Download Role of the mitotic cyclin Clb2 in mitotic regulation

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Masters Theses
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8-2010
Role of the mitotic cyclin Clb2 in mitotic
regulation
Dustin K. Stutts
[email protected]
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Stutts, Dustin K., "Role of the mitotic cyclin Clb2 in mitotic regulation. " Master's Thesis, University of Tennessee, 2010.
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To the Graduate Council:
I am submitting herewith a thesis written by Dustin K. Stutts entitled "Role of the mitotic cyclin Clb2 in
mitotic regulation." I have examined the final electronic copy of this thesis for form and content and
recommend that it be accepted in partial fulfillment of the requirements for the degree of Master of
Science, with a major in Biochemistry and Cellular and Molecular Biology.
Ana A Kitazono, Major Professor
We have read this thesis and recommend its acceptance:
Mariano Labrador, Sundar Venkatachalam
Accepted for the Council:
Dixie L. Thompson
Vice Provost and Dean of the Graduate School
(Original signatures are on file with official student records.)
To the Graduate Council:
I am submitting herewith a thesis written by Dustin Kimbrough Stutts entitled “Role of
the mitotic cyclin Clb2 in regulation of mitosis.” I have examined the final electronic
copy of this thesis for form and content and recommend that it be accepted in partial
fulfillment of the requirements for the degree of Master of Science, with a major in
Biochemistry and Cellular and Molecular Biology.
Ana Kitazono, Major Professor
We have read this thesis
and recommend its acceptance:
Mariano Labrador
Sundar Venkatachalam
Accepted for the Council:
Carolyn R. Hodges
Vice Provost and Dean of the Graduate School
Role of the mitotic cyclin Clb2 in regulation of mitosis
A Thesis Presented for
The Master of Science Degree
The University of Tennessee, Knoxville
Dustin Kimbrough Stutts
August 2010
ABSTRACT
In the budding yeast Saccharomyces cerevisiae, the mitotic cell cycle is regulated
by the cyclin-dependent kinase (CDK) Cdc28. Cdc28 is activated by binding to one of
nine cyclins, which then directs Cdc28’s function and localization. Clb2 is the main
mitotic cyclin, promoting entry into mitosis and progression from the metaphase to
anaphase transition. In order for cells to exit mitosis, CDK activity must decrease; CDK
activity is regulated through Clb2 degradation. Degradation of Clb2 is mediated by the
Anaphase-Promoting Complex (APC), which is an E3 ubiquitin ligase that is regulated
by the spindle assembly checkpoint. The APC has two activators: Cdc20 and Cdh1;
APC-Cdc20 recognizes an N-terminal destruction box (D box) motif in Clb2 during
anaphase, and APC-Cdh1 recognizes a KEN box motif at the onset of telophase.
Activation of the spindle assembly checkpoint or inactivation of the APC causes
cells to arrest in metaphase and, thus, results in accumulation of Clb2. While performing
analysis of the function of Clb2 in regulation of mitosis, a lower molecular weight
population of Clb2 was identified that seems to correspond to a cleaved form of Clb2
(p45Clb2). Both full-length Clb2 (p56Clb2) and truncated Clb2 (p45Clb2) have different
accumulation patterns during either activation of the spindle assembly checkpoint or
inactivation of the APC. Our current hypothesis is that p45Clb2 is cleaved between the D
box and the KEN box and thus cannot be recognized by APC-Cdc20 but can still be
ubiquitinated by APC-Cdh1. p45Clb2 also lacks a nuclear-export signal (NES), which
causes its accumulation in the nucleus. We hypothesize that Clb2 cleavage constitutes a
mechanism to ensure presence of high CDK activity to delay exit from mitosis.
ii
TABLE OF CONTENTS
I. INTRODUCTION
Regulation of cyclin-dependent kinases..............................................................................1
Functions of the Cdc28-Clb2 complex................................................................................2
The Anaphase Promoting Complex regulates Clb2............................................................4
The APC is regulated by the Spindle Assembly Checkpoint..............................................5
Cleavage of vertebrate cyclins............................................................................................6
Current dilemma.................................................................................................................7
II. MATERIALS AND METHODS
Yeast strains.......................................................................................................................10
Time courses......................................................................................................................10
Protein extract preparations...............................................................................................10
Western blotting................................................................................................................10
Flow cytometry..................................................................................................................11
Spot tests...........................................................................................................................12
III. RESULTS
Lower molecular weight band is specific to Clb2............................................................13
iii
Characterization of p45Clb2 accumulation patterns............................................................15
p45Clb2 remains associated with Cdc28.............................................................................20
Hypothesis: Clb2 is cleaved between D box and KEN box..............................................21
p45Clb2 levels correlate with p56Clb2 levels throughout the cell cycle..........................23
Activation of Spindle Assembly Checkpoint or inactivation of the
Anaphase-Promoting Complex causes more p45Clb2 to accumulate.................................25
Ectopic activation of the spindle checkpoint induces accumulation of p45Clb2................27
Cells expressing Clb2 lacking the first 79 amino acids are more resistant
to benomyl........................................................................................................................28
Overexpression of Clb2 lacking the first 79 amino acids delays mitotic exit..................30
IV. DISCUSSION
Presence or absence of Clb2 is essential for proper mitotic progression..........................32
Lower molecular weight band is specific to Clb2.............................................................32
p45Clb2 has distinct accumulation patterns........................................................................33
Cells expressing only clb2-Δ79N are more resistant to mitotic inhibitors
and delay mitotic exit........................................................................................................34
p45Clb2-Cdc28 complexes may have different roles from p56Clb2-Cdc28
complexes.........................................................................................................................34
p45Clb2 may function to maintain high CDK activity to prevent premature
mitotic exit........................................................................................................................35
Conclusions about p45Clb2................................................................................................35
Future directions..............................................................................................................36
LIST OF REFERENCES................................................................................................37
iv
APPENDIX....................................................................................................................42
VITA..............................................................................................................................44
v
LIST OF FIGURES
Fig I-1.
Structural model of a CDK-cyclin complex................................................1
Fig I-2.
Localization signals and motifs located within Clb2’s sequence................3
Fig I-3.
Amon and Rahal’s model for how levels of CDK activity contribute
to the order of mitotic events and exit from mitosis...................................5
Fig III-1.
The lower molecular weight band shifted when a C-terminal HA
tag was added to Clb2................................................................................14
Fig III-2.
Hydroxyurea treatment caused p45Clb2 to accumulate to low levels.........17
Fig III-3.
.
Fig III-4.
Nocodazole treatment caused p45Clb2 to accumulate................................18
Fig III-5.
p45Clb2 remains bound to Cdc28...............................................................20
Fig III-6.
Proposed site of Clb2 cleavage.................................................................21
Fig III-7.
Model of Clb2 cleavage with respect to the cell cycle.............................22
Fig III-8.
Levels of p56Clb2 and p45Clb2 appear in the same pattern
during the cell cycle..................................................................................24
Fig III-9.
p45Clb2 accumulates at higher levels than p56Clb2 upon activation
of the spindle assembly checkpoint or inactivation of the APC...............26
Fig III-10
Ectopic activation of spindle checkpoint causes low levels of
p45Clb2 accumulation.................................................................................28
Fig III-11.
Cells expressing Clb2 lacking the first 79 amino acids are more
resistant to benomyl..................................................................................29
Fig III-12.
Overexpression of clb2-Δ79N delayed mitotic exit.................................31
Fig A-1.
Alignment of mitotic B-type cyclins........................................................42
Alpha factor treatment caused p45Clb2 to disappear until the
arrest was broken......................................................................................19
vi
CHAPTER 1 - INTRODUCTION
Regulation of cyclin-dependent kinases
In eukaryotic cells, cyclin-dependent kinases (CDKs) regulate cell cycle
progression. In the budding yeast Saccharomyces cerevisiae, Cdc28 is the main CDK,
and thus the main cell cycle regulator. In order for Cdc28 to be active, it must be
phosphorylated at Thr169 by the CDK-activating kinase CAK1 and bound to one of nine
cyclins, as shown in Figure I-1. Different cyclins appear in waves during the cell cycle at
specific times and bind to Cdc28, giving the complex specific functions at different
stages of the cell cycle. Cdc28-cyclin complexes are also regulated via interactions with
activators and inhibitors. Cks1/Suc1, a positive regulator, associates with active Cdc28cyclin complexes and plays a role in targeting the complex to its substrates (Tang 1993).
Negative regulation is achieved through the phosphorylation of Tyr19 of Cdc28 by the
inhibitory kinase Swe1.
Cyclin
Cdk
CDKactivating
kinase
(Cak1)
Fig. I-1: Structural model of a CDK-cyclin complex. The green star represents
Threonine 169, which is phosphorylated by CAK1 to fully activate the Cdc28cyclin complex.
1
All of the cyclin genes, except CLN3, have a homologous cyclin gene, and those
pairs of homologous cyclins have overlapping functions (Mendenhall 1998). Cyclins
Cln1, 2, 3 appear in G1 and promote the transition into S phase. The six B-type cyclins
promote cell cycle progression from S phase through M phase. Clb5 and Clb6 appear in
late G1 to initiate DNA synthesis (Schwob 1993) and help assemble the mitotic spindle
along with Clb3 and Clb4, which appear in S phase and may also play a role in DNA
replication (Richardson 1992). Clb1 and Clb2 appear in G2 and promote cell cycle
progression from G2 to M phase. Clb1 is the major B-type cyclin regulating meiosis,
while Clb2 is the most important in regulating mitosis (Grandin 1993).
Although Clb2 is the most important mitotic cyclin, it is not essential. clb2
mutants remain viable but show a significant mitotic delay and have elongated cells. In
Δclb1,3,4 null mutants, cells are still viable and have no discernable phenotype, which
demonstrates that Clb2 can perform all mitotic functions necessary for viability.
However, Δclb2 combined with either Δclb1 or Δclb3 is synthetically lethal, further
indicating some overlap in function of the late B-type cyclins (Hood 2000).
Functions of the Cdc28-Clb2 complex
The functions of the Cdc28-Clb2 complex are dependent in part on the complex’s
subcellular localization (Eluere 2007). Cdc28-Clb2 localizes to the mother-bud neck
(Hood 2000), as well as to the spindle pole body (SPB) and along the mitotic spindle
(Bailly 2003). This localization to both the nucleus and cytoplasm can occur because
Clb2 contains one nuclear localization signal (NLS) and two nuclear export signal (NES)
sequences as seen in Figure I-2. Clb2 also contains a hydrophobic patch, and its
2
interactions are important for bud-neck localization of Clb2 as well as Clb2’s movement
between the nucleus and cytoplasm (Bailly 2003).
Cytoplasmic Clb2 plays an important role in bud morphogenesis. At the G1 to S
phase transition, Cdc28-Cln1,2 triggers apical cell growth; this induces polarized growth
from the bud tip, allowing the new bud to grow. Once the budding cell reaches G2,
cytoplasmic Cdc28-Clb1,2 levels reach a threshold to induce the switch from apical to
isotropic growth, allowing uniform growth over the bud surface; this results in a round
bud shape (Eluere 2007). Cytoplasmic Cdc28-Clb2 is also required to inactivate Swe1, a
kinase that inhibits Cdc28, via phosphorylation at the G2 to M transition; inactivation of
Swe1 is required to ensure the timely onset of mitosis. Cdc28-Clb2 and other kinases
hyperphosphorylate Swe1; this hyperphosphorylated Swe1 relocalizes from the nucleus
to the bud neck and is then degraded (Longtine 2000). A portion of cytoplasmic Cdc28Clb2 localizes to the mother-bud neck and is thought to contribute to the inactivation of
Swe1, although it is not essential for Swe1 inactivation (Hood-DeGrenier 2007).
Fig. I-2: Localization signals and motifs located within Clb2’s sequence. Clb2
contains one nuclear localization signal (NLS) and two nuclear export signals (NES).
Two cyclin box domains are present, which are essential for binding and activating
Cdc28. Clb2 also contains two destruction recognition motifs, the D box and the KEN
box.
3
Nuclear Cdc28-Clb2 promotes entry into mitosis (Surana 1991; Fitch 1992;
Richardson 1992), but its activity is also required throughout mitosis. Cdc28-Clb2
activity is necessary for the degradation of the anaphase inhibitor Pds1/securin, resulting
in cleavage of sister chromatid cohesion, and for anaphase spindle elongation (Rahal
2008). Cdc28-Clb2 also promotes the transition from metaphase to anaphase by
phosphorylating several components of the Anaphase Promoting Complex (APC).
The Anaphase Promoting Complex regulates Clb2
The APC is an E3 ubiquitin ligase that targets several proteins for degradation,
including Clb2. The APC associates with two activating subunits, resulting in two
isoforms of the APC: APCCdc20 and APCCdh1. Cdc28-Clb2 phosphorylates and activates
Cdc16, Cdc23, and Cdc27, and this stimulates Cdc20 binding to the APC and thus
APCCdc20 activity (Shirayama 1998; Kramer 2000; Rudner 2000). Cdc28-Clb2 also
phosphorylates Cdh1 to prevent its activation of the APC; however, when Cdc28-Clb2
levels drop in late mitosis, the phosphatase Cdc14 is active and dephosphorylates and
activates Cdh1 (Zachariae 1998; Jaspersen 1999; Azzam 2004).
The substrate specificity of the APC depends on which activator it is associated
with (Schwab 1997; Visintin 1997). APCCdc20 ubiquitinates Pds1, which inhibits anaphase
by binding the Esp1 separase to prevent sister chromatid separation. Once Pds1 is
degraded, Esp1 cleaves the cohesin complex binding the sister chromatids, which allows
them to move to opposing spindle pole bodies (Uhlmann 1999). APCCdc20 also
ubiquitinates a portion of Clb2 during anaphase; however, APCCdh1 ubiquitinates the
majority of Clb2 at the onset of telophase (Amon 1997; Baumer 2000; Yeong 2000;
Sullivan 2007). APCCdc20 mediates Clb2’s degradation through recognition of a
4
Destruction box (D box) motif, which is contained within Clb2’s first NES (residues
Arg26-Asn33) (Glotzer 1991). APCCdh1-mediated degradation does not rely on the D box,
but instead depends on a KEN box about 70 amino acids downstream of the D box
(Pfleger 2000; Burton 2001; Hendrickson 2001; Schwab 2001).
A model demonstrating the effect of CDK activity levels on cell cycle progression
was constructed by Amon and her colleagues; this model shows a peak in CDK activity
at the metaphase to anaphase transition, followed by a sharp decrease in CDK activity,
which is necessary for mitotic exit (Rahal 2008). Thus, this degradation of Clb2 is
necessary to inactivate Cdc28 to allow cells to exit mitosis (Wasch 2002). If Clb2 is not
degraded and CDK activity remains high, mitotic exit is delayed (Cross 2005).
Fig. I-3: Amon and Rahal’s model for how levels of CDK
activity contribute to the order of mitotic events and exit
from mitosis.
5
The APC is regulated by the Spindle Assembly Checkpoint
The APC is also regulated by the spindle assembly checkpoint; if the microtubule
spindle is not assembled correctly or if the kinetochores are not correctly attached to the
microtubules, this checkpoint is activated and the APC is inhibited (Chan 2003;
Acquaviva 2004). Inhibition of the APC prevents ubiquitination and degradation of Clb2,
thus preventing the cell from entering anaphase and allowing the cell time to correct the
defects to ensure proper chromosome segregation. This APC can be inhibited by
introducing microtubule toxins, such as nocodazole or benomyl; these drugs destabilize
the microtubules, which activates the spindle checkpoint and arrests the cells in
metaphase. Mutations preventing activation of the spindle checkpoint allow the cell to
continue through mitosis without any repair, resulting in chromosome instability and
ultimately cell death.
Genetic screens identified two sets of genes in parallel pathways involved in
regulating the spindle checkpoint: MAD1, 2, 3 (Mitotic arrest deficient) (Li 1991) and
BUB1, 2, 3 (Budding uninhibited by benzimidazole) (Hoyt 1991). Of these, deletions of
MAD2 and BUB2 result in the strongest phenotypes and seem to be the most important
based on previous studies (Alexandru 1999). Mad2 binds to unattached kinetochores and
complexes with Cdc20 (along with Mad1, Mad3, and Bub1) to prevent premature sister
chromatid separation by inhibiting APCCdc20 (Hwang 1998). Bub2 is proposed to regulate
the other isoform of the APC, APCCdh1 (Alexandru 1999). Bub2 is also proposed to
regulate cytokinesis by preventing actin ring formation in metaphase cells (Lee 2001).
Vertebrate cells lacking Mad2 and Bub2 are not viable; however, yeast cells lacking
Mad2 and Bub2 are able to survive. This indicates that an alternate mechanism exists to
6
arrest yeast cells in metaphase in the absence of these spindle checkpoint regulators;
currently, this mechanism has not yet been identified.
Cleavage of vertebrate cyclins
We recently identified a lower molecular weight population of Clb2 that seems to
correspond to a cleaved form of Clb2, and it accumulates under conditions that induce
mitotic arrest, such as activation of the spindle assembly checkpoint or inactivation of the
APC; importantly, these conditions require a high level of CDK activity to prevent
premature exit from mitosis.
Interestingly, cleavage of vertebrate cyclins during mitotic catastrophe has been
reported in several organisms. Chan et al. recently discovered that mammalian cyclin B1
is cleaved by caspases during mitotic catastrophe, such as disruption of the DNA damage
checkpoint, and that the truncated cyclin B1 remained associated with CDK1 (Chan
2009). The truncated form of cyclin B lacked the N-terminus, which contained the D box
necessary for the first wave of cyclin B1 degradation, resulting in a stable form of cyclin
B1 that is not degraded by the APC. Truncated cyclin B1 is also missing part of the
cytoplasmic retention signal as well, although it is still mostly cytoplasmic, as is fulllength cyclin B1. Their studies showed that ectopic expression of truncated cyclin B1
prevented mitotic exit and induced apoptosis, and they proposed that truncated cyclin B1
allows prolonged activation of CDK1 during mitotic catastrophe, which contributes to the
apoptotic pathway. Cyclin A is also cleaved in Xenopus embryos when treated with
hydroxyurea under conditions that induce apoptosis; truncated cyclin A also lacks the D
box (Stack 1997). These two examples of cyclin cleavage create more stable forms of the
7
cyclin and are hypothesized to maintain high CDK activity to prevent premature mitotic
exit.
Since Saccharomyces cerevisiae is a well-established model for eukaryotes, it
seems likely that yeast have a similar cleavage mechanism and that cleavage of Clb2
functions to prevent premature mitotic exit. N-terminal cleavage of Clb2 would remove
the D box motif recognized by Cdc20 and would result in a more stable form of Clb2 that
is resistant to Cdc20-mediated ubiquitination. This Cdc20-resistant form of Clb2 would
help explain why such a small portion of Clb2 is susceptible to ubiquitination via Cdc20.
Current dilemma
An ongoing dilemma in this area of study is: How yeast cells are able to survive
in the absence of Mad2 and Bub2, especially when the spindle assembly checkpoint is
compromised? We believe that the truncated form of Clb2 may provide some answers to
this important question.
Mad2 and Bub2 are required for cell viability in vertebrates, but, interestingly,
Mad2 and Bub2 are not essential for viability in S. cerevisiae. Since yeast cells are able to
survive in the absence of Mad2 and Bub2, actually growing similar to wild type cells
under normal conditions (data not shown), another mechanism must be present to activate
the spindle assembly checkpoint. In vertebrates, truncated cyclin B1 results in apoptosis,
but in yeast, truncated Clb2 only delays mitotic exit (data to follow) and may actually
contribute to survival. It is possible that truncated Clb2 functions to supplement the
spindle assembly checkpoint by maintaining high enough CDK activity to prevent
premature entry into anaphase. Thus, truncated Clb2 may be the key to explaining how
yeast cells are able to survive in the absence of Mad2 and Bub2.
8
Evidence for the role of truncated Clb2 in this pathway is presented in the results
sections. Interestingly, we found that truncated Clb2 is present throughout the cell cycle,
but accumulates to high levels in response to spindle checkpoint activation. Since
truncated Clb2 is present throughout the cell cycle, it may be performing a role that may
or may not be distinct from that of full-length Clb2. Upon activation of the spindle
assembly checkpoint, truncated Clb2 accumulates to higher levels than full-length Clb2;
this indicates that truncated Clb2 may be more important than full-length Clb2 in the
event of spindle assembly checkpoint activation. All of this data suggests that Clb2 may
be contributing to cell cycle arrest in response to activation of the spindle assembly
checkpoint.
9
CHAPTER II – MATERIALS AND METHODS
Yeast strains
All yeast strains used are from the W303 background.
Time courses
Cells were grown at 25°C except for experiments where temperature-sensitive
mutants were used to induce spindle checkpoint activation or APC inactivation; these
cells were treated with 5 µM alpha-factor at 25°C, cells were washed twice with 10 ml
0.5X YPD to wash out the alpha factor, then cells were transferred to media prewarmed
to 36°C. Cells were collected and washed twice with 8M Urea/1X PBS/10% glycerol.
Protein extraction
For denaturing conditions, cells were resuspended in 8M Urea/1X PBS/10%
glycerol/1 mM PMSF/1X Complete protease inhibitor cocktail (Roche). For affinity
purification and non-denaturing conditions, cells were resuspended in 1X PBS/10%
glycerol/1mM PMSF/1X Complete protease inhibitor cocktail (Roche)/50 mM NaF/60
mM beta-glycerophosphate. Glass beads were added to slightly less than half the volume.
Cells were vortexed for 15 minutes at 4°C, and then centrifuged for 40 minutes at 4°C.
Bradford assays were performed to determine protein concentrations.
Western blotting
Sample buffer was added to 100 µg protein of each sample and boiled at 95°C for
5 minutes. 12% SDS-PAGE was run using 100 µg of protein in each lane. Protein was
transferred to nitrocellulose membrane at 16V overnight or at 35V for 5 hours at 4°C.
Membranes were blocked in 5% non-fat milk. Polyclonal anti-Clb2 antibody (generated
by Covance) was added at 1:2000 dilution for 1 hour at room temperature (RT) with
10
rotating, and then membranes were washed three times with 1X TBS-T. Anti-rabbit
antibody conjugated to horseradish peroxidase was added at 1:2000 dilution for 1 hour at
RT with rotating, and then membranes were washed three times with 1X TBS-T.
SuperSignal West Pico Chemiluminescent Substrate was incubated on membranes for 5
minutes prior to exposure to x-ray film. Monoclonal anti-HA antibody was used at 1:500
dilution and secondary anti-mouse antibody was used at 1:1000 dilution. Monoclonal
anti-PSTAIRE antibody was used at 1:7000 dilution and secondary anti-mouse antibody
was used at 1:2000 dilution.
Flow cytometry
All centrifugations were at 14,000 rpm for 2 minutes at room temperature. Cells
were centrifuged at and the media was removed. The cells were resuspended in 1 ml of
70% ethanol and allowed to incubate either at room temperature for 1 hour or overnight
at 4°C. Cells were centrifuged, the supernatant was discarded, cells were resuspended in
500 µl of 50 mM Tris containing 20 µg/ml RNase A, and incubated overnight at 37°C.
Cells were centrifuged, the supernatant was removed, cells were resuspended in 500 µl of
0.05 M HCl containing 5 mg/ml pepsin, and incubated for 1 hour at 37°C. Cells were
centrifuged, the supernatant was removed, and cells were resuspended in 500 µl of PBS.
Cells were sonicated and counted using a hemocytometer. An aliquot of cells and PBS
was made up to 500 µl, containing approximately 5 X 106 cells. 500 µl of 5 µM sytox
green was added, giving a final concentration of 2.5 µM, and incubated overnight at 4°C.
DNA content was normalized using wild-type cells in log phase (actively progressing
through the cell cycle) and then analyzed using the FACS Calibur.
11
Spot tests
Cells were grown to saturation in 1 ml of YPD for three days at room temperature
with shaking. Once cell growth was saturated, cells were sonicated and six five-fold
serial dilutions were prepared with 80 µl of sterile distilled water and 20 µl of cells from
the previous dilution. 5 µl of each of the six dilutions was then placed on the appropriate
plate in order of increasing dilution. YPD and hydroxyurea (0.2 M) plates were grown at
25°C, and benomyl plates were grown at 30°C.
12
CHAPTER III: RESULTS
Lower molecular weight band is specific to Clb2
To determine if this lower molecular weight band was specific to Clb2, wild type
cells and cells with a hemagglutinin (3HA) epitope tag at the C-terminus of Clb2 (Clb23HA) were treated with 15 µg/ml Nocodazole for three hours. Nocodazole is a drug that
destabilizes microtubules, which activates the spindle assembly checkpoint and causes
cells to arrest at metaphase. Figure III-1A shows that the lower molecular weight band is
shifted up in Clb2-3HA and that the band corresponding to Clb2 accumulates as
expected. Interestingly, the lower molecular weight band also accumulates to a lesser
degree than full length Clb2 in response to Nocodazole; together these data indicate that
the lower band is specific to Clb2.
To further verify that the lower molecular weight band was specific to Clb2, wild
type, Clb2-3HA, and Clb2 deletion strains were treated with 15 µg/ml Nocodazole for
three hours and western blotting was performed using both anti-Clb2 and anti-HA
antibodies. As shown in Figure III-1B, the anti-HA antibody detected full length Clb2 as
well as the lower molecular weight band, which further indicated that the lower
molecular weight band was specific to Clb2. The anti-Clb2 antibody was unable to detect
either band in the Clb2 deletion cells, which confirmed that the lower molecular weight
band was specific to Clb2. The current hypothesis is that this lower molecular weight
band represents a truncated form of Clb2. Since the 3HA epitope tag is at the C-terminus
of Clb2 and an anti-HA antibody also detected Clb2 and the lower band, the truncation
must occur at the N-terminus of Clb2.
13
A.
anti-Clb2
B.
anti-HA
anti-Clb2
Fig. III-1: The lower molecular weight band shifted when a C-terminal HA
tag was added to Clb2. (A) An anti-Clb2 antibody confirmed that both bands
shift upward in Clb2-3HA cells, indicating the lower molecular weight band was
specific to Clb2. (B) Anti-HA antibody showed specificity to lower molecular
weight band. In Clb2 deletion cells, neither band was detectable using an antiClb2 antibody.
14
Characterization of p45Clb2 accumulation patterns
To gain insight into the mechanism behind appearance of p45Clb2, we followed
Clb2 protein during treatment of cells with 0.2 M hydroxyurea (S-phase arrest), 5 µM
alpha factor (G1 arrest), and 15 µg/ml nocodazole (metaphase arrest). All cells treated
were haploid and contained a C-terminal GFP-tagged Spc42; Spc42 is a core component
of the spindle pole body. The spindle pole body (SPB) begins duplicating in early G1,
and by the end of G1, two SPBs are visible side by side and are connected by a bridge.
As the cell cycle progresses, the SPBs continue to grow, move farther apart, and form a
bipolar spindle (Castillo 2002). Therefore, progression through the cell cycle can be
followed by observing Spc42-GFP.
Hydroxyurea inhibits the enzyme ribonucleotide reductase, which is responsible
for reducing ribonucleoside diphosphate (rNDP) to deoxyribonucleoside diphosphate
(dNDP); ultimately, hydroxyurea leads to a reduction in the amount of
deoxyribonucleotides (dNTPs) produced, and cells arrest in S phase before DNA can be
completely duplicated. In response to hydroxyurea treatment, some p45Clb2 accumulated
(Fig. III-2-A), which may be in response to activation of the DNA damage checkpoint, as
Clb2 has been shown to interact with several proteins involved in the DNA repair
pathway. Fluorescence microscopy revealed that by three hours after treatment,
representative cells had not duplicated their DNA (Fig. III-2-B, DAPI) and some still had
not duplicated the SPB (Fig. III-2-B, GFP). Cells also had only produced small buds,
indicative of cells that have not completed S phase (Fig. III-2-B, DIC). By five hours
after treatment, representative cells contained duplicated spindle pole bodies in only the
mother cell, but not the daughter cell, indicating that cells had completed G1 (Fig. III-215
B, GFP). Although some cells had duplicated their DNA (Fig. III-2-B, DAPI) and had
developed larger buds (Fig. III-2-B, DIC), indicating that cells were beginning to
overcome the arrest.
Nocodazole destabilizes microtubules, preventing proper attachment of the
microtubule spindles to chromosomes at metaphase, resulting in activation of the spindle
assembly checkpoint, and, thus, cells arrest in metaphase. The levels of p45Clb2
accumulation were significantly higher in nocodazole-treated cells (Fig. III-3-A).
Microscopy revealed that cells were large-budded, indicating that cells were arrested
before exiting mitosis (Fig. III-3-B, DIC). Only the mother cells contained DNA at one
and three hours, indicating that cells had not yet exited mitosis (Fig. III-3-B, DAPI).
Alpha factor is a mating pheromone from mating type alpha cells (MATalpha) that
causes mating type a cells (MATa) to arrest in G1 in preparation for conjugation
(Bucking-Throm 1973). Upon treatment with alpha factor, both Clb2 forms disappear, as
expected (Fig. III-4-A), and both forms of Clb2 reappear when cells break the arrest at 5
hours. As expected, microscopy showed that cells were mostly unbudded and took on a
slightly altered morphology known as “shmooing” in response to the alpha-factor
treatment (Fig. III-4-B, DIC). At 5 hours after treatment, the SPB had been duplicated in
the majority of cells and the SPBs remained close together, indicating that cells had
completed G1 and were beginning to progress through the cell cycle (Fig. III-4-B, GFP).
16
A.
B.
Fig. III-2: Time course with hydroxyurea treatment to arrest cells in S
phase. (A) Anti-Clb2 western blot showing accumulation of full-length and
truncated Clb2 for 5 hours after treatment. (B) Fluorescence microscopy
images following DNA content and duplication of the spindle pole body
throughout the 5 hour time course.
17
A.
B.
Fig. III-3: Time course with nocodazole treatment to arrest cells at
metaphase. (A) Anti-Clb2 western blot showing accumulation of full-length
and truncated Clb2 for 5 hours after treatment. (B) Fluorescence microscopy
images following DNA content and duplication of the spindle pole body
throughout the 5 hour time course.
18
A.
B.
Fig. III-4: Time course with alpha-factor treatment to arrest cells in G1.
(A) Anti-Clb2 western blot showing accumulation of full-length and
truncated Clb2 for 5 hours after treatment. Alpha-factor arrests cells in G1
when all of Clb2 has been degraded. Once cells overcome the arrest, Clb2
populations begin to increase as cells enter mitosis. (B) Fluorescence
microscopy images following DNA content and duplication of the spindle
pole body throughout the 5 hour time course.
19
p45Clb2 remains associated with Cdc28
Since vertebrate cyclins remain bound to CDK, the next step was to confirm that
p45Clb2 also remains bound to Cdc28. WT and Clb2 deletion strains were treated with 15
µg/ml nocodazole. Crude protein was extracted from cells after 3 hours of treatment, and
affinity purification was performed using agarose beads conjugated to Suc1. Suc1 binds
CDKs very strongly with a Kd of 100-200 nM (Morris 1998; Landrieu 2001); therefore,
Suc1 beads provide a good means of isolating CDKs and any proteins bound to CDKs.
Figure III-5 shows that the Suc1 beads pulled down Cdc28 (bottom panel) bound to both
p56Clb2 and p45Clb2 (top panel), demonstrating that p45Clb2 remains bound to Cdc28. A
faint band appears in the clb2Δ input lane, but is not pulled down using Suc1 beads. This
band runs slightly higher than the band corresponding to the truncation and is
nonspecific.
anti-Clb2
Cdc28→
anti-PSTAIRE
Clb2
Fig. III-5: p45
remains bound to Cdc28. Cells were treated with
15 µg/ml nocodazole for 3 hours and protein extracts were incubated
with Suc1-agarose beads. Suc1 beads pulled down Cdc28 bound to
p56Clb2 and p45Clb2.
20
Hypothesis: Clb2 is cleaved between D box and KEN box
Because a C-terminal HA tag on Clb2 was detectable in both the full-length and
the cleaved form of Clb2, the cleavage must occur at the N-terminus. In vertebrate
cyclins, this cleavage occurred after the D box and resulted in a more stable form of the
cyclin that can still be ubiquitinated at the onset of telophase. Therefore, our current
hypothesis is that Clb2 is cleaved between the D box and the KEN box before Anaphase,
as shown in Figures III-6 and III-7, and that this constitutes a mechanism to ensure the
presence of high CDK activity in mitosis. This cleavage would remove the first NES
along with the D box, thus preventing ubiquitination of Clb2 via APCCdc20 and causing
this truncated form of Clb2 to be mostly nuclear. One advantage of having a nuclear
population of Clb2 that is resistant to ubiquitination by APCCdc20 would be the ability to
maintain high CDK activity to prevent the premature exit from mitosis. This truncation of
Clb2 could also help ensure that enough Clb2 was in the nucleus to carry out its nuclear
functions necessary for the completion of mitosis. Since this truncated form of Clb2 still
contains an intact KEN box, it can still be targeted for degradation via APCCdh1 in
telophase to allow cells to exit mitosis.
Fig. III-6: Proposed site of Clb2 cleavage. Schematic of Clb2. The current hypothesis is
that p56Clb2 is cleaved between the D box and the KEN box.
21
Fig. III-7: Model of Clb2 cleavage with respect to the cell cycle. Model showing when
Clb2 is proposed to be cleaved, as well as when and how Clb2 is ubiquitinated. Metaphase
arrest is induced by spindle checkpoint activation, APC inactivation, and microtubuledestabilizing drugs, such as nocodazole and benomyl. At the onset of anaphase, APCCdc20
recognizes the D box within Clb2 and ubiquitinates Clb2. Also at metaphase, the N-terminus
is cleaved from a portion of Clb2, especially under conditions that induce metaphase arrest.
At the onset of telophase, APCCdh1 recognizes both full-length and truncated Clb2 via the
KEN box and ubiquitinates both forms of Clb2.
22
p45Clb2 levels correlate with p56Clb2 levels throughout the cell cycle
Since low levels of p45Clb2 were present before any treatment (Figures III-2, 3, 4),
it seemed likely that some p45Clb2 may be present throughout the normal cell cycle to
perform a role distinct from p56Clb2. In order to further characterize the levels of p45Clb2
during the cell cycle, it is beneficial to first deplete Clb2. This can be achieved by treating
cells with alpha-factor, a S. cerevisiae mating pheromone secreted by MATalpha cells.
Alpha-factor affects cells of the opposite mating type, MATa cells, by arresting the cells
in G1 before DNA synthesis in preparation for conjugation (Bucking-Throm 1973). At
G1, Clb2 has been depleted since Clb2 from the previous mitotic cycle has been degraded
and Clb2 for the next mitotic cycle has yet to be synthesized.
Wild type mat a cells containing a GFP-tagged Spc42 were treated with 5 µM
alpha-factor for three hours. Cells were washed twice to remove alpha-factor, and then
grown at 25°C for four hours to follow levels of p56Clb2 and p45Clb2. Figure III-8-A shows
that p56Clb2 and p45Clb2 levels rise and fall in the same pattern as cells go through the cell
cycle.
Spc42 was followed via its GFP tag to follow cell cycle progression; images from
a few time points are shown in Figure III-8-B. At 15 and 45 minutes after release into
YPD, cells only had one GFP signal, indicating that cells had not yet duplicated the
spindle pole body and had not entered G2. By 75 minutes after release, two GFP signals
were observed in cells, indicating that cells had duplicated the spindle pole body and
were progressing through the cell cycle.
23
A.
anti-Clb2
anti-PSTAIRE
B.
Fig. III-8: Levels of p56Clb2 and p45Clb2 appear in the same pattern during the cell
cycle. To deplete Clb2, SPC42-GFP cells were treated with alpha-factor for 3 hours,
and then washed twice with YPD to remove the alpha-factor. Cells were then
resuspended in YPD and grown at 25°C for 4 hours. (A) Aliquots were taken every 30
minutes to follow levels of p56Clb2 and p45Clb2. (B) Fluorescence microscopy was used
to observe Spc42-GFP.
24
Activation of Spindle Assembly Checkpoint or inactivation of the AnaphasePromoting Complex causes more p45Clb2 to accumulate
As shown in Figure III-3, nocodazole-induced metaphase arrest caused full length
Clb2 and truncated Clb2 to accumulate at different levels. This led to the question: Do the
accumulation patterns of full length Clb2 and truncated Clb2 change when different
factors cause a metaphase arrest?
To begin addressing this question, a temperature sensitive strain that activates the
spindle assembly checkpoint at the restrictive temperature of 36°C was chosen to observe
the accumulation patterns of both forms of Clb2. This strain carried a temperature
sensitive allele of CTF13, called ctf13-30. Ctf13 is a core component of the kinetochore
and has been shown to be required for proper chromosome segregation (Pietrasanta
1999). ctf13-30 cells accumulated p56Clb2, but accumulated more p45Clb2, Figure III-9-A.
To inactivate the APC, a different temperature sensitive strain was used that lacks
Cdc26, which is important for assembly of the APC at high temperatures. Cdc26 has also
been shown to stabilize the interaction of three core subunits of the APC with the rest of
the APC complex (Zachariae 1996; Zachariae 1998). cdc26 cells accumulated p56Clb2,
and interestingly, accumulated much higher levels of p45Clb2. cdc26 cells most likely
accumulated more p45Clb2 than the ctf13-30 cells because inactivation of the APC caused
the cells to arrest more quickly than activation of the spindle assembly checkpoint. Flow
cytometry measures DNA content and confirmed that cells arrested after DNA
duplication as shown in Figure III-9-B for both ctf13-30 and cdc26 strains.
25
A.
⎯
anti-Clb2
B.
1N 2N
ctf13-­30 1N 2N
cdc26 Fig. III-9: p45Clb2 accumulates at higher levels than p56Clb2 upon activation of the
spindle assembly checkpoint or inactivation of the APC. Cells were treated with 5
µM alpha-factor for three hours to deplete Clb2 and then shifted to the restrictive
temperature of 36°C for five hours. Aliquots were taken each hour after the shift to
36°C for western blotting and every 30 minutes after the shift to 36°C for flow
cytometry. (A) Anti-Clb2 western blots show a drastic increase in p45Clb2 compared to
p56Clb2 upon activation of the spindle assembly checkpoint or inactivation of the APC.
(B) Flow cytometry showed that cells arrested after DNA duplication, indicating
mitotic arrest.
26
Ectopic activation of the spindle checkpoint induces accumulation of p45Clb2
The next step was to determine if ectopic activation of the spindle checkpoint
would have a similar effect as the treatments mentioned above on the accumulation of
p45Clb2. Work by the Murray and Winey laboratories has demonstrated that
overexpression of the Mps1 kinase leads to ectopic activation of the spindle checkpoint,
in a manner that is dependent on another checkpoint component, Mad2. Levels of p45Clb2
and p56Clb2 were followed in cells that were induced to overexpress Mps1 under the
control of the GAL1 promoter, either with or without MAD2.
Cells were grown overnight in YEP, 2% raffinose to an OD600 of 0.3. Then, 2%
galactose was added and the cells were allowed to grow for 5 hours to induce
overexpression of Mps1. The anti-Clb2 western blot shown in Figure III-10 shows that
both p56Clb2 and p45Clb2 accumulate upon overexpression of Mps1, although at much
lower levels than seen previously in Figure III-9. As expected, Clb2 does not accumulate
in cells overexpressing Mps1 that lack Mad2, as these cells fail to activate the spindle
assembly checkpoint and, therefore, fail to arrest at metaphase.
The levels of Clb2 accumulation were lower than what was observed with the
methods shown previously; this may be due to suboptimal growth conditions. For the
other methods, cells were grown in YEP containing glucose, but for the galactose time
course, cells were grown overnight in YEP containing raffinose. The cells grown in
raffinose grew much more slowly than cells grown in glucose, which may account for the
difference in Clb2 levels.
27
Fig. III-10: Ectopic activation of spindle checkpoint causes low levels of p45Clb2
accumulation. Anti-Clb2 western blot showing accumulation of low levels of p56Clb2 and
p45Clb2 when Mps1 is overexpressed and Mad2 is absent.
Cells expressing Clb2 lacking the first 79 amino acids are more resistant to benomyl
Since the cleavage site has yet to be identified, we chose to simulate p45Clb2 by
removing the first 79 amino acids, as this would leave the KEN box intact while
removing the D box, and retaining the endogenous promoter. Spot tests were performed
to investigate the effect of accumulation of p45Clb2 in regulation of mitosis. As controls,
we used a strain lacking the whole CLB2 ORF (clb2Δ) or expressing only the aminoterminal 360 amino acids (clb2-ΔC), or the DNA-damage checkpoint deficient strain
rad53Δ sml1Δ, which grows poorly in the presence of hydroxyurea.
On YPD, growth of clb2Δ and clb2-Δ79N were comparable and both grew less
than wild type (Figure III-11, top left panel). Surprisingly, this truncated protein
conferred increased benomyl resistance to otherwise wild type cells, shown in Figure III11 (top right and bottom left panels). No significant difference was observed when cells
were treated with hydroxyurea (Figure III-11, bottom right panel). Overall, this indicates
that truncated Clb2 slows progression through the cell cycle, which fits with our proposed
hypothesis that truncated Clb2 functions to prevent premature mitotic exit.
28
Fig. III-11: Cells expressing Clb2 lacking the first 79 amino acids are more
resistant to benomyl. (Top left) clb2Δ and clb2-Δ79N growth was comparable and
less than wild type on rich YPD media. (Top right) clb2-Δ79N cells grew better
than wild type on plates containing 10 µg/ml benomyl. (Bottom left) clb2-Δ79N
cells grew markedly better than wild type on plates containing 20 µg/ml benomyl.
(Bottom right) clb2-Δ79N cells showed no difference in growth from wild type on
0.2 M hydroxyurea plates.
29
Overexpression of Clb2 lacking the first 79 amino acids delays mitotic exit
Constructs were generated to add a pGALL promoter and an N-terminal GFP tag to either
wild type Clb2 (GFP-Clb2) or to an N-terminal truncation of Clb2 lacking the first 79
amino acids (GFP-Clb2-Δ79N). Haploid and diploid strains expressing either GFP-Clb2
or GFP-Clb2-Δ79N were constructed; the diploid strains maintained one wild type CLB2
allele.
Cells were grown overnight in YEP containing 2% raffinose, and overexpression
was induced by adding galactose to 2%. Anti-Clb2 western blots shown in Figure III-12A show that more GFP-Clb2-Δ79N accumulated than GFP-Clb2 in both haploid and
diploid strains. Flow cytometry analysis in Figure III-12-B shows that haploid cells
expressing GFP-Clb2 began exiting mitosis by about four hours after galactose induction,
indicated by peaks at both 1N and 2N DNA content; however, cells expressing GFPClb2-Δ79N were unable to exit mitosis by five hours after galactose induction, as
evidenced by the absence of the 1N peak. This inability to normally exit mitosis further
supports our hypothesis that p45Clb2 functions to prevent premature mitotic exit.
Figure III-12-C shows that in cells containing only full-length GFP-Clb2, GFPClb2 did not localize strongly to any particular part of the cell; however, GFP-Clb2-Δ79N
localized to the nucleus, which supports our hypothesis that the truncated form of Clb2 is
nuclear.
30
A.
anti-Clb2
anti-Clb2
B.
C.
Fig. III-12: Overexpression of clb2-Δ79N delays mitotic exit. (A) Anti-Clb2 western
blot showed greater accumulation of GFP-Clb2-Δ79N than GFP-Clb2. (B) Flow
cytometry showed GFP-Clb2 cells exited mitosis by about four hours after galactose
induction, while GFP- Clb2-Δ79N cells did not exit mitosis by five hours after
galactose induction. (C) GFP-Clb2-Δ79N accumulated in the nucleus, but GFP-Clb2
did not show strong localization.
31
CHAPTER IV: DISCUSSION
Presence or absence of Clb2 is essential for proper mitotic progression
Clb2 is the major mitotic cyclin in Saccharomyces cerevisiae, and the presence or
absence of Clb2 directly affects Cdc28 CDK activity. A decrease in CDK activity is
necessary for cells to exit mitosis. This is accomplished through two waves of Clb2
ubiquitination by the APC; the first wave of ubiquitination occurs at anaphase and is
mediated through Cdc20’s recognition of the D box, and the second wave occurs from
telophase through G1 and is mediated through Cdh1’s recognition of the KEN box.
Lower molecular weight band is specific to Clb2
We have identified what appears to be a truncated form of Clb2 (p45Clb2) with a
distinct pattern of accumulation when compared to full-length Clb2 (p56Clb2). p45Clb2
accumulates under conditions that induce mitotic arrest; several vertebrate cyclins are
known to be cleaved during mitotic catastrophe.
Since a C-terminal hemagglutinin tag on Clb2 is still detectable on the truncated
form of Clb2, cleavage must occur at the N-terminus of Clb2. Our current hypothesis is
that p56Clb2 is cleaved between the D box and KEN box. This cleavage removes the first
NES along with the D box, preventing ubiquitination of Clb2 via APCCdc20. This results
in a form of Clb2 resistant to APCCdc20-mediated degradation and allows p45Clb2 to
accumulate, as has been observed in vertebrate cyclins. In contrast to cleaved vertebrate
cyclins, which are only missing part of the cytoplasmic retention signal, Clb2 loses the
entire first NES, and, subsequently, p45Clb2 localizes to the nucleus. Since this truncated
form of Clb2 still contains an intact KEN box, it can still be targeted for degradation via
APCCdh1, allowing the cells to still effectively degrade Clb2 and exit mitosis.
32
p45Clb2 has distinct accumulation patterns
Cleavage of vertebrate cyclins occurs during mitotic catastrophe, so it seems
reasonable to think that cleavage of Clb2 would also occur under conditions that induced
mitotic arrest. When cells were arrested in metaphase with nocodazole, both p56Clb2 and
p45Clb2 accumulated in response. When cells were arrested in G1 with alpha factor, when
Clb2 has been completely degraded, both forms of Clb2 disappeared; once the cells
overcame the arrest five hours after treatment, both forms of Clb2 returned at low levels.
Cells were also arrested in S phase by using hydroxyurea, and p45Clb2 accumulated,
although at lower levels than resulted from nocodazole treatment. This low level of
p45Clb2 accumulation may be in response to activation of the DNA damage checkpoint as
Clb2 has been shown to interact with several proteins involved in the DNA repair
pathway.
High levels of p45Clb2 also accumulate in response to inactivation of the APC and
activation of the spindle checkpoint by inactivating or removing necessary components of
the APC or spindle checkpoint, respectively, which shows that this effect is not an artifact
of treatment with nocodazole. By accumulating a form of Clb2 resistant to Cdc20mediated degradation, the cell is maintaining a population of Clb2 that remains bound to
Cdc28, and thus, may provide a means for the cell to prevent premature exit from mitosis.
With p45Clb2 still bound to Cdc28, Cdc28 will remain active and the cell will not exit
mitosis. Therefore, generating this truncated form a Clb2 may be a failsafe to prevent
premature mitotic exit, which otherwise could lead to cell death.
33
Cells expressing only clb2-Δ 79N are more resistant to mitotic inhibitors and delay
mitotic exit
Cells expressing only clb2-Δ79N displayed several interesting phenotypes. First,
these cells were more resistant to mitotic inhibitors, such as benomyl. Second,
overexpression of clb2-Δ79N, to simulate high levels of accumulation, delayed exit from
mitosis. These results together indicate that clb2-Δ79N is functioning to delay mitotic
exit, specifically under conditions that activate the spindle assembly checkpoint or
inactivate the APC. By preventing premature mitotic exit, the cell is able to protect the
integrity of its genetic material.
p45Clb2-Cdc28 complexes may have different roles from p56Clb2-Cdc28 complexes
Also, similar to cleaved vertebrate cyclins, both cyclin boxes of p45Clb2 are still
present and p45Clb2 remains associated with Cdc28. This indicates that the Cdc28-p45Clb2
complex may be functional, and it is possible that the Cdc28-p45Clb2 and Cdc28-p56Clb2
complexes have distinct functions. As seen in Figure A-1, the N-termini of cyclins are not
very conserved and are thought to contribute to the specificity of each cyclin, which lends
credence to the idea that these two complexes may have different substrates. Additional
support for these differing roles is shown in Figure III-8, which shows that p45Clb2 levels
correlate with p56Clb2 levels throughout the cell cycle. Since p45Clb2 is present throughout
the cell cycle and peaks when p56Clb2 levels peak, it seems likely that p45Clb2 also affects
cell cycle progression. Unlike vertebrates, yeast cells are viable in the absence of Mad2
and Bub2, which indicates that p45Clb2 may be performing a role in the spindle assembly
checkpoint.
34
p45Clb2 may function to maintain high CDK activity to prevent premature mitotic
exit
Activation of the spindle assembly checkpoint or inactivation of the anaphasepromoting complex results in a significant increase in Clb2 accumulation, with much
higher levels of p45Clb2 than p56Clb2. An increase in the nuclear population of Clb2 that is
resistant to ubiquitination by APCCdc20 would allow cells to maintain high CDK activity
to prevent premature exit from mitosis. p45Clb2 could also help ensure that enough Clb2 is
in the nucleus to carry out nuclear functions necessary for completion of mitosis. In this
regard, a recent model proposed by Amon and colleagues suggests that thresholds of
CDK activity are needed to ensure that the myriad of mitotic processes take place in a
coordinated manner (Rahal 2008). We believe that cleavage and accumulation of p45Clb2
is a mechanism that allows yeast cells to finely regulate mitotic progression to allow the
presence of high levels of CDK activity in the nucleus when needed.
Conclusions about p45Clb2
Similar to vertebrate cyclins, p45Clb2 remains associated with Cdc28, indicating
that this complex is functional. Since the N-termini of cyclins dictates their specificity
and p45Clb2 levels parallel p56Clb2 levels, it is logical to think that Cdc28-p45Clb2
complexes are functional and most likely have distinct functions from Cdc28-p56Clb2
complexes. Since p45Clb2 accumulates in response to activation of the spindle assembly
checkpoint and inactivation of the APC and overexpression clb2-Δ79N cells delays
mitotic exit, it can be inferred that p45Clb2 may function to prevent premature mitotic
exit under conditions that activate the spindle assembly checkpoint. Since, unlike
vertebrates, yeast cells are viable in the absence of Mad2 and Bub2, p45Clb2 may be
35
performing a role in either regulating the spindle assembly checkpoint. These inferences
suggest that p45Clb2 may be acting as a “failsafe” in case the spindle assembly checkpoint
is compromised.
Future directions
The next step is to identify the cleavage site via mass spectrometry analysis. Once
the cleavage site is identified, a more accurate truncation can be created to repeat many of
the above experiments to confirm the results. Kinase assays will also be performed to
compare the activity of Cdc28-p56Clb2 and Cdc28-p45Clb2. In addition, the cleavage site
will also be mutated to create an uncleavable form of Clb2, which will then be
characterized to provide more insight into the role of truncated Clb2.
36
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APPENDIX
42
Abbreviations:
APC: Anaphase-Promoting Complex
CAK1: CDK-activating kinase 1
CDK: Cyclin-dependent kinase
Clb2: B-type cyclin
D box: Destruction box
KEN: Lysine, Glutamate, Asparagine motif
NES: Nuclear export signal
NLS: Nuclear localization signal
p45Clb2: truncated Clb2 (~45 kDa)
p56Clb2: Full-length Clb2 (56 kDa)
SPB: Spindle pole body
WT: Wild type
YPD: rich media (yeast extract, bactopeptone, glucose)
3HA: three tandem hemagglutinin epitope (hemagglutinin residues 98-106)
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Fig. A-1: Alignment of mitotic B-type cyclins. The C-termini,
containing the cyclin boxes, are very conserved among all of the
mitotic cyclins. However, the N-termini are not very conserved and the
variation in the N-termini is thought to contribute to each cyclin’s
specificity.
44
VITA
Dustin Kimbrough Stutts was born in Knoxville, TN. She graduated from Central
High School in Knoxville, TN in 2000. She developed an interest for science early in her
college career and graduated from the University of Tennessee, Knoxville in 2006 with a
bachelor’s degree in Biochemistry and Cellular and Molecular Biology. In the fall of
2006, she began graduate school at the University of Tennessee, Knoxville in the
Biochemistry and Cellular and Molecular Biology department. She graduated with her
Masters of Science in August 2010 at the University of Tennessee, Knoxville.
45