Non-proteolytic ubiquitylation regulates the APC/C-inhibitory function of XErp1

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Non-‐proteolytic ubiquitylation regulates the APC/C-‐inhibitory

function of XErp1

Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.)

vorgelegt von Eva Beate Hörmanseder

an der

Mathematisch-‐Naturwissenschaftliche Sektion Fachbereich Biologie

Tag der mündlichen Prüfung: 16. Dezember 2011

1. Referent: Prof. Dr. Thomas U. Mayer 2. Referent: Prof. Dr. Martin Scheffner

3. Referent: Prof. Dr. Olaf Stemmann

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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

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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

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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

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7 breakdown, the assembly of the mitotic spindle and chromosome 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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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

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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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11 1.4. Regulation of APC/C^Cdc20 activity in meiosis

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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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

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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

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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 SCF^β^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 SCF^β^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

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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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19 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/C^Cdc20 interaction is regulated and how the binding of XErp1 to the APC/C leads to its inactivation.

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UbcX dissociates XErp1 from the APC/C Results

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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/C^Cdc20 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 degraded upon calcium addition (Figure 2.1. b, panel 1), suggesting that APC/C

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inhibition was due to SAC-‐ but not CSF-‐activity. Addition of recombinant wild type UbcX (UbcX^wt) 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 (UbcX^ci) 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 UbcX^wt overrides SAC-‐arrest in Xenopus egg extract. (a) CSF-‐extracts containing ³⁵S-‐securinwas 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 UbcX^wt 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 the decondensation of the sperm nuclei chromatin (Figure 2.2. b) and by the degradation of the APC/C substrates ³⁵S-‐securin and cyclin B2 (Figure 2.2. c,

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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 UbcX^wt causes CSF inactivation by different means than XErp1 degradation. The addition of UbcX^ci 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.

Figure 2.2. Ectopic UbcX^wt overrides CSF arrest in Xenopus egg extract. (a) To CSF-‐extract the indicated reagents were added and (b) at the 90 minute time point chromatin structures were analyzed or (c) at the indicated time points samples were taken and ³⁵S-‐securin was detected by autoradiography and XErp1 and α-‐tubulin by WB. (d) Experiment described in (a) was repeated using UbcH10 instead of UbcX. CSF, cytostatic factor; ³⁵S-‐securin, in vitro translated,

35S-‐labeled securin; wt, wild type; ci, catalytical inactive.

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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 meiosis by microscopic analysis and by westernblotting for cyclin B2 and XErp1 (Figure 2.3. a). Oocytes injected with buffer performed germinal vesicle breakdown (GVBD), which is indicative of the resumption of meiosis I and exited meiosis I 60 min after GVBD, visible by a decline in cyclin B2 levels. Oocytes progressed through meiosis II and finally entered CSF arrest about 120 min after GVBD where XErp1 levels and are highest and cyclin B2 levels peak (Figure 2.3. b). In contrast, oocytes injected with UbcX^wt failed to re-‐accumulate cyclin B2 after progressing through meiosis I despite the presence of XErp1 (Figure 2.3. b). In addition, similar to XErp1 depleted oocytes, the pigmentation of the animal pole was disrupted and no small, defined spot indicative of a CSF arrested egg could be observed (Figure 2.3. c). Thus, consistent with results in Xenopus egg extract, we conclude that also in vivo CSF arrest is sensitive to changes in the activity of the E2 enzyme UbcX.

Figure 2.3. Ectopic UbcX^wt prevents Meiosis I -‐Meiosis II transition in oocytes. (a) Stage VI oocytes were injected with buffer or 80 ng UbcX^wt, resulting in 8,9 µM exogenous UbcX, which is about 11 fold of the endogenous protein. Maturation was induced by progesterone treatment and (b) at the indicated time points after GVBD samples were taken for WB analysis or (c) a picture was taken at the 90 min time point. wt, wild type; PG, Progesterone; GVBD, Germinal vesicle breakdown.

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25 2.4. UbcH10 – Induced CSF release requires proteasome and APC/C^Cdc20

activity

Addition of UbcX or UbcH10 to CSF arrested egg extracts inactivated the CSF as indicated by the degradation of the APC/C substrates securin and cyclin B (Figure 2.2. c). This degradation was proteasome dependent, as the addition of the potent proteasome inhibitor MG262 to CSF arrested extract inhibited the degradation of APC/C substrates in the presence of UbcH10^wt when compared to the DMSO control (Figure 2.4. a).

Figure 2.4. UbcH10^wt-‐induced CSF-‐release requires APC/C activity. (a) CSF arrested extract was supplemented with MG262 or DMSO and treated with calcium, buffer, UbcH10^wt or

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UbcH10^ci and the stability of ³⁵S-‐securin was analyzed by autoradiography. (b) Cdc20 was depleted from CSF-‐extracts in three rounds of immunodepletion. Cdc20 was detected by WB in the input fraction, in the extract after the third round of Cdc20-‐depletion (ΔCdc20) or control-‐

depletion as well as on the α-‐Cdc20 and control-‐beads (IP) after the indicated rounds of depletion. (c) Cdc20-‐ or control-‐depleted extract was supplemented with calcium, buffer, UbcH10^wt, or UbcH10^ci and the stability of ³⁵S-‐securin was analyzed by autoradiography. (d) Cdc20 was depleted from CSF-‐extracts as in (b) and recombinant Cdc20 was added to a final concentration of 170nM as indicated. Cdc20 was detected by WB after the third round of Cdc20-‐depletion (ΔCdc20). Note that recombinant Cdc20 is 8 kDa bigger than endogenous due to a 10xhis-‐2xTEV-‐tag. (e) Cdc20-‐depleted or Cdc20 depletion/add-‐back extract was supplemented with calcium, buffer or UbcX^wt and the stability of ³⁵S-‐securin was analyzed by autoradiography. (f) CSF-‐extract depleted of Cdc27 or control-‐depleted extract was supplemented with the indicated reagents and ³⁵S-‐securin was detected at the time points indicated. Ctrl, control; IP, immunoprecipitation; IgG, immunoglobulin G; Cdc, cell division cycle; ³⁵S-‐securin, in vitro translated, ³⁵S-‐labeled securin; wt, wild type; ci, catalytical inactive.

To understand whether this degradation was APC/C^Cdc20 dependent, we depleted the APC/C co-‐activating subunit Cdc20 from CSF arrested egg extracts by three rounds of immunodepletion (Figure 2.4. b, d). As expected, ³⁵S-‐securin was not degraded in Cdc20 depleted extracts supplemented with calcium when compared to the control depleted extracts. Cdc20 depletion also inhibited the degradation of ³⁵S-‐securin in extracts incubated with recombinant UbcH10^wt (Figure 2.4. c, e). The addition of recombinant Cdc20 was able to restore the degradation of ³⁵S-‐securin in both calcium and UbcH10 supplemented extracts, confirming the specificity of the Cdc20 depletion (Figure 2.4. e).

To corroborate this finding, we depleted the APC/C from CSF arrested egg extract by immunoprecipitating the APC/C core-‐subunit Cdc27. Also in these extracts, neither calcium addition nor UbcH10^wt addition induced ³⁵S-‐securin degradation (Figure 2.4. f). Together, these results suggest that APC/C^Cdc20 dependent ubiquitylation and proteasome dependent degradation of the APC/C^Cdc20 substrates is essential for the UbcX/UbcH10-‐dependent and calcium-‐ independent induction of CSF release.

2.5. Does USP44 counteract UbcX to maintain CSF arrest?

Reportedly, during SAC arrest the activity of UbcH10 needs to be antagonized

Cdc20

Non-proteolytic ubiquitylation regulates the APC/C-inhibitory function of XErp1