counted for each time-point. Right: the
mean and standard deviation of three independent experiments
are shown. *P50.022; **P,0.005; ***P,0.0007.
Fig. 7. Self-regulatory control of Sty1 kinase activity.
(A) Potential conservations of the Sty1-controlled Pyp2
phosphorylation sites within the linker of human DUSP6.
(B) Diagram of the proposed Sty1-dependent regulation of Pyp2
stability. Arrows indicate direct interactions.
Journal of Cell Science 126 (15)3330
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deactivation of the ERK1/2 MAPKs that drive proliferation, are
reduced or absent (Furukawa et al., 2003). Similarly, in breast
neoplasms, high levels of DUSP6 are associated with resistance
to tamoxifen, a drug commonly used to treat oestrogen receptor
positive breast cancer patients (Cui et al., 2006). Therefore, not
surprisingly, the potential for MAPK phosphatases as anti-cancer
targets is being exploited in preclinical trials (reviewed in Nunes-
Xavier et al., 2011). Our data suggests that the Pyp2 linker region
is a destabilisation domain important for correct control of
MAPK phosphatase levels/activity. Interestingly five of the six
DUSP6 mutations reported in the Sanger Institute Catalogue Of
Somatic Mutations In Cancer (COSMIC; http://www.sanger.ac.
uk/cosmic) are found in the DUSP6 linker region (Bamford et al.,
2004). Finally, the MAPK-controlled Pyp2 phosphorylation sites
that we report here may be conserved in DUSP6 (Fig. 7A).
Therefore, a similar linker-dependent control of DUSP6 stability,
to fine-tune MAPK activity, is likely to be conserved in
mammalian cells.
Materials and MethodsCell cultures and strains
Strains used in this study are listed in supplementary material Table S1. Cells wereexponentially grown for 48 hours at 28 C in EMM2-N (Formedium) minimalmedium supplemented with L-glutamic acid (EMM-G) as a nitrogen source.Where necessary, L-leucine was added to a final concentration of 150 mg/ml. Forspot tests, cells were grown to a density of 3.56106 cells/ml. For western blotanalysis cells were grown to a density of 26106 cells/ml. For nutritional-stress cellswere grown as described previously (Petersen and Nurse 2007). Before heat stressof liquid cultures, cells were grown as described previously (Hartmuth andPetersen, 2009).
Microscopy
In order to determine septation index, calcofluor white (Sigma Aldrich) staining ofsepta was performed as described previously (Moreno et al., 1991). 500 cells werecounted for each time-point.
Cycloheximide and MAPK inhibitor treatment
Cultures were exponentially grown in EMM-G to a density of 26106 cells/ml andtreated with cycloheximide (Sigma-Aldrich) dissolved in DMSO at a finalconcentration 100 mg/ml or DMSO as a control. The cells were incubated furtherat 28 C. The MAPK inhibitor was used as described previously (Hartmuth andPetersen, 2009). At the indicated time-points samples were collected.
Biochemistry
Total protein extracts were prepared by TCA precipitation (Caspari et al., 2000).Myc-dependent immunoprecipitation of Pyp2 was carried out using protein GDynabeads (Invitrogen). The proteins were extracted by TCA precipitation andresolubilised in 200 ml sample buffer (80 mM Tris-HCl pH 6.8, 5 mM DTT,5 mM EDTA) plus 2% SDS. Samples were boiled for 3 minutes diluted with900 ml of sample buffer plus 1% Triton X-100 (Sigma-Aldrich). For l-phosphatase treatment the immunocomplexes were washed with (50 mM Tris-HCl pH 7.5, 100 mM sodium chloride, 0.1 EGTA, 2 mM DTT, 0.01% Brij 35)and l-protein phosphatase was added for 30 minutes at 30 C. For co-immunoprecipitation proteins were extracted and washed with IP buffer(50 mM Hepes pH 8.0, 100 mM sodium chloride, 0.1% Tween20, 1 mMEDTA, 50 mM sodium fluoride, 1 mM DTT, 1 mM PMSF, 2 mM Na3V04,20 mM sodium b-glycerophosphate, and complete protease inhibitor; Roche).Sty1 kinase assay was carried out as described previously (Nguyen and Shiozaki,1999). As substrate a 30 amino acid long Pyp2 peptide (with Ser234 located inthe middle) fused to GST (pET-41a; Novagen) was used. This fusion protein andthe GST control were expressed in E. coli. Proteins were detected using thefollowing antibodies: 1:500 4A6 anti-myc (Millipore), 1:500 anti-phospho-Sty1(raised in rabbit by Eurogentec), 1:200 anti-Hog1 antibodies (Santa CruzBiotechnology INC), 1:500 anti-phospho-pyp2.S234 (Eurogentec), 1:1500 anti-phospho-pyp2.T279 (Eurogentec), anti-ubiquitin (Dako UK Limited). Alkaline-phosphatase-coupled secondary antibodies (Sigma Aldrich) were used for allblots, followed by direct detection with NBT/BCIP (VWR) substrates on PVDFmembranes (Millipore). Co-immune-precipitates were separated on NU-PAGEgels (Invitrogen) and analysed by mass spectrometry. Signal intensities werequantified using ImageJ software. Unless otherwise stated, the graphs representthe quantified levels from three individual experiments.
AcknowledgementsWe thank the Biological Mass Spectrometry facility at ManchesterUniversity for protein identification, members of the laboratory forstimulating discussions and Elizabeth Davie for valuable commentson the manuscript.
Author contributionsK.K., S.H. and D.P. performed the experiments. K.K., S.H., D.P. andJ.P. analysed the data. P.S. performed preliminary experiments. K.K.and J.P. wrote the manuscript.
FundingThis work was supported by a Cancer Research UK project grant[grant number C10888/A9015 to J.P.]; a Cancer Research UK SeniorFellowship [grant number C10888/A11178 to J.P.]; and TheUniversity of Manchester.
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