Rum1, an inhibitor of cyclin-dependent kinase in fission yeast,
is negatively regulated by mitogen-activated protein kinase-mediated
phosphorylation at Ser and Thr residues
Kentaro Matsuoka
1,2
, Nobutaka Kiyokawa
1
, Tomoko Taguchi
1
, Jun Matsui
1
, Toyo Suzuki
1
, Kenichi Mimori
1
,
Hideki Nakajima
1
, Hisami Takenouchi
1
, Tang Weiran
1
, Yohko U. Katagiri
1
and Junichiro Fujimoto
1
1
Department of Pathology, National Children’s Medical Research Center, Tokyo, Japan;
2
Department of Pathology,
Keio University, School of Medicine, Tokyo, Japan

phorylation of Thr13 and Ser19 negatively regulates the
function of p25
rum1
. Further evidence indicates that phos-
phorylation of Thr13 and Ser19 may retain a negative effect
on the function of p25
rum1
even in vivo. Therefore, MAPK
may regulate the function of p25
rum1
via phosphorylation of
its Thr and Ser residues and thus participate in cell cycle
control in fission yeast.
Keywords: cell cycle; Rum1; Cdc2; mitogen-activated pro-
tein kinase; phosphorylation.
The yeasts have been the favored organisms for investiga-
tion of the basic biology, genetics, and biochemistry of the
cell cycle [1]. Studies of the fission yeast Schizosaccharomy-
ces pombe have played an instrumental role in the discovery
of proteins that regulate the mitotic cycle. S. pombe appears
to be able to control its cell cycle with considerably fewer
components than are used by other eukaryotes, including
the budding yeast Saccharomyces cerevisiae. For example,
S. pombe relies on a single cyclin-dependent kinase (CDK),
Cdc2, to coordinate its mitotic cell-cycle events. Only four
cyclins, Cdc13, Cig1, Cig2, and Puc1, and only one CDK
inhibitor (CKI), namely p25
rum1
, have been identified in
S. pombe [1]. Therefore, S. pombe is considered to provide a

during cell cycling by transcription and ubiquitin-mediated
proteolysis [11]. Second, the kinase activity of Cdc2 is also
regulated by the phosphorylation state of specific amino-
acid residues. In S. pombe, the phosphorylations at Tyr15
and Thr167 residues of Cdc2 regulate its kinase activity
negatively and positively, respectively. The phosphoryla-
tion of Tyr15 is regulated by a combination of protein
kinases, Wee1 and Mik1, and protein phosphatase, Cdc25,
whereas the regulatory mechanism of Thr167 is largely
unknown [12–16]. In addition to the above biochemical
events, CKI also plays an important role in the regulation
of CDK activity [1].
p25
rum1
, the only known CKI in S. pombe, was originally
isolated by Moreno & Nurse in a screen for genes that, when
Correspondence to N. Kiyokawa, Department of Pathology,
National Children’s Medical Research Center, 3-35-31, Taishido,
Setagaya-ku, Tokyo 154-8567, Japan. Fax/Tel.: + 81 3 3487 9669,
E-mail:
Abbreviations: CDK, cyclin-dependent kinase; CKI, CDK inhibitor;
MAPK, mitogen-activated protein kinase; MAP3K, MAPK kinase
kinase; MAP2K, MAPK kinase; ERK, extracellular signal-regulated
kinase; JNK, c-Jun amino-terminal kinase; GST, glutathione-
S-transferase; EMM, Edinburgh minimal medium; YES, yeast extract
+ supplements; PI, propidium iodide.
(Received 26 February 2002, revised 8 May 2002,
accepted 31 May 2002)
Eur. J. Biochem. 269, 3511–3521 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03033.x
overproduced, would induce extra rounds of DNA replica-

1
phase interval. By
inhibiting mitotic CDK activity, p25
rum1
also prevents the
premature-onset of mitosis in cells that have not initiated
DNA replication [17].
Although a number of studies have clarified the function
and regulatory mechanism of p25
rum1
[17–30], details
remain unclear. For example, the amino acid sequence of
p25
rum1
has revealed the presence of five putative phos-
phorylation sites for mitogen-activated protein kinase
(MAPK), i.e. Thr5, Thr13, Thr16, Ser19, and Thr58 [17],
while the precise roles of these sites are largely unknown.
Whether these sites are indeed phosphorylated by MAPK,
and have any functional importance in regulating p25
rum1
activity, has not yet been determined.
MAPK is a central component of the evolutionarily
conserved growth-promoting signaling pathway. The
MAPK cascade, which transmits extracellular signals from
cell surface receptors to nuclear transcription factors [31],
consists of a module of three sequentially activated protein
kinases, namely, MAPK kinase kinase (MAP3K), MAPK
kinase (MAP2K), and MAPK. The extracellular signals
are mediated intracellularly as an activation of MAP3K

demonstrate the possibility that this regulatory mechanism
of p25
rum1
also plays a role in vivo.
MATERIALS AND METHODS
Materials
The 796-bp fragment of rum1 cDNA (nucleotides 107–902)
was amplified by PCR from cDNA prepared from S. pombe
(Library-in-a-Tube
TM
S. pombe, log phase, BIO 101, Inc.,
Vista, CA, USA) using the primers, 5¢-GTTTTTGG
ATTGTCAGTTCG-3¢ (sense) and 5¢-CATGAATAAGG
CAGAAGAGT-3¢ (antisense). PCR was performed using
high-fidelity UlTma
TM
DNA polymerase (PerkinElmer Co.,
Foster City, LA, USA). The PCR product was subcloned
into the EcoRV site of pGEMÒ-5zf(+) vector (Promega,
Madison, WI, USA). The obtained cDNA was sequenced
and used as a template for PCR in the following experi-
ments. Enzymes used for molecular biological manipulation
were obtained from New England Biolabs, Inc. (New
EnglandBiolabs,Bevery,MA,USA).Allchemicalreagents
were obtained from Sigma–Aldrich Fine Chemicals (St
Louis, MO, USA), unless otherwise indicated.
Plasmid construction
All plasmids generated and used in this study are listed in
Table 1. Oligonucleotides used for PCR primers were as
follows: antisense primer 5¢-GTGATTG

pGEX-rum1-13A16A
pGEX-rum1-13A19A
pGEX-rum1-16A19A
pGEX-rum1-13A16A19A
pGEX-rum1-13E19E
pESP-rum1-WT (DN2)
pESP-rum1-13E19E
3512 K. Matsuoka et al. (Eur. J. Biochem. 269) Ó FEBS 2002
877) were amplified by PCR using the antisense primer
and DN2-sense primer. A 741-bp BamHI and blunt-ended
BclI fragment of the cDNA that corresponding to amino
acids 3–230 of p25
rum1
was excised and subcloned into
pGEX-3X bacterial expression vector (Pharmacia Biotech,
Uppsala, Sweden) at BamHI and blunt-ended EcoRI
sites. The consequent pGEX-rum1-D2 vector was desig-
nated wild type vector in this study for convenience. To
generate rum1 mutants containing different N-terminal
deletions, rum1 cDNA fragments were amplified by PCR
using the antisense primer and eitherDN13-sense (nucleo-
tides 157–877), DN16-sense (nucleotides 166–877), or
DN41-sense primer (nucleotides 244–877). The BamHI
and EcoRI fragments (493 bp, 484 bp, and 406 bp,
respectively) were excised from the PCR products and
were subcloned into a pGEX-rum1-WT vector at BamHI
and EcoRI sites. The consequent plasmids were designa-
ted pGEX-rum1-DN13, -DN16, and -DN41 corresponding
to amino acids 14–230, 17–230, and 42–230 of p25
rum1

CACCACCTATGCGAGGG-3¢; 13A, 5¢-GCGAGGGTT
GTGT
GCTCCATCTACCCCAGAGTCTCCTGGG-3¢;
16A, 5¢-GGGTTGTGTACTCCATCT
GCCCCAGAGTC
TCCTGGG-3¢; 19A, 5¢-CTACCCCAGAG
GCTCCTGG
GAG-3¢;58A,5¢-GCACATTTCCACCT
GCACCTGCT
AAAACTCCC-3¢;13A19A,5¢-GCGAGGGTTGTGT
G
CTCCATCTACCCCAGAG
GCTCCTGGG-3¢; 13E19E,
5¢-CACCACCTATGCGAGGGTTGTGT
GAGCCATC
TACCCCAGAG
GAGCCTGGGAGTTTTAAAG-3¢.
The underlined nucleotides contain the mutated sequence.
All mutants were sequenced after the mutagenesis.
To generate a glutathione S-transferase (GST)-fusion
protein expression vector for fission yeast, a BamHI and
blunt-ended BclI fragment was excised from either pGEX-
rum1-WT or -E13E19 and subcloned into pESP-1 (Strata-
gene Cloning Systems, La Jolla, CA, USA) at BamHI and
SmaI sites. To obtain an in-frame sequence between GST
and rum1 genes, subsequent plasmids were digested with
BamHI and re-ligated after blunt-ending with klenow DNA
polymerase. The consequent plasmids were designated
pESP-rum1-WT and -E13E19, respectively, and were
sequenced after construction.

by in vitro kinase assay. GST-p25
rum1
fusion
proteins were bound on glutathione–SepharoseÒ4B. After
intensive washing with NaCl/P
i
and kinase assay buffer
(50 m
M
Tris/HCl, pH 7.5, 10 m
M
MgCl
2
,1m
M
dithiothre-
itol, 1 m
M
EGTA, 100 l
M
ATP), the precipitates were
mixed with 100 ng of either purified sea star Pisaster
ochraceus oocyte MAPK, p44
mpk
(Seikagaku Co., Tokyo,
Japan) or recombinant murine Erk2 prepared from E. coli
(New England Biolabs) and incubated for 15 min at room
temperature in 30 lL of kinase assay buffer with 10 lCi of
[c-
32

examined using histone H1 as a substrate in 30 lLof
kinase assay buffer with 10 lCi of [c-
32
P] ATP, essentially as
described above. To test the effects on phosphorylation
activity of Cdc2 kinase against Histone H1, GST-fusion
proteins of wild-type and various mutants of p25
rum1
purified on glutathione–SepharoseÒ4B were added to each
kinase reaction mixture. As a negative control not affecting
Cdc2 kinase activity, GST was also tested.
To test whether phosphorylation of p25
rum1
affects
electrophoretic mobility and the activity as a Cdc2 kinase
inhibitor of this protein, GST–p25
rum1
proteins bound on
glutathione–SepharoseÒ4B were nonisotopically phos-
phorylated by MAPK as described above with an exception
Ó FEBS 2002 MAP kinase negatively regulates Rum1 (Eur. J. Biochem. 269) 3513
of the absence of [c-
32
P]ATP. After intensive washing,
nonisotopically prephosphorylated GST–p25
rum1
were used
for following SDS/PAGE and Cdc2 kinase assays.
Binding of GST–p25
rum1

-E13E19, were transformed into SP-Q01 S. pombe cells
using a YEASTMAKER
TM
Yeast Transformation System
(Clontech Laboratories, Inc., Palo Alto, CA, USA). After
being grown on Edinburgh minimal medium (EMM) agar
supplemented with thiamine at 30 °C, positive clones were
selected by PCR using specific primers for the pESP vector
(Stratagene); sense, 5¢-GTACTTGAAATCCAGCAAGT
ATATAGC-3¢;antisense,5¢-CAAAATCGTAATATGCA
GCTTGAATGGGCTTCC-3¢. The selected positive colony
was grown in yeast extract plus supplements (YES) medium
to D
600
¼ 1.0 at 30 °C. After intensive washing in H
2
O,
yeast cells were further cultured in EMM broth without
thiamine at 30 °C for 24 h to induce the expression of GST
fusion proteins of either the p25
rum1
-WT or the -E13E19
mutant.
To determine DNA contents, 1 · 10
7
cells were harvested
andwashedwithH
2
O. After fixation in 70% ethanol, cells
were stained with propidium iodide and analyzed by flow

clonal anti-GST antibody (Boeringer Manheim Biochemica,
Manheim, Germany) as described previously [35].
RESULTS
In vitro
phosphorylation of p25
rum1
by MAPK
As putative phosphorylation sites for MAPK were found in
the amino acid sequence of p25
rum1
[17], we first examined
whether MAPK can phosphorylate p25
rum1
in vitro by
generating GST fusion proteins of p25
rum1
. When recom-
binant GST-fusion p25
rum1
-WT prepared from E. coli
(Fig. 1A) was incubated with MAPK purified from sea
star oocytes in the presence of
32
P-labeled ATP, apparent
incorporation of
32
P into GST–p25
rum1
was observed
(Fig. 1C). We also similarly used recombinant murine

N-Terminal deletion does not influence the activity
of p25
rum1
as a Cdc2 kinase inhibitor
The p25
rum1
protein is known to inhibit the kinase activity
of a complex of Cdc2 and B type cyclin and thereby regulate
the cell cycle of fission yeast. Consistently, GST fusion
p25
rum1
-WT effectively inhibited the phosphorylation of
histone H1 mediated by a Cdc2 kinase complex purified
from sea star oocytes in vitro (Fig. 2, lane 2) and
32
P
incorporation into histone H1 was reduced to less than 30%
of that in the absence of p25
rum1
-WT, as assessed by
Fig. 2. Inhibition of Cdc2 kinase activity by
GST fusion proteins of p25
rum1
. (A) GST
fusion proteins of various C-terminal deletion
mutants of p25
rum1
, as indicated, were gener-
ated and examined by SDS/PAGE essentially
as in Fig. 1A. (B) GST fusion proteins shown

inhibition of Cdc2 kinase activity comparable to that of
p25
rum1
-WT was observed (Fig. 2C,D, compare lanes 1 and
2). In contrast, a C-terminal deletion induced apparent loss
of the activity of p25
rum1
as a Cdc2 kinase inhibitor. As
shown in Fig. 2C,D, functional reduction of p25
rum1
depends on the length of C-terminal deletion and longer
deletions yielded greater recovery of the phosphorylation of
histone H1 mediated by Cdc2 kinase in an in vitro kinase
assay. These data indicate that the catalytic domain of
p25
rum1
is located in its C-terminal portion and deletion
of 74 N-terminal amino acids does not affect the activity of
p25
rum1
as an inhibitor of Cdc2 kinase.
Effect of N-terminal phosphorylation
on p25
rum1
function
Although an N-terminal deletion does not directly affect the
Cdc2 kinase inhibitor function of p25
rum1
, it is possible that
the N-terminal portion contributes to functional regulation

tion, could be ruled out. In contrast, pretreatment with a
combination of MAPK and nonisotopic ATP did not affect
the activity of GST–p25
rum1
-DN74proteinsasaCdc2
kinase inhibitor (Fig. 3B). Therefore, the data indicate that
N-terminal phosphorylation of p25
rum1
indeed reduces the
Cdc2 kinase inhibitor activity of this protein.
Determination of the MAPK-mediated phosphorylation
sites in p25
rum1
In the experiment presented in Fig. 1,  60 pmol phosphate
were incorporated in 35 pmols p25
rum1
, suggesting the
presence of two phosphorylation sites for MAPK in the
molecule. Next we determined which Ser and Thr residues
in p25
rum1
are responsible for MAPK-mediated phosphory-
lation. To test this, we first generated various N-terminal
deletion mutants of p25
rum1
as indicated in Table 1, and
examined
32
P incorporation into these mutants mediated by
MAPK. The DN41 mutant showed almost no

Thr13 and Ser19 were replaced with Ala, respectively, both
showed an  50% reduction of
32
P incorporation as
compared with that of wild-type (Fig. 4A,B), indicating
that these two amino-acid residues are major phosphoryl-
ation sites in p25
rum1
mediated by MAPK. To confirm this,
we generated an A13A19 mutant (Fig. 4C) in which both
Thr13 and Ser19 were replaced with Ala (Fig. 4D), and
found that this mutant showed only a faint incorporation of
32
PmediatedbyMAPK(Fig.4E).Basedonthesedata,
Thr13 and Ser19 were assumed to be major MAPK-
mediated phosphorylation sites in p25
rum1
.
Fig. 3. Functional modulation of p25
rum1
by MAPK-mediated phos-
phorylation. (A) The same amounts of GST–p25
rum1
-WT proteins were
nonisotopically phosphorylated (P, lanes 2 and 3) or not phosphory-
lated (lane 1) by MAPK prior to the assay. Cdc2 kinase activity against
histone H1 was examined by in vitro kinase assay in the presence or
absence (lane 4) of either untreated (lane 1) or prephosphorylated (lane
2) GST–p25
rum1

-WT prephos-
phorylated by MAPK had a mobility shift on SDS/PAGE
gel similar to that observed for the GST–E13E19 mutant
(Fig. 5A). In contrast, pretreatment with a combination of
MAPK and nonisotopic ATP did not affect the electropho-
retic mobility of GST–p25
rum1
-D74 that lacks N-terminal
portion (Fig. 5C). Considering the above, it is most likely
that replacement of Thr13 and Ser19 with Glu mimics the
change in electrophoretic mobility of p25
rum1
mediated by
phosphorylation at Thr13/Ser19.
Next, we examined whether Glu13/Glu19 mutation alters
the function of p25
rum1
as a Cdc2 kinase inhibitor using an
in vitro kinase assay. In comparison with the significant
inhibition achieved by wild-type, the E13E19 mutant
showed only a limited inhibition of the
32
P translocation
in histone H1 mediated by Cdc2 kinase (Fig. 5D). As shown
in Fig. 5D, wild-type was sufficient to inhibit Cdc2 kinase
activity even at lower concentration, whereas E13E19
mutant presented a weak inhibition of Cdc2 kinase activity
only at higher concentration, indicating that replacement of
Thr13/Ser19 with Glu inactivates the function of p25
rum1

were examined as in Fig. 1A,C (upper
panels and lower panels, respectively).
(B) Subsequent phosphorylation of GST
fusion proteins in each experiment was
quantified and expressed as a percentage of
wild-type proteins as in Fig. 2D. (C) GST
fusion proteins of wild-type (lane 1) and the
A13A19 mutant (lane 2) of p25
rum1
were
generated and examined by SDS/PAGE,
essentially as in Fig. 1A. (D) The GST fusion
proteins are schematically presented. In the
A13A19 mutant of p25
rum1
(lane 2), both the
Thr13 and the Ser19 residues of the WT were
replaced with Ala. (E) Phosphorylation activ-
ities of MAPK against GST-fusion proteins of
either the wild-type (lane 1) or the A13A19
mutant of p25
rum1
(lane 2) were examined as in
Fig. 1(C).
Ó FEBS 2002 MAP kinase negatively regulates Rum1 (Eur. J. Biochem. 269) 3517
GST–p25
rum1
was similarly tested, no reduction of the
binding between p25
rum1

, it is suggested that the GST–
E13E19 mutant did not function as a Cdc2 kinase inhibitor
in yeast cells.
DISCUSSION
Our data clearly indicate that p25
rum1
is a potent substrate
for MAPK. In vitro experiments also revealed that
MAPK mediated phosphorylation negatively regulates the
activity of p25
rum1
as a Cdc2 inhibitor. Direct in vitro kinase
assay using GST fusion proteins of wild type as well as
various mutants of p25
rum1
demonstrated that residues
Thr13/Ser19 are major phosphorylation sites for MAPK.
Since the weak but visible phosphorylation of A13A19
mutant by MAPK was observed (Fig. 4E), the other Ser or
Thr residue(s) might also be involved in the phosphorylation
by MAPK. However, Glu13/Glu19 mutation (E13E19
mutant), which mimics the phosphorylated state of Thr13/
Ser19, significantly abolishes p25
rum1
function in in vitro,itis
suggested that residues Thr13/Ser19 are essential for
phosphorylation-mediated inactivation of the protein by
MAPK. Given that E13E19 mutant also abolishes p25
rum1
function in yeast cells, it is most likely that the Thr13/Ser19

assay in the presence or absence (lane 7) of
different concentrations (lanes 1 and 4,
0.45 lg; lanes 2 and 5, 0.9 lg; lanes 3 and 6,
1.8 lg) of wild-type (lanes 1–3) and the
E13E19 mutant (lanes 4–6) proteins. (E)
Either untreated (lane 4) or prephosphoryl-
ated (lane 5) GST–p25
rum1
immobilized on the
glutathion–Sepharose beads were incubated
with Cdc2-cyclin B complex. The Cdc2 pro-
teins bound to GST p25
rum1
were detected by
immunoblotting as described in Materials and
methods. As negative controls for coprecipi-
tation, GST proteins were similarly examined
(lanes 2 and 3). As a positive control for im-
munoblotting, Cdc2–cyclin B complex was
appliedinlane1(CNT).
3518 K. Matsuoka et al. (Eur. J. Biochem. 269) Ó FEBS 2002
p25
rum1
is a catalytic domain of the protein while the
N-terminal portion is a regulatory domain mediating a
reduction in activity upon phosphorylation of Thr13/Ser19
by MAPK.
At this moment, the precise mechanism of negative
regulation of the function of p25
rum1

1
period. Secondly, it prevents mitosis from occurring in
early G
1
cells [17]. As the regulation of CDK activity must
be accurate, the function of p25
rum1
must also be regulated
tightly in the cell-cycle process. The regulatory mechanism
of p25
rum1
function has been well characterized by Correa-
Bordes et al. and Benito et al. [20,24]. According to their
observations, p25
rum1
protein levels are regulated sharply
and periodically during the cell cycle and can hence
contribute to appropriate control of the cell cycle. The
p25
rum1
begins to accumulate in anaphase, persisting in G
1
and disappearing during S phase. As p25
rum1
is stabilized
and polyubiquitinated in a mutant with a defective 26S
proteosome, it is suggested that degradation normally
occurs via the ubiquitin-dependent 26S proteosome
pathway.
Interestingly, these authors also observed that p25

rum1
in G
1
phase play a role in setting a
threshold of cyclin levels important in determining the
length of the pre-Start G
1
phase and in ensuring the correct
order of cell-cycle events [20].
In contrast to their observation, we found MAPK-
phosphorylated p25
rum1
to show reduced activity as a Cdc2
kinase inhibitor in vitro, indicating that negative regulation
of the p25
rum1
function mediated by Thr13/Ser19 phos-
phorylation is independent of ubiquitination, rather being
induced by some conformational change of the protein.
Considering all of the above evidence together, we
speculate that p25
rum1
has, at a minimum, two distinct
and independent mechanisms of functional regulation,
both of which are mediated by phosphorylation. The
Fig. 6. Effect of E13E19 mutant of p25
rum1
on mitosis of S. pombe.
(A) The pESP expression vectors of GST fusion proteins of either the
wild-type (WT) (mid panels) or the E13E19 mutant (lower panels) of

consequence of a delay in the timing of mitotic initiation,
which is exacerbated in response to stresses such as high
osmolarity and nutritional limitation [32]. Although such
mutants still undergo cell-cycle arrest in response to stress,
they are unable to resume proliferation and die [32,33].
These lines of evidence suggest that the Sty1/Spc1 MAPK
pathway is required for recovery from stress-induced cell-
cycle arrest in S. pombe. Activation of Sty1/Spc1 induces
nuclear translocation and subsequent phosphorylation of
the bZIP transcription factor Atf1, a homologue of ATF2
that is targeted by the mammalian SAPKs, whereas it is
currently unclear exactly how Sty1/Spc1 influences basal
cell-cycle machinery. However, several possibilities have
been raised [31]. One possibility is that Sty1/Spc1
promotes the expression of B-type cyclin Cdc13. Alter-
natively, Sty1/Spc1 may be required for the assembly or
stability of mitotic cyclin–CDK complexes or other cell-
cycle components. In relation to the second possibility, it
is plausible that Sty1/Spc1 phosphorylates p25
rum1
, inhib-
iting its activity as a negative regulator of Cdc2 kinase
and thereby stabilizes cyclin–CDK complexes. Interest-
ingly, it was observed in mammalian cells that MAPK
ERK is able to phosphorylate p27
KIP1
, a member of the
p21
CIP1/WAF1
CKI family, in vitro, preventing the CKI

Technology Promotion Bureau, STA for Organized Research Combi-
nation System.
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