non$proteolytic-ubiquitylation regulates-the-apc/c
TRANSCRIPT
Non-proteolytic ubiquitylation regulates the APC/C-inhibitory
function of XErp1function of XErp1
Doktors der Naturwissenschaften (Dr. rer. nat.)
vorgelegt von
2. Referent: Prof. Dr. Martin Scheffner
3. Referent: Prof. Dr. Olaf Stemmann
1.2. The APC/C counteracts the activity of Cdk1 7
1.3. The “wait anaphase signal”: The SAC inhibits the APC/C in mitosis 9
1.4. Regulation of APC/CCdc20 activity in meiosis 11
1.5. The postulation of MPF and CSF 12
1.6. The discovery of Mos as a CSF component 13
1.7. Identification of the CSF component XErp1 14
1.8. XErp1 inactivation upon CSF release 15
1.9. The molecular mechanism of XErp1 mediated APC/C inhibition 16
1.10. Feedback loops controlling XErp1 activity during CSF arrest 18
1.11. Aim of this project 20
2. RESULTS 21
2.1. UbcX can suppress SAC activity in Xenopus egg extract 21
2.2. UbcX can suppress CSF activity in Xenopus egg extract 22
2.3. Elevated UbcX activity prevents meiosis I - meiosis II transition in
Xenopus oocytes 24
activity 25
2.5. Does USP44 counteract UbcX to maintain CSF arrest? 26
2.6. An eight-fold increase in UbcX activity is required for CSF release. 27
2.7. UbcX levels increase during oocyte maturation and remain constant
during CSF release and embryonic cell cycles 28
2.8. UbcX dependent CSF release can be suppressed by XErp1 29
2.9. UbcX mediated ubiquitylation disrupts the APC/C - XErp1 complex 30
2
2.10. XErp1 is the main target of UbcX mediated ubiquitylation in CSF
extract 32
2.11. Ubiquitylation of XErp1 is dependent on the APC/C and independent
of SCFβ TRCP 33
2.12. Dissociation of XErp1 upon Cdk1 phosphorylation does not require
ubiquitylation 35
2.13. Cdc20 degradation is not required for CSF arrest maintenance 36
3. DISCUSSION 38
3.1. Regulation of spindle checkpoint signaling by UbcH10/UbcX 39
3.1.1. The spindle assembly checkpoint can be inactivated by UbcX in
Xenopus egg extract 39
3.1.2. Is an APC/C inhibitor targeted for ubiquitylation during SAC
signaling? 41
3.2. UbcX mediated ubiquitylation of XErp1 regulates its APC/C inhibitory
activity 43
3.2.1. Cdc20 is not destabilized in CSF arrested egg extract 43
3.2.2. UbcX mediated ubiquitylation of XErp1 regulates its APC/C inhibitory
activity 44
3.2.3. Are ubiquitin hydrolases counteracting the activity of UbcX during CSF
arrest? 46
3.3. Is the regulation of UbcX activity important during the meiotic cell
cycle? 48
3.3.2. Could UbcX participate in the inactivation of XErp1 upon
fertilization? 48
pathways regulating the activity of XErp1 49
3.4. Could ubiquitylation of XErp1 be required for its APC/C inhibitory
activity? 50
5.2. Plasmids 55
5.2.3. Cloning and Mutagenesis 57
5.3. Proteins 57
5.3.2. His-tagged protein expression in SF9 cells 58
5.3.3. His-tagged protein purification from bacteria and SF9 cells 58
5.3.4. Coupled in vitro transcription/translation (IVT) 59
5.4. Antibodies 59
5.4.2. Affinity purification of antibodies 59
5.5. Gel electrophoresis and immunoblot analysis 60
5.6. Xenopus egg extracts 61
5.6.1. Xenopus CSF egg extract preparation 61
5.6.2. Extract manipulations 62
5.7. Xenopus oocyte injections 64
6. LITERATURE 65
7. APPENDIX 75
7.1. Summary 75
7.2. Zusammenfassung 75
7.3. Acknowledgements 76
1. INTRODUCTION
Most eukaryotes reproduce sexually, where cells from two parents fuse to
generate a single cell, the zygote, which develops into a new organism (Figure
1.1.). Since the combination of two diploid cells would lead to the duplication
of the chromosomal content at every generation, sexual reproduction depends
on a process called meiosis.
Figure 1.1. The life cycle of vertebrates. Cells in vertebrates proliferate mitotically in the diploid phase to form a multicellular organism. Sexual reproduction begins with meiosis to generate haploid cells, which fuse upon fertilization to form a new organism.
1.1. Meiosis and meiotic maturation
Meiosis is a specialized form of nuclear division that leads to the generation of
cells containing half the normal complement of chromosomes from diploid
oocytes (Figure 1.2. a, Alberts et al., 2002). (Alberts et al., 2002).
Before entering the meiotic program, oocytes are diploid like somatic cells and
contain two copies of each chromosome, one of them inherited from each
parent. Meiosis begins with an S-phase (Petronczki et al., 2003) in which
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chromosomes are replicated to produce sister chromatid pairs tightly linked by
cohesion (Klein et al., 1999). Next, the duplicated homologues pair to form
tetrads and undergo homologues recombination, a process important for
generating genetic variation and to guarantee accurate segregation of the
homologues at the following nuclear division. Homologous recombination
starts with the introduction of DNA double-strand breaks (DSB) at almost
variable positions along the chromosome (Sun et al., 1989). In most of the
cases, DSBs are repaired without rendering the DNA sequence of the two
homologs. Sometimes however, the repair leads to the formation of a
continuous DNA strand between two homologous chromatids, which can lead
to a reciprocal DNA exchange or crossover (Allers and Lichten, 2001). The
result is a strong physical linkage between the two homologous chromosomes
as long as the sister chromatid arms are held together by cohesion. As a result,
the homologous chromosomes become bioriented on the first meiotic spindle
and after cohesin cleavage at the chromosome arms at anaphase I, exactly one
of the two homologous chromosomes is segregated into each daughter cell
(Buonomo et al., 2000). After the completion of meiosis I, cells enter directly
the next division cycle without replicating the chromosomes. In meiosis II,
similar to mitosis, sister chromatids are divided into the two daughter cells by
the cleavage of centromeric cohesion upon anaphase II onset. Together,
meiotic divisions result in the production of four haploid cells, which can be
differentiated into special reproductive cells, i.e. the egg and the sperm.
In animals, oocytes arrest before the first meiotic division at prophase I, and
these immature oocytes or stage VI oocytes can stop at this point for decades
(Hunt, 1989). The production of a fertilizable egg from such an immature
oocyte involves a process called oocyte maturation (Figure 1.2. b). Upon
hormonal induction, immature oocytes resume meiosis I and undergo germinal
vesicle breakdown (GVBD) which is visible on the surface of the oocytes by the
appearance of a white dot. Meiosis I is completed with the extrusion of the
first polar body after which the oocytes proceed directly through meiosis II
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where the second polar body is extruded and haploid gametes are produced.
In vertebrates like Xenopus laevis, oocytes complete meiotic maturation with
an arrest at metaphase of meiosis II, in which they await fertilization. From the
viewpoint of cell-cycle control, the major questions are concerning the
mechanisms underlying the induction and regulation of oocyte maturation as
well as the arrest of mature oocytes at metaphase of meiosis II and its release
upon fertilization (Tunquist and Maller, 2003).
Figure 1.2. The meiotic program. (a) In meiosis, after DNA replication, two divisions generate haploid gametes. For clarity, only one chromosome is depicted. (b) Meiosis in vertebrates is arrested at two stages. After DNA synthesis, the oocytes grow to their final size and arrest at meiotic prophase I. Progesterone induces meiotic maturation and the production of an egg arrested at meiotic metaphase II. Fertilization triggers the completion of Meiosis II and a diploid zygote is formed (Adapted from Morgan, 2007).(Morgan, 2007)
1.1. Cdk1/cyclin B drives the meiotic cell cycle
The ordered progression of the meiotic cell cycle, like the mitotic cell cycle, is
mediated mainly by the activity of cyclin dependent kinases (Cdks) and
ubiquitin ligases (Murray, 2004). Cdks are serine-threonine kinases that are
activated by their regulatory subunit, the cyclins. In mitotic G1, low Cdk1
activity is important for the resetting of the origins of DNA replication. Rising
Cdk activity triggers the firing of DNA replication origins and as S-phase
progresses and DNA replication continues, the activity of Cdk1/CylinB1
promotes entry into mitosis, which is characterized by nuclear envelope
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condensation. After the successful division of the replicated chromosomes into
two daughter cells, the cell needs again low Cdk1 activity to exit mitosis and to
enter G1. Therefore, low Cdk activity followed by high activity links DNA
replication to progression through mitosis (Porter, 2008) – the basis for the
mitotic cell cycle.
In Xenopus meiosis, the hormone progesterone induces entry into metaphase I
by the activation and amplification of Cdk1/cyclin B by inducing both the
dephosphorylation of inhibitory residues on Cdk1 and the accumulation of
cyclin B (Tunquist and Maller, 2003). Progression from metaphase I to
anaphase I is accompanied by a drop in cyclin B levels and decreasing Cdk1
activity. But unlike in mitotic cells, cyclin B is not completely degraded upon
anaphase onset but appears to be reduced to half (Furuno et al., 1994;
Iwabuchi et al., 2000). While it remains controversial whether this drop in
cyclin B levels is required for meiotic progression (Peter et al., 2001; Taieb et
al., 2001), the inhibition of complete cyclin B degradation is essential for the
persistence of M-phase and the inhibition of DNA replication (Ohe et al., 2007).
Thus, the oocytes directly enter a second M-phase, where the stabilization of
cyclin B levels is important for establishing the second meiotic arrest. Upon
fertilization, cyclin B is degraded, Cdk1 is inactivated and the zygotes enter
mitotic cell cycles.
1.2. The APC/C counteracts the activity of Cdk1
Anaphase onset requires the inactivation of both Cdk1 kinase and the
inactivation of the anaphase inhibitory protein securin. Securin prevents
cohesin cleavage and thus the irreversible step of sister chromatid separation
by keeping the cohesin directed protease separase inactive (Uhlmann et al.,
1999; Uhlmann et al., 2000). Both, Cdk1/cyclin B and securin activity is
regulated by the E3 ubiquitin ligase anaphase promoting complex/cyclosome
(APC/C). It mediates the specific ubiquitylation of cyclin B and securin (Sudakin
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et al., 1995; Zou et al., 1999) thereby targeting them for destruction by the 26
S proteasome at anaphase onset.
The APC/C is an unusual large E3 ubiquitin ligase that consists of at least 13
subunits including proteins with cullin and RING-finger domains (Zachariae and
Nasmyth, 1999). In addition, the APC/C associates with coactivator proteins
called Cdc20 and Cdh1 (Pesin and Orr-Weaver, 2008), which bind transiently to
the APC/C core complex and are thought to regulate both the activity and
substrate specificity of the APC/C. While in somatic mitotic cell cycles, the
coactivator of the APC/C alternates between Cdc20 and Cdh1, the main
coactivator required for meiosis and early embryonic cell cycles has been
reported to be Cdc20 (Lorca et al., 1998). The APC/C together with its
coactivator is responsible for substrate recognition and thus confers specificity
to the ubiquitylation reaction (Peters, 2006). It functions at the last step of a
cascade of enzymes that sequentially act to transfer ubiquitin to the target
protein (Hershko and Ciechanover, 1998). Free ubiquitin is first covalently
attached to an ubiquitin-activating enzyme E1 via a thioester bond. It is then
transferred to an ubiquitin-conjugating enzyme E2 where it forms a thioester
bond with the active site cystein. The main E2 enzyme cooperating with the
APC/C has been identified in clam as E2-C (Hershko et al., 1994) and orthologs
were found in Xenopus named UbcX (Yu et al., 1996), and in humans named
UbcH10 (Townsley et al., 1997). In Xenopus, UbcX is essential for APC/C
activity, since a dominant negative mutation in the active site cystein (C114S)
inhibits APC/C dependent substrate ubiquitylation (Townsley et al., 1997), and
the depletion of UbcX inhibits APC/C substrate degradation (data not shown).
In the final step of APC/C dependent ubiquitylation, the E2-bound ubiquitin is
covalently attached to a lysine residue in the target protein. In this reaction,
the APC/C is thought to approximate the substrate and the E2-ubiquitin and to
position them for efficient ubiquitin transfer (Peters, 2006). Recently, it has
been shown that in human cells, UbcH10 forms an E2-enzyme module with
Ube2S, and both enzymes were shown to be important for the formation of
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ubiquitin chains on APC/C substrates, where UbcH10 conjugates the first
ubiquitin to the lysine residue of the substrate and Ube2S then elongates the
ubiquitin chain (Garnett et al., 2009; Williamson et al., 2009; Wu et al., 2010).
As a consequence, ubiquitylation can target proteins to the 26 S proteasome, a
high molecular weight protease complex that hydrolyses its substrates into
short peptides and thus inactivates them irreversibly. Alternatively,
ubiquitylation can act as a reversible posttranslational modification of a
protein to regulate its activity (Hershko and Ciechanover, 1998).
1.3. The “wait anaphase signal”: The SAC inhibits the APC/C in mitosis
Mitotically and meiotically dividing cells depend on ubiquitin-mediated
proteolysis of key cell-cycle regulators at the correct time (Pesin and Orr-
Weaver, 2008). In mitosis, a conserved mechanism called the spindle assembly
checkpoint (SAC) guarantees an equal segregation of the chromosomes to the
two nascent daughter cells (Musacchio and Salmon, 2007). The SAC is activated
by missattached or unattached kinetochores (Nicklas et al., 1995; Rieder et al.,
1995; Rieder et al., 1994) and prevents the APC/C from ubiquitylating cyclin B
and securin. Although it is not yet completely understood how the SAC
inactivates the APC/C, it is well accepted that the primary target of the SAC is
the APC/C coactivator Cdc20 (Hwang et al., 1998; Kim et al., 1998) and that
SAC activity is propagated by a number of conserved proteins including Mad1,
Mad2 and Bub3/BubR1 (Hoyt et al., 1991; Li and Murray, 1991). Current
models of SAC mediated APC/C inactivation suggest that Mad2 binds to Cdc20
in conjunction with BubR1 and Bub3 to form the “Mitotic Checkpoint Complex”
(MCC), which binds to the APC/C and renders it inactive (Sudakin et al., 2001).
Once all kinetochores are properly attached, it has been suggested that the
inhibitory MCC complexes have to be actively dissociated by APC/C dependent,
non-proteolytic ubiquitylation of Cdc20 to turn off the SAC. Specifically, it has
been shown that addition of the E2 ubiquitin conjugating enzyme UbcH10 to
SAC-arrested cell extract triggers the APC/C-dependent multi-ubiquitylation of
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Cdc20, and possibly other components of the APC/C–Cdc20-MCC complex,
resulting in the release of Mad2 and BubR1 from Cdc20 (Reddy et al., 2007). In
checkpoint arrest conditions, this ubiquitylation reaction is antagonized by the
activity of the ubiquitin hydrolase USP44 (Figure 1.3.), which removes ubiquitin
from Cdc20 (Stegmeier et al., 2007). As soon as the last kinetochore is
attached, ubiquitylation of Cdc20 is thought to exceed its deubiquitylation,
Cdc20 is freed from the MCC and the APC/C can be rapidly activated in a
switch-like manner.
Figure 1.3. Dynamic ubiquitylation and deubiquitylation regulate SAC activity. During mitotic checkpoint arrest, ubiquitylation of Cdc20 by UbcX, which leads to the dissociation of the APC/C inhibitors Mad2 and BubR1, needs to be counteracted by USP44 dependent deubiquitylation of Cdc20 to maintain SAC mediated APC/C inhibition.
A different model contradicts this view of SAC arrest and instead suggests that
in cells with an active SAC, Cdc20 in complex with the MCC proteins is
ubiquitylated and targeted for destruction, and this degradation is important
for inactivating the APC/C (Ge et al., 2009; Nilsson et al., 2008). Supporting this
model, experiments in budding yeast and human cells have shown that Cdc20
is ubiquitylated and degraded during SAC arrest and overexpression of Cdc20
could overcome the SAC mediated inhibition of the APC/C (King et al., 2007;
Pan and Chen, 2004). Importantly, a non-ubiquitylatable form of Cdc20 where
every lysine was mutated to an arginine was insensitive to the checkpoint
arrest and activated the APC/C (Nilsson et al., 2008). These results contradict a
model where Cdc20 ubiquitylation causes its activation and rather support the
latter model where ubiquitylation inactivates Cdc20.
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The regulation of APC/C activity is especially important during oocyte
maturation in vertebrates where meiosis is arrested twice to coordinate oocyte
development with the events of meiosis (Figure 1.4.).
In prophase I, the APC/C has to be inactive to maintain chromosome cohesion
(Pesin and Orr-Weaver, 2008). When oocytes mature, the APC/C needs to
become active at the metaphase I - anaphase I transition to allow the
degradation of securin and the separation of the homologous chromosomes
(Buonomo et al., 2000; Siomos et al., 2001). In contrast to all organisms tested,
the requirement of the APC/C for meiosis I - meiosis II transition is
controversial in Xenopus. Although microinjections of Xenopus oocytes with
inhibitory antibodies or antisense oligonucleotides directed against the APC/C
coactivator Cdc20 did not disrupt progression through meiosis I (Peter et al.,
2001; Taieb et al., 2001), it is possible that these approaches did not eliminate
APC/C activity completely. Nevertheless, the complete degradation of cyclin B
must be prevented also in Xenopus to maintain M-phase and to inhibit S-phase
(Ohe et al., 2007), suggesting that the APC/C needs to be regulated to
contribute to this modulation of cyclin B levels.
Figure 1.4. Oocyte maturation on a molecular level: Cdk1 and APC/C. The cell cycle in meiosis is driven by the activity of Cdk1/cyclin B which is counteracted by the APC/C, the relative activities of which through the maturation process are illustrated (adapted from Wu and Kornbluth, 2008).
At the second meiotic arrest at metaphase II, the APC/C needs to be inhibited
to stabilize cyclin B and securin to prevent premature anaphase onset and
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parthenogenetic activation of the egg. Upon fertilization, APC/C activation is
required to induce the exit from the metaphase II arrest (Lorca et al., 1998;
Peter et al., 2001) and thereby allowing entry into early embryonic cell cycles.
While the spindle checkpoint is important for the metaphase arrest and APC/C
inhibition in mitotic cells in the presence of unattached kinetochores, it is
unlikely that the SAC mediates the metaphase arrest observed in mature
vertebrate eggs. Evidence against such a hypothesis includes the fact that CSF
arrest is terminated by fertilization and the following elevation in cytoplasmic
calcium levels, but calcium addition does not overcome SAC arrest (Minshull et
al., 1994). Additionally, the SAC requires kinetochores and microtubule
depolymerization, whereas neither is required for meiotic metaphase II arrest
(Tunquist and Maller, 2003). What inhibits oocytes at metaphase of Meiosis II?
1.5. The postulation of MPF and CSF
In 1971, Yoshio Masui and Clement L. Markert performed experiments in Rana
pipiens oocytes and embryos that became fundamental for the identification of
the mechanisms mediating the metaphase II arrest in mature oocytes (Masui
and Markert, 1971).
Specifically, they observed that injection of immature oocytes with endoplasm
of mature oocytes induced meiotic maturation. Therefore they postulated that
maturation is induced by a maturation promoting factor (MPF) which is
released by hormonal induction and remains active in the mature egg (Figure
1.5.). To analyze whether the same activity could accelerate cell divisions in
embryonic cells, they injected endoplasm of the mature egg into one cell of a
two-cell stage embryo. Surprisingly, they found that the injected blastomere
arrested at the next mitosis, prompting them to propose the existence of a
cytostatic factor (CSF) present in the mature egg that is responsible for
inducing the metaphase II arrest (Figure 1.5.). Additionally, this activity is
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inactivated upon fertilization, since injection of blastomeres with endoplasm of
fertilized embryos did not cause cell-cycle arrest.
Figure 1.5. The discovery of MPF and CSF. Illustration of the oocyte- and blastomere-injection assays originally performed by Masui and Markert in 1971 that led to the identification of the maturation promoting factor MPF and the cytostatic factor CSF.
While MPF was soon identified to be the activity of cyclin dependent kinase
Cdk1 together with its regulatory subunit cyclin B (Gautier et al., 1990; Gautier
et al., 1988; Lohka et al., 1988; Murray et al., 1989), the discovery of the
molecular identity of the CSF took more than three decades.
1.6. The discovery of Mos as a CSF component
To identify the CSF activity that mediates the metaphase II arrest, three criteria
were proposed for a protein or an activity to be a CSF: (1) The activity emerges
during oocyte maturation and peaks in the metaphase II arrested egg. (2)
Injection of blastomeres with the activity induces mitotic arrest and (3)
fertilization triggers the inactivation of the factor (Masui and Markert, 1971).
The first protein identified meeting these criteria was the kinase Mos. Mos is
expressed during oocyte maturation (Sagata et al., 1988); Figure 1.6.), it could
induce mitotic arrest when injected into blastomeres of a dividing embryo
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(Sagata et al., 1989) and it was degraded upon fertilization (Lorca et al., 1991).
To understand the detailed molecular mechanism linking Mos to the
metaphase II arrest, the signaling pathway of the kinase was investigated.
Biochemical analysis revealed that Mos can activate the mitogen activated
protein kinase (MAPK) pathway (Posada et al., 1993) resulting in the activation
of the ribosomal S6 kinase (Rsk), and functional analysis of the members of this
pathway showed that they are required for CSF arrest (Abrieu et al., 1996;
Bhatt and Ferrell, 1999; Cross and Smythe, 1998; Gotoh and Nishida, 1995;
Gross et al., 1999; Haccard et al., 1993; Kosako et al., 1994a, b). Therefore, the
Mos activated MAPK-pathway was proposed to be a molecular component of
the CSF. Since both, the Mos-MAP kinase pathway and APC/C inhibition are
responsible for CSF arrest, it seemed possible that these two pathways are
interconnected. However, it remained unclear how Rsk as the terminal kinase
in this cascade was communicating with the cell-cycle machinery to establish
the CSF arrest.
1.7. Identification of the CSF component XErp1
Reportedly, polo-like kinase Plx1 is required CSF inactivation and APC/C
activation (Descombes and Nigg, 1998). Specifically, it has been shown that
Xenopus egg extracts depleted of Plx1 fail to release the CSF arrest upon
increasing cytoplasmic calcium levels. Therefore, a yeast two-hybrid screen
was performed to identify proteins that interacted with Plx1 (Schmidt et al.,
2005), and this approach led finally to the identification of the sought after
component of CSF, the XErp1 protein. XErp1 nicely satisfied the Masui and
Markert criteria proposed for CSF. First, XErp1 is synthesized during Xenopus
oocyte maturation; it starts to be detectable at the MI-MII transition and it
accumulates as oocytes proceed through meiosis II where it reaches highest
levels at metaphase II (Figure 1.6.); second, exogenous introduction of XErp1
into one blastomere of a two-cell stage embryo promoted a cell-cycle arrest
and third, XErp1 was degraded after fertilization in a Plx1 dependent manner.
15
Importantly, XErp1 is essential for CSF arrest as Xenopus egg extracts arrested
at metaphase II depleted of XErp1 were unable to maintain CSF arrest and
entered interphase.
Further characterization XErp1 revealed the C-terminus of the protein, which is
sufficient for CSF arrest maintenance, shares high sequence similarity with the
mitotic APC/C inhibitor Emi1 and like Emi1, XErp1 was shown to inhibit the
APC/C directly (Schmidt et al., 2005). Therefore, XErp1 is a CSF specific APC/C
inhibitor.
Figure 1.6. Oocyte maturation and CSF on a molecular level. Oocyte maturation is driven by the activities of Cdk1/cyclin B, the APC/C and CSF factors Mos and XErp1, ad the relative activities during oocyte maturation are depicted on the left (adapted from Kornbluth, 2008).
Since XErp1 was shown to be a substrate of Rsk, the Mos-MAPK pathway could
finally be linked to the regulation of the APC/C. Rsk phosphorylation was
shown to increase the inhibitory activity of XErp1 in CSF arrested eggs, which
will be described later.
1.8. XErp1 inactivation upon CSF release
As proposed by Masui and Markert, fertilization causes the inactivation of CSF.
The first response of an egg to fertilization is an elevation in cytoplasmic
calcium levels, which results in the activation of calcium/calmodulin dependent
kinase II (CaMKII;(Lorca et al., 1993). The identification of XErp1 as a CaMKII
16
substrate provided insights into how fertilization is connected with CSF
inactivation (Figure 1.7.;(Hansen et al., 2006; Liu and Maller, 2005; Rauh et al.,
2005).
Figure 1.7. Fertilization mediated CSF inactivation. Fertilization (1) triggers the activation of CaMKII (2) which phosphorylates XErp1 (3) creating a docking site for Plx1 (4). Plx1 in turn phosphorylates XErp1 creating a phosphodegron (5), which is recognized by the ubiquitin ligase SCFβ
TRCP. XErp1 ubiquitylation targets it for degradation (6) and thus CSF inactivation, the APC/C becomes active (7) and cells complete meiosis II (adapted from Rauh et al., 2005).
CaMKII mediated phosphorylation of XErp1 provides a docking site for Plx1 on
XErp1. Through Plx1 mediated phosphorylation of XErp1 a phosphodegron is
created and XErp1 is recognized by the SCFβ TRCP complex, an ubiquitin E3 ligase
that ubiquitylates and targets XErp1 for degradation. Consequently, calcium
triggers CSF inactivation resulting in APC/C activation and the fertilized egg can
proceed with embryonic cell divisions.
1.9. The molecular mechanism of XErp1 mediated APC/C inhibition
In CSF arrested eggs, XErp1 maintains the metaphase II arrest by directly
inhibiting the APC/C. The binding of XErp1 to the APC/C is essential for its
inhibitory activity as mutants defective in APC/C binding are inefficient in
17
inhibiting the APC/C (Wu et al., 2007b). The well-conserved C-terminal peptide
sequence of XErp1, termed the RL tail, was reported to mediate the
recruitment of XErp1 by serving as a docking site to the APC/C (Ohe et al.,
2010). Binding to the APC/C allows and enhances the inhibitory interactions of
two other sequence elements of XErp1, the D-box and the ZBR-domain. While
it is well established that all three elements are critical for APC/C inhibition,
the specific contribution of the D-box and the ZBR domain to the inhibition of
the APC/C by XErp1 remain elusive (Nishiyama et al., 2007; Ohe et al., 2010;
Tang et al., 2010).
Notably, all three elements are conserved between XErp1 and Emi1, a somatic
paralog of XErp1, whose APC/C inhibitory activity is required to prevent DNA
re-replication (Di Fiore and Pines, 2007; Machida and Dutta, 2007) suggesting
that XErp1 and Emi1 share the same mode of APC/C inhibition. Emi1, when
bound to the APC/C together with the E2 enzyme UbcH10, was shown to
inhibit the correct engagement of the substrate to the APC/C thereby reducing
substrate ubiquitylation (Summers et al., 2008). Further studies on Emi1
suggested that it acts as an APC/C pseudosubstrate and the D-box mediates
APC/C binding, while its ZBR mediates APC/C inhibition (Miller et al., 2006).
Consistently, it has been shown that Emi1 mutated in its ZBR does not inhibit
the APC/C but rather is quickly targeted for destruction by the APC/C. Given
that XErp1 – like Emi1 – contains a D-box and ZBR, it is tempting to speculate
that XErp1 acts as well as a pseudosubstrate. However, previous studies
suggest that XErp1 does not compete with substrates for APC/C binding but
rather interferes with the transfer of ubiquitin to substrate proteins bound to
the APC/C (Tang et al., 2010). Furthermore, our preliminary experiments
revealed that in contrast to Emi1, mutation of the ZBR of XErp1 does not
convert it into an APC/C substrate corroborating the idea that XErp1 inhibits
the APC/C by a mechanism distinct to the one of Emi1.
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Together, although it is established that XErp1 needs to be recruited to the
APC/C to exert its inhibitory function, the exact molecular mechanism of XErp1
mediated APC/C inhibition remains elusive.
1.10. Feedback loops controlling XErp1 activity during CSF arrest
During metaphase II arrest, the Mos-MAPK pathway was shown to activate
XErp1 by upregulating both the stability and activity of XErp1 (Isoda et al.,
2011; Wu et al., 2007a; Wu et al., 2007b). The Mos-MAPK pathway activates
the kinase Rsk (Bhatt and Ferrell, 1999; Gross et al., 1999), which
phosphorylates XErp1 at residues in the central region (Inoue et al., 2007;
Nishiyama et al., 2007) leading to the recruitment of the protein phosphatase
PP2A containing the regulatory subunit B56β or B56ε to XErp1 (Wu et al.,
2007a). PP2A- B56β,ε antagonizes N-terminal and C-terminal inhibitory
phosphorylations of XErp1 by Cdk1 (Isoda et al., 2011). Cdk1 phosphorylations
destabilize XErp1 and decrease its affinity for the APC/C (Wu et al., 2007a; Wu
et al., 2007b).
Figure 1.8. Oocyte maturation and CSF on a molecular level. Oocyte maturation is driven by the activities of Cdk1/cyclin B, the APC/C and CSF factors Mos and XErp1, ad the relative activities during oocyte maturation are depicted on the left (adapted from Kornbluth, 2008). On the right, a simplified signaling network controlling the activity of XErp1 is illustrated (adapted from Isoda et al., 2011).
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Specifically, it has been shown that multiple N-terminal Cdk1 phosphorylation
motifs bind cyclin B1-Cdk1 itself as well as Plk1 and CK1 δ/ε to inhibit XErp1
(Isoda et al., 2011). While Plk1 phosphorylation was shown to partially
destabilize XErp1, Cdk1 and CK1δ/ε phosphorylations are thought to
cooperatively inhibit XErp1 binding to the APC/C (Figure 1.8.). Since Cdk1 levels
are high during the Metaphase II arrest, constant phosphorylation of XErp1
would lead to gradual XErp1 inactivation and CSF release. By recruiting PP2A-
B56β,ε to counteract the inhibitory phosphorylations, the Mos MAPK- pathway
keeps XErp1 active and therefore maintains CSF arrest (Figure 1.8.). At the
same time, this mechanism allows to maintain Cdk1 activity at the correct level
during CSF arrest (Figure 1.9.(Wu and Kornbluth, 2008; Wu et al., 2007b).
Continuous cyclin B synthesis during CSF arrest leads to a temporal increase in
Cdk1/cyclin B activity, which in turn leads to an increase in the phosphorylation
of XErp1, since the activity of PP2A on XErp1 remain equal. XErp1
phosphorylated by Cdk1 dissociates from the APC/C leading to a transient
APC/C activation and slow degradation of cyclin B.
Figure 1.9. Cdk1/cyclin B2 and PP2A regulate XErp1’s association with the APC/C. Phosphorylation of XErp1 by Cdk1/cyclin B2 leads to the dissociation of XErp1 from the APC/C, which is counteracted by PP2A, which dephosphorylates XErp1 and promotes XErp1 association with the APC/C.
Therefore, the continuous synthesis of cyclin B induces a slow degradation of
cyclin B during CSF arrest. Otherwise, continuous synthesis would create an
amount of cyclin B that cannot be degraded by the APC/C anymore in a short
time. This would result in a slow and gradual rather than a switch-like exit from
CSF arrest as observed upon fertilization.
20
1.11. Aim of this project
XErp1 is an APC/C inhibitor operating in CSF arrested oocytes. However, the
exact molecular mechanism of APC/C inhibition and its regulation is unknown.
The D-box and the RL-tail of XErp1 mediate the binding of XErp1 to the APC/C,
most likely to position the ZBR of XErp1 correctly to inactivate the APC/C.
However, the interaction with the APC/C needs to be dynamic to allow slow
cyclin B degradation during CSF arrest. Phosphorylation and dephosphorylation
of XErp1 can regulate its association with the APC/C, and the Mos-MAPK
pathway was shown to promote XErp1 association. Intrigued by the findings on
APC/C regulation by the spindle checkpoint, we would like to understand if a
dynamic balance of ubiquitylation/deubiquitylation of Cdc20, XErp1 and/or
other components of the APC/C is also required for CSF arrest. In addition, we
would like to test whether Cdc20 turnover is required for CSF arrest and if
XErp1 regulates this potential turnover. Thus, these studies will provide a
deeper understanding of how the XErp1-APC/CCdc20 interaction is regulated and
2. RESULTS
In this study, we show that non-proteolytic ubiquitylation of XErp1 regulates its
APC/C inhibitory function during CSF arrest in Xenopus egg extracts. This
section describes the experiments demonstrating that ectopic UbcX, the E2
enzyme of the APC/C, induces release from SAC- and CSF arrest. The release
from CSF arrest is APC/CCdc20 dependent and in the presence of elevated UbcX
activity, XErp1 is ubiquitylated resulting in the dissociation of XErp1 from the
APC/C. Hence, the APC/C inhibitory activity of XErp1 in CSF arrest can be
modulated in an UbcX-dependent manner. Furthermore, evidence is provided
that in contrast to SAC arrested somatic cells, Cdc20 is not degraded during
meiotic CSF arrest suggesting that CSF arrest is not mediated by the
destabilization of Cdc20.
2.1. UbcX can suppress SAC activity in Xenopus egg extract
The finding that in human somatic cells, the APC/C can liberate itself from
inhibition by the SAC (Reddy et al., 2007) prompted us to analyze whether a
similar mechanism operates in Xenopus eggs or egg extracts to regulate APC/C
activity during SAC and - more interestingly - during CSF arrest. In Xenopus
eggs, SAC activity was reported to be absent but can be induced by increasing
the ration of nucleus to cytoplasm in the presence of spindle poisons (Minshull
et al., 1994). To analyze the effect of UbcX on SAC arrest in Xenopus eggs, we
prepared CSF arrested egg extract and triggered SAC arrest by the microtubule
poison nocodazole in the presence of high concentrations of sperm nuclei
(Figure 2.1. a). Under these conditions, calcium addition did not result in APC/C
activation as in vitro translated 35S-securin remained stable (Figure 2.1. b,
panel 1). Westernblot (WB) analysis revealed that XErp1 was efficiently
22
inhibition was due to SAC- but not CSF-activity. Addition of recombinant wild
type UbcX (UbcXwt) to SAC arrested extracts caused APC/C activation and 35S-
securin degradation (Figure 2.1. b, panel 2). This effect was dependent on the
catalytic activity of UbcX, as the addition of a catalytic inactive form of UbcX
(UbcXci) had no effect on 35S-securin stability (Figure 2.1. b, panel 3). Therefore,
the mechanism of UbcX mediated SAC inactivation is conserved between
humans and Xenopus.
Figure 2.1. Ectopic UbcXwt overrides SAC-arrest in Xenopus egg extract. (a) CSF-extracts containing 35S-securin was supplemented with nocodazole and high concentrations of sperm to activate the SAC. CSF arrest was released by calcium addition. (b) At the indicated time points after the addition of the specified reagents samples were taken and 35S-securin was detected by autoradiography and XErp1 and α-tubulin by WB. CSF, cytostatic factor; SAC, spindle assembly checkpoint; 35S-securin, in vitro translated, 35S-labeled securin; wt, wild type; ci, catalytical inactive.
2.2. UbcX can suppress CSF activity in Xenopus egg extract
To analyze if an increase in the activity of UbcX similarly influences CSF
mediated APC/C inhibition, ectopic UbcXwt was added to CSF arrested egg
extract supplemented with a low concentration of sperm nuclei and 35S-securin
(Figure 2.2. a). Interestingly, also in these extracts ectopic UbcX caused APC/C
activation and CSF release in the absence of the calcium signal, as indicated by
23
panel 2). However - unlike in extracts treated with calcium - XErp1 remained
stable and showed an increase in its electrophoretic mobility following exit
from meiosis (Figure 2.2. c, panel 1 and 2), suggesting that UbcXwt causes CSF
inactivation by different means than XErp1 degradation. The addition of UbcXci
or dialysis buffer had no effect on CSF arrest (Figure 2.2. b, c, panel 3 and 4),
suggesting that the observed CSF override is dependent on an increase in the
catalytic activity of UbcX.
Additionally, the human homologue of UbcX was equivalent in the ability to
overcome CSF arrest in Xenopus egg extract, as the addition of catalytic active
UbcH10 triggered premature CSF release (Figure 2.2. d, panel 3),
demonstrating that both UbcX and UbcH10 are interchangeable in inducing
CSF release.
24
2.3. Elevated UbcX activity prevents meiosis I - meiosis II transition in
Xenopus oocytes
To collect evidence for UbcX mediated regulation of CSF arrest in vivo, we
injected recombinant UbcX into Xenopus stage VI oocytes arrested at prophase
of meiosis I. We induced oocyte maturation by the addition of progesterone
and followed the resumption of
Doktors der Naturwissenschaften (Dr. rer. nat.)
vorgelegt von
2. Referent: Prof. Dr. Martin Scheffner
3. Referent: Prof. Dr. Olaf Stemmann
1.2. The APC/C counteracts the activity of Cdk1 7
1.3. The “wait anaphase signal”: The SAC inhibits the APC/C in mitosis 9
1.4. Regulation of APC/CCdc20 activity in meiosis 11
1.5. The postulation of MPF and CSF 12
1.6. The discovery of Mos as a CSF component 13
1.7. Identification of the CSF component XErp1 14
1.8. XErp1 inactivation upon CSF release 15
1.9. The molecular mechanism of XErp1 mediated APC/C inhibition 16
1.10. Feedback loops controlling XErp1 activity during CSF arrest 18
1.11. Aim of this project 20
2. RESULTS 21
2.1. UbcX can suppress SAC activity in Xenopus egg extract 21
2.2. UbcX can suppress CSF activity in Xenopus egg extract 22
2.3. Elevated UbcX activity prevents meiosis I - meiosis II transition in
Xenopus oocytes 24
activity 25
2.5. Does USP44 counteract UbcX to maintain CSF arrest? 26
2.6. An eight-fold increase in UbcX activity is required for CSF release. 27
2.7. UbcX levels increase during oocyte maturation and remain constant
during CSF release and embryonic cell cycles 28
2.8. UbcX dependent CSF release can be suppressed by XErp1 29
2.9. UbcX mediated ubiquitylation disrupts the APC/C - XErp1 complex 30
2
2.10. XErp1 is the main target of UbcX mediated ubiquitylation in CSF
extract 32
2.11. Ubiquitylation of XErp1 is dependent on the APC/C and independent
of SCFβ TRCP 33
2.12. Dissociation of XErp1 upon Cdk1 phosphorylation does not require
ubiquitylation 35
2.13. Cdc20 degradation is not required for CSF arrest maintenance 36
3. DISCUSSION 38
3.1. Regulation of spindle checkpoint signaling by UbcH10/UbcX 39
3.1.1. The spindle assembly checkpoint can be inactivated by UbcX in
Xenopus egg extract 39
3.1.2. Is an APC/C inhibitor targeted for ubiquitylation during SAC
signaling? 41
3.2. UbcX mediated ubiquitylation of XErp1 regulates its APC/C inhibitory
activity 43
3.2.1. Cdc20 is not destabilized in CSF arrested egg extract 43
3.2.2. UbcX mediated ubiquitylation of XErp1 regulates its APC/C inhibitory
activity 44
3.2.3. Are ubiquitin hydrolases counteracting the activity of UbcX during CSF
arrest? 46
3.3. Is the regulation of UbcX activity important during the meiotic cell
cycle? 48
3.3.2. Could UbcX participate in the inactivation of XErp1 upon
fertilization? 48
pathways regulating the activity of XErp1 49
3.4. Could ubiquitylation of XErp1 be required for its APC/C inhibitory
activity? 50
5.2. Plasmids 55
5.2.3. Cloning and Mutagenesis 57
5.3. Proteins 57
5.3.2. His-tagged protein expression in SF9 cells 58
5.3.3. His-tagged protein purification from bacteria and SF9 cells 58
5.3.4. Coupled in vitro transcription/translation (IVT) 59
5.4. Antibodies 59
5.4.2. Affinity purification of antibodies 59
5.5. Gel electrophoresis and immunoblot analysis 60
5.6. Xenopus egg extracts 61
5.6.1. Xenopus CSF egg extract preparation 61
5.6.2. Extract manipulations 62
5.7. Xenopus oocyte injections 64
6. LITERATURE 65
7. APPENDIX 75
7.1. Summary 75
7.2. Zusammenfassung 75
7.3. Acknowledgements 76
1. INTRODUCTION
Most eukaryotes reproduce sexually, where cells from two parents fuse to
generate a single cell, the zygote, which develops into a new organism (Figure
1.1.). Since the combination of two diploid cells would lead to the duplication
of the chromosomal content at every generation, sexual reproduction depends
on a process called meiosis.
Figure 1.1. The life cycle of vertebrates. Cells in vertebrates proliferate mitotically in the diploid phase to form a multicellular organism. Sexual reproduction begins with meiosis to generate haploid cells, which fuse upon fertilization to form a new organism.
1.1. Meiosis and meiotic maturation
Meiosis is a specialized form of nuclear division that leads to the generation of
cells containing half the normal complement of chromosomes from diploid
oocytes (Figure 1.2. a, Alberts et al., 2002). (Alberts et al., 2002).
Before entering the meiotic program, oocytes are diploid like somatic cells and
contain two copies of each chromosome, one of them inherited from each
parent. Meiosis begins with an S-phase (Petronczki et al., 2003) in which
5
chromosomes are replicated to produce sister chromatid pairs tightly linked by
cohesion (Klein et al., 1999). Next, the duplicated homologues pair to form
tetrads and undergo homologues recombination, a process important for
generating genetic variation and to guarantee accurate segregation of the
homologues at the following nuclear division. Homologous recombination
starts with the introduction of DNA double-strand breaks (DSB) at almost
variable positions along the chromosome (Sun et al., 1989). In most of the
cases, DSBs are repaired without rendering the DNA sequence of the two
homologs. Sometimes however, the repair leads to the formation of a
continuous DNA strand between two homologous chromatids, which can lead
to a reciprocal DNA exchange or crossover (Allers and Lichten, 2001). The
result is a strong physical linkage between the two homologous chromosomes
as long as the sister chromatid arms are held together by cohesion. As a result,
the homologous chromosomes become bioriented on the first meiotic spindle
and after cohesin cleavage at the chromosome arms at anaphase I, exactly one
of the two homologous chromosomes is segregated into each daughter cell
(Buonomo et al., 2000). After the completion of meiosis I, cells enter directly
the next division cycle without replicating the chromosomes. In meiosis II,
similar to mitosis, sister chromatids are divided into the two daughter cells by
the cleavage of centromeric cohesion upon anaphase II onset. Together,
meiotic divisions result in the production of four haploid cells, which can be
differentiated into special reproductive cells, i.e. the egg and the sperm.
In animals, oocytes arrest before the first meiotic division at prophase I, and
these immature oocytes or stage VI oocytes can stop at this point for decades
(Hunt, 1989). The production of a fertilizable egg from such an immature
oocyte involves a process called oocyte maturation (Figure 1.2. b). Upon
hormonal induction, immature oocytes resume meiosis I and undergo germinal
vesicle breakdown (GVBD) which is visible on the surface of the oocytes by the
appearance of a white dot. Meiosis I is completed with the extrusion of the
first polar body after which the oocytes proceed directly through meiosis II
6
where the second polar body is extruded and haploid gametes are produced.
In vertebrates like Xenopus laevis, oocytes complete meiotic maturation with
an arrest at metaphase of meiosis II, in which they await fertilization. From the
viewpoint of cell-cycle control, the major questions are concerning the
mechanisms underlying the induction and regulation of oocyte maturation as
well as the arrest of mature oocytes at metaphase of meiosis II and its release
upon fertilization (Tunquist and Maller, 2003).
Figure 1.2. The meiotic program. (a) In meiosis, after DNA replication, two divisions generate haploid gametes. For clarity, only one chromosome is depicted. (b) Meiosis in vertebrates is arrested at two stages. After DNA synthesis, the oocytes grow to their final size and arrest at meiotic prophase I. Progesterone induces meiotic maturation and the production of an egg arrested at meiotic metaphase II. Fertilization triggers the completion of Meiosis II and a diploid zygote is formed (Adapted from Morgan, 2007).(Morgan, 2007)
1.1. Cdk1/cyclin B drives the meiotic cell cycle
The ordered progression of the meiotic cell cycle, like the mitotic cell cycle, is
mediated mainly by the activity of cyclin dependent kinases (Cdks) and
ubiquitin ligases (Murray, 2004). Cdks are serine-threonine kinases that are
activated by their regulatory subunit, the cyclins. In mitotic G1, low Cdk1
activity is important for the resetting of the origins of DNA replication. Rising
Cdk activity triggers the firing of DNA replication origins and as S-phase
progresses and DNA replication continues, the activity of Cdk1/CylinB1
promotes entry into mitosis, which is characterized by nuclear envelope
7
condensation. After the successful division of the replicated chromosomes into
two daughter cells, the cell needs again low Cdk1 activity to exit mitosis and to
enter G1. Therefore, low Cdk activity followed by high activity links DNA
replication to progression through mitosis (Porter, 2008) – the basis for the
mitotic cell cycle.
In Xenopus meiosis, the hormone progesterone induces entry into metaphase I
by the activation and amplification of Cdk1/cyclin B by inducing both the
dephosphorylation of inhibitory residues on Cdk1 and the accumulation of
cyclin B (Tunquist and Maller, 2003). Progression from metaphase I to
anaphase I is accompanied by a drop in cyclin B levels and decreasing Cdk1
activity. But unlike in mitotic cells, cyclin B is not completely degraded upon
anaphase onset but appears to be reduced to half (Furuno et al., 1994;
Iwabuchi et al., 2000). While it remains controversial whether this drop in
cyclin B levels is required for meiotic progression (Peter et al., 2001; Taieb et
al., 2001), the inhibition of complete cyclin B degradation is essential for the
persistence of M-phase and the inhibition of DNA replication (Ohe et al., 2007).
Thus, the oocytes directly enter a second M-phase, where the stabilization of
cyclin B levels is important for establishing the second meiotic arrest. Upon
fertilization, cyclin B is degraded, Cdk1 is inactivated and the zygotes enter
mitotic cell cycles.
1.2. The APC/C counteracts the activity of Cdk1
Anaphase onset requires the inactivation of both Cdk1 kinase and the
inactivation of the anaphase inhibitory protein securin. Securin prevents
cohesin cleavage and thus the irreversible step of sister chromatid separation
by keeping the cohesin directed protease separase inactive (Uhlmann et al.,
1999; Uhlmann et al., 2000). Both, Cdk1/cyclin B and securin activity is
regulated by the E3 ubiquitin ligase anaphase promoting complex/cyclosome
(APC/C). It mediates the specific ubiquitylation of cyclin B and securin (Sudakin
8
et al., 1995; Zou et al., 1999) thereby targeting them for destruction by the 26
S proteasome at anaphase onset.
The APC/C is an unusual large E3 ubiquitin ligase that consists of at least 13
subunits including proteins with cullin and RING-finger domains (Zachariae and
Nasmyth, 1999). In addition, the APC/C associates with coactivator proteins
called Cdc20 and Cdh1 (Pesin and Orr-Weaver, 2008), which bind transiently to
the APC/C core complex and are thought to regulate both the activity and
substrate specificity of the APC/C. While in somatic mitotic cell cycles, the
coactivator of the APC/C alternates between Cdc20 and Cdh1, the main
coactivator required for meiosis and early embryonic cell cycles has been
reported to be Cdc20 (Lorca et al., 1998). The APC/C together with its
coactivator is responsible for substrate recognition and thus confers specificity
to the ubiquitylation reaction (Peters, 2006). It functions at the last step of a
cascade of enzymes that sequentially act to transfer ubiquitin to the target
protein (Hershko and Ciechanover, 1998). Free ubiquitin is first covalently
attached to an ubiquitin-activating enzyme E1 via a thioester bond. It is then
transferred to an ubiquitin-conjugating enzyme E2 where it forms a thioester
bond with the active site cystein. The main E2 enzyme cooperating with the
APC/C has been identified in clam as E2-C (Hershko et al., 1994) and orthologs
were found in Xenopus named UbcX (Yu et al., 1996), and in humans named
UbcH10 (Townsley et al., 1997). In Xenopus, UbcX is essential for APC/C
activity, since a dominant negative mutation in the active site cystein (C114S)
inhibits APC/C dependent substrate ubiquitylation (Townsley et al., 1997), and
the depletion of UbcX inhibits APC/C substrate degradation (data not shown).
In the final step of APC/C dependent ubiquitylation, the E2-bound ubiquitin is
covalently attached to a lysine residue in the target protein. In this reaction,
the APC/C is thought to approximate the substrate and the E2-ubiquitin and to
position them for efficient ubiquitin transfer (Peters, 2006). Recently, it has
been shown that in human cells, UbcH10 forms an E2-enzyme module with
Ube2S, and both enzymes were shown to be important for the formation of
9
ubiquitin chains on APC/C substrates, where UbcH10 conjugates the first
ubiquitin to the lysine residue of the substrate and Ube2S then elongates the
ubiquitin chain (Garnett et al., 2009; Williamson et al., 2009; Wu et al., 2010).
As a consequence, ubiquitylation can target proteins to the 26 S proteasome, a
high molecular weight protease complex that hydrolyses its substrates into
short peptides and thus inactivates them irreversibly. Alternatively,
ubiquitylation can act as a reversible posttranslational modification of a
protein to regulate its activity (Hershko and Ciechanover, 1998).
1.3. The “wait anaphase signal”: The SAC inhibits the APC/C in mitosis
Mitotically and meiotically dividing cells depend on ubiquitin-mediated
proteolysis of key cell-cycle regulators at the correct time (Pesin and Orr-
Weaver, 2008). In mitosis, a conserved mechanism called the spindle assembly
checkpoint (SAC) guarantees an equal segregation of the chromosomes to the
two nascent daughter cells (Musacchio and Salmon, 2007). The SAC is activated
by missattached or unattached kinetochores (Nicklas et al., 1995; Rieder et al.,
1995; Rieder et al., 1994) and prevents the APC/C from ubiquitylating cyclin B
and securin. Although it is not yet completely understood how the SAC
inactivates the APC/C, it is well accepted that the primary target of the SAC is
the APC/C coactivator Cdc20 (Hwang et al., 1998; Kim et al., 1998) and that
SAC activity is propagated by a number of conserved proteins including Mad1,
Mad2 and Bub3/BubR1 (Hoyt et al., 1991; Li and Murray, 1991). Current
models of SAC mediated APC/C inactivation suggest that Mad2 binds to Cdc20
in conjunction with BubR1 and Bub3 to form the “Mitotic Checkpoint Complex”
(MCC), which binds to the APC/C and renders it inactive (Sudakin et al., 2001).
Once all kinetochores are properly attached, it has been suggested that the
inhibitory MCC complexes have to be actively dissociated by APC/C dependent,
non-proteolytic ubiquitylation of Cdc20 to turn off the SAC. Specifically, it has
been shown that addition of the E2 ubiquitin conjugating enzyme UbcH10 to
SAC-arrested cell extract triggers the APC/C-dependent multi-ubiquitylation of
10
Cdc20, and possibly other components of the APC/C–Cdc20-MCC complex,
resulting in the release of Mad2 and BubR1 from Cdc20 (Reddy et al., 2007). In
checkpoint arrest conditions, this ubiquitylation reaction is antagonized by the
activity of the ubiquitin hydrolase USP44 (Figure 1.3.), which removes ubiquitin
from Cdc20 (Stegmeier et al., 2007). As soon as the last kinetochore is
attached, ubiquitylation of Cdc20 is thought to exceed its deubiquitylation,
Cdc20 is freed from the MCC and the APC/C can be rapidly activated in a
switch-like manner.
Figure 1.3. Dynamic ubiquitylation and deubiquitylation regulate SAC activity. During mitotic checkpoint arrest, ubiquitylation of Cdc20 by UbcX, which leads to the dissociation of the APC/C inhibitors Mad2 and BubR1, needs to be counteracted by USP44 dependent deubiquitylation of Cdc20 to maintain SAC mediated APC/C inhibition.
A different model contradicts this view of SAC arrest and instead suggests that
in cells with an active SAC, Cdc20 in complex with the MCC proteins is
ubiquitylated and targeted for destruction, and this degradation is important
for inactivating the APC/C (Ge et al., 2009; Nilsson et al., 2008). Supporting this
model, experiments in budding yeast and human cells have shown that Cdc20
is ubiquitylated and degraded during SAC arrest and overexpression of Cdc20
could overcome the SAC mediated inhibition of the APC/C (King et al., 2007;
Pan and Chen, 2004). Importantly, a non-ubiquitylatable form of Cdc20 where
every lysine was mutated to an arginine was insensitive to the checkpoint
arrest and activated the APC/C (Nilsson et al., 2008). These results contradict a
model where Cdc20 ubiquitylation causes its activation and rather support the
latter model where ubiquitylation inactivates Cdc20.
11
The regulation of APC/C activity is especially important during oocyte
maturation in vertebrates where meiosis is arrested twice to coordinate oocyte
development with the events of meiosis (Figure 1.4.).
In prophase I, the APC/C has to be inactive to maintain chromosome cohesion
(Pesin and Orr-Weaver, 2008). When oocytes mature, the APC/C needs to
become active at the metaphase I - anaphase I transition to allow the
degradation of securin and the separation of the homologous chromosomes
(Buonomo et al., 2000; Siomos et al., 2001). In contrast to all organisms tested,
the requirement of the APC/C for meiosis I - meiosis II transition is
controversial in Xenopus. Although microinjections of Xenopus oocytes with
inhibitory antibodies or antisense oligonucleotides directed against the APC/C
coactivator Cdc20 did not disrupt progression through meiosis I (Peter et al.,
2001; Taieb et al., 2001), it is possible that these approaches did not eliminate
APC/C activity completely. Nevertheless, the complete degradation of cyclin B
must be prevented also in Xenopus to maintain M-phase and to inhibit S-phase
(Ohe et al., 2007), suggesting that the APC/C needs to be regulated to
contribute to this modulation of cyclin B levels.
Figure 1.4. Oocyte maturation on a molecular level: Cdk1 and APC/C. The cell cycle in meiosis is driven by the activity of Cdk1/cyclin B which is counteracted by the APC/C, the relative activities of which through the maturation process are illustrated (adapted from Wu and Kornbluth, 2008).
At the second meiotic arrest at metaphase II, the APC/C needs to be inhibited
to stabilize cyclin B and securin to prevent premature anaphase onset and
12
parthenogenetic activation of the egg. Upon fertilization, APC/C activation is
required to induce the exit from the metaphase II arrest (Lorca et al., 1998;
Peter et al., 2001) and thereby allowing entry into early embryonic cell cycles.
While the spindle checkpoint is important for the metaphase arrest and APC/C
inhibition in mitotic cells in the presence of unattached kinetochores, it is
unlikely that the SAC mediates the metaphase arrest observed in mature
vertebrate eggs. Evidence against such a hypothesis includes the fact that CSF
arrest is terminated by fertilization and the following elevation in cytoplasmic
calcium levels, but calcium addition does not overcome SAC arrest (Minshull et
al., 1994). Additionally, the SAC requires kinetochores and microtubule
depolymerization, whereas neither is required for meiotic metaphase II arrest
(Tunquist and Maller, 2003). What inhibits oocytes at metaphase of Meiosis II?
1.5. The postulation of MPF and CSF
In 1971, Yoshio Masui and Clement L. Markert performed experiments in Rana
pipiens oocytes and embryos that became fundamental for the identification of
the mechanisms mediating the metaphase II arrest in mature oocytes (Masui
and Markert, 1971).
Specifically, they observed that injection of immature oocytes with endoplasm
of mature oocytes induced meiotic maturation. Therefore they postulated that
maturation is induced by a maturation promoting factor (MPF) which is
released by hormonal induction and remains active in the mature egg (Figure
1.5.). To analyze whether the same activity could accelerate cell divisions in
embryonic cells, they injected endoplasm of the mature egg into one cell of a
two-cell stage embryo. Surprisingly, they found that the injected blastomere
arrested at the next mitosis, prompting them to propose the existence of a
cytostatic factor (CSF) present in the mature egg that is responsible for
inducing the metaphase II arrest (Figure 1.5.). Additionally, this activity is
13
inactivated upon fertilization, since injection of blastomeres with endoplasm of
fertilized embryos did not cause cell-cycle arrest.
Figure 1.5. The discovery of MPF and CSF. Illustration of the oocyte- and blastomere-injection assays originally performed by Masui and Markert in 1971 that led to the identification of the maturation promoting factor MPF and the cytostatic factor CSF.
While MPF was soon identified to be the activity of cyclin dependent kinase
Cdk1 together with its regulatory subunit cyclin B (Gautier et al., 1990; Gautier
et al., 1988; Lohka et al., 1988; Murray et al., 1989), the discovery of the
molecular identity of the CSF took more than three decades.
1.6. The discovery of Mos as a CSF component
To identify the CSF activity that mediates the metaphase II arrest, three criteria
were proposed for a protein or an activity to be a CSF: (1) The activity emerges
during oocyte maturation and peaks in the metaphase II arrested egg. (2)
Injection of blastomeres with the activity induces mitotic arrest and (3)
fertilization triggers the inactivation of the factor (Masui and Markert, 1971).
The first protein identified meeting these criteria was the kinase Mos. Mos is
expressed during oocyte maturation (Sagata et al., 1988); Figure 1.6.), it could
induce mitotic arrest when injected into blastomeres of a dividing embryo
14
(Sagata et al., 1989) and it was degraded upon fertilization (Lorca et al., 1991).
To understand the detailed molecular mechanism linking Mos to the
metaphase II arrest, the signaling pathway of the kinase was investigated.
Biochemical analysis revealed that Mos can activate the mitogen activated
protein kinase (MAPK) pathway (Posada et al., 1993) resulting in the activation
of the ribosomal S6 kinase (Rsk), and functional analysis of the members of this
pathway showed that they are required for CSF arrest (Abrieu et al., 1996;
Bhatt and Ferrell, 1999; Cross and Smythe, 1998; Gotoh and Nishida, 1995;
Gross et al., 1999; Haccard et al., 1993; Kosako et al., 1994a, b). Therefore, the
Mos activated MAPK-pathway was proposed to be a molecular component of
the CSF. Since both, the Mos-MAP kinase pathway and APC/C inhibition are
responsible for CSF arrest, it seemed possible that these two pathways are
interconnected. However, it remained unclear how Rsk as the terminal kinase
in this cascade was communicating with the cell-cycle machinery to establish
the CSF arrest.
1.7. Identification of the CSF component XErp1
Reportedly, polo-like kinase Plx1 is required CSF inactivation and APC/C
activation (Descombes and Nigg, 1998). Specifically, it has been shown that
Xenopus egg extracts depleted of Plx1 fail to release the CSF arrest upon
increasing cytoplasmic calcium levels. Therefore, a yeast two-hybrid screen
was performed to identify proteins that interacted with Plx1 (Schmidt et al.,
2005), and this approach led finally to the identification of the sought after
component of CSF, the XErp1 protein. XErp1 nicely satisfied the Masui and
Markert criteria proposed for CSF. First, XErp1 is synthesized during Xenopus
oocyte maturation; it starts to be detectable at the MI-MII transition and it
accumulates as oocytes proceed through meiosis II where it reaches highest
levels at metaphase II (Figure 1.6.); second, exogenous introduction of XErp1
into one blastomere of a two-cell stage embryo promoted a cell-cycle arrest
and third, XErp1 was degraded after fertilization in a Plx1 dependent manner.
15
Importantly, XErp1 is essential for CSF arrest as Xenopus egg extracts arrested
at metaphase II depleted of XErp1 were unable to maintain CSF arrest and
entered interphase.
Further characterization XErp1 revealed the C-terminus of the protein, which is
sufficient for CSF arrest maintenance, shares high sequence similarity with the
mitotic APC/C inhibitor Emi1 and like Emi1, XErp1 was shown to inhibit the
APC/C directly (Schmidt et al., 2005). Therefore, XErp1 is a CSF specific APC/C
inhibitor.
Figure 1.6. Oocyte maturation and CSF on a molecular level. Oocyte maturation is driven by the activities of Cdk1/cyclin B, the APC/C and CSF factors Mos and XErp1, ad the relative activities during oocyte maturation are depicted on the left (adapted from Kornbluth, 2008).
Since XErp1 was shown to be a substrate of Rsk, the Mos-MAPK pathway could
finally be linked to the regulation of the APC/C. Rsk phosphorylation was
shown to increase the inhibitory activity of XErp1 in CSF arrested eggs, which
will be described later.
1.8. XErp1 inactivation upon CSF release
As proposed by Masui and Markert, fertilization causes the inactivation of CSF.
The first response of an egg to fertilization is an elevation in cytoplasmic
calcium levels, which results in the activation of calcium/calmodulin dependent
kinase II (CaMKII;(Lorca et al., 1993). The identification of XErp1 as a CaMKII
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substrate provided insights into how fertilization is connected with CSF
inactivation (Figure 1.7.;(Hansen et al., 2006; Liu and Maller, 2005; Rauh et al.,
2005).
Figure 1.7. Fertilization mediated CSF inactivation. Fertilization (1) triggers the activation of CaMKII (2) which phosphorylates XErp1 (3) creating a docking site for Plx1 (4). Plx1 in turn phosphorylates XErp1 creating a phosphodegron (5), which is recognized by the ubiquitin ligase SCFβ
TRCP. XErp1 ubiquitylation targets it for degradation (6) and thus CSF inactivation, the APC/C becomes active (7) and cells complete meiosis II (adapted from Rauh et al., 2005).
CaMKII mediated phosphorylation of XErp1 provides a docking site for Plx1 on
XErp1. Through Plx1 mediated phosphorylation of XErp1 a phosphodegron is
created and XErp1 is recognized by the SCFβ TRCP complex, an ubiquitin E3 ligase
that ubiquitylates and targets XErp1 for degradation. Consequently, calcium
triggers CSF inactivation resulting in APC/C activation and the fertilized egg can
proceed with embryonic cell divisions.
1.9. The molecular mechanism of XErp1 mediated APC/C inhibition
In CSF arrested eggs, XErp1 maintains the metaphase II arrest by directly
inhibiting the APC/C. The binding of XErp1 to the APC/C is essential for its
inhibitory activity as mutants defective in APC/C binding are inefficient in
17
inhibiting the APC/C (Wu et al., 2007b). The well-conserved C-terminal peptide
sequence of XErp1, termed the RL tail, was reported to mediate the
recruitment of XErp1 by serving as a docking site to the APC/C (Ohe et al.,
2010). Binding to the APC/C allows and enhances the inhibitory interactions of
two other sequence elements of XErp1, the D-box and the ZBR-domain. While
it is well established that all three elements are critical for APC/C inhibition,
the specific contribution of the D-box and the ZBR domain to the inhibition of
the APC/C by XErp1 remain elusive (Nishiyama et al., 2007; Ohe et al., 2010;
Tang et al., 2010).
Notably, all three elements are conserved between XErp1 and Emi1, a somatic
paralog of XErp1, whose APC/C inhibitory activity is required to prevent DNA
re-replication (Di Fiore and Pines, 2007; Machida and Dutta, 2007) suggesting
that XErp1 and Emi1 share the same mode of APC/C inhibition. Emi1, when
bound to the APC/C together with the E2 enzyme UbcH10, was shown to
inhibit the correct engagement of the substrate to the APC/C thereby reducing
substrate ubiquitylation (Summers et al., 2008). Further studies on Emi1
suggested that it acts as an APC/C pseudosubstrate and the D-box mediates
APC/C binding, while its ZBR mediates APC/C inhibition (Miller et al., 2006).
Consistently, it has been shown that Emi1 mutated in its ZBR does not inhibit
the APC/C but rather is quickly targeted for destruction by the APC/C. Given
that XErp1 – like Emi1 – contains a D-box and ZBR, it is tempting to speculate
that XErp1 acts as well as a pseudosubstrate. However, previous studies
suggest that XErp1 does not compete with substrates for APC/C binding but
rather interferes with the transfer of ubiquitin to substrate proteins bound to
the APC/C (Tang et al., 2010). Furthermore, our preliminary experiments
revealed that in contrast to Emi1, mutation of the ZBR of XErp1 does not
convert it into an APC/C substrate corroborating the idea that XErp1 inhibits
the APC/C by a mechanism distinct to the one of Emi1.
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Together, although it is established that XErp1 needs to be recruited to the
APC/C to exert its inhibitory function, the exact molecular mechanism of XErp1
mediated APC/C inhibition remains elusive.
1.10. Feedback loops controlling XErp1 activity during CSF arrest
During metaphase II arrest, the Mos-MAPK pathway was shown to activate
XErp1 by upregulating both the stability and activity of XErp1 (Isoda et al.,
2011; Wu et al., 2007a; Wu et al., 2007b). The Mos-MAPK pathway activates
the kinase Rsk (Bhatt and Ferrell, 1999; Gross et al., 1999), which
phosphorylates XErp1 at residues in the central region (Inoue et al., 2007;
Nishiyama et al., 2007) leading to the recruitment of the protein phosphatase
PP2A containing the regulatory subunit B56β or B56ε to XErp1 (Wu et al.,
2007a). PP2A- B56β,ε antagonizes N-terminal and C-terminal inhibitory
phosphorylations of XErp1 by Cdk1 (Isoda et al., 2011). Cdk1 phosphorylations
destabilize XErp1 and decrease its affinity for the APC/C (Wu et al., 2007a; Wu
et al., 2007b).
Figure 1.8. Oocyte maturation and CSF on a molecular level. Oocyte maturation is driven by the activities of Cdk1/cyclin B, the APC/C and CSF factors Mos and XErp1, ad the relative activities during oocyte maturation are depicted on the left (adapted from Kornbluth, 2008). On the right, a simplified signaling network controlling the activity of XErp1 is illustrated (adapted from Isoda et al., 2011).
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Specifically, it has been shown that multiple N-terminal Cdk1 phosphorylation
motifs bind cyclin B1-Cdk1 itself as well as Plk1 and CK1 δ/ε to inhibit XErp1
(Isoda et al., 2011). While Plk1 phosphorylation was shown to partially
destabilize XErp1, Cdk1 and CK1δ/ε phosphorylations are thought to
cooperatively inhibit XErp1 binding to the APC/C (Figure 1.8.). Since Cdk1 levels
are high during the Metaphase II arrest, constant phosphorylation of XErp1
would lead to gradual XErp1 inactivation and CSF release. By recruiting PP2A-
B56β,ε to counteract the inhibitory phosphorylations, the Mos MAPK- pathway
keeps XErp1 active and therefore maintains CSF arrest (Figure 1.8.). At the
same time, this mechanism allows to maintain Cdk1 activity at the correct level
during CSF arrest (Figure 1.9.(Wu and Kornbluth, 2008; Wu et al., 2007b).
Continuous cyclin B synthesis during CSF arrest leads to a temporal increase in
Cdk1/cyclin B activity, which in turn leads to an increase in the phosphorylation
of XErp1, since the activity of PP2A on XErp1 remain equal. XErp1
phosphorylated by Cdk1 dissociates from the APC/C leading to a transient
APC/C activation and slow degradation of cyclin B.
Figure 1.9. Cdk1/cyclin B2 and PP2A regulate XErp1’s association with the APC/C. Phosphorylation of XErp1 by Cdk1/cyclin B2 leads to the dissociation of XErp1 from the APC/C, which is counteracted by PP2A, which dephosphorylates XErp1 and promotes XErp1 association with the APC/C.
Therefore, the continuous synthesis of cyclin B induces a slow degradation of
cyclin B during CSF arrest. Otherwise, continuous synthesis would create an
amount of cyclin B that cannot be degraded by the APC/C anymore in a short
time. This would result in a slow and gradual rather than a switch-like exit from
CSF arrest as observed upon fertilization.
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1.11. Aim of this project
XErp1 is an APC/C inhibitor operating in CSF arrested oocytes. However, the
exact molecular mechanism of APC/C inhibition and its regulation is unknown.
The D-box and the RL-tail of XErp1 mediate the binding of XErp1 to the APC/C,
most likely to position the ZBR of XErp1 correctly to inactivate the APC/C.
However, the interaction with the APC/C needs to be dynamic to allow slow
cyclin B degradation during CSF arrest. Phosphorylation and dephosphorylation
of XErp1 can regulate its association with the APC/C, and the Mos-MAPK
pathway was shown to promote XErp1 association. Intrigued by the findings on
APC/C regulation by the spindle checkpoint, we would like to understand if a
dynamic balance of ubiquitylation/deubiquitylation of Cdc20, XErp1 and/or
other components of the APC/C is also required for CSF arrest. In addition, we
would like to test whether Cdc20 turnover is required for CSF arrest and if
XErp1 regulates this potential turnover. Thus, these studies will provide a
deeper understanding of how the XErp1-APC/CCdc20 interaction is regulated and
2. RESULTS
In this study, we show that non-proteolytic ubiquitylation of XErp1 regulates its
APC/C inhibitory function during CSF arrest in Xenopus egg extracts. This
section describes the experiments demonstrating that ectopic UbcX, the E2
enzyme of the APC/C, induces release from SAC- and CSF arrest. The release
from CSF arrest is APC/CCdc20 dependent and in the presence of elevated UbcX
activity, XErp1 is ubiquitylated resulting in the dissociation of XErp1 from the
APC/C. Hence, the APC/C inhibitory activity of XErp1 in CSF arrest can be
modulated in an UbcX-dependent manner. Furthermore, evidence is provided
that in contrast to SAC arrested somatic cells, Cdc20 is not degraded during
meiotic CSF arrest suggesting that CSF arrest is not mediated by the
destabilization of Cdc20.
2.1. UbcX can suppress SAC activity in Xenopus egg extract
The finding that in human somatic cells, the APC/C can liberate itself from
inhibition by the SAC (Reddy et al., 2007) prompted us to analyze whether a
similar mechanism operates in Xenopus eggs or egg extracts to regulate APC/C
activity during SAC and - more interestingly - during CSF arrest. In Xenopus
eggs, SAC activity was reported to be absent but can be induced by increasing
the ration of nucleus to cytoplasm in the presence of spindle poisons (Minshull
et al., 1994). To analyze the effect of UbcX on SAC arrest in Xenopus eggs, we
prepared CSF arrested egg extract and triggered SAC arrest by the microtubule
poison nocodazole in the presence of high concentrations of sperm nuclei
(Figure 2.1. a). Under these conditions, calcium addition did not result in APC/C
activation as in vitro translated 35S-securin remained stable (Figure 2.1. b,
panel 1). Westernblot (WB) analysis revealed that XErp1 was efficiently
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inhibition was due to SAC- but not CSF-activity. Addition of recombinant wild
type UbcX (UbcXwt) to SAC arrested extracts caused APC/C activation and 35S-
securin degradation (Figure 2.1. b, panel 2). This effect was dependent on the
catalytic activity of UbcX, as the addition of a catalytic inactive form of UbcX
(UbcXci) had no effect on 35S-securin stability (Figure 2.1. b, panel 3). Therefore,
the mechanism of UbcX mediated SAC inactivation is conserved between
humans and Xenopus.
Figure 2.1. Ectopic UbcXwt overrides SAC-arrest in Xenopus egg extract. (a) CSF-extracts containing 35S-securin was supplemented with nocodazole and high concentrations of sperm to activate the SAC. CSF arrest was released by calcium addition. (b) At the indicated time points after the addition of the specified reagents samples were taken and 35S-securin was detected by autoradiography and XErp1 and α-tubulin by WB. CSF, cytostatic factor; SAC, spindle assembly checkpoint; 35S-securin, in vitro translated, 35S-labeled securin; wt, wild type; ci, catalytical inactive.
2.2. UbcX can suppress CSF activity in Xenopus egg extract
To analyze if an increase in the activity of UbcX similarly influences CSF
mediated APC/C inhibition, ectopic UbcXwt was added to CSF arrested egg
extract supplemented with a low concentration of sperm nuclei and 35S-securin
(Figure 2.2. a). Interestingly, also in these extracts ectopic UbcX caused APC/C
activation and CSF release in the absence of the calcium signal, as indicated by
23
panel 2). However - unlike in extracts treated with calcium - XErp1 remained
stable and showed an increase in its electrophoretic mobility following exit
from meiosis (Figure 2.2. c, panel 1 and 2), suggesting that UbcXwt causes CSF
inactivation by different means than XErp1 degradation. The addition of UbcXci
or dialysis buffer had no effect on CSF arrest (Figure 2.2. b, c, panel 3 and 4),
suggesting that the observed CSF override is dependent on an increase in the
catalytic activity of UbcX.
Additionally, the human homologue of UbcX was equivalent in the ability to
overcome CSF arrest in Xenopus egg extract, as the addition of catalytic active
UbcH10 triggered premature CSF release (Figure 2.2. d, panel 3),
demonstrating that both UbcX and UbcH10 are interchangeable in inducing
CSF release.
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2.3. Elevated UbcX activity prevents meiosis I - meiosis II transition in
Xenopus oocytes
To collect evidence for UbcX mediated regulation of CSF arrest in vivo, we
injected recombinant UbcX into Xenopus stage VI oocytes arrested at prophase
of meiosis I. We induced oocyte maturation by the addition of progesterone
and followed the resumption of