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Molecular and Cellular Biology, August 2001, p. 5071-5081, Vol. 21, No. 15
Institute of Molecular and Cell Biology,
Singapore 117609, Singapore
Received 2 March 2001/Returned for modification 17 April
2001/Accepted 8 May 2001
Periodic accumulation and destruction of mitotic cyclins are
important for the initiation and termination of M phase. It is known
that both APCCdc20 and APCHct1 collaborate to
destroy mitotic cyclins during M phase. Here we show that this
relationship between anaphase-promoting complex (APC) and Clb proteins
is reversed in S phase such that the early Clb kinases (Clb3, Clb4, and
Clb5 kinases) inactivate APCHct1 to allow Clb2
accumulation. This alternating antagonism between APC and Clb proteins
during S and M phases constitutes an oscillatory system that generates
undulations in the levels of mitotic cyclins.
Oscillation in the abundance of
mitotic cyclins is a major driving force for the progression through
mitosis. While mitotic entry requires the accumulation of these
cyclins, the exit is dependent on their destruction.
Generally, a combination of transcriptional and posttranslational
controls determines the appropriate levels of cyclins at particular
stages of the cell cycle. In the budding yeast Saccharomyces cerevisiae, the transcription of mitotic cyclins Clb1 and Clb2 is
cell cycle regulated such that their mRNA levels rise in late S phase,
peak in G2/M, and finally decline at the end of M
phase (4, 5, 16). Crucial to the posttranslational
regulation of cyclin abundance is the ubiquitin-dependent proteolytic
machinery. A ubiquitin ligase called the anaphase-promoting complex
(APC) is essential for the mitotic cyclin degradation necessary for the
exit from mitosis (26). In yeast, the APC is a multimeric complex consisting of at least 12 subunits (24) which
requires the activator protein Cdc20 or its homolog Hct1 (also known
as Cdh1) (9, 12, 18). While the APC activated by
Cdc20 (APCCdc20) promotes chromosome
segregation by causing destruction of the anaphase inhibitor Pds1,
APCHct1 plays a role in the final exit from
mitosis by mediating proteolytic degradation of the mitotic cyclin Clb2
(12, 18). Hct1 phosphorylation by mitotic kinases prevents
it from activating APC (6, 23). The removal of this
inhibitory phosphorylation by Cdc14 phosphatase (19) is a
critical step which allows Hct1 to bind to and activate APC (7,
23). The activity of Cdc14 itself is under the control of the
mitotic exit network pathway that includes regulatory proteins such as
Tem1, Cdc15, and Net1 (25). Interestingly, Cdc20 function is also essential for the exit from mitosis (9, 21). It
has been suggested elsewhere that Cdc20 promotes exit from mitosis solely by catalyzing the destruction of S-phase cyclin Clb5
(14). However, according to a less deterministic scheme,
APCCdc20 facilitates activation of
APCHct1 by causing progressive lowering of the
inhibitory Clb-Cdc28 kinase activities by partial destruction of Clb
proteins (3, 22). A reduction in the collective Clb kinase
activities would allow a net increase in the dephosphorylation of Hct1
by Cdc14 phosphatase. A fully active APCHct1 can
then target the remaining Clb2 for degradation during telophase, thus
easing the cells out of mitosis. It is widely believed that, once
activated in mitosis, this proteolytic machinery is turned off by the
Cln kinases at the onset of the next cycle (2).
Although Clb5 along with other Clbs may prevent untimely exit from
mitosis, we show here that the early Clbs collectively also have an
essential role during S phase. We demonstrate that it is the early Clb
kinase complexes (Clb3, Clb4, and Clb5) that inactivate
APCHct1 in S phase. Their ability to suppress the
APCHct1 activity early in the cycle is necessary
for the effective expression and accumulation of late mitotic cyclins
(such as Clb2) critical for the onset of mitosis. This regulation is a
reversal of the scenario in M phase, where APC ensures the inactivation
of Clb proteins. We suggest that such a periodic reversal of antagonism between APC and Clb proteins during S and M phases is critical in
generating basic oscillations in the levels of major mitotic cyclins.
Yeast media and reagents.
All strains used in this study are
congenic to the wild-type strain W303. Cells were grown in yeast
extract-peptone (YEP) or selective medium supplemented with 2% glucose
(+Glu) or 2% raffinose plus galactose (+Raff +Gal).
Kanamycin-resistant colonies were selected on plates containing G418
(200 mg/liter).
Strains and plasmids.
The strains (Table
1) were constructed by a combination of
standard molecular genetic techniques such as gene transplacement, gene
disruption, and tetrad dissection. Southern blot analysis was performed
to confirm gene disruptions and transplacements.
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.15.5071-5081.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Early Expressed Clb Proteins Allow Accumulation of
Mitotic Cyclin by Inactivating Proteolytic Machinery during S
Phase
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Strains used in this study
Cell synchronization, cell extracts, kinase assays, and Western
blot analysis.
To obtain synchronous cultures, exponential-phase
cells were grown in medium at 24°C containing either 
factor (5 µg/ml for BAR1 cells and 0.8 µg/ml for bar1
cells) or 30 mg of hydroxyurea (HU) or nocodazole (15 µg/ml) per ml
for 3 to 4 h. Lysates for whole-cell extracts were prepared, and
kinase assays were performed according to the method of Surana et al.
(17). Western blot analyses were performed as described in
the work of Yeong et al. (22).
Northern blot hybridization, flow cytometry, and immunofluorescence staining. The RNA extraction procedure, Northern blot analysis, flow cytometry analysis, and immunofluorescence staining were carried out as described by Lim et al. (8). Immunofluorescence signals were detected using the Leica DM microscope, and images were acquired using an attached Hamamatsu charge-coupled device camera driven by the MetaMorph (Universal Imaging Corporation) software.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Absence of Clb5 limits Clb2 accumulation in late S phase.
We
have previously reported that Cdc20 function is essential for the final
exit from mitosis (9). We had also shown previously that
Cdc20's role in mitotic exit is to catalyze Clb protein destruction to
varying degrees during the cell's approach to telophase, thus lowering
the collective Clb kinase activities to facilitate Hct1 activation by
Cdc14 phosphatase (22). Interestingly, a mutation in the
S-phase cyclin CLB5 gene alone was found to be sufficient to
allow cells to exit mitosis in the absence of the CDC20 gene (14), leading to the proposal that Cdc20 allows mitotic
exit solely by causing destruction of Clb5 at metaphase. However, it seems puzzling that an S-phase cyclin, which is normally destroyed by
the time that cells are in metaphase (11), could be
responsible for inhibiting Hct1 just prior to exit from mitosis. To
address this, we asked whether the absence of the CLB5 gene
causes an overall change in the levels of mitotic cyclin. The wild-type and clb5
cells carrying CDC14-GFP (at its
native locus) were synchronized in G1 by
-factor treatment and then released into -factor-free medium at
24°C. In the wild-type strain, Clb2 protein rose approximately
15-fold relative to its level in -factor arrest before beginning its
decline at 120 min (Fig. 1, left panels). In clb5 cells, on the other hand, Clb2 levels increased
only three- to fourfold compared to the level in
G1 (Fig. 1, right panels). This is clearly not
due to a difference in the extent of synchrony, because the two
cultures showed comparable degrees of synchrony (see graphs). These
results suggest that a lack of Clb5 function compromises significantly
the cells' ability to build up Clb2 protein (and most likely Clb1),
though the reduced amount is still sufficient to allow onset of
mitosis. They also imply that the reason why deletion of the
CLB5 gene allows exit from mitosis in the Cdc20-deficient
cells is that, in the absence of Clb5, the collective Clb kinase
activities do not build up to the wild-type level; consequently, Cdc20
function is no longer required to reduce the overall Clb kinase
activities any further for the effective activation of Hct1 by Cdc14
phosphatase.
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Early transcribed CLB3, CLB4, and
CLB5 genes are collectively essential for accumulation
of late mitotic cyclin Clb2.
It has been reported previously that,
while both clb3 clb4 and clb5
strains are viable (5, 13), clb3
clb4 clb5 triple mutant cells fail to
survive (13); they arrest prior to mitosis with 2N DNA
content and a single nucleus but completely lack a mitotic spindle. The
explanation put forward for the inviability is that, in the absence of
Clb3 and Clb4, Clb5 becomes essential for the biogenesis of mitotic
spindle (13); thus, the clb3 clb4 clb5 mutant fails to form a spindle
and eventually loses viability. Prompted by our finding that
clb5 cells are compromised in their ability to accumulate
Clb2, we determined the extent to which the collective deficiency in
CLB3, CLB4, and CLB5 might affect the
level of Clb2 protein. Wild-type and clb3
clb4 clb5 strains kept alive by
GAL-CLB5 integrated at the TRP1 locus were synchronized in G1 by -factor treatment and
then allowed to resume cell cycle progression in glucose medium at
24°C. As expected, the abundance of both CLB2 transcript
and the protein showed oscillatory behavior in the wild-type strain
(Fig. 2A,
left panel). CLN1 transcript appears slightly earlier and
also shows characteristic fluctuation as cells continue to course
through the cycle. The clb3 clb4 clb5 cells were released normally from their
G1 arrest but soon accumulated at
G2/M with an elongated bud, 2N DNA content (data not shown), and an undivided nucleus lacking a mitotic spindle (Fig.
2B, right panel). Surprisingly, the levels of both CLB2 transcript and the Clb2 protein remained low in these cells throughout the experiment (Fig. 2A). The CLN1 transcript appeared
within 20 min of release from G1 arrest but,
unlike that in wild-type cells, persisted throughout the time course.
This is consistent with an earlier report that, in the absence of Clb
kinases, cells are unable to turn off CLN1 transcription
effectively (1). These results suggest that cells lacking
CLB3, CLB4, and CLB5 can neither
effectively transcribe the CLB2 gene nor accumulate Clb2
protein. Most likely, this is also true of the CLB1 gene since CLB1 and CLB2 are similarly regulated
(4, 5).
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clb4 clb5 cells may be
due to a severe deficiency of Clb1 and Clb2 proteins. Although the
native promoter-driven CLB2 gene on a 2µm plasmid cannot
rescue clb3 clb4 clb5 cells (13), strikingly, CLB2 expressed from the
MET3 promoter on a CEN vector allows them to form
healthy colonies (Fig. 2C). This observation is consistent with the
notion that the clb3 clb4 clb5 cells die due to a lack of Clb2 protein (and perhaps
Clb1) and suggests that, in the absence of Clb3, Clb4, and Clb5, the native promoter of CLB2 is not fully active.
Clb2 is unstable in clb3 clb4
clb5 cells.
It is known that the native
CLB2 promoter is under positive feedback control; i.e.,
unlike the MET3 promoter, efficient transcription from the
native promoter requires Clb2-Cdc28 activity (1). One
reason why the clb3 clb4 clb5
mutant is unable to effectively transcribe CLB2 and
accumulate Clb2 may be because Clb2 is unstable in these cells. To test
this notion, wild-type and clb3 clb4 clb5 cells carrying
MET3-CLB2-HA3 on a CEN vector
were synchronized in early S phase by HU treatment in the presence of
methionine (+Met) and then transferred to 
Met medium to induce
Clb2-HA3 protein. The induction was terminated
after 40 min by the addition of methionine, and the stability of the
protein was monitored by Western blot analysis. The cell cycle arrest
was maintained throughout the experiment by the presence of HU in the
medium at all times. The Clb2-HA3 protein,
ectopically expressed during the short pulse period, remained stable in
wild-type cells (Fig. 2D, left panel), indicated by its unchanged
abundance at various time points. In clb3
clb4 clb5 cells, however,
Clb2-HA3 begins to dissipate at 40 min and is
barely detectable by 60 min, suggesting that the absence of Clb3, Clb4,
and Clb5 makes Clb2 highly unstable (Fig. 2D, right panel).
clb3 clb4
clb5 cells are unable to phosphorylate Hct1
efficiently.
Since APCHct1 targets Clb2 for
proteolytic degradation in telophase and in G1 of
the subsequent division cycle, we suspected that the instability of
Clb2 in clb3 clb4 clb5 cells
might be due to hyperactive Hct1 during S and G2
phase. This may result either from premature release of Cdc14 from the
nucleolus or from incomplete inactivation of
APCHct1 (due to underphosphorylation of Hct1)
during S phase. Therefore, we first examined the localization of Cdc14
in clb5 cells described for the experiment whose results
are shown in Fig. 1. In both wild-type and clb5 cells,
Cdc14-GFP remained sequestered in the nucleolus until the onset of
sister chromatid separation, after which it was dispersed for a short
period (Fig.
3A). The
dispersion of Cdc14 from nucleolus during the metaphase-anaphase
transition has been well documented elsewhere (15, 20).
Hence, the deficiency of CLB5 does not lead to premature
dispersion of Cdc14.
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cells carrying
GAL-HA3-HCT1 on a CEN
plasmid were subjected to the experimental conditions described for
Fig. 1, and the extent of Hct1 phosphorylation was monitored at various
times. During -factor arrest, Hct1 is predominantly in
nonphosphorylated form in both wild-type and clb5 cells
(Fig. 3B). In wild-type cells, Hct1 appears as a single band at a
higher position on the gel within 90 min after the release, indicating
modification. However, clb5 cells fail to fully convert Hct1 to a single, low-mobility band (Fig. 3B); instead, multiple bands
are seen throughout the course of the experiment. Since Hct1 contains
at least nine potential phosphorylation sites (23), these
multiple bands most likely represent different phosphorylated forms.
These results suggest that clb5 cells are compromised in
their ability to modify Hct1 completely. It is possible that a
significant proportion of Hct1, which remains underphosphorylated in
clb5 cells, is sufficient for the continuous destruction
of Clb2 throughout the cell cycle, thereby preventing Clb2 from rising to the wild-type level (Fig. 1).
To determine if Hct1 is also underphosphorylated in the absence of
CLB3, CLB4, and CLB5 genes,
clb3 clb4 clb5 cells carrying one copy of GAL-CLB5 (integrated at the TRP1
locus) and MET3-HA3-HCT1 on a 2µm
vector were synchronized in G1 by -factor
treatment for 3 h at 24°C. The last 1.5 h of -factor
treatment was carried out in Met+Raff medium to induce the synthesis
of HA3-Hct1. Cells were then released in either
YEP+Raff+Met (no Clb5) or YEP+Raff+Gal+Met medium (Clb5 induction).
Hct1 first appears as a nonphosphorylated, single band (Fig. 3C,
-factor lane). In the culture where Clb5 was induced (Fig. 3C, right
panel), Hct1 phosphorylated to different extents can be seen within 60 min, as indicated by the appearance of low-mobility bands (Fig. 3C,
right panel). By 90 min, most of Hct1 shifts upward. This is
accompanied by the appearance of Clb2 protein (90- and 120-min lanes).
In cells devoid of Clb5, a discernible amount of unphosphorylated Hct1
is still present even at 120 min (Fig. 3C, left panel). In addition, no
Clb2 protein is detected in these cells. These findings suggest that
cells deficient in Clb3, Clb4, and Clb5 are inefficient in
phosphorylating Hct1 and are therefore unable to inactivate it
completely. This results in the inability of these cells to accumulate
Clb2. It is noteworthy that, in the absence of Clb3, Clb4, and Clb5,
Hct1 is still phosphorylated to some extent though not sufficiently to
allow growth. We suspect that this could be due to fully functional Clb6-Cdc28 kinase and Cln kinases present in these cells. However, the
severe consequences elicited by the absence of Clb3, Clb4, and Clb5
suggest that phosphorylation by Clb6 and Cln kinases is clearly not
sufficient to inactivate Hct1. These results imply that early Clb
kinases (Clb3-Cdc28, Clb4-Cdc28, and Clb5-Cdc28) play a critical role,
by inhibiting Hct1 through phosphorylation, in the accumulation of Clb2
to an appropriate level.
Deletion of HCT1 relieves G2/M arrest in
clb3 clb4 clb5
cells.
The data presented in the preceding sections argued that
clb3 clb4 clb5 cells fail to
enter mitosis because of their inability to inactivate Hct1. An
important prediction of this claim is that a deletion of the
HCT1 gene should allow clb3 clb4
clb5 cells to undergo nuclear division. We therefore
constructed an hct1 clb3 clb4
clb5 quadruple-deletion mutant carrying one copy of
GAL-CLB5 integrated at the TRP1 locus. While the
clb3 clb4 clb5 triple mutant
completely fails to grow on glucose plates, the hct1
clb3 clb4 clb5 mutant forms
viable colonies (Fig. 4B). Microscopic
examination revealed that not all quadruple mutant cells survived;
however, they did appear to have undergone a few divisions before
losing viability. We deemed it necessary to establish more clearly the
ability of the quadruple mutant to undergo nuclear division. Since
cells deficient in Hct1 are somewhat resistant to pheromone treatment,
clb3 clb4 clb5 and
hct1 clb3 clb4 clb5 cells carrying GAL-CLB5 were synchronized
in a pre-nuclear-division state by treatment with the
microtubule-disrupting agent nocodazole in galactose medium. Cells were
then released into glucose medium to allow them to progress through the
cycle in the absence of Clb5. As expected, clb3
clb4 clb5 cells reassembled mitotic spindles, underwent nuclear division within 40 min, and then exited mitosis as indicated by the disappearance of Clb2 protein (Fig. 4A,
left panel). Within the next 60 min, they entered the next cycle (fresh
buds) but eventually arrested with an elongated bud and an undivided
nucleus devoid of mitotic spindle (Fig. 4C, left panels). As described
in a previous section (Fig. 3), the level of Clb2 protein remained
barely detectable after 100 min in these cells (Fig. 4A, left panel).
The clb3 clb4 clb5 cells
lacking HCT1 also reassembled the mitotic spindle, divided
the nucleus, and exited mitosis. They entered the next cycle,
reassembled the mitotic spindle, and continued through the cycle as
indicated by the presence of divided nuclei with an elongated spindle
(Fig. 4C). Moreover, the Clb2 protein also accumulates in these cells (Fig. 4A, left panel). However, the reaccumulation of Clb2 is not
dramatic, perhaps due to a low degree of synchrony in cells released
from nocodazole arrest. Nevertheless, these results suggest that
HCT1 deletion indeed allows mitotic entry in
clb3 clb4 clb5 cells. Thus,
the collective essential function of these early transcribed
CLB genes is to inactivate Hct1, making way for the
accumulation of Clb2 (and Clb1) necessary for the onset of mitosis.
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Clb-Cdc28 complexes are critical for switching off proteolysis in
early S phase.
It is generally believed that the proteolytic
machinery responsible for mitotic cyclin destruction is turned off
early in the subsequent cycle by Cln-Cdc28 kinase (1).
However, in direct contrast with this, our data suggest that
APCHct1 activated during late mitosis is
inactivated by Clb (Clb3, Clb4, and Clb5) kinases in the subsequent
cycle. Consequently, clb3 clb4
clb5 cells are unable to inactivate
APCHct1 and die due to a severe deficiency of
Clb2 (and most likely of Clb1). If so, then the cyclin kinase that
inactivates the proteolytic machinery would be expected to restore the
accumulation of Clb2 protein in clb3 clb4
clb5 cells. We therefore compared the abilities of
GAL-CLB5 (single copy) and MET3-CLN2 (on a 2µm
vector) to reinstate the expression of CLB2. clb3
clb4 clb5 GAL-CLB5 cells were
synchronized by -factor treatment in YEP+Raff at 24°C and allowed
to resume the cell cycle in YEP+Raff, while clb3 clb4 clb5 GAL-CLB5 cells
carrying MET3-HA3-CLN2 on a 2µm
plasmid were treated with factor in YEP+Glu medium and were
released in YEP+Glu+Met medium. After 2 h, galactose was added to
cells without MET3-HA3-CLN2 to
induce synthesis of Clb5, whereas those with
MET3-HA3-CLN2 were shifted to
Met+Glu medium to induce Cln2. While both Clb2 protein and
CLB2 transcript began to accumulate within 40 min of Clb5
induction, expression of Cln2 did not alter Clb2 expression, which
remained at a basal level throughout the experiment (Fig.
5A). These results strengthen the notion
that it is the early Clb kinases which make Clb2 accumulation possible. This is further supported by the fact that, while both
GAL-CLB5 and MET3-CLB2 can efficiently rescue
clb3 clb4 clb5 cells, a
chronic expression of Cln2 from the MET3-CLN2 construct on a 2µm vector is unable to do so (Fig. 5B). However, it must be noted that our results do not completely rule out the possibility that Cln
kinases may contribute toward APCHct1
inactivation to some extent, perhaps early in the cell cycle prior to S
phase.
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Contribution of transcriptional regulation to oscillations in Clb2
abundance.
The results described above suggest that, while APCs
act to inactivate Clb kinase complexes in M phase, some of the Clb
kinases cooperate to inactivate APCHct1 during S
phase to allow accumulation of mitotic cyclins. This also implies that
such an alternating antagonism between APC and Clb kinases in S and M
phases should be able to generate oscillations in Clb2 levels. If so,
then to what extent does the fluctuating transcription of
CLB2 contribute to these oscillations? We addressed this in
a strain where CLB2 is transcribed, not periodically from its native promoter, but continuously from the ADH promoter.
The wild-type strain and a strain in which the endogenous
CLB2 gene had been replaced by ADH-CLB2 were
synchronized in G1 by pheromone treatment at
24°C. Cells were allowed to resume cell cycle progression in
pheromone-free medium. As expected, Clb2 protein showed oscillatory behavior for two synchronous cycles in wild-type cells (Fig.
6; lower panels, solid diamonds). Cells
carrying ADH-CLB2 also exhibited the oscillatory pattern in
the abundance of Clb2 (Fig. 6, lower panels, open circles). However,
compared to the wild type, the amplitude of the oscillation in these
cells was lower in the first cycle; the reduction in the amplitude was
even more pronounced in the second division cycle. These observations
imply that while the mutually repressive behavior of APC and Clb
proteins is sufficient for producing basic oscillations in Clb2
protein, transcriptional regulation modulates the amplitude of these
oscillations.
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) and clb2
) cells, synchronized in
G1 by | |
DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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A quick rise and a rapid fall in mitotic cyclin levels within a division cycle are important for the orderly progression through M phase. The cellular logistics which give rise to this oscillatory behavior are also of great interest because they promise a glimpse into the regulatory dynamics that generate such a pattern. The cyclic fashion in which the major mitotic cyclins appear and disappear has been attributed to the onset of cyclin transcription in late S phase and destruction in late telophase, respectively. As a coarse approximation, this indeed appears to be true. However, such a simplistic view overlooks entirely the dynamic realities of cellular events that generate such a periodic pattern.
It has been well documented elsewhere that the high mitotic kinase (such as Clb2 kinase) activity keeps Hct1 in an inactive state (6) and thus prevents cells from exiting mitosis. In vitro assays have shown that Hct1 can also be phosphorylated by Clb5 kinase (23). Consistent with these findings, it has been reported elsewhere that Clb5 destruction at the onset of anaphase is required to pave the way for Hct1 activation and hence mitotic exit (14). However, these studies do not in any way address the role that Clb5 (and other early Clb proteins) may play in inactivating proteolytic machinery during early S phase. Although APCHct1 activated in telophase is responsible for keeping mitotic cyclin levels to a minimum during G1, it becomes a detriment to cells as they attempt to reaccumulate mitotic cyclins (Clb1 and Clb2) at the onset of mitosis. In this report, we show that the inactivation of APCHct1 in early S phase is accomplished by the kinase activities associated with early transcribed Clb3, Clb4, and Clb5, which begin to accumulate soon after cells' passage through start. This role of early Clbs is essential such that, in the absence of Clb3, Clb4, and Clb5, cells fail to enter mitosis due to a lack of Clb2 (and Clb1) proteins.
The inability of the clb5 mutant to build up Clb2 protein
to wild-type levels and to phosphorylate Hct1 efficiently provided the
initial clue (Fig. 1 and 3). The severity of this defect becomes starkly apparent in cells deficient in Clb3, Clb4, and Clb5. The clb3 clb4 clb5 cells fail to
accumulate Clb2 (and most likely Clb1) and therefore arrest in
G2/M without mitotic spindles (Fig. 2). The fact
that the deletion of the HCT1 gene restores viability and
Clb2 accumulation in these cells strongly suggests that the low level
of Clb2 in clb3 clb4 clb5
cells is due to their inability to inactivate Hct1 (Fig. 4). The
instability of ectopically expressed Clb2 and insufficient
phosphorylation of Hct1 in clb3 clb4
clb5 cells are both consistent with this hypothesis (Fig.
2D and 3). It should be noted that a sizable proportion of
clb3 clb4 clb5 hct1 cells divide several times but die before forming a
colony. This may be due to the buildup of a physiological imbalance
caused by growth in the presence of multiple deletions to which some cells are unable to develop tolerance. Nevertheless, these results underscore the acute importance of shutting down proteolytic machinery in early S phase to allow the initial accumulation of Clb2 necessary for the initiation of the positive feedback loop for CLB2
transcription (1). They also strongly argue that the early
Clb kinases collectively play a critical role of allowing accumulation
of late mitotic cyclin for the onset of mitosis. The involvement of
cyclin A in the accumulation of B-type cyclin (late cyclins) in
mammalian cells has been reported earlier (10).
Our findings have a number of important implications. First, it has
been suggested that Cdc20 facilitates mitotic exit by mediating Clb5
destruction in metaphase. This was based on the observation that a
mutation in CLB5 allows a cdc20
pds1 double mutant to exit mitosis (14). We
suspect that the reason why a mutation in CLB5 allows
cdc20 pds1 cells to exit mitosis is that,
in the absence of Clb5 function, the collective Clb kinase activities
do not rise to the wild-type level (Fig. 1); consequently, Hct1 is not
completely inactivated and cells no longer require Cdc20 function to
reduce the kinase activities any further in order to effectively
activate Hct1. We have also found that, in the absence of Pds1, cells'
ability to build up Clb2-Cdc28 kinase is also compromised (data not
shown). Second, our results also suggest that clb3
clb4 clb5 cells are inviable not because Clb5 is required for mitotic spindle formation in the absence of Clb3
and Clb4, as previously suggested (13); instead, a
collectively essential function of the early expressed Clb3, Clb4, and
Clb5 cyclins is to inactivate APCHct1 to make way
for the accumulation of late mitotic cyclins. Third, the early
Clb-Cdc28 kinases play a critical role in the inactivation of the
mitotic proteolytic machinery in early S phase (Fig. 5); however, the
possibility that Cln-Cdc28 kinases may contribute toward this
inactivation during the early part of the division cycle cannot be
completely ruled out. Last, the accumulation of Clb2 (and Clb1) is not
a simple consequence of turning on CLB2 transcription; the
inactivation of the cyclin destruction machinery plays a critical role
in bringing the transcriptional positive feedback loop into effect.
Complex processes such as mitosis are quite finely tuned to allow cells
to adapt to changing intra- and extracellular contexts. For a process
to be well honed, its on-off controls have to be overlaid with
mechanisms that regulate the thresholds of activities of various
effectors. Very often, the critical threshold of an effector is a
result of interactions between multiple events. Our investigations
suggest that the oscillatory pattern in cyclin abundance results from
the interwoven functional relationships between various players.
Central to these interactions is the antagonistic relationship between
the APC and the Clb proteins. In our previous work, we had established
that APCCdc20 and APCHct1
together inactivate the Clb kinases during mitosis (22).
Here we have shown that APCHct1 is inactivated in
early S phase of the subsequent cycle by early transcribed Clb3, Clb4,
and Clb5 kinases to allow accumulation of late mitotic cyclin Clb2. In
combination, these two sets of regulation can be seen to constitute a
two-stroke engine. The forward stroke occurs in S phase when Clb2
accumulates as a result of APCHct1 inhibition by
early Clb kinases. During the second (reverse) stroke,
APCHct1 (activated by
APCCdc20 and Cdc14) destroys Clb2 during mitosis.
The occurrence of these two steps in tandem would therefore generate
oscillations in Clb2 abundance. In a two-stroke process, it is
important that, at the end of one stroke, the reverse stroke is
initiated efficiently. In G2/M, the reverse
stroke is set in motion by the active APCCdc20,
which begins the process of APCHct1 activation.
This stroke leads to full activation of APCHct1
and complete destruction of Clb2. However, it is reversed again in
early S phase when early transcribed Clb3, Clb4, and Clb5 inactivate APCHct1, making way for Clb2 accumulation. These
functional relationships are schematically depicted in Fig.
7.
|
This study has taken a renewed look at the familiar oscillatory pattern in mitotic cyclins and has revealed the dynamic relationships that generate it. It also suggests that events which occur early in the division cycle (such as the inhibition of APCHct1 by Clb3, Clb4, and Clb5) have profound influence on the events in late mitosis. We suspect that many apparently simple patterns of cellular behavior emerge from such nonlinear functional interactions. These intertwined relationships are what make cells resilient and adaptable systems.
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ACKNOWLEDGMENTS |
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We are grateful to Wolfgang Zachariae, Bruce Futcher, and David Morgan for various plasmids and constructs. We thank Shannon Allan for the CDC14-GFP construct and Bor Luen Tang for 12CA5 ascites.
This work was supported by the National Science and Technology Board, Singapore.
F.M.Y. and H.H.L. contributed equally to the work.
U.S. is an adjunct staff member of the Department of Pharmacology, National University of Singapore.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institute of Molecular and Cell Biology, 30 Medical Dr., Singapore 117609, Singapore. Phone: (65) 7743612, 8743518, or 8746680. Fax: (65) 7791117. E-mail: mcbucs{at}imcb.nus.edu.sg.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Molecular and Cellular Biology, August 2001, p. 5071-5081, Vol. 21, No. 15
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.15.5071-5081.2001
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