Download The Cdk inhibitors p25rum1 and p40SIC1 are functional

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Journal of Cell Science 111, 843-851 (1998)
Printed in Great Britain © The Company of Biologists Limited 1998
JCS3685
The Cdk inhibitors p25rum1 and p40SIC1 are functional homologues that play
similar roles in the regulation of the cell cycle in fission and budding yeast
Alberto Sánchez-Díaz, Isabel González*, Manuel Arellano and Sergio Moreno†
Instituto de Microbiología Bioquímica and Departamento de Microbiología y Genética, CSIC/Universidad de Salamanca, Edificio
Departamental, Campus Miguel de Unamuno, 37007 Salamanca, Spain
*Present address: I.M.P. Research Institute of Molecular Pathology, Dr Böhr-Gasse 7, A-1030 Vienna, Austria
†Author for correspondence (e-mail: [email protected])
Accepted 7 January 1998: published on WWW 23 February 1998
SUMMARY
p25rum1 and p40SIC1 are specific inhibitors of p34cdc2/CDC28
kinase complexes with B-type cyclins that play a central
role in the regulation of the G1 phase of the cell cycle. We
show here that low levels of expression of SIC1 in
Schizosaccharomyces pombe rescues all the phenotypes of
cells lacking the rum1+ gene. In addition, high level
expression of SIC1 in S. pombe induces extra rounds of
DNA replication without mitosis, a phenotype very similar
to the overexpression of rum1+. Transient expression of
rum1+ in S. cerevisiae restores the G1 arrest phenotype of
cdc4 sic1∆ double mutants. Overproduction of rum1+ in
Saccharomyces cerevisiae causes a cell cycle block in G1
with a phenotype similar to inactivation of all the Clb
cyclins. Finally, we have mapped the cyclin interacting
domain and Cdk inhibitory domain to a region of about 80
amino acids in p25rum1 that has significant homology to the
C-terminal domain of p40SIC1. All these observations
suggest that fission yeast p25rum1 and budding yeast
p40SIC1 define a family of Cdk inhibitors that specifically
down regulate cyclin B/Cdk1 during the G1 phase of the
cell cycle.
INTRODUCTION
inhibitors (CKI). These are proteins that induce cell cycle
arrest or delay in response to intracellular or extracellular
signals. In mammalian cells, addition of transforming growth
factor β (TGFβ) arrests the cultures in late G1 (Laiho et al.,
1990). A similar situation occurs in budding yeast after
addition of sexual pheromones (Herskowitz, 1995). Some
CKIs function in response to extracellular signals, but others
appear to be part of the intrinsic cell cycle machinery. CKIs
are able to associate with the Cdk subunit, the cyclin or the
Cdk/cyclin complex to inhibit its kinase activity. In animal
cells, two structurally defined classes of Cdk inhibitors have
been described so far (Sherr and Roberts, 1995; Elledge et al.,
1996; Martín-Castellanos and Moreno, 1997). Members of the
Ink4 family (p15, p16, p18 and p19) specifically bind to Cdk4
and Cdk6, acting as inhibitors of cyclin Ds. The Kip/Cip
family members (p27, p57 and p21), however, seem to
function only in heterotrimeric complexes with the Cdk and
the cyclin. They bind to and inhibit Cdk2, Cdk3, Cdk4 and
Cdk6 kinases but are less effective towards Cdc2/cyclinB,
suggesting that p21 and p27 are not universal inhibitors of
Cdks and display selectivity for G1/S Cdk/cyclin complexes
(Harper et al., 1995).
In budding yeast, two Cdk inhibitors have been described,
Far1 and p40SIC1. Far1 binds to and inhibits Cdc28/Cln
complexes to mediate pheromone cell cycle blockage (Chang
and Herskowitz, 1990; Peter et al., 1993; Peter and Herskowitz,
1994). In contrast to Far1, p40SIC1 functions as an intrinsic
Cell cycle transitions are regulated by the activation and
inactivation of cyclin-dependent kinases (Cdks). Cdks are
regulated by association with positive regulatory subunits
known as cyclins, negative regulators known as Cdk
inhibitors and by phosphorylation (reviewed by MartínCastellanos and Moreno, 1997). In yeast a single Cdk,
encoded by CDC28 in S. cerevisiae or cdc2+ in S. pombe,
regulates cell cycle progression in association with different
cyclins (Nurse and Bissett, 1981; Piggott et al., 1982; Reed
and Wittenberg, 1990). Budding yeast G1 cyclins Cln1, Cln2
and Cln3 drive cells through G1 by activating the Cdc28
protein kinase; Clb5 and Clb6, promote S phase and Clb1
through Clb4 entry into mitosis (reviewed by Nasmyth,
1996). In fission yeast, cig2 is the cyclin that regulates entry
into S-phase, while cdc13 is the mitotic cyclin (reviewed by
Stern and Nurse, 1996). In both yeasts, mitotic cyclins can
perform the function of S-phase cyclins which means that
there is some degree of functional redundancy between Clb16 in budding yeast and between cig2 and cdc13 in fission
yeast (Fisher and Nurse, 1996; Monderset et al., 1996;
Schwob et al., 1994). Cdk/cyclin activity in S-phase and G2
is also necessary to prevent another round of DNA replication
within the same cell cycle (Hayles et al., 1994; Dahmann et
al., 1995).
Cdk activity is negatively regulated by binding of Cdk
Key words: Cell Cycle, Cdk, Cyclin, rum1, SIC1
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A. Sánchez-Díaz and others
component of the cell cycle machine in that its protein levels
or activity is not dependent on external factors. p40SIC1 was
first described as a tightly bound substrate of Cdc28
(Mendenhall et al., 1987; Nugroho and Mendenhall, 1994;
Reed et al., 1985). p40SIC1 accumulates as cells exit from
mitosis and disappears just before the initiation of DNA
replication (Donovan et al., 1994; Schwob et al., 1994).
Destruction of p40SIC1 is dependent on the activity of the
ubiquitin conjugating enzyme, Cdc34 (Goebl et al., 1988;
Schwob et al., 1994). It has been shown that removal of p40SIC1
results in premature entry into S-phase in a high percentage of
unbudded cells. In fact, DNA replication and budding occur at
different times in sic1 null mutants (Schneider et al., 1996).
p40SIC1 inactivation is essential for S phase initiation since it
exerts its inhibitory activity on the Clb5-Cdc28 and Clb6Cdc28 complexes (Schwob et al., 1994). It has been proposed
that p40SIC1 couples DNA replication to the completion of Start
(Schneider et al., 1996; Tyers, 1996).
In fission yeast, p25rum1 plays a central role in the
regulation of the G1 phase (Moreno and Nurse, 1994;
reviewed by Labib and Moreno, 1996). Lack of rum1+ in
small cells is lethal because cells undergo premature S-phase
immediately after mitosis (Moreno and Nurse, 1994). p25rum1
is a potent inhibitor of the cdc2/cdc13 mitotic kinase (CorreaBordes and Nurse, 1995; Jallepalli and Kelly, 1996; MartínCastellanos et al., 1996). Recently it has been shown that
p25rum1 is also required to promote cdc13 proteolysis in G1
cells (Correa-Bordes et al., 1997). Similarly to p40SIC1 in S.
cerevisiae, p25rum1 also oscillates during the cell cycle and is
present from anaphase until the end of G1 where it is
destroyed through the ubiquitin-dependent proteasome
pathway (Benito et al., 1998).
In this paper we show that the Cdk inhibitors p25rum1 and
p40SIC1 are functional and structural homologues that may
perform a similar function in the regulation of the cell cycle in
fission yeast and budding yeast.
MATERIALS AND METHODS
Yeast strains and methods
The fission yeast strains used in this study were (Sp1) h− 972, (Sp20)
h− leu1-32, (Sp21) h+ ade6-704 leu1-32 ura4-d18, (Sp282) h−
rum1::ura4+ ura4-d18 leu1-32 ade6-M216, (Sp544) h−/h+
rum1::ura4+/rum1::ura4+ ura4-d18/ura4-d18 leu1-32/leu1-32
ade6-M216/ade6-M216, (Sp177) h+ cdc10-129, (Sp224) h90 cdc10129 rum1::ura4+ ura4-d18 leu1-32, (Sp63) h+ ade6-704 leu1-32
ura4-d18 integ-nmt-rum1+ (sup3.5), (Sp507) h+ ade6-704 leu1-32
ura4-d18 integ-nmt-SIC1-(sup3-5), (Sp563) h+ rum1::ura4+ ura4d18 leu1-32 intpJK148-81X, (Sp564) h+ rum1::ura4+ ura4-d18
leu1-32 intpJK148-3X-rum1+, (Sp565) h+ rum1::ura4+ ura4-d18
leu1-32 int pJK148-3X-SIC1. The budding yeast strains used were
all generated from W303 (Sc20, a ade2 trp1 can1 leu2 his3 ∆ura3),
15 Dau (Sc55, α ade1 leu2 his3 trp1 ∆ura3), 15 Dau cdc4-1 (Sc55,
α cdc4-1 leu2 his3 ∆ura3) and 15 Dau cdc4-1 sic1::URA3 (Sc56, α
cdc4-1 sic1::URA3 leu2 his3 ∆ura3). Growth conditions and strain
manipulations for fission yeast and budding yeast were as previously
described (Moreno et al., 1991; Sherman, 1991). Yeast
transformation was carried out using the lithium acetate
transformation protocol (Moreno et al., 1991) and integrants were
confirmed by Southern blotting.
All experiments in liquid culture with fission yeast were carried out
in minimal medium (EMM) containing the required supplements,
starting with a cell density of 2-4×106 cells/ml, corresponding to midexponential phase growth. To induce expression from the nmt1
promoter, cells were grown to mid-exponential phase in minimal
medium (EMM) containing 5 µg/ml thiamine, then spun down and
washed four times with minimal medium, and resuspended in minimal
medium lacking thiamine at a density calculated to produce 4×106
cells/ml at the time of peak expression from the nmt1 promoter.
Overexpression of rum1+ deletion fragments in S. pombe was carried
out transferring the different rum1 truncates alleles from pAS2 to
pREP1 (Durfee et al., 1993; Maundrell, 1989, 1993) after digesting
with NdeI-BamHI. For galactose induction, budding yeast cells were
grown in YEP supplemented with 2% raffinose (YEPR) at 28ºC until
the culture reached mid-exponential phase. Then galactose was added
to a final concentration of 2.5%.
Plasmids
The plasmids used in this work are the following: pREP3X-rum1+
contains the 1.5 kb cDNA cloned in pREP3X as a SalI-BamHI
fragment (Moreno and Nurse, 1994). pREP3X-SIC1 contains a 1.6 kb
BamHI-BglII from pb148 (a gift of M. Mendenhall, University of
Kentucky) cloned at the BamHI site of pREP3X. pREP3X-FAR1
contains a 3.2 kb XhoI-NotI from pRS129 (a gift of M. Peter,
University of California, San Francisco) cloned in pREP3X. pREP3XCIP1 contains a 2 kb XhoI fragment from pBS-CIP1 (a gift of S.
Elledge, Baylor College of Medicine) in pREP3X. pREP1-KIP1
contains a 0.6 kb NheI-BamHI fragment from pMEP4 (a gift from
Kornelia Polyak and Joan Massagué, Memorial Sloan-Kettering
Cancer Center) cloned pREP1 digested with XbaI-BamHI. pNURIINK4a contains a 1 kb fragment from pBS-p16 (a gift of Manuel
Serrano and David Beach, Cold Spring Harbor Laboratory) cloned in
SpeI-XhoI in pNURI. pIRT2-rum1+ contains a 3.8 kb PstI-BamHI
rum1+ genomic fragment cloned in pIRT2. S. cerevisiae plasmid
YipG3-SIC1 contains a 1.6 kb BamHI-BglII from pb148 (a gift of M.
Mendenhall, University of Kentucky) cloned at the BamHI site of
YipG3. YipG3-rum1+ contains a 0.8 kb BamHI fragment amplified
by PCR with the oligonucleotides 5′-GGTTTTTGGATCCTCAGTTCG-3′ and 5′-GGGCATTTATATAAACGGGATCCAACAC-3′
cloned at the BamHI site in YipG3.
Flow cytometry and microscopy
About 107 cells were spun down, washed once with water, fixed in
70% ethanol and processed for flow cytometry or DAPI staining as
described previously (Moreno et al., 1991; Sazer and Sherwood,
1990). A Becton-Dickinson FACScan was used for flow cytometry.
For microscopy, cells were photographed using a phase contrast Zeiss
axiophot photomicroscope.
Protein-protein interactions using the two hybrid system
Protein interactions were analysed using the two hybrid system
(Durfee et al., 1993). rum1+ fragments were amplified by PCR, cloned
into the NcoI-BamHI sites of pACT2 and sequenced. PCR
amplifications were performed using different oligonucleotides
including the NcoI and BamHI restriction sites. For rum1 (1-147) we
used the primers S1 (5′-ATATACCATGGGAACCTTCAACACC-3′)
and SD (5′-TATATGGATCCTTAGTCAGCAAACAAGAGTTTGGG-3′); for rum1 (52-147), S6 (5′-ATATACCATGGTTAGCACATTTCCACCTACACC-3′) and SD; for rum1 (67-147), SC (5′ATTACCATGGTACACCAAATTTAATG-3′) and SD; and for rum1
(84-147), SA (5′-ATATACCATGGAAGAAGAAGACAGAGAA-3′)
and SD. A 924 bp fragment coding for the cdc13 307 carboxyterminal amino acids was amplified by PCR using the primers S18
(5′-ATATACCATGGATAGTGTTGAGAGTCCCG-3′) and S20 (5′TATATGGATCCTTAGTCACGAACAAAAAGACTAGC-3′).
The
NcoI-BamHI fragment was introduced in the same restriction sites of
pAS2. The S. cerevisiae Y190 strain was transformed and βgalactosidase activity was analysed in the transformants as described
(Guarante, 1983).
Cell cycle regulation by rum1 and Sic1
RESULTS
Complementation of rum1 deletion by Cdk inhibitors
The first goal of these studies was to stablish if Cdk inhibitors
from other organisms were able to rescue the phenotypes of
fission yeast cells deleted for the rum1+ gene. Deletion of the
rum1+ in the temperature sensitive cdc10-129 mutant
background reduces the maximal temperature at which the
cdc10-129 mutant can grow from 32ºC to 28ºC (Fig. 1A), since
at higher temperatures cells enter mitosis inappropiately
without having replicated their chromosomes. This defect can
be rescued by expressing the rum1+ gene from its own
promoter (Fig. 1B; pIRT2-rum1+) or from the thiamine
repressible nmt1 promoter in minimal medium containing 5
µg/ml of thiamine (Fig. 1B; pREP3X-rum1+). This promoter
allows moderate levels of gene expression in the presence of
thiamine and high levels of gene expression in the absence of
thiamine (Maundrell, 1989, 1993). To test whether other Cdk
inhibitors from budding yeast or animal cells were able to
rescue the rum1∆ phenotype, we subcloned the cDNAs of
FAR1, SIC1, CIP1 and KIP1 in pREP3X and transformed each
of these constructions into the cdc10-129 rum1∆ strain. We
found that expression of SIC1 in the presence of thiamine was
sufficient to restore growth of the double mutant at 32ºC (Fig.
1B). The rest of the Cdk inhibitors did not rescue this
phenotype even when produced at high level in minimal
medium without thiamine.
In fission yeast there is functional redundancy between cig2
and cdc13 cyclins in G1 and inhibition of both cdc2/cig2 and
cdc2/cdc13 kinase activities is required for G1 arrest (Stern and
Nurse, 1997). Cells lacking rum1+ do not arrest in G1 in
response to nitrogen starvation and to mating pheromones
(Moreno and Nurse, 1994). Therefore, rum1∆ cells are sterile
and unable to undergo meiosis (Fig. 2B). The rum1∆ mating
and meiotic deficiencies were completely suppressed when a
rum1+ cDNA was expressed using the nmt1 promoter under
Fig. 1. Expression of the S. cerevisiae SIC1
gene rescues the growth defect at 32ºC of the
cdc10-129 rum1∆ mutant. (A) Deletion of
the rum1+ gene reduces the maximal
temperature at which the cdc10-129 mutant
can grow from 32ºC to 28ºC. Wild-type 972
h− (Sp1), cdc10-129 (Sp177) and cdc10-129
rum1∆ (Sp224) cells were grown at 25ºC in
YES medium until early exponential phase
and then plated out in YES agar and
incubated for 3 days at 25ºC, 28ºC, 30ºC,
32ºC and 37ºC. (B) The cdc10-129 rum1∆
leu1-32 strain (Sp224) was transformed with
pREP3X, pREP3X-rum1+, pIRT2-rum1+,
pREP3X-SIC1, pREP3X-FAR1, pREP3XCIP1 and pREP1-KIP1. Transformants were
allowed to grow at 25ºC in minimal medium
containing 5 µg/ml thiamine and then
streaked out onto the same medium and
incubated at 25ºC and 32ºC for 3 days.
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repressing conditions (Fig. 2C). We used the constructions
described above containing the S. cerevisiae and animal cells
Cdk inhibitors cDNAs to test if these genes were able to rescue
the conjugation and meiotic defects of cells deleted for the
rum1+ gene. Expression of the SIC1 gene was able to
complement the mating defect of the h− rum1∆ strain (Fig. 2D,
left) and the meiotic defect of a h−/h+ rum1∆/rum1∆
homozygous diploid (Fig. 2D, right). SIC1 also partially
rescued the inability of rum1+ deleted cells to arrest in G1 in
response to nitrogen starvation (Fig. 3). In summary, these
experiments show that the S. cerevisiae SIC1 gene
complements all the phenotypes of the rum1+ deletion strain
and that none of the other Cdk inhibitors analysed (Far1,
p21CIP1 and p27KIP1) rescued any of these defects. We have
also tried the p16INK4a Cdk inhibitor and did not find
complementation of any of the phenotypes of the rum1∆
described above (data not shown).
Overproduction of SIC1 in fission yeast generates a
phenotype very similar to the overproduction of
rum1+
In fission yeast, the cdc2/cdc13 complex plays a dual role
during the cell cycle. First, it promotes entry into mitosis and,
second, it inhibits re-initiation of extra rounds of DNA
replication within a single cell cycle (Fisher and Nurse, 1996;
Hayles et al., 1994). Overproduction of rum1+ in G2 inhibits
the protein kinase activity associated with cdc2/cdc13 blocking
entry into mitosis, allowing multiple rounds of DNA
replication leading to an increase in DNA content (Moreno and
Nurse, 1994; Fig. 4A). Constitutive low level expression of
rum1+ from the nmt1 promoter in minimal medium containing
thiamine induces some degree of diploidization (about 2-5%
of the cells are diploids) These diploids can be detected by
plating the cells in medium containing phloxin B, where
haploid colonies are pink while diploid colonies are red
(Moreno et al., 1991). Low level expression of SIC1 in S.
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A. Sánchez-Díaz and others
Fig. 2. S. cerevisiae SIC1 gene rescues the mating and meiotic defect
of fission yeast cells deleted for rum1+. Haploid h− rum1∆ leu1-32
(Sp282) and diploid h−/h+ rum1∆/rum1∆ leu1-32/leu1-32 (Sp544)
cells were transformed with pREP3X, pREP3X-rum1+ and pREP3XSIC1. Cells were plated out in minimal medium containing 5 µg/ml
thiamine and incubated at 32ºC. Crosses were set for the haploid
strains and cells were photographed after 3 days using a Zeiss
axiophot phase contrast microscope. (A) Wild-type controls.
(B) Mutant cells transformed with pREP3X. (C) Mutant cells
transformed with pREP3X-rum1+. (D) Mutant cells transformed
with pREP3X-SIC1.
pombe also causes a similar degree of diploidization and high
level expression of SIC1 induces re-replication (Fig. 4B; see
also Jallepalli and Kelly, 1996). The cells started to elongate
after 18 hours of induction and showed a more prominent
nucleus after DAPI staining (Fig. 5D). The re-replication
phenotype induced by p40SIC1 is delayed by about 4 hours
compared to the one induced by p25rum1 (Fig. 4B). After
prolonged incubation, these cells presented a nucleus of a
similar size to cells overexpressing rum1+ (compare Fig. 5B
and D). We have also induced either rum1+ or SIC1 for 14
hours at 32ºC and then plated cells on medium containing
thiamine and phloxin B (at this temperature expression from
the nmt1 promoter begins 12-14 hours after the removal of
thiamine from the medium). This short pulse of induction
Fig. 3. Expression of the S. cerevisiae SIC1 gene rescues the inability
of rum1∆ mutants to arrest in G1 in response to nitrogen starvation.
Cells deleted for the rum1 gene were transformed with the plasmids
pJK148-81X, pJK148-3X-rum1+ and pJK148-3X-SIC1 and
integrants were isolated. Wild-type control 972 h− (Sp1) and the
different integrants in the rum1∆ background were grown at 25ºC in
minimal medium containing 5 µg/ml thiamine until early exponential
phase and then shifted to minimal medium lacking nitrogen. Samples
for FACS analysis were taken at the indicated times.
generates in both cases more than 50% of diploid colonies. We
did not observe either diploidization or re-replication when
Far1, p21CIP1, p27KIP1 or p16INK4a Cdk inhibitors where
expressed in fission yeast (data not shown).
Overproduction of rum1+ in S. cerevisiae causes a
phenotype similar to the inactivation of all the Clb
cyclins
The experiments described above indicate that p40SIC1 when
expressed in fission yeast can carry out the functions of
p25rum1. That is, it can rescue the sterility and meiotic defects
of the rum1∆ and the synthetic lethality of the cdc10-129
rum1∆ strain at 32ºC. In addition, overproduction of SIC1 in
S. pombe produces phenotypes similar to the overproduction
of rum1+.
Next, we decided to study if p25rum1 behaves as a cell cycle
inhibitor when expressed in S. cerevisiae. For this purpose,
we expressed rum1+ or SIC1 from the GAL1 promoter in the
S. cerevisiae strain W303. Integrants were selected in
minimal medium containing glucose and confirmed by
Southern blot analysis. Induction of the SIC1 gene in
Cell cycle regulation by rum1 and Sic1
847
see also Nugroho and Mendenhall, 1994). Cells
overexpressing rum1+ arrested both with unbudded and large
budded cells (Fig. 7D). Some of the large budded cells have
multiple buds (Fig. 7D). This phenotype is reminiscent of the
lack of all the Clb cyclins or the cdc4, cdc34, cdc53 or skp1
mutants (Bai et al., 1996; Mathias et al., 1996; Schwob et al.,
1994; Yochem and Byers, 1987). Although the bud arrest
morphology is not homogeneous, these cells are
homogeneously arrested in G1 with 1C DNA content (Fig.
6B). This result suggests that high level expression of p25rum1
in budding yeast generates a phenotype consistent with a
preferential inhibition of Cdc28/Clb kinase complexes in
vivo.
S. cerevisiae cdc4-1 mutants lacking SIC1 do not arrest in
G1 when shifted to the restristive temperature (Schwob et al.,
1994). We found that expression of rum1+ in this double
mutant background was able to restore the G1 arrest of cdc41 mutants at 37ºC (Fig. 8), suggesting that at least in this
situation rum1 is able functionally replace Sic1 in vivo,
inhibiting Cdc28/Clb activity and preventing entry into S
phase.
Fig. 4. Overexpression of SIC1 in fission yeast induces rereplication. Cells from the h+ ade6-704 ura4-d18 leu1-32 strain
(Sp21) were transformed with either pREP3X-(sup3-5)-rum1+ or
pREP3X-(sup3-5)-SIC1. Transformants were allowed to grow at
32ºC in minimal medium with thiamine and then replica plated to the
same medium containing 10 µg/ml adenine. Fast growing white
colonies were selected as putative integrants and tested for stability.
Integrants were confirmed by Southern blot. FACS analysis of cells
overproducing rum1+ (A) or SIC1 (B) at different times after the
removal of thiamine from the medium.
galactose causes a cell cycle delay both in G1 and G2 with
cells containing elongated multi-budded cells (Figs 6A, 7B;
Fig. 5. Phenotype of fission yeast cells
overexpressing SIC1. DAPI staining of integrant cells
expressing nmt-rum1+ (A,B) and nmt-SIC1 (C,D)
growing in minimal medium with thiamine (A,C) and
24 hours (B,D) after the removal of thiamine.
(B) Cells overexpressing rum1+ were mixed with
wild-type cells for comparison of the nuclear
staining.
The Cdk inhibitory domain of p25rum1 is conserved
in p40SIC1
We have mapped the cyclin interacting domain and the Cdk
inhibitory domain in p25rum1 to a region of 80 amino acids in
the middle of the protein, from amino acid 67 to 147. To map
the cyclin binding domain in p25rum1 we used the two hybrid
interaction system. p25rum1 strongly interacts with p56cdc13
cyclin. Indeed, expression of either rum1+ or cdc13+
individually in S. cerevisiae from the ADH promoter is lethal
but cells co-expressing rum1+ (or certain deletions of rum1+)
and cdc13+ are alive. Using different deletions of the rum1+
gene we have mapped the interaction domain between p25rum1
and p56cdc13 to a central region in p25rum1 extending from
amino acids 67 to 147 (Fig. 9B). For the mapping of the Cdk
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A. Sánchez-Díaz and others
Fig. 7. Phenotype of budding yeast cells overexpressing rum1+.
Cultures from integrant-GAL1-SIC1 (A,B) and integrant-GAL1rum1+ (C,D) growing in glucose (A,C) or 8 hours after the addition
of galactose (B,D).
Fig. 6. Overexpression of rum1+ in budding yeast induces a cell
cycle block in G1. The S. cerevisiae W303 strain was transformed
with the plasmids YipG3-SIC1 and YipG3-rum1+. Integrants were
selected in minimal medium with glucose and confirmed by Southern
blot. FACS analysis of cells overproducing SIC1 (A) or rum1+ (B) at
different times after the addition of galactose.
inhibitory domain, we overexpressed the same deletions of the
rum1+ gene in S. pombe cells from the nmt1 promoter. In this
case, we found that expression of a truncated version of
p25rum1 from amino acid 67 to 147 was sufficient to block the
cell cycle (Fig. 9A). A similar result was obtained when we
expressed the mutant 52 to 147 (Fig. 9A). Overexpression of
the 84 to 147 mutant did not show any phenotype (Fig. 9A).
We could detect more elongated cells and diploids when we
Fig. 8. Expression of rum1+ restores
the G1 arrest of cdc4-1 sic1∆* at
37ºC. S. cerevisiae 15 Dau cdc4-1
sic1∆ cells were transformed with the
plasmids YipG2-SIC1 and YipG2rum1+. Integrants were selected in
minimal medium with glucose and
confirmed by Southern blot. Cells
were grown in YP raffinose at 25ºC.
Galactose was added to a final
concentration of 2.5% and incubated
at 25ºC to induce a transient
expression of rum1+ and SIC1 genes.
After 150 minutes, the cells were
centrifugated and resuspended in
YPD medium at 25ºC or at 37ºC.
expressed the 52 to 147 or the 67 to 147 truncations than with
wild-type rum1+ in medium containing thiamine (Fig. 9A),
suggesting that these truncated proteins are either more active
as inhibitors or more stable than full length p25rum1.
p25rum1 and p40SIC1 seem to perform similar functions as
cell cycle regulators in fission and budding yeast. But are they
true homologues? Protein sequence alignment between
p25rum1 and p40SIC1 revealed only a 23% identity over the
whole protein (Fig. 10). However, this homology rises to 33%
within the 80 amino acids domain we have defined in p25rum1
to interact with cdc13 and to inhibit the cell cycle in vivo.
Interestingly, this domain corresponds to the C-terminal region
of p40SIC1 that has recently been shown to be required for
binding of Clb5/Cdc28 (Verma et al., 1997a) (Fig. 10).
Cell cycle regulation by rum1 and Sic1
849
Fig. 9. Mapping of the cdc13
interacting domain and the Cdk
inhibitory domain in p25rum1.
(A) Over-production of a truncated
form of p25rum1 from residues 67 to
147 is sufficient to block the cell
cycle in fission yeast. A h− leu1-32
strain (Sp20) was transformed with
pREP3X-rum1-52-147, pREP3Xrum1-67-147 and pREP3X-rum1-84147 and grown in the presence (+Th)
or in the absence (−Th) of thiamine
for 20 hours. (B) p25rum1 interacts
with cdc13 in the two hybrid system.
pAS2 vectors containing different
deletions of the rum1+ gene were cotransformed with the pACT2-cdc13+
vector. Transformants containing
both plasmids were selected in
minimal medium and β-galactosidase
activity was measured.
DISCUSSION
Our results show that p25rum1 and p40SIC1 are structurally and
functionally related Cdk inhibitors. Furthermore, expression of
SIC1 rescues the phenotypes of the deletion of rum1+ gene in
fission yeast and overproduction of SIC1 in fission yeast also
induces re-replication, suggesting that p40SIC1 like p25rum1 has
specificity for the inhibition of S-phase and mitotic Cdk/cyclin
Fig. 10. Amino acid sequence alignment of p25rum1
and p40SIC1. Identical residues in both proteins are
indicated with an asterisk (*). The p25rum1 Cdk
inhibitory domain (residues 67 to 147) is included in
the box.
complexes in fission yeast. None of the other Cdk inhibitors
used in this study (budding yeast Far1 and animal cells p21CIP1,
p27KIP1 and p16INK4) were able to rescue the phenotype of the
rum1 deletion nor did they generate any significant phenotype
when overexpressed in fission yeast. S. cerevisiae Far1 inhibits
only the G1 Cdc28/Cln complexes (Peter and Herskowitz,
1994), and animal cells p16INK4a and p21CIP1 and p27KIP1 are
known to inhibit preferentially the Cdk/cyclin complexes with
850
A. Sánchez-Díaz and others
a role in G1 or S-phase (Harper et al., 1995). The Cdk inhibitors
described so far in animal cells have a low affinity for the
mitotic Cdc2/cyclinB kinase complexes (Elledge et al., 1996;
Harper et al., 1995), suggesting that either animal cells have
evolved in such a way that they do not need a rum1/Sic1
homologue or that this homologue is still to be discovered.
There are many parallels between the function and the
regulation of p25rum1 and p40SIC1. First, they both have a
specificity for S-phase and mitotic Cdk/cyclin complexes. In
budding yeast, p40SIC1 has been shown to inhibit specifically
Cdc28/Clb associated kinases, preventing the initiation of Sphase and mitosis during G1 (Schwob et al., 1994). In fission
yeast, p25rum1 inhibits cdc2/cig2 and cdc2/cdc13 complexes
(Benito et al., 1998; Correa-Bordes and Nurse, 1995; MartínCastellanos et al., 1996). Second, p40SIC1 is stabilised during
anaphase and is degraded at the end of G1 through the Cdc4Cdc34-Cdc53-Skp1 ubiquitin-dependent proteolytic pathway
(Bai et al., 1996; Donovan et al., 1994; Schwob et al., 1994;
Verma et al., 1997a,b). Recently, it has been shown that
p25rum1 is also stable from anaphase until the onset of Sphase (Benito et al., 1998) and that p25rum1 is stabilised in
the fission yeast mutant pop1-1, defective in a protein related
to budding yeast Cdc4 (Kominami and Toda, 1997),
suggesting that this proteolytic pathway may be conserved in
both yeasts. Cdk/cyclin phosphorylation appears to be
necessary and to serve as the trigger for p25rum1 proteolysis,
because p25rum1 is transiently phosphorylated in vivo on two
threonine residues within Cdk consensus sites (T58 and T62)
and a non-phosphorylatable mutant (rum1-A58A62) is stable
in vivo (Benito et al., 1998). Degradation of p40SIC1 is also
dependent on Cdc28 phosphorylation by the Cdc28/Cln
kinases (Verma et al., 1997b; Skowyra et al., 1997; Feldman
et al., 1997). A similar mechanism appears to regulate the
stability of the G1 cyclins Cln2 and Cln3 in S. cerevisiae
(Deshaies et al., 1995; Lanker et al., 1996; Willems et al.,
1996; Yaglom et al., 1995).
p25rum1 and p40SIC1 a common function in inhibiting
Cdk activity during G1
p25rum1 and p40SIC1 play an important function in the G1 phase
of the fission yeast and budding yeast cell cycle (Bai et al.,
1996; Correa-Bordes and Nurse, 1995; Donovan et al., 1994;
Jallepalli and Kelly, 1996; Labib et al., 1995; MartínCastellanos et al., 1996; Moreno and Nurse, 1994; Nugroho
and Mendenhall, 1994; Schwob et al., 1994; Schneider et al.,
1996). Both proteins oscillate during the cell cycle being
present from anaphase until the end of G1 (Benito et al., 1998;
Donovan et al., 1994; Schwob et al., 1994). This pattern is the
exact opposite of the pattern of cyclin B. During anaphase
when Clb1-6 and cdc13 proteolysis takes place, p40SIC1 and
p25rum1 begin to accumulate. Recently it has been shown that
p25rum1 associates with the cdc2/cdc13 complex during G1 and
is necessary to promote cdc13 degradation (Correa-Bordes et
al., 1997). Proteolysis of mitotic cyclins in G1 is important to
keep low Cdk/cyclin B activity in G1 cells, ensuring that Sphase initiation is coordinated with cell size and with other cell
cycle events such as budding in S. cerevisiae.
It has been argued that inhibition of Cdk/cyclin B complexes
in G1 cells may be necessary to establish DNA pre-replication
complexes (pre-RC) (Dahmann et al., 1995; Nasmyth, 1996;
Stern and Nurse, 1996; Wuarin and Nurse, 1996). At the end
of G1, phosphorylation of p40SIC1 in S. cerevisiae and p25rum1
in fission yeast by Cdc28/Cln and cdc2/cig1 kinase activity,
respectively, target them for degradation and allow the switch
from a state of low Cdk/cyclin B kinase activity to a state of
high Cdk/cyclin B kinase activity (Benito et al., 1998; Verma
et al., 1997b), which triggers the DNA replication process.
Cdk/cyclin B complexes do not only trigger initiation from
origins that have formed pre-RC but also prevent the de novo
assembly of pre-RC (Dahmann et al., 1995). Thus, alternation
of these two states of low and high Cdk/cyclin B kinase activity
allows for the correct assembly of proteins at the DNA
replication origin and that origins are fired only once per cell
cycle.
We thank Cristina Martín-Castellanos for the data shown in Fig.
1A; Tamar Enoch and Karim Labib for comments to the manuscript;
Michael Mendenhall for the sequence alignment between p25rum1 and
p40SIC1 shown in Fig. 10; and Michael Mendenhall, Manuel Serrano,
David Beach, Etienne Schwob, Kim Nasmyth, Kornelia Polyak, Joan
Massagué, Randy Poon, Steve Elledge and Mathias Peter for the gift
of plasmids and strains. A.S.-D. is a fellow of the CSIC-Glaxo
predoctoral fellowship program. This work was supported by the
DGICYT, the Human Frontier Science Program and the Ramón
Areces Foundation.
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