Arch Microbiol (2010) 192:157165

DOI 10.1007/s00203-009-0531-6
1 3
ORI GI NAL PAPER
IdentiWcation of protein kinase disruptions as suppressors
of the calcium sensitivity of S. cerevisiae ptp2 msg5
protein phosphatase double disruptant
Hermansyah Walter A. Lavia Minetaka Sugiyama
Yoshinobu Kaneko Satoshi Harashima
Received: 10 August 2009 / Revised: 10 November 2009 / Accepted: 30 November 2009 / Published online: 7 January 2010
Springer-Verlag 2010
Abstract The double disruptant of the S. cerevisiae pro-
tein phosphatase (PPase) genes, PTP2 (phosphotyrosine-
speciWc PPase) and MSG5 (phosphotyrosine and phospho-
threonine/serine-PPase) causes calcium-sensitive growth
(Ca
s
). Previous study using Fluorescent-activated cell sort-
ing (FACS) analysis showed that this growth defect with
calcium occurs at G1S transition in the cell cycle. We dis-
covered that six non-essential protein kinase (PKase) dis-
ruptions (bck1, mkk1, slt2/mpk1, mck1, ssk2 and
yak1) suppressed the Ca
s
-phenotype of the ptp2 msg5
double disruptant. Bck1p, Mkk1p and Slt2p are compo-
nents of the mitogen-activated protein kinase (MAPK) cas-
cade of cell wall integrity pathway (Slt2 pathway), and
Mck1p is its down regulator. Ssk2p is the MAPK kinase
kinase of the high-osmolarity glycerol (HOG) pathway,
while Yak1p is a negative regulator for the cAMP-depen-
dent PKA pathway. FACS analysis revealed that only the
disruption of ssk2 and yak1 but not bck1, mkk1, slt2
and mck1 was able to suppress the delayed G1S transi-
tion, suggesting that suppression of the growth defect is not
always accompanied by suppression of the G1S transition
delay. The discovery of these PKases as suppressors
revealed that in addition to the previously anticipated Slt2
pathway, HOG, Yak1p and Mck1p regulatory pathways
may also be involved in the calcium sensitivity of the ptp2
msg5 double disruptant.
Keywords PTP2 MSG5 Protein phosphatase Protein
Kinase Calcium sensitive Saccharomyces cerevisiae
Abbreviations
PPase Protein phosphatase
PKase Protein kinase
MAPK Mitogen-activated protein kinase
Ca
s
Calcium-sensitive growth
FACS Fluorescent-activated cell sorting
HOG High-osmolarity glycerol
Introduction
In Saccharomyces cerevisiae, at least 37 genes encoding
protein phosphatases (PPases) and 117 genes encoding pro-
tein kinases (PKases) are responsible for reversible phos-
phorylation/dephosphorylation activity that regulates the
majority of cellular pathways, including cell signaling, cell
cycle and gene expression (Zolnierowicz and Bollen 2000;
http://proteome.com). Our laboratory is concerned with
elucidating the function of these PPases involved in various
biological processes. In our previous studies (Sakumoto
et al. 1999, 2002), we have constructed 435 PPase double
disruptants in all possible combinations of the 30 PPases
(known at that time) to study redundant functions of
PPases. Through this work, we discovered that the ptp2
msg5 double disruptant showed a calcium sensitive (Ca
s
)-
phenotype whereas the single disruptant of either PTP2 or
MSG5 did not (Sakumoto et al. 2002). Ptp2p and Msg5p
belong to diVerent subfamilies of PPases, namely, protein
tyrosine phosphatase and dual speciWcity PPase (DSPs),
respectively (Guan et al. 1992; Doi et al. 1994). Ptp2p
plays an important role in the negative regulation of Slt2p
in the mitogen-activated protein kinase (MAPK) Mpk1/Slt2
Communicated by Axel Brakhage.
Hermansyah W. A. Lavia M. Sugiyama Y. Kaneko
S. Harashima (&)
Department of Biotechnology,
Graduate School of Engineering, Osaka University,
2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
e-mail: harashima@bio.eng.osaka-u.ac.jp
158 Arch Microbiol (2010) 192:157165
1 3
cell wall integrity pathway and Hog1p in the high-osmolar-
ity glycerol (HOG) MAPK pathway while Msg5p nega-
tively regulates Mkk1p and Slt2p of the Slt2 pathway and
Fus3p of the MAPK pheromone pathway (Mattison et al.
1999; Jacoby et al. 1997; Zhan and Guan 1999; Watanabe
et al. 1995; Flandez et al. 2004). Thus, double disruption of
PTP2 and MSG5 is expected to trigger the activation of the
Slt2p MAP kinase of cell integrity pathway (Mattison et al.
1999; Flandez et al. 2004). In fact, our previous work (Her-
mansyah et al. 2009) has shown that the Slt2p pathway was
activated in the ptp2 msg5 double disruptant even in the
absence of calcium. This activation was further stimulated
by the addition of calcium in which the level of activation
of the Slt2 pathway caused growth defect. We have also
revealed that this growth defect occurs in the G1S transi-
tion of the cell cycle, which is a rare observation since pre-
vious studies dealing with calcium-triggered signaling in S.
cerevisiae implicate calcium with the G2M transition
(Mizunuma et al. 1998). We believe that the phenotypic
eVect is brought about by a combined eVect of calcium
exposure and the double disruption of these two PPases. In
order to gain further insight into the molecular mechanism
of Ca
s
-phenotype of the ptp2 msg5 double disruptant,
we attempted in this study to identify PKase genes
implicated in the Ca
s
-phenotype by systematically con-
structing 101 triple disruptants having the genotype of
ptp2::CgHIS3 msg5::CgLEU2 pkase::kanMX (disrup-
tion of each of 101 non-essential PKase). The idea behind
the construction of these triple disruptants was based on the
hypothesis that the sensitivity is caused either by accumula-
tion of unknown phosphorylated substrate(s) of Ptp2p and
Msg5p or by depletion of unphosphorylated substrate(s) of
Ptp2p and Msg5p in the ptp2 msg5 double disruptant
(Hirasaki et al. 2008). We discovered from this analysis
that six non-essential PKase disruptions (bck1, mkk1,
slt2, mck1, ssk2 and yak1) that have been reported to
be involved in a variety of pathways suppressed the Ca
s
-
phenotype of the ptp2 msg5 double disruptant. Bck1p,
Mkk1p which is functionally redundant with Mkk2p, and
Slt2p have been identiWed as components of the Pkc1p-
mediated signal transduction pathway that functions to
maintain cell wall integrity in yeast (Irie et al. 1993).
Mck1p, a glycogen synthase kinase (GSK-B) homolog,
has been classiWed as a downstream regulator of the Slt2
pathway (Mizunuma et al. 2001). On the other hand, Ssk2p
is a MAPKKK involved in HOG signal transduction path-
way (Posas and Saito 1998), while Yak1p has been identi-
Wed as a recessive suppressor of the cAMP-dependent
protein kinase (PKA)-deWcient growth defect that is antag-
onistic to PKA and mediator of PKA-dependent inhibition
of stress responses (Smith et al. 1998; Lee et al. 2008). The
discovery of these PKases as suppressors revealed that in
addition to the previously anticipated Slt2 and HOG path-
ways, Yak1p and Mck1p regulatory pathways are also
involved in calcium sensitivity caused by the disruption of
the PTP2 and MSG5 protein phosphatase genes.
Materials and methods
Yeast strains, plasmids and culture conditions
Strains used in this study are described in Table 1. Yeast
strains SH5209 (=FY833) and SH5210 (=FY834) (Winston
et al. 1995) were used as a wild-type and parental strains.
The 101 MAT pkase::KanMX4 S. cerevisiae non-essen-
tial gene disruptants (Table 2), generated by the Saccharo-
myces Genome Deletion Project (Winzeler et al. 1999),
were obtained from Research Genetics/Invitrogen. The rich
medium YPAD was prepared by supplementing YPD broth
(SigmaAldrich Co.) with 0.4 mg/ml adenine. SC medium
consisted of 0.67% yeast nitrogen base without amino
acids, 2% glucose and the required auxotrophic supple-
ments. SPM medium contained 0.30% potassium acetate,
0.02% raYnose and was supplemented with 10 g/ml of
adenine, arginine, histidine, isoleucine, leucine, lysine,
methionine, phenylalanine, threonine, tryptophan, uracil
and valine. Unless indicated otherwise, yeast strains were
grown at 30C. Plasmids were propagated in Escherichia
coli strain DH5 cultivated on LB medium containing
100 g/ml ampicillin at 37C. Plasmid pCgHIS3 (Sakum-
oto et al. 1999; Kitada et al. 1995) and pCgTRP1 (Sakum-
oto et al. 2002; Kitada et al. 1995) carrying a 1.8-kbp
fragment of the Candida glabrata HIS3 (CgHIS3) gene and
a 1.6-kbp fragment CgTRP1 gene, respectively. Plasmid
p3005 was constructed by subcloning a 1.7-kbp BamHI
XhoI fragment containing CgLEU2 (a kind gift from K.
Kitada) into pT7Blue-T vector (Novagen) and was used as
template for the PCR ampliWcation of PTP2 and MSG5 dis-
ruption cassettes.
Construction of yeast double and triple disruptants
Single and double gene disruptants were constructed by tar-
geted gene replacement, crossing and tetrad analysis as
described previously (Hermansyah et al. 2009). The triple
disruptants ptp2 msg5 pkase were constructed by tet-
rad analysis of the diploid strains resulting from crossing
ptp2::CgHIS3 msg5::CgLEU2 (SH6793) with each of
the 101 MAT pkase::kanMX4 disruptants. The corre-
sponding heterozygous diploid strain was dissected with a
micromanipulator (Sherman and Hicks 1991), and the tetr-
ads were analyzed to screen for His
+
Leu
+
Kan
r
segregants.
The correct disruptions of the six PKases suppressor for
the Ca
2+
sensitivity of the ptp2 msg5 background were
veriWed by PCR ampliWcation using primers in Table 3.
Arch Microbiol (2010) 192:157165 159
1 3
Spot assay of Ca
2+
sensitivity
From yeast cells grown in YPAD medium to mid logarith-
mic phase, suspensions containing equal cell numbers were
prepared on the basis of OD
660
, and ten-fold serial dilutions
were spotted onto YPAD plates with or without 0.6 M
CaCl
2
that were incubated for 2 days.
FACS analysis and vacuolar staining with FM4-64
FACS analysis and vacuolar staining with FM4-64 were
carried out as described previously (Hermansyah et al.
2009).
Immunoblot analysis of (phosphorylated) Slt2p and
(phosphorylated) Hog1p
Immunoblot analysis was performed as described previ-
ously (Hermansyah et al. 2009). Samples were probed with
antiphospho-p38 MAPK (Thr
180
/Tyr
182
) (Cell Signaling
Technology) or anti-Hog1(yC-20):sc-6815 (Santa Cruz
Biotechnology, Inc.) antibodies at 1:1000 dilution to detect
phosphorylated or total Hog1p. These primary antibodies
were detected using 1:10,000 diluted horseradish peroxi-
dase-conjugated anti-rabbit or anti-goat antibodies, respec-
tively. Actin was probed with mouse anti-Actin monoclonal
antibody (Chemicon International, USA) at 1:5,000 dilu-
tion. This primary antibody was detected using 1:10,000
diluted horseradish peroxidase-linked species-speciWc
whole antibody from sheep (GE Healthcare). Blots were
detected using Western Lightning
TM
Chemiluminescence
Reagent Plus (PerkinElmer LAS, Inc.).
Results
Screening for PKases as suppressors of the ptp2 msg5
double disruptant and their genetic characterization
In order to gain insight into the mechanism of Ca
s
-pheno-
type of the ptp2 msg5 double disruptant, we screened
for PKases whose disruption can suppress the Ca
s
-pheno-
type of the ptp2 msg5 double disruptant. Single disrup-
tants of ptp2::CgHIS3 (SH6790) and msg5::CgLEU2
Table 1 Strains used in this study
a
NBRP/YGRC, National BioResource Project/Yeast Genetic Research Center, Japan (http://yeast.lab.nig.ac.jp/nig/index_en/html)
Strains Genotype Source
BY4739 MAT leu20 lys20 ura30 Invitrogen
BY4742 MAT his31 leu20 lys20 ura30 Invitrogen
SH5209 MATa ura3-52 his3200 leu21 lys2202 trp163 NBRP, YGRC
a
SH5210 MAT ura3-52 his3200 leu21 lys2202 trp163 NBRP, YGRC
a
SH6790 MATa ptp2::CgHIS3 ura3-52 his3200 leu21 lys2202 trp163 SH5209 disruptant
SH6791 MAT msg5::CgLEU2 ura3-52 his3200 leu21 lys2202 trp163 SH5210 disruptant
SH6792 MAT ptp2::CgHIS3 msg5::CgLEU2 ura3-52 his3200 leu21
lys2202 trp163
SH6790 SH6791
SH6793 MATa ptp2::CgHIS3 msg5::CgLEU2 ura3-52 his3200 leu21
lys2202 trp163
SH6790 SH6791
SH7995 MATa ptp2::CgHIS3 msg5::CgLEU2 bck1::kanMX4 ura3-52
(or ura30) his3200 (or his31) leu21 (leu20) lys2202
(lys20) trp163
SH6793 BY4742 bck1::kanMX4
SH7996 MAT ptp2::CgHIS3 msg5::CgLEU2 slt2::kanMX4 ura3-52
(or ura30) his3200 (or his31) leu21 (leu20) lys2202
(lys20) trp163
SH6793 BY4742 slt2::kanMX4
SH7997 MAT ptp2::CgHIS3 msg5::CgLEU2 ssk2::kanMX4 ura3-52
(or ura30) his3200 (or his31) leu21 (leu20) lys2202
(lys20) trp163
SH6793 BY4742 ssk2::kanMX4
SH7998 MAT ptp2::CgHIS3 msg5::CgLEU2 mck1::kanMX4 ura3-52
(or ura30) his3200 (or his31) leu21 (leu20) lys2202
(lys20) trp163
SH6793 BY4742 mck1::kanMX4
SH7999 MAT ptp2::CgHIS3 msg5::CgLEU2 yak1::kanMX4 ura3-52
(or ura30) his3200 (or his31) leu21 (leu20) lys2202
(lys20) trp163
SH6793 BY4742 yak1::kanMX4
SH8517 MAT ptp2::CgHIS3 msg5::CgLEU2 mkk1::CgTRP1 ura3-52
his3200 leu21 lys2202 trp163
Derived from SH6792
160 Arch Microbiol (2010) 192:157165
1 3
(SH6791) were constructed by PCR-mediated gene disrup-
tion as described in the previous report (Hermansyah et al.
2009). The double disruptant ptp2 msg5 (SH6793) was
isolated by tetrad analysis of the diploid strain resulting
from the cross of ptp2::CgHIS3 (SH6790) and
msg5::CgLEU2 (SH6791). To construct the ptp2 msg5
pkase triple disruptant, we crossed the double disruptant,
SH6793 (MATa ptp2::CgHIS3 msg5::CgLEU2), with
each of 101 MAT pkase::kanMX4 S. cerevisiae non-
essential gene disruptants (Table 2) that were derived
mostly from BY4742 (Brachmann et al. 1998), except for
kin3::kanMX4 and psk1::kanMX4 that were derived
from the BY4739 background (Brachmann et al. 1998).
Diploid cells showing the His
+
Leu
+
Kan
r
-phenotype were
selected, sporulated on SPM medium and subjected to tet-
rad analysis. By using this method, we obtained triple dis-
ruptants for all pkases, except for the triple disruptants that
contained each of ste7, ste11, bub1 and mkk1 disrup-
tions. We constructed ptp2 msg5 ste7, ptp2 msg5
ste11, ptp2 msg5 bub1 and ptp2 msg5 mkk1 tri-
ple disruptants by using PCR-mediated gene disruption
method starting from the ptp2 msg5 double disruptants.
Finally, we obtained the ptp2 msg5 pkase triple dis-
ruptants for all of the 101 PKases. We then examined the
Ca
s
-phenotype of all of the triple disruptants in the presence
of 0.6 M CaCl
2
. The result showed that six pkase disrup-
tions, namely, BCK1, MKK1, SLT2, MCK1, SSK2 and
YAK1 (Fig. 1) suppressed the Ca
s
-phenotype of the ptp2
msg5 double disruptant, suggesting that the role of Bck1p,
Mkk1p, Slt2p, Mck1p, Ssk2p and Yak1p as a growth antag-
onist of the ptp2 msg5 double disruptant in the presence
of high Ca
2+
concentration.
To further conWrm the suppression of the Ca
s
-phenotype,
we conducted backcrosses between the triple disruptant
ptp2 msg5 bck1, ptp2 msg5 slt2, ptp2 msg5
ssk2, ptp2 msg5 mck1 and ptp2 msg5 yak1 triple
disruptants and the ptp2 msg5 double disruptant. Results
of the tetrad analysis showed that calcium tolerance (Ca
t
)
and His
+
Leu
+
Kan
r
-phenotype co-segregated in all of the
tetrads so far tested and Ca
t
/Ca
s
-phenotype segregated in a
2:2 fashion (data not shown). Based on these results, we
Table 2 pkase::kanMX4 disruptions used in this study (Invitrogen)
All disruptants were derived from SH4742 (MAT his31 leu20
lys20 ura30), except for kin3 and psk1 that were derived from
SH4739 (MAT leu20 lys20 ura30)
1. kin3 26. hsl1 51. ptk1 76. dbf2
2. psk1 27. prr1 52. ykt9 77. yak1
3. kns1 28. ypk1 53. vhs1 78. hal5
4. kin2 29. ykl161c 54. gcn2 79. ctk1
5. hog1 30. tpk3 55. cka1 80. ssk22
6. sky1 31. kkq8 56. prk1 81. rim11
7. ymr291w 32. ykl171w 57. cmk1 82. hrk1
8. mck1 33. fmp48 58. pkh2 83. pbs2
9. ckb2 34. twf1 59. skm1 84. ypk2
10. cka2 35. mkk1 60. ygk3 85. ydl025c
11. mek1 36. kin4 61. cmk2 86. mrk1
12. psk2 37. swe1 62. smk1 87. pkh3
13. ypl236c 38. tpk1 63. ime2 88. pkh1
14. tpk2 39. ste20 64. bck1 89. gin4
15. ypl150w 40. rck2 65. prr2 90. sps1
16. ypl141c 41. ssn3 66. chk1 91. rck1
17. mkk2 42. pho85 67. ptk2 92. tos3
18. kin1 43. sks1 68. dun1 93. apg1
19. bub1 44. isr1 69. ste7 94. yck3
20. slt2 45. dbf20 70. ybr028c 95. pak1
21. ire1 46. kin82 71. akl1 96. snf1
22. ksp1 47. fus3 72. ark1 97. ste11
23. kcc4 48. ckb1 73. ssk2 98. rim15
24. sat4 49. npr1 74. fpk1 99. vps15
25. elm1 50. yck2 75. kss1 100. bud32
101. cla4
Table 3 Oligonucleotides used
in this study
Primer Primer sequences
Kf conWrm bck1 5-ATCAGAACTGAGTATGAACT-3
Kr conWrm bck1 5-GTTGGTTTATCAGATACTGC-3
Kf conWrm mkk1 5-GAAAGATACCGTACACCTGC-3
Kr conWrm mkk1 5-ACTCATGGGAGTTACGTTTG-3
Kf conWrm slt2 5-CTATTTAGCTAAGCCTACGT-3
Kr conWrm slt2 5-ATATTCTAAGCGCTTGGTTT-3
Kf conWrm mck1 5-GGGGGATCCTCCCCTCTTGCTGCCTTCCTA-3
Kr conWrm mck1 5-GGGCTCGAGTTCTGAAGAAATGGTTCTGTT-3
Kf conWrm ssk2 5-TAGAAAGAAGCCAAATCTGC-3
Kr conWrm ssk2 5-TGTTAAAAGCGATGTCTTCT-3
Kf conWrm yak1 5-AGGCCTAATAAAAATATCAA-3
Kr conWrm yak1 5-G CTAGCCTCCTTTACGTTTTT-3
Arch Microbiol (2010) 192:157165 161
1 3
concluded that disruption of each of BCK1, MKK1, SLT2,
MCK1, SSK1 and YAK1 suppresses the Ca
s
-phenotype of
the ptp2 msg5 double disruptant.
Disruption of SSK2 and YAK1 restores the G1-S transition
delay of the ptp2 msg5 double disruptant
Our previous study revealed that the ptp2 msg5 double
disruptant displayed delay in G1S transition (Fig. 2a, Her-
mansyah et al. 2009), and exogenous Ca
2+
pronounced this
retardation (Hermansyah et al. 2009). Since the disruption
of each of the six PKases (BCK1, MKK1, SLT2, SSK2,
MCK1 and YAK1) suppressed the growth inhibition of the
ptp2 msg5 double disruptant in the presence of 0.6 M
CaCl
2
, we investigated whether these PKase disruptions
also suppressed the delayed G1S transition. Results of
FACS analysis revealed that in the presence of Ca
2+
, cells
of two triple disruptants, ptp2 msg5 ssk2 and ptp2
msg5 yak1 accumulated as 2C (diploid) cells while cells
of remaining four triple disruptants, ptp2 msg5 bck1,
ptp2 msg5 mkk1, ptp2 msg5 slt2, and ptp2
msg5 mck1 accumulated as 1C (haploid) cells (Fig. 2b).
This indicates that suppression of delayed G1S transition
occurred in the ptp2 msg5 ssk2 and ptp2 msg5
yak1 triple disruptants but not in the ptp2 msg5 bck1,
ptp2 msg5 mkk1, ptp2 msg5 slt2 and ptp2
msg5 mck1 triple disruptants. We also investigated the
FACS proWles of the ptp2 msg5 bck1, ptp2 msg5
mkk1, ptp2 msg5 slt2, ptp2 msg5 ssk2, ptp2
msg5 mck1 and ptp2 msg5 yak1 triple disruptants
as well as the ptp2 msg5 double disruptant in the
absence of Ca
2+
. Results of FACS analysis showed that the
double disruptant and all the triple disruptants had the simi-
lar proWle (Fig. 2b) in which they showed accumulated 1C
cells, suggesting that Ca
2+
is required for restoring the G1
S transition in the ptp2 msg5 ssk2 and ptp2 msg5
yak1 triple disruptants.
As described earlier, disruptions of either SSK2 or YAK1
in the ptp2 msg5 double disruptant displayed a diVerent
FACS proWle compared to that of the other pkase disrup-
tant suppressors in the presence of calcium. Ssk2p is the
MAPKKK of the HOG pathway (Posas and Saito 1998),
which is located upstream of MSN2/MSN4, a transcription
factor for stress response genes. On the other hand, Yak1p,
which is activated by Msn2p/Msn4p (Smith et al. 1998),
can also activate Msn2 possibly by direct phosphorylation
(Lee et al. 2008). Since Msn2p/Msn4p is reported to be reg-
ulated by the HOG pathway (Rep et al. 2000), we next
investigated the involvement of the HOG pathway in the
mechanism of the Ca
s
-phenotype of the ptp2 msg5 dou-
ble disruptant.
Slt2 and HOG pathways are involved in suppression
of Ca
s
-phenotype of the ptp2 msg5 double disruptant
We analyzed whether the lack of these PKases indeed inXu-
ences the activation of the Slt2 and HOG pathway in the
Fig. 1 Calcium sensitivity of the ptp2 msg5 double disruptant and
ptp2 msg5 pkase suppressor. Ten-fold serial dilutions of wild type
(SH5209), ptp2 (SH6790), msg5 (SH6791), ptp2 msg5
(SH6793), bck1, ptp2 msg5 bck1 (SH7995), mkk1, ptp2
msg5 mkk1 (SH8517), slt2, ptp2 msg5 slt2 (SH7996),
mck1, ptp2 msg5 mck1 (SH7998), ssk2, ptp2 msg5 ssk2
(SH7997), yak1 and ptp2 msg5 yak1 (SH7999) spotted on
YPAD plates with and without 0.6 M CaCl
2
were incubated at 30C for
2 days
Fig. 2 FACS proWle of propidium iodide stained cells. a wild type
(SH5209); ptp2 msg5 (SH6793) and b ptp2 msg5 bck1
(SH7995), ptp2 msg5 mkk1 (SH8517), ptp2 msg5 slt2
(SH7996), ptp2 msg5 mck1 (SH7998), ptp2 msg5 ssk2
(SH7997) and ptp2 msg5 yak1 (SH7999). Cells were cultivated in
YPAD with and without 0.3 M CaCl
2
at 30C to OD
660
= 1.0 and sub-
jected to FACS analysis. 1C and 2C represent haploid and diploid cells,
respectively
162 Arch Microbiol (2010) 192:157165
1 3
ptp2 msg5 double disruptant (Fig. 3a, b) at the protein
level speciWcally through changes in the phosphorylation
levels of the Slt2p and Hog1p proteins. Results of the West-
ern blot showed that even in the absence of calcium, the
phosphorylated form of Slt2p is detectable in the ptp2
msg5 double disruptant as well as in four triple disrup-
tants, i.e. ptp2 msg5 mkk1, ptp2 msg5 mck1,
ptp2 msg5 ssk2 and ptp2 msg5 yak1 triple disrup-
tant. Interestingly, phosphorylated Slt2p was completely
undetectable in the ptp2 msg5 bck1 (Fig. 3a). In the
presence of calcium, a signiWcant increase in the phosphor-
ylated form of Slt2p was observed in the ptp2 msg5 dou-
ble disruptant. Compared to the double disruptant, Slt2p
phosphorylation was decreased in the ptp2 msg5 mkk1,
ptp2 msg5 mck1, ptp2 msg5 ssk2 and ptp2
msg5 yak1 triple disruptant, suggesting that the disrup-
tion of BCK1, MKK1 and SLT2 suppressed the Ca
s
-pheno-
type through the repression of activation of the Slt2
pathway (Fig. 3a).
As for the involvement of the HOG pathway in the Ca
s
-
phenotype of the ptp2 msg5, Western blot using anti-
phospho-Hog1p antibody revealed that there was a slight
phosphorylation of the Hog1p in all the strains tested in the
absence of calcium. Upon addition of calcium, a signiWcant
increase in the level of phosphorylated Hog1p was
observed in the ptp2 msg5 double disruptant. Among
the suppressors, ptp2 msg5 bck1, ptp2 msg5
mkk1, ptp2 msg5 slt2, ptp2 msg5 mck1 and
ptp2 msg5 yak1 showed phosphorylation levels com-
parable to that of the double disruptant. Only, ptp2 msg5
ssk2 displayed reduced Hog1p phosphorylation when
compared to ptp2 msg5, indicating that the HOG path-
way is aVected only in the suppression mechanism where
SSK2 is involved.
EVect of pkase suppression on vacuolar morphology of
the ptp2 msg5 double disruptant
In the previous study, we demonstrated that double disrup-
tion of PTP2 and MSG5 caused fragmented vacuole that
was not observed either in wild type, ptp2 or msg5 sin-
gle disruptant (Fig. 4) (Hermansyah et al. 2009; Seeley
et al. 2002). It is reported that strains that undergo vacuole
fragmentation display sensitivity to high extracellular cal-
cium concentration (HoVman-Sommer et al. 2005). Our
observation suggests that fragmented vacuole enhanced the
sensitivity of the ptp2 msg5 double disruptant to high
Ca
2+
concentration. Therefore, we then investigated
whether disruption of PKases also abolished this pheno-
type, i.e. abnormal morphology of the vacuole. Results
revealed that abnormal vacuolar morphology of the ptp2
msg5 double disruptant was suppressed by the mkk1,
slt2 and yak1 disruption, while the number of frag-
mented vacuoles was signiWcantly reduced with the disrup-
tion of ssk2 and mck1. However, almost the same
number of fragmented vacuoles as that of the ptp2 msg5
double disruptant was observed in ptp2 msg5 bck1 tri-
ple disruptant, suggesting that the suppression mechanism
of calcium sensitivity and the fragmentation of vacuole are
not always associated (Fig. 4).
Fig. 3 Detection of Slt2p phosphorylation and Hog1p phosphoryla-
tion levels. a Anti-phospho-Slt2p immunoblot analysis of wild type
(SH5209), ptp2 msg5 (SH6793), ptp2 msg5 bck1 (SH7995),
ptp2 msg5 mkk1 (SH8517), ptp2 msg5 slt2 (SH7996), ptp2
msg5 mck1 (SH7998), ptp2 msg5 ssk2 (SH7997) and ptp2
msg5 yak1 (SH7999). Proteins extracted from cells grown in media
with or without 0.3 M CaCl
2
to an OD
660
= 1.0 at 30C were separated
on SDSPAGE, and immunoblotted with anti-antiphospho-p44/42
MAPK (Thr
202
/Tyr
204
) antibody. Identical samples were used to detect
total Slt2p by immunoblot with anti-Mpk1p/Slt2p. B) Identical sam-
ples were immunoblotted with antiphospho-p38 MAPK (Thr
180
/
Tyr
182
) and anti-Hog1(yC-20):sc-6815 to detect phosphorylated and
total Hog1p, respectively
Arch Microbiol (2010) 192:157165 163
1 3
Discussion
The connection of G1S delay with Ca
s
-phenotype depends
on the PKase and the corresponding MAPK pathway
involved. Slt2p functions as a positive regulator of the SBF
transcriptional factor composed of the Swi4p/Swi6p hetero-
dimeric complex that plays an essential role in the regula-
tion of transition from G1 to S cell cycle (Madden et al.
1997). Therefore, it is expected that the disruption of com-
ponents of MAPK Slt2 pathway (bck1, mkk1, slt2)
cannot restore the G1 delay of the ptp2 msg5 double dis-
ruptant. In the case of mck1, retention of G1 delay might
be due to the stabilization of Cln proteins by mck1 disrup-
tion. This is based on the assumption that Mck1p functions
similarly with its mammalian homolog glycogen synthase
kinase (GSK-B) by regulating the stability of cyclin D, a
mammalian Cln homolog (Zhang et al. 2002). It might be
possible that the stable nature of Cln proteins in the ptp2
msg5 mck1 triple disruptants resulted in the interruption
of the normal oscillation in Cln protein levels during the
cell cycle thereby causing G1 delay (Mizunuma et al.
2005).
It is noted that phosphorylated Slt2p was detectable in
the ptp2 msg5 mkk1 triple disruptant although Mkk1p
is located upstream of Slt2p. This observation could be
explained by the fact that Mkk1p has a MAPKK homolog,
Mkk2p, which functions in the PKC-mediated pathway
(Irie et al. 1993). Mkk1p and Mkk2p have high homology
(80.42%) in C conserved C-terminal kinase domain that
possibly confers the functional redundancy although
Mkk1p seems to play a more dominant role in the Slt2 path-
way (Martin et al. 2000).
HOG pathway and its MAPK cascade (Ste11, Ssk2p,
Ssk22p, Pbs2p and Hog1p) in budding yeast that plays an
important and somewhat specialized role in adapting to
hyperosmotic stress seem to participate in Ca
s
-phenotype
(Shitamukai et al. 2004). However, unexpectedly, disrup-
tion of the other components of the HOG MAPK pathway
except for SSK2, namely, SSK22, STE11, PBS2 and HOG1
did not suppress the Ca
s
-phenotype of the ptp2 msg5
double disruptant (Lavina et al., unpublished observation).
One possibility is that there might be a speciWc interaction
between Ssk2p and both Ptp2p and Msg5p in the regulation
of the calcium signaling pathway. Further study to eluci-
date the detailed mechanism of the suppression of the cal-
cium sensitivity by Ssk2p is currently underway.
In conclusion, we revealed in this study that disruptions
of six PKase (bck1, mkk1, slt2, mck1, ssk2 and
yak1) suppress Ca
s
-phenotype of the ptp2 msg5 double
disruptant, and this suppression is not always accompanied
by suppression of G1 delay in which G1S transition is
restored only by PKases that are not involved in the Slt2
pathway. In addition, inhibition of vacuole fragmentation is
also not always observed in the Ca
s
suppressors, prompting
Fig. 4 Vacuole fragmentation
in the ptp2 msg5 double dis-
ruptant. Wild type (SH5209),
ptp2 msg5 (SH6793), ptp2
msg5 bck1 (SH7995), ptp2
msg5 mkk1 (SH8517), ptp2
msg5 slt2 (SH7996), ptp2
msg5 mck1 (SH7998), ptp2
msg5 ssk2 (SH7997) and
ptp2 msg5 yak1 (SH7999)
were stained with FM4-64 and
photographed as described in the
Materials and methods. Bar
5 m
164 Arch Microbiol (2010) 192:157165
1 3
us to think that at least two independent or parallel mecha-
nisms contribute to the suppression of the ptp2 msg5
double disruptant. In summary, we have described some
characteristics of the PKase suppressors of the ptp2
msg5 double disruptant, and our results suggest that the
mechanism of Ca
s
-phenotype of the ptp2 msg5 double
disruptant might implicate some novel pathways operating
with redundant function of diVerent classes of protein phos-
phatases in S. cerevisiae. These possibilities are currently
under investigated.
Acknowledgments This work was supported by a Grant-in-Aid for
ScientiWc Research B, 20072009, to S.H. from the Ministry of Educa-
tion, Science, Sports and Culture of Japan.
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