No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Dynamic regulation of Emi2 by Emi2-bound Cdk1/Plk1/CK1 and PP2A-B56 in meiotic arrest of Xenopus eggs
Abstract
In vertebrates, unfertilized eggs are arrested at metaphase of meiosis II by Mos and Emi2, an inhibitor of the APC/C ubiquitin ligase. In Xenopus, Cdk1 phosphorylates Emi2 and both destabilizes and inactivates it, whereas Mos recruits PP2A phosphatase to antagonize the Cdk1 phosphorylation. However, how Cdk1 phosphorylation inhibits Emi2 is largely unknown. Here we show that multiple N-terminal Cdk1 phosphorylation motifs bind cyclin B1-Cdk1 itself, Plk1, and CK1δ/ε to inhibit Emi2. Plk1, after rebinding to other sites by self-priming phosphorylation, partially destabilizes Emi2. Cdk1 and CK1δ/ε sequentially phosphorylate the C-terminal APC/C-docking site, thereby cooperatively inhibiting Emi2 from binding the APC/C. In the presence of Mos, however, PP2A-B56β/ε bind to Emi2 and keep dephosphorylating it, particularly at the APC/C-docking site. Thus, Emi2 stability and activity are dynamically regulated by Emi2-bound multiple kinases and PP2A phosphatase. Our data also suggest a general role for Cdk1 substrate phosphorylation motifs in M phase regulation.
... Previous studies showed that MPF (Maturation-promoting factor) activity remains constant during Xenopus CSF-arrest despite ongoing cyclin B1 synthesis (Isoda et al., 2011;Wu et al., 2007b). At high MPF activity, Cdk1/cyclin B1 phosphorylates the N-terminus of Emi2 resulting in initial recruitment of Plk1, which then creates its own high affinity docking sites by phosphorylating Emi2 at T170 and T195 ( Figure S3B). ...
... Subsequently, Plk1 targets Emi2 for degradation by SCF b-TRCP through phosphorylation of two phosphodegrons (DSGX3S 38 and DSAX2S 288 ). The drop in Emi2 levels and the resulting transient APC/C activation reduces MPF activity, tipping the balance in favor of PP2A-B56, which dephosphorylates the inhibitory sites on Emi2 causing its restabilization Isoda et al., 2011;Nishiyama et al., 2007;Wu et al., 2007a;Wu et al., 2007b). Hence, transitory phosphorylation of Emi2 controls the APC/C to maintain MPF activity at constant levels while cyclin B1 synthesis continues. ...
... Mutation of T170 (T170A), but not of T195 (T195A), also resulted in stabilization of Emi2 ZBR ( Figure 4C). During CSF-arrest, Plk1 itself phosphorylates T170 and this depends on Cdk1/cyclin B1 phosphorylation-dependent recruitment of Plk1 to Emi2's N-terminus ( Figure S3B) (Isoda et al., 2011). Crucially, Emi2 ZBR carrying non-phosphorylatable mutations at all four N-terminal Cdk1 sites (4 T/SP > 4A) was stable in presence of cyclin B3 WT ( Figure S3C). ...
To ensure successful offspring ploidy, vertebrate oocytes must halt the cell cycle in meiosis II until sperm entry. Emi2 is essential to keep oocytes arrested until fertilization. However, how this arrest is implemented exclusively in meiosis II and not prematurely in meiosis I has until now remained enigmatic. Using mouse and frog oocytes, we show here that cyclin B3, an understudied B-type cyclin, is essential to keep Emi2 levels low in meiosis I. Direct phosphorylation of Emi2 at an evolutionarily highly conserved site by Cdk1/cyclin B3 targets Emi2 for degradation. In contrast, Cdk1/cyclin B1 is inefficient in Emi2 phosphorylation, and this provides a molecular explanation for the requirement of different B-type cyclins for oocyte maturation. Cyclin B3 degradation at exit from meiosis I enables Emi2 accumulation and thus timely arrest in meiosis II. Our findings illuminate the evolutionarily conserved mechanisms that control oocyte arrest for fertilization at the correct cell-cycle stage, which is essential for embryo viability.
... PP2A phosphatase activity counteracts the destabilizing phosphorylation by kinases (Cdk1, Plx1, Ck1) (Hertz et al. 2016;Isoda et al. 2011;Nishiyama, Ohsumi, and Kishimoto 2007;Inoue et al. 2007;Gross et al. 2000Gross et al. , 1999Bhatt and Ferrell 1999). A similar mechanism is observed for mouse oocytes with a phosphorylation of T337 that is important for Emi2 CSF activity. ...
... This provides correct MII entry. Because Cdk1 activity is lower in meiosis II than in meiosis I, Emi2 can act as an APC/C inhibitor (Isoda et al. 2011;Tang et al. 2008). However, it is tempting to speculate that the ability of Emi2 to act as an APC/C inhibitor is determined by absence (meiosis II) or presence (meiosis I) of cyclin B3. ...
... As seen in Xenopus oocytes, Cdk1 phosphorylates Emi2 on specific sites on the N-terminus to destabilize it and in the C-terminus to inactivate it. These phosphorylation sites have been identified in Xenopus oocytes (Isoda et al. 2011 Phosphorylation on certain sites on the N-terminus of Xerp1/Emi2 recruits Plk1 to Xerp1/Emi2 leading to its degradation. Cyclin B3 could regulate Plk1 activity or/and cyclin B1-Cdk1. ...
Meiosis is a tightly regulated process made up of two successive divisions, meiosis I and II. They must be completed in an orderly manner to obtain haploid gametes with the correct number of chromosomes. Female mammalian meiosis is an error-prone process where errors in segregation create aneuploid gametes. In addition, incidence of aneuploidy increases in correlation with age. Understanding the regulation of female mammalian meiosis is therefore essential. Meiotic cell divisions are regulated by cyclins associated to their binding catalytic partners Cdks. I investigated the role of a unique cyclin, cyclin B3, through the use of cyclin B3 KO female mice. I found that lack of cyclin B3-Cdk1 activity in KO oocytes affects APC/C activity and induces an arrest at metaphase I due to high cyclin B1 levels, high Cdk1 activity, and inactive separase. Surprisingly, cyclin B3 from other species was able to rescue mouse cyclin B3 KO oocytes. I was also able to show that cyclin B3 is able to inhibit CSF arrest. My recent data suggests that cyclin B3 KO oocytes put in place a precocious CSF arrest, leading to the metaphase I arrest observed. Hence, my PhD work has shown that cyclin B3 is essential for female meiosis I and to prevent precocious CSF arrest in meiosis I instead of meiosis II.
... Before fertilization, Erp1/Emi2 inhibits Cdc20-activated anaphase-promoting complex (APC/C), and consequently M phase promoting factor (MPF, complex of Cdk1and Cyclin B), to maintain meiotic arrest (Inoue et al., 2007;J.Q. Wu et al., 2007;Isoda et al., 2011;Tischer et al., 2012;H€ ormanseder et al., 2013). In this pathway, PP2Ac-B56 contribute to the maintenance of meiotic arrest by stabilizing and activating Erp1/Emi2 by dephosphorylating it (Isoda et al., 2011). ...
... Wu et al., 2007;Isoda et al., 2011;Tischer et al., 2012;H€ ormanseder et al., 2013). In this pathway, PP2Ac-B56 contribute to the maintenance of meiotic arrest by stabilizing and activating Erp1/Emi2 by dephosphorylating it (Isoda et al., 2011). On the other hand, in ascidian Ciona robusta (former C. intestinalis, type A), it is likely that PP2A does not have a role in maintaining meiotic arrest. ...
... In this molecular pathway, PP2Ac (catalytic subunit)-B56 (regulatory subunit) complex stabilizes and activates Emi2 by dephosphorylating it. Mutation of Emi2 protein at PP2A-B56 recruitment sites (S335A/T336A) cause degradation and inactivation of Emi2 (Isoda et al., 2011;. Thus, PP2A is essential for maintenance of meiotic arrest in vertebrates. ...
... In Xenopus oocytes, activation of XErp1 requires its phosphorylation by the 90-kDa ribosomal protein S6 kinase (p90 RSK ), the downstream kinase of the c-Mos-/mitogen-activated protein kinase (MAPK) pathway [5,6]. Upon phosphorylation of XErp1 by p90 RSK , protein phosphatase PP2A in complex with the regulatory B 0 56 subunit binds to XErp1 and protects it from inhibitory phosphorylation by Cdk1/cyclin B and other kinases [10,11]. In Xenopus oocytes, the transient rise in calcium levels associated with fertilization causes the activation of the kinase CaMKII and the phosphatase calcineurin (CaN, also called PP2B) [12][13][14]. ...
... Using a non-degradable XErp1 version, we could demonstrate that CaN inhibition unexpectedly accelerated the dephosphorylation of XErp1 during meiotic exit. We could demonstrate that CaN dephosphorylates XErp1 at a site that is part of a phosphorylation-dependent recruiting motif for PP2-B'56, which protects XErp1 from inactivating and destabilizing phosphorylation events [5,6,10,11]. In the case of Cdc20, CaN inhibition delayed the calcium-induced dephosphorylation. ...
... As expected, in the absence of ectopic XErp1 and at high calcium concentrations, CaN inhibition had only a mild effect on cyclin B2 destruction ( Fig 1D, lane 1-3 vs. 4-6). However, when the amount of XErp1 in the extract was roughly doubled (1:160 IVT Myc-XErp1 wt ), cyclin B2 degradation was significantly inhibited in CsA-treated extracts compared to the DMSO control (lane 7-9 vs. [10][11][12]. Further increases in XErp1 wt interfered with cyclin B2 degradation in control-as well as in CsA-treated extracts (lanes [13][14][15][16][17][18][19][20][21][22][23][24]. ...
Vertebrate oocytes await fertilization arrested at metaphase of the second meiotic division. Fertilization triggers a transient calcium wave, which induces the activation of the anaphase-promoting complex/cyclosome (APC/C) and its co-activator Cdc20 resulting in the destruction of cyclin B and hence meiotic exit. Two calcium-dependent enzymes are implicated in fertilization-induced APC/CCdc20 activation: calcium-/calmodulin-dependent kinase type II (CaMKII) and calcineurin (CaN). While the role of CaMKII in targeting the APC/C inhibitor XErp1/Emi2 for destruction is well-established, it remained elusive how CaN affects APC/CCdc20 activation. Here, we discover that CaN contributes to APC/CCdc20 activation in Xenopus laevis oocytes by two independent but interrelated mechanisms. First, it facilitates the degradation of XErp1 by dephosphorylating it at a site that is part of a phosphorylation-dependent recruiting motif for PP2A-B′56, which antagonizes inhibitory phosphorylation of XErp1. Second, it dephosphorylates Cdc20 at an inhibitory site, thereby supporting its APC/C-activating function. Thus, our comprehensive analysis reveals that CaN contributes to timely APC/C activation at fertilization by both negatively regulating the APC/C inhibitory activity of XErp1 and positively regulating the APC/C-activating function of Cdc20.
... Mos activates the MAPKK/MAPK/p90 Rsk cascade and p90 Rsk directly phosphorylates Ser335, Thr336 and Ser342 of XErp1 (Bhatt and Ferrell, 1999;Gross et al., 1999;Gross et al., 2000;Inoue et al., 2007;Nishiyama et al., 2007a). Phosphorylation of these residues creates a docking site for the phosphatase PP2A in complex with a regulatory subunit of the B56 type (PP2A-B56) (Hertz et al., 2016;Isoda et al., 2011;Wu et al., 2007a). PP2A-B56 actively counteracts destabilizing phosphorylation events in the N-terminus and inactivating phosphorylation events in the C-terminus of XErp1, which are catalyzed by the kinases cyclin-B/Cdk1, Plx1 and casein kinase 1 δ/ε (CK1δ/ε) (Isoda et al., 2011;Wu et al., 2007b). ...
... Phosphorylation of these residues creates a docking site for the phosphatase PP2A in complex with a regulatory subunit of the B56 type (PP2A-B56) (Hertz et al., 2016;Isoda et al., 2011;Wu et al., 2007a). PP2A-B56 actively counteracts destabilizing phosphorylation events in the N-terminus and inactivating phosphorylation events in the C-terminus of XErp1, which are catalyzed by the kinases cyclin-B/Cdk1, Plx1 and casein kinase 1 δ/ε (CK1δ/ε) (Isoda et al., 2011;Wu et al., 2007b). ...
... This leads to an increase in APC/C Cdc20 activity, which will then decrease the cyclin-B levels again. The resulting drop in Cdk1 activity allows PP2A-B56 to prevail over it and to increase XErp1 stability and activity (Isoda et al., 2011). Additionally, Figure 5 Regulation of XErp1 during metaphase II 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 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). ...
... 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 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). ...
... Cdk1 phosphorylations destabilize XErp1 and decrease its affinity for the APC/C (Wu et al., 2007a;Wu et al., 2007b). 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 time. ...
... The N-terminal Cdk1 cluster contributes to XErp1 stability (Wu et al, 2007b). When XErp1 is phosphorylated here by Cdk1, it leads to the recruitment of Introduction Casein kinase 1 (CK1) and both kinases conjoinedly create a binding site for Xenopus Plk1 (Plx1) (Figure 3 B) (Isoda et al, 2011). Subsequently, Plx1 phosphorylates Ser-33 and Ser-38 as well as Ser-284 and Ser-288, leading to SCF β-TRCP dependent ubiquitylation and destruction of XErp1. ...
... As described before, the cMos pathway via p90Rsk is essential for XErp1 function. The kinase activates XErp1 by phosphorylating it at Ser-335 and Thr-336 and possibly at Ser-342 and Ser-344 as well Nishiyama et al, 2007a;Wu et al, 2007a), which leads to the binding of PP2A-B'56 (Isoda et al, 2011;Wu et al, 2007a) and subsequent removal of the inhibitory phosphorylations done by Cdk1 on XErp1 (Figure 3 A and B). responsible for its stability. ...
... Therefore a Cdk1 counteracting activity, namely a phosphatase, might be necessary to keep XErp1 active. From meiosis it is known that PP2A can bind to and activate XErp1 by dephosphorylation (Isoda et al, 2011;Wu et al, 2007b). If this also applies for embryonic cell cycles, inhibition of PP2A should lead to XErp1 inactivation. ...
... Later, however, at least two other proteins, Wee1 and Boi1, were also shown to be phosphorylated by Cdk1 on non-S/T-P motifs 15,16 . More recently, we showed that the C-terminal tail of Emi2, an inhibitor of the anaphase-promoting complex/cyclosome (APC/C), is phosphorylated by Cdk1 on a non-S/T-P motif 17 . As these (not well-defined) non-S/T-P motifs are evolutionarily well-conserved at least in their respective protein species, they might have some structural similarity to each other, possibly forming a ''consensus'' sequence(s). ...
... More than two decades ago, three intermediate filament proteins (vimentin, desmin and GFAP) and myosin II (regulatory light chain) were shown to be phosphorylated by Cdk1 on their N-terminal non-S/T-P motifs [11][12][13][14]25 . More recently, we showed that Emi2, an inhibitor of the APC/C, is phosphorylated on a C-terminal non-S/T-P motif to be inactivated in Xenopus eggs 17 . To determine whether the non-S/T-P motifs in these proteins (Fig. 1a) make any consensus sequence for phosphorylation by Cdk1, we performed in vitro Cdk1 kinase assays of the respective non-S/T-P motif-spanning peptides (WT or Ala-mutants, fused to glutathione S-transferase or GST) in the presence of [U-32 P]ATP. ...
... 43 and references therein). Furthermore, and interestingly, multiple S/T-P motifs are often clustered in the disordered regions of mitotic proteins 44 and, in some cases, their Cdk1 phosphorylation is required for Cdk1 phosphorylation of the non-S/ T-P motifs in the same protein [15][16][17] ; notably, in the case of Emi2, the N-terminal S/T-P motifs (phosphorylated by Cdk1) bind cyclin B-Cdk1 itself, which in turn phosphorylates a C-terminal non-S/T-P motif 17 . Thus, in these cases, the S/T-P motifs could function to locally concentrate Cdk1 for phosphorylation of the non-S/T-P motifs. ...
The cyclin B-dependent protein kinase Cdk1 is a master regulator of mitosis and phosphorylates numerous proteins on the minimal consensus motif Ser/Thr-Pro (S/T-P). At least in several proteins, however, not well-defined motifs lacking a Pro in the +1 position, referred herein to as non-S/T-P motifs, have been shown to be phosphorylated by Cdk1. Here we show that non-S/T-P motifs in fact form consensus sequences for Cdk1 and probably play roles in mitotic regulation of physiologically important proteins. First, we show, by in vitro kinase assays, that previously identified non-S/T-P motifs all harbour one or more C-terminal Arg/Lys residues essential for their phosphorylation by Cdk1. Second, using Arg/Lys-scanning oriented peptide libraries, we demonstrate that Cdk1 phosphorylates a minimal sequence S/T-X-X-R/K and more favorable sequences (P)-X-S/T-X-[R/K]2-5 as its non-S/T-P consensus motifs. Third, on the basis of these results, we find that highly conserved linkers (typically, T-G-E-K-P) of C2H2 zinc finger proteins and a nuclear localization signal-containing sequence (matching P-X-S-X-[R/K]5) of the cytokinesis regulator Ect2 are inhibitorily phosphorylated by Cdk1, well accounting for the known mitotic regulation and function of the respective proteins. We suggest that non-S/T-P Cdk1 consensus motifs identified here may function to regulate many other proteins during mitosis.
... In addition, the well-conserved C-terminal tail, termed the RL tail, directly binds to the APC/C and helps the inhibitory interactions of the D-box and the ZBR with the APC/C 18 . By recruiting PP2A-B56 phosphatase, the Mos-MAPK pathway antagonizes Emi2 inhibitory phosphorylations by cyclin B-Cdk1, which occur on both the N-terminal and C-terminal regions, including the RL tail 19,20 . ...
... Consistent with this idea, merely immunodepleting Emi2 from CSF extracts induced a fivefold increase in APC/C-Ube2S interactions, similar to calcium treatment of the extracts (Fig. 4a). Furthermore, treating CSF extracts with okadaic acid (OA), which can dissociate APC/C-Emi2 complexes (without degrading Emi2) by inhibiting Emi2-associated PP2A-B56 phosphatase 19,20 , also induced an increase of APC/C-Ube2S interactions, while dissociating the APC/C-Emi2 complexes (Fig. 4b). Thus, these results support the idea that the increase of APC/C-Ube2S interactions after CSF release is a result of the disappearance of Emi2 proteins or APC/C-Emi2 interactions on CSF release. ...
... The RL tail peptides, but not its ultimate dipeptide RL-AA peptides, strongly inhibited the increase of APC/C-Ube2S interactions after CSF release (as well as inhibiting the weak APC/ C-Ube2S interactions during CSF arrest and also APC/C-Emi2 interactions) (Fig. 4d). Because RL tail peptides, but not AA peptides, can directly bind to the APC/C 18,19 , these results strongly suggest that APC/C binding of the Emi2 RL tail can by itself inhibit Ube2S binding to the APC/C, thereby impairing Ube2S function and, hence, APC/C activity. Consistent with this, adding RL tail (but not AA) peptides, together with calcium, to CSF extracts was able to delay the degradation of cyclin B (Fig. 4e), similar to immunodepletion of Ube2S (Fig. 1c). ...
In vertebrates, unfertilized eggs are arrested at metaphase of meiosis II by Emi2, a direct inhibitor of the APC/C ubiquitin ligase. Two different ubiquitin-conjugating enzymes, UbcH10 and Ube2S, work with the APC/C to target APC/C substrates for degradation. However, their possible roles and regulations in unfertilized/fertilized eggs are not known. Here we use Xenopus egg extracts to show that both UbcH10 and Ube2S are required for rapid cyclin B degradation at fertilization, when APC/C binding of Ube2S, but not of UbcH10, increases several fold, coincidently with (SCF(β-TrCP)-dependent) Emi2 degradation. Interestingly, before fertilization, Emi2 directly inhibits APC/C-Ube2S binding via the C-terminal tail, but on fertilization, its degradation allows the binding mediated by the Ube2S C-terminal tail. Significantly, Emi2 and Ube2S bind commonly to the APC/C catalytic subunit APC10 via their similar C-terminal tails. Thus, Emi2 competitively inhibits APC/C-Ube2S binding before fertilization, while its degradation on fertilization relieves the inhibition for APC/C activation.
... The MII arrest is maintained despite a constitutively high Cdk1-Cyclin B is activated by calcium-ions and directly phosphorylates XErp1 at T195 promoting strong binding of Plk1 in a polo-box domain (PBD) dependent manner (43)(44)(45). A pool of Plk1 is weakly bound to N-terminal Cdk1 sites on XErp1 and phosphorylates residues T170 and T195 which turn into strong docking sites for more Plk1 molecules (3). In the end, Plk1 targets residues in the destruction box motifs DSG (DS33GX3S38) and DSA (DS275AX2S279) marking XErp1 for SCF β-TRCP mediated proteasomal destruction (2,37). ...
... In summary, we showed that the protein levels of PP2A-C and the investigated B56 isoforms do not contribute to a temporal regulation of PP2A neither in a cell cycle specific manner nor during the rapid cleavage cycles (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) and that an upregulation of a subset of PP2A subunits occurs only during or after MBT. Figure 9C). Despite performing several rounds of depletion with high antibody amounts we have never been able to completely deplete B56αβɛ from CSF extract ( Figure 9C). ...
... AF3.1 and B60 were used for immunoprecipitation or immunodepletion of APC/C and Cdc20, respectively [36]. Plasmids for the expression of X. laevis PP2A regulatory B subunits and PP2A (Aa, B56c and Cb) subunits are gifts from Drs N. Sagata [37] and S. Mochida [17], respectively. ...
... Apc1-loop 500 binding assaysFor binding assay with [ 35 S]-labelled PP2A B subunits and Apc1-Loop 500 fragments, N-terminally 6xHis-2xFlag-tagged X. laevis PP2A regulatory B subunits (B56c, B55d or B″/PR70)[37] were labelled with [ 35 S]methionine (Hartmann Analytic, UK) in a coupled in vitro transcription-translation system (Promega, UK). Purified Apc1-Loop 500 fragment proteins were bound to amylose beads (New England Biolabs) by incubating at 4°C for 0.5-1 h. ...
Cell cycle progression and genome stability are regulated by a ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C). Cyclin-dependent kinase 1 (Cdk1) has long been implicated in APC/C activation; however, the molecular mechanisms of governing this process in vivo are largely unknown. Recently, a Cdk1-dependent phosphorylation relay within Apc3-Apc1 subunits has been shown to alleviate Apc1-mediated auto-inhibition by which a mitotic APC/C co-activator Cdc20 binds to and activates the APC/C. However, the underlying mechanism for dephosphorylation of Cdc20 and APC/C remains elusive. Here, we show that a disordered loop domain of Apc1 (Apc1-loop500 ) directly binds the B56 regulatory subunit of protein phosphatase 2A (PP2A) and stimulates Cdc20 loading to the APC/C. Using the APC/C reconstitution system in Xenopus egg extracts, we demonstrate that mutations in Apc1-loop500 that abolish B56 binding decrease Cdc20 loading and APC/C-dependent ubiquitylation. Conversely, a non-phosphorylatable mutant Cdc20 can efficiently bind the APC/C even when PP2A-B56 binding is impeded. Furthermore, PP2A-B56 preferentially dephosphorylates Cdc20 over the Apc1 inhibitory domain. These results indicate that Apc1-loop500 plays a role in dephosphorylating Cdc20, promoting APC/C-Cdc20 complex formation in mitosis.
... As aforementioned, XErp1 inhibits the APC/C, and this activity is essential to arrest mature oocytes at metaphase of meiosis II Shoji et al. 2006 ;Rauh et al. 2005 ). Phosphorylation of XErp1 by the mitotic kinase Cdk1/cyclin-B primes XErp1 for further phosphorylations by Polo-like kinase 1 (Plk1) and casein kinase 1 (CK1), and these phosphorylations result in the destabilisation and inhibition of XErp1 (Isoda et al. 2011 ). The inhibitory effect of Cdk1/cyclin-B on the APC/C inhibitory activity of XErp1 is antagonised by PP2A-B`56-mediated dephosphorylation of XErp1. ...
... During the MII arrest of Xenopus eggs, this mechanism maintains the activity of Cdk1/cyclin-B at constant levels despite ongoing synthesis of cyclin-B (Wu et al. 2007 ): once cyclin-B levels reach a critical upper threshold, XErp1 is inactivated by Cdk1/cyclin-B resulting in APC/C activation. APC/C activation leads to cyclin-B destruction until PP2A-B`56 prevails over Cdk1/cyclin-B resulting in XErp1 dephosphorylation and the re-inhibition of the APC/C (Isoda et al. 2011 ). In somatic human cells, PP2A-B`56 has been shown to be important for fine-tuning the APC/C-mediated destruction of securin. ...
The challenging task of mitotic cell divisions is to generate two genetically identical daughter cells from a single precursor cell. To accomplish this task, a complex regulatory network evolved, which ensures that all events critical for the duplication of cellular contents and their subsequent segregation occur in the correct order, at specific intervals and with the highest possible fidelity. Transitions between cell cycle stages are triggered by changes in the phosphorylation state and levels of components of the cell cycle machinery. Entry into S-phase and M-phase are mediated by cyclin-dependent kinases (Cdks), serine-threonine kinases that require a regulatory cyclin subunit for their activity. Resetting the system to the interphase state is mediated by protein phosphatases (PPs) that counteract Cdks by dephosphorylating their substrates. To avoid futile cycles of phosphorylation and dephosphorylation, Cdks and PPs must be regulated in a manner such that their activities are mutually exclusive.
... Independent of the identity of the most downstream component of the MAPK signaling pathway, it is likely that the common target in Xenopus and mammalian eggs is EMI2, an APC inhibitor. EMI2 appears to be a meiosisspeci fi c inhibitor of the APC, whose activity is fi rst seen at the end of meiosis I [77][78][79][80][81][82] . Importantly, the relationship between the pathways involving c-MOS-MAPK and APC EMI2 has been established in Xenopus , by the fi nding that p90RSK-induced phosphorylation of EMI2 is needed to keep this APC inhibitor stable [ 83,84 ] . ...
... At fertilization in mouse, sperm calcium signals activate the calcium-sensitive kinase calmodulin-dependent protein kinase II (CAMKII) [ 88,89 ] . This signal also occurs in frog, where it has been established that CAMKII phosphorylates EMI2, which in so doing causes its degradation and thereby releasing the APC from its arrest [77][78][79][80][81][82] . Interestingly, in the mouse, this CAMKII signal also activates WEE1B, which contributes to switching off CDK1 activity, necessary for meiotic exit [ 90 ] . ...
Oocytes are made in the fetal ovary and are only ever fertilized some considerable time later in the adult. During this time, they have to undergo two meiotic divisions (meiosis I and II), which must be executed faithfully and on time so as to produce a mature egg, with a haploid chromosome content, that is ovulated into the fallopian tube ready to be fertilized. The two meiotic divisions are controlled by both internal and external (hormonal) triggers, principally executed by changes in the activity of the kinase CDK1 in the oocyte. Here, we focus on how the oocyte controls CDK1 activity at three important time points: (1) the arrest at prophase I in the ovary and the hormone-driven release from this arrest, (2) the progression through meiosis I, and finally (3) the rearrest at metaphase II and subsequent completion of meiosis triggered by a sperm calcium signal.
... A control morpholino (GeneTools) was used as injection control. 91-Securin harboring an N-terminal 91 amino acid deletion was PCR-cloned from human Securin into pRN3 (34). ...
... This suggests that TAM could regulate OSD1 to prevent precocious meiotic exit. Alternatively, phosphorylation by TAM could inactivate OSD1 and thus allow exit from meiosis I. Interestingly, the activity and stability of Emi2/Erp1 -the vertebrate meiotic APC/C inhibitoris regulated by phosphorylation [33,34]. Further functional analysis of the OSD1 putative phosphorylation sites is required to establish the role of this CYCA1;2/TAM-CDKA;1-mediated phosphorylation in meiotic cell cycle progression. ...
Women in industrialized countries tend to postpone childbearing, leading to a 70% increase intrisomic pregnancies over 20 years. Meiosis in females is error prone, with rates of meiotic chromosome missegregations strongly increasing towards the end of the reproductive lifespan. A strong reduction of BubR1 has been observed in oocytes of women approaching menopause and in ovaries of aged mice, which led to the hypothesis that deterioration of spindle assembly checkpoint fidelity contributes to age-related aneuploidization. However, this idea has remained controversial since transient knock-down of BubR1 was found to prevent meiotic prophase arrest and chromosome segregation in a checkpoint independent manner. We employed a conditional knockout approach in mouse oocytes to dissect the meiotic roles of BubR1. We show that BubR1 is required for diverse meiotic functions, including persistent spindle assembly checkpoint activity, timing of meiosis I, and establishment of robust kinetochore-microtubule attachments in a meiosis specific manner, but not prophase I arrest. These data reveal that BubR1 plays a multi-faceted role in chromosome segregation during the first meiotic division and suggest that age-related loss of BubR1 is a key determinant of formation of aneuploid oocytes as women approach menopause. Using mouse oocytes, a second aspect of my thesis reveals that cyclin A2 promotes entry into meiosis, as well as an additional unexpected role, namely, its requirement for separase- dependent sister chromatid separation in meiosis II.
... XErp1/ Emi2, a highly conserved F-box protein, accumulates in . XErp1/Emi2 is phosphorylated by p90Rsk on Ser-335, Thr-336, Ser-342 and Ser-344, and subsequently associates with the B'56 subtype of PP2A (PP2A-B'56), which in turn removes Cdk1mediated inhibitory phosphorylation from amino-and carboxy-terminal regions of XErp1/Emi2 (Inoue et al, 2007;Nishiyama et al, 2007a;Wu et al, 2007a,b;Isoda et al, 2011) ( Figure 3). Phosphorylation of the XErp1/Emi2 C-terminal region interferes with its ability to bind and inhibit the APC/C ( Figure 4A) (Wu et al, 2007a), similar to the situation of the activator Cdc20 that can also only associate with the APC/C when dephosphorylated (Labit et al, 2012). ...
... Phosphorylation in the amino-terminal Cdk1 site cluster, on the other hand, controls XErp1/Emi2 stability ( Figure 4A), by serving as priming event for the recruitment of Polo-like kinase 1 (Plk1). Further phosphorylation by this kinase creates a phosphorylation-dependent recognition motif (or phosphodegron) for the ubiquitin ligase SCF b-TRCP , which then ubiquitylates and targets XErp1/Emi2 for proteasomal degradation (Wu et al, 2007a;Isoda et al, 2011). Thus, p90Rsk-mediated recruitment of PP2A-B'56 downstream of the c-Mos/MAPK pathway activates XErp1/Emi2, both by stabilizing it and by directly impinging on its APC/C inhibitory properties. ...
Ex ovo omnia-all animals come from eggs-this statement made in 1651 by the English physician William Harvey marks a seminal break with the doctrine that all essential characteristics of offspring are contributed by their fathers, while mothers contribute only a material substrate. More than 360 years later, we now have a comprehensive understanding of how haploid gametes are generated during meiosis to allow the formation of diploid offspring when sperm and egg cells fuse. In most species, immature oocytes are arrested in prophase I and this arrest is maintained for few days (fruit flies) or for decades (humans). After completion of the first meiotic division, most vertebrate eggs arrest again at metaphase of meiosis II. Upon fertilization, this second meiotic arrest point is released and embryos enter highly specialized early embryonic divisions. In this review, we discuss how the standard somatic cell cycle is modulated to meet the specific requirements of different developmental stages. Specifically, we focus on cell cycle regulation in mature vertebrate eggs arrested at metaphase II (MII-arrest), the first mitotic cell cycle, and early embryonic divisions.
... This caused a decrease in the level of cyclin synthesized when proteasome activity was permitted ( Figure 4C, left, boxes), whereas blocking the proteasome ( Figure 4C, left, open circles) caused accumulation of cyclin that matched levels seen in extracts with unaltered CDK1 activities. This confirmed that increasing CDK1 activity can increase APC activity in CSF extracts-which had been reported previously (Hansen et al., 2007;Wu et al., 2007;Isoda et al., 2011)-but did not slow the production of cyclin B. This is the opposite of what we observed prior to and during M phase of mitotic cycles and demonstrates that CDK1 activity does not exert the same inhibition on cyclin synthesis in mature oocytes/eggs (CSF extract) that it does in embryos (cycling extract). ...
... The control of meiotic cyclin B translation in the unfertilized egg, however, is different from that of mitotic cycles. Polyadenylation of cyclin B1 mRNA has been reported in the unfertilized egg (Sheets et al., 1994;Ballantyne et al., 1997;Kim and Richter, 2007;Belloc et al., 2008), and we confirmed that meiotic CSF extracts are capable of continuous cyclin translation in the presence of elevated CDK1 activity, in agreement with previously published studies (Hansen et al., 2007;Wu et al., 2007;Isoda et al., 2011). Therefore, although translation of cyclin B in meiotic M phase is continuous despite CDK1 activity, it is attenuated during M phase of mitotic cycles. ...
Cyclin B activates cyclin-dependent kinase 1 (CDK1) at mitosis, but conflicting views have emerged on the dynamics of its synthesis during embryonic cycles, ranging from continuous translation to rapid synthesis during mitosis. Here we show that a CDK1-mediated negative-feedback loop attenuates cyclin production before mitosis. Cyclin B plateaus before peak CDK1 activation, and proteasome inhibition caused minimal accumulation during mitosis. Inhibiting CDK1 permitted continual cyclin B synthesis, whereas adding nondegradable cyclin stalled it. Cycloheximide treatment before mitosis affected neither cyclin levels nor mitotic entry, corroborating this repression. Attenuated cyclin production collaborates with its destruction, since excess cyclin B1 mRNA accelerated cyclin synthesis and caused incomplete proteolysis and mitotic arrest. This repression involved neither adenylation nor the 3' untranslated region, but it corresponded with a shift in cyclin B1 mRNA from polysome to nonpolysome fractions. A pulse-driven CDK1-anaphase-promoting complex (APC) model corroborated these results, revealing reduced cyclin levels during an oscillation and permitting more effective removal. This design also increased the robustness of the oscillator, with lessened sensitivity to changes in cyclin synthesis rate. Taken together, the results of this study underscore that attenuating cyclin synthesis late in interphase improves both the efficiency and robustness of the CDK1-APC oscillator.
... Further, recent studies indicate functional relationship between PP2A-B56 and CLK2 in different physiological and pathological context 50,51 . Lastly, it was recently reported that RSK2-mediated phosphorylation of Emi2 promotes its well-known interaction with PP2A-B56 52,53 . Finally, in a pathway analysis, CIP2A-regulated phosphoproteins were enriched among senescence-associated proteins ( Fig. 6F and Supplementary Data 5), which with the previous evidence linking CIP2A to senescence evasion 18 , provides a plausible mechanistic explanation for the potent tumour growth inhibition in CIP2A K21A mutant clones. ...
The protein phosphatase 2A (PP2A) heterotrimer PP2A-B56α is a human tumour suppressor. However, the molecular mechanisms inhibiting PP2A-B56α in cancer are poorly understood. Here, we report molecular level details and structural mechanisms of PP2A-B56α inhibition by an oncoprotein CIP2A. Upon direct binding to PP2A-B56α trimer, CIP2A displaces the PP2A-A subunit and thereby hijacks both the B56α, and the catalytic PP2Ac subunit to form a CIP2A-B56α-PP2Ac pseudotrimer. Further, CIP2A competes with B56α substrate binding by blocking the LxxIxE-motif substrate binding pocket on B56α. Relevant to oncogenic activity of CIP2A across human cancers, the N-terminal head domain-mediated interaction with B56α stabilizes CIP2A protein. Functionally, CRISPR/Cas9-mediated single amino acid mutagenesis of the head domain blunted MYC expression and MEK phosphorylation, and abrogated triple-negative breast cancer in vivo tumour growth. Collectively, we discover a unique multi-step hijack and mute protein complex regulation mechanism resulting in tumour suppressor PP2A-B56α inhibition. Further, the results unfold a structural determinant for the oncogenic activity of CIP2A, potentially facilitating therapeutic modulation of CIP2A in cancer and other diseases.
... EMI2 is an inhibitor of APC/C, which is involved in regulating the progression of cell cycle-related proteins [32]. To explore the influence of EMI2, we detected the changes in certain molecules that are involved in 19 different signaling pathways in stable transgenic RBE cells after silencing EMI2, and the expression of CDKN1B and P53 was found to be increased. ...
Background
Cholangiocarcinoma (CCA) is one of the deadliest cancers of the digestive tract. The prognosis of CCA is poor and the 5-year survival rate is low. Bioinformatic analysis showed that early mitotic inhibitor 2 (EMI2) was overexpressed in CCA but the underlying mechanism is not known.
Methods
The data on bile duct carcinoma from TCGA and GEO databases were used to detect the expression of EMI2. The transcription factors of EMI2 were predicted using JASPAR and PROMO databases. Among the predicted transcription factors, YY1 has been rarely reported in cholangiocarcinoma, and was verified using the luciferase reporter gene assay. RT-PCR was performed to predict the downstream pathway of EMI2, and PI3K/Akt was suspected to be associated with it. Subsequently, in vivo and in vitro experiments were conducted to verify the effects of silencing and overexpressing EMI2 and YY1 on the proliferation, invasion, and metastasis of the bile duct cancer cells.
Results
EMI2 was highly expressed in CCA. Silencing EMI2 inhibited the proliferation, invasion, and migration of CCA cells, arrested cell cycle in the G1 phase, and promoted of apoptosis. The luciferase reporter gene assay showed that YY1 bound to the promoter region of EMI2, and after silencing YY1, the expression of EMI2 decreased and the progression of CCA was inhibited. Moreover, key proteins in the PI3K/Akt signaling pathway decreased after silencing EMI2.
Conclusion
EMI2 may be one of the direct targets of YY1 and promotes the progression of CCA through the PI3K/Akt signaling pathway.
... EMI2 is an inhibitor of APC/C, which is involved in regulating the progression of cell cycle-related proteins [32] . To explore the in uence of EMI2, we detected the changes in certain molecules that are involved in 19 different signaling pathways in stable transgenic RBE cells after silencing EMI2, and the expression of CDKN1B and P53 was found to be increased. ...
Background: Cholangiocarcinoma (CCA) is one of the deadliest cancers of the digestive tract. The prognosis of CCA is poor and the 5-year survival rate is low. Bioinformatic analysis showed that early mitotic inhibitor 2 (EMI2) was overexpressed in CCA but the underlying mechanism is not known.
Methods: The data on bile duct carcinoma from TCGA and GEO databases were used to detect the expression of EMI2. The transcription factors of EMI2 were predicted using JASPAR and PROMO databases. Among the predicted transcription factors, YY1 has been rarely reported in cholangiocarcinoma, and was verified using the luciferase reporter gene assay. RT-PCR was performed to predict the downstream pathway of EMI2, and PI3K/Akt was suspected to be associated with it. Subsequently, in vivo and in vitro experiments were conducted to verify the effects of silencing and overexpressing EMI2 and YY1 on the proliferation, invasion, and metastasis of the bile duct cancer cells.
Results: EMI2 was highly expressed in CCA. Silencing EMI2 inhibited the proliferation, invasion, and migration of CCA cells, arrested cell cycle in the G1 phase, and inhibition of apoptosis. The luciferase reporter gene assay showed that YY1 bound to the promoter region of EMI2, and after silencing YY1, the expression of EMI2 decreased and the progression of CCA was inhibited. Moreover, key proteins in the PI3K/Akt signaling pathway decreased after silencing EMI2.
Conclusion: EMI2 may be one of the direct targets of YY1 and promotes the progression of CCA through the PI3K/Akt signaling pathway.
... It has been established that PP2A-B56 localizes specifically to kinetochores and centromeres, participating in chromatid-microtubule interactions and silencing of the spindle assembly checkpoint 37,38 . Likely relating to this M-phase role, PP2A-B56 is involved in the metaphase IIarrest of Xenopus oocyte by regulating the APC/C inhibitor, XErp1 39,40 . These M-phase roles of PP2A-B56 are not directly connected to Cdk1 regulation and do not involve Arpp19. ...
Ma thèse a visé à comprendre comment la cellule germinale femelle, ou ovocyte, reprend la division méiotique. Dans tout le règne animal, l’ovocyte est bloqué en prophase de première division méiotique et reprend la division au moment de l’ovulation, sous l’effet d’un signal externe, la progestérone chez le xénope. Cette hormone déclenche une voie de signalisation qui aboutit après 3 à 5 heures à l’activation de la kinase Cdk1. Les premières molécules activées de Cdk1 déclenchent une boucle d’auto-amplification permettant son activation totale et l’entrée en division. Cette boucle repose sur des phosphorylations catalysées par Cdk1 et ne fonctionne que si la phosphatase qui contrecarre son activité, PP2A-B55δ, est inhibée. Cette inhibition dépend de la protéine Arpp19, un inhibiteur spécifique de PP2A-B55δ quand elle est phosphorylée sur la S67 par la kinase Greatwall. Arpp19 a un second rôle lors de la reprise de la méiose. Chez tous les vertébrés, l’arrêt en prophase est maintenu par une forte activité de PKA. Chez le xénope, Arpp19 est phosphorylée par PKA sur un résidu distinct de celui ciblé par Greatwall, la S109. En réponse à la progestérone, l’inhibition de PKA provoque la déphosphorylation d’Arpp19, ce qui permet d'enclencher la voie de signalisation conduisant à l'activation de Cdk1. Une phosphatase est donc requise pour déphosphoryler Arpp19 sur S109 et lever le verrou exercé sur l'activation de Cdk1 en prophase. Mon travail a visé à élucider l’identité moléculaire de cette phosphatase inconnue. Par des approches de biochimie et protéomique, j’ai identifié cette phosphatase comme étant PP2A-B55δ. Par des approches fonctionnelles dans des extraits acellulaires et des ovocytes, j’ai établi que PP2A-B55δ est active en prophase. La phosphorylation d’Arpp19 sur la S109 dépend d'une balance entre les activités de PKA et PP2A-B55δ, en faveur de la kinase. En réponse à la progestérone, l’activité de PP2A-B55δ n’est pas affectée et l’inhibition de PKA suffit à la déphosphorylation d’Arpp19. PP2A-B55δ orchestre donc la levée de l’arrêt en prophase et l'entrée en phase M en agissant à deux périodes et sur deux sites distincts d’Arpp19, respectivement ciblés par PKA et Greatwall.
... It has been established that PP2A-B56 localizes specifically to kinetochores and centromeres, participating in chromatidmicrotubule interactions and silencing of the spindle assembly checkpoint 45,46 . Likely relating to this M-phase role, PP2A-B56 is involved in the metaphase II-arrest of Xenopus oocyte by regulating the APC/C inhibitor, XErp1 47,48 . These M-phase roles of PP2A-B56 are not directly connected to Cdk1 regulation and do not involve Arpp19. ...
Oocytes are held in meiotic prophase for prolonged periods until hormonal signals trigger meiotic divisions. Key players of M-phase entry are the opposing Cdk1 kinase and PP2A-B55δ phosphatase. In Xenopus , the protein Arpp19, phosphorylated at serine 67 by Greatwall, plays an essential role in inhibiting PP2A-B55δ, promoting Cdk1 activation. Furthermore, Arpp19 has an earlier role in maintaining the prophase arrest through a second serine (S109) phosphorylated by PKA. Prophase release, induced by progesterone, relies on Arpp19 dephosphorylation at S109, owing to an unknown phosphatase. Here, we identified this phosphatase as PP2A-B55δ. In prophase, PKA and PP2A-B55δ are simultaneously active, suggesting the presence of other important targets for both enzymes. The drop in PKA activity induced by progesterone enables PP2A-B55δ to dephosphorylate S109, unlocking the prophase block. Hence, PP2A-B55δ acts critically on Arpp19 on two distinct sites, opposing PKA and Greatwall to orchestrate the prophase release and M-phase entry.
... Substrate binding involves specific consensus motifs: PP2A-B56 recognizes the SLiM "LxxIxE" while PP2A-B55 preferentially targets substrates with positively charged polybasic units on either side of the residue to be dephosphorylated [93][94][95]. Furthermore, phosphorylation of residues inside or adjacent to PP2A-B56 recognition motifs potentiates its binding to the substrate [94,96]. ...
Cell division is orchestrated by the phosphorylation and dephosphorylation of thousands of proteins. These post-translational modifications underlie the molecular cascades converging to the activation of the universal mitotic kinase, Cdk1, and entry into cell division. They also govern the structural events that sustain the mechanics of cell division. While the role of protein kinases in mitosis has been well documented by decades of investigations, little was known regarding the control of protein phosphatases until the recent years. However, the regulation of phosphatase activities is as essential as kinases in controlling the activation of Cdk1 to enter M-phase. The regulation and the function of phosphatases result from post-translational modifications but also from the combinatorial association between conserved catalytic subunits and regulatory subunits that drive their substrate specificity, their cellular localization and their activity. It now appears that sequential dephosphorylations orchestrated by a network of phosphatase activities trigger Cdk1 activation and then order the structural events necessary for the timely execution of cell division. This review discusses a series of recent works describing the important roles played by protein phosphatases for the proper regulation of meiotic division. Many breakthroughs in the field of cell cycle research came from studies on oocyte meiotic divisions. Indeed, the meiotic division shares most of the molecular regulators with mitosis. The natural arrests of oocytes in G2 and in M-phase, the giant size of these cells, the variety of model species allowing either biochemical or imaging as well as genetics approaches explain why the process of meiosis has served as an historical model to decipher signalling pathways involved in the G2-to-M transition. The review especially highlights how the phosphatase PP2A-B55δ critically orchestrates the timing of meiosis resumption in amphibian oocytes. By opposing the kinase PKA, PP2A-B55δ controls the release of the G2 arrest through the dephosphorylation of their substrate, Arpp19. Few hours later, the inhibition of PP2A-B55δ by Arpp19 releases its opposing kinase, Cdk1, and triggers M-phase. In coordination with a variety of phosphatases and kinases, the PP2A-B55δ/Arpp19 duo therefore emerges as the key effector of the G2-to-M transition.
... Although Ssa1 S151 does not conform to the (S/T)-P (phosphorylated serine or threonine) consensus phosphorylation motif that is normally found for Cdk1 substrates, our data from in vitro as well as in vivo assays indicate that Cdk1 participates in Ssa1 phosphorylation at S151 (Fig. 3). Non-(S/T)-P sites for Cdk kinases have been reported in other biological contexts (79)(80)(81)(82)(83)(84). Ssa1 S151 was also identified in a phosphorylation screen for Cdk1 targets, although the extent of cell cycle dependence was not very high compared to other targets, suggesting that there could also be other kinases responsible for this modification (14). ...
Maintenance of protein homeostasis in eukaryotes during normal growth and stress conditions requires the functions of Hsp70 chaperones and associated co-chaperones. Here we investigate an evolutionarily-conserved serine phosphorylation that occurs at the site of communication between the nucleotide-binding and substrate-binding domains of Hsp70. Ser151 phosphorylation in yeast Hsp70 (Ssa1) is promoted by cyclin-dependent kinase (Cdk1) during normal growth. Phospho-mimic substitutions at this site (S151D) dramatically down-regulate heat shock responses, a result conserved with HSC70 S153 in human cells. Phospho-mimic forms of Ssa1 also fail to relocalize in response to starvation conditions, do not associate in vivo with Hsp40 co-chaperones, Ydj1 and Sis1, and do not catalyze refolding of denatured proteins in vitro in cooperation with Ydj1 and Hsp104. Despite these negative effects on HSC70/HSP70 function, the S151D phospho-mimic allele promotes survival of heavy metal exposure and suppresses the Sup35-dependent [ PSI ⁺ ] prion phenotype, consistent with proposed roles for Ssa1 and Hsp104 in generating self-nucleating seeds of misfolded proteins. Taken together, these results suggest that Cdk1 can downregulate Hsp70 function through phosphorylation of this site, with potential costs to overall chaperone efficiency but also advantages with respect to reduction of metal-induced and prion-dependent protein aggregate production.
... It has been established that PP2A-B56 localizes specifically to kinetochores and centromeres, participating in chromatid-microtubule interactions and silencing of the spindle assembly checkpoint 37,38 . Likely relating to this Mphase role, PP2A-B56 is involved in the metaphase II-arrest of Xenopus oocyte by regulating the APC/C inhibitor, XErp1 39,40 . These M-phase roles of PP2A-B56 are not directly connected to Cdk1 regulation and do not involve Arpp19. ...
Oocytes are held in meiotic prophase for prolonged periods until hormonal signals trigger meiotic divisions. Key players of M-phase entry are the opposing Cdk1 kinase and PP2A-B55δ phosphatase. In Xenopus, the protein Arpp19, phosphorylated at serine 67 by Greatwall, plays an essential role in inhibiting PP2A-B55δ, promoting Cdk1 activation. Furthermore Arpp19 has an earlier role in maintaining the prophase arrest through a second serine (S109) phosphorylated by PKA. Prophase release, induced by progesterone, relies on Arpp19 dephosphorylation at S109, owing to an unknown phosphatase. Here we identified this phosphatase as PP2A-B55δ. In prophase, PKA and PP2A-B55δ are simultaneously active, suggesting the presence of other important targets for both enzymes. The drop in PKA activity induced by progesterone decreases S109 phosphorylation, unlocking the prophase block. Hence, PP2A-B55δ acts critically on Arpp19 on two distinct sites, opposing PKA and Greatwall to orchestrate the prophase release and M-phase entry.
... Although Ssa1 S151 does not conform to the S/T-P consensus phosphorylation motif that is normally found for Cdk1 substrates, our data from in vitro as well as in vivo assays indicates that Cdk1 participates in Ssa1 phosphorylation at S151 (Fig. 3). Non-S/T-P sites for Cdk kinases have been reported in other biological contexts (Suzuki et al, 2015;Kusubata et al, 1993;Satterwhite, 1992;Harvey et al, 2005;Isoda et al, 2011;McCusker et al, 2007). Ssa1 S151 was also identified in a phosphorylation screen for Cdk1 targets, although the extent of cell cycle dependence was not very high compared to other targets, suggesting that there could also be other kinases responsible for this modification (Holt et al, 2009). ...
Maintenance of protein homeostasis in eukaryotes during normal growth and stress conditions requires the functions of Hsp70 chaperones and associated co-chaperones. Here we investigate an evolutionarily-conserved serine phosphorylation that occurs at the site of communication between the nucleotide-binding and substrate-binding domains of Hsp70. Ser151 phosphorylation in yeast Hsp70 (Ssa1) is promoted by cyclin-dependent kinase (Cdk1) during normal growth and dramatically affects heat shock responses, a function conserved with HSC70 S153 phosphorylation in human cells. Phospho-mimic forms of Ssa1 (S151D) also fail to relocalize in response to starvation conditions, do not associate in vivo with Hsp40 co-chaperones, Ydj1 and Sis1, and do not catalyze refolding of denatured proteins in vitro in cooperation with Ydj1 and Hsp104. S151 phosphorylation strongly promotes survival of heavy metal exposure and reduces Sup35-dependent [PSI+] prion activity, however, consistent with proposed roles for Ssa1 and Hsp104 in generating self-nucleating seeds of misfolded proteins. Taken together, these results suggest that Cdk1 downregulates Hsp70 function during periods of active growth, reducing propagation of aggregated proteins despite potential costs to overall chaperone efficiency.
... Hrr25 belongs to the casein kinase 1 (CK1) family of protein kinases and is the yeast ortholog of the mammalian CK1δ enzyme (Demaggio et al. 1992). The Hrr25/CK1δ proteins have conserved roles in ribosome maturation, vesicle trafficking, DNA repair and chromosome segregation during meiosis (Hoekstra et al. 1991;Petronczki et al. 2006;Schafer et al. 2006;Ray et al. 2008;Grozav et al. 2009;Biswas et al. 2011;Isoda et al. 2011;Lord et al. 2011). We report here that this critical regulator of cell proliferation is efficiently recruited to P-bodies in both yeast and mammalian cells under all conditions examined. ...
The eukaryotic cytoplasm contains a variety of ribonucleoprotein (RNP) granules in addition to the better-understood membrane-bound organelles. These granules form in response to specific stress conditions and contain a number of signaling molecules important for the control of cell growth and survival. However, relatively little is known about the mechanisms responsible for, and the ultimate consequences of, this protein localization. Here, we show that the Hrr25/CK1δ protein kinase is recruited to cytoplasmic Processing-bodies (P-bodies) in an evolutionarily-conserved manner. This recruitment requires Hrr25 kinase activity and the Dcp2 decapping enzyme, a core constituent of these RNP granules. Interestingly, the data indicate that this localization sequesters active Hrr25 away from the remainder of the cytoplasm and thereby shields this enzyme from the degradation machinery during these periods of stress. Altogether, this work illustrates how the presence within an RNP granule can alter the ultimate fate of the localized protein.
... One possible explanation is that simultaneous binding of two Plk1-PBDs via Thr 170 and Thr 195 may facilitate multiple phosphorylation of Plk1-PBD in complex with Emi2. Because two degradation motifs of Emi2 are prone to phosphorylation by Plk1 and to recognition by a ubiquitin ligase of the SCF family [38][39][40] , simultaneous binding and recruitment of two Plk1-PBDs to Emi2 may facilitate Plk1-mediated degradation of Emi2. Nonetheless, the exact physiological significance of the two possible Plk1-PBD-binding sites for Emi2 degradation needs further research. ...
In a mammalian oocyte, completion of meiosis is suspended until fertilization by a sperm, and the cell cycle is arrested by a biochemical activity called cytostatic factor (CSF). Emi2 is one of the CSFs, and it maintains the protein level of maturation promoting factor (MPF) by inhibiting ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Degradation of Emi2 via ubiquitin-mediated proteolysis after fertilization requires phosphorylation by Polo-like kinase 1 (Plk1). Therefore, recognition and phosphorylation of Emi2 by Plk1 are crucial steps for cell cycle resumption, but the binding mode of Emi2 and Plk1 is poorly understood. Using biochemical assays and X-ray crystallography, we found that two phosphorylated threonines (Thr 152 and Thr 176) in Emi2 are each responsible for the recruitment of one Plk1 molecule by binding to its C-Terminal polo box domain (PBD). We also found that meiotic maturation and meiosis resumption via parthenogenetic activation were impaired when Emi2 interaction with Plk1-PBD was blocked by a peptidomimetic called 103-8. Because of the inherent promiscuity of kinase inhibitors, our results suggest that targeting PBD of Plk1 may be an effective strategy for the development of novel and specific contraceptive agents that block oocyte maturation and/or fertilization.
... At metaphase II of meiosis in Xenopus eggs APC/C activity is kept in an inactive state and cell cycle progression is inhibited by Emi2 competing with Cdc20 for APC/C binding. Emi2 is stabilized and activated by the MAPK-mos-rsk pathway, which recruits PP2A, bound to its regulatory subunit B56β/ε, to Emi2 to counteract the inactivating phosphorylations of the mitotic kinases [39]. PP2A, with the B55δ regulatory subunit, is largely responsible for counteracting CDK dependent phosporylations during mitotic exit. ...
Many important discoveries in cell cycle research have been made using cell-free extracts prepared from the eggs of the South African clawed frog Xenopus laevis. These extracts efficiently support the key nuclear functions of the eukaryotic cell cycle in vitro under apparently the same controls that exist in vivo. The Xenopus cell-free system is therefore uniquely suited to the study of the mechanisms, dynamics and integration of cell cycle regulated processes at a biochemical level. Here, we describe methods currently in use in our laboratory for the preparation of Xenopus egg extracts and demembranated sperm nuclei. We detail how these extracts can be used to study the key transitions of the eukaryotic cell cycle and describe conditions under which these transitions can be manipulated by addition of drugs that either retard or advance passage. In addition, we describe in detail essential techniques that provide a practical starting point for investigating the function of proteins involved in the operation of the eukaryotic cell cycle.
... This suggests that TAM could regulate OSD1 to prevent precocious meiotic exit. Alternatively, phosphorylation by TAM could inactivate OSD1 and thus allow exit from meiosis I. Interestingly, the activity and stability of Emi2/Erp1 -the vertebrate meiotic APC/C inhibitor -is regulated by phosphorylation (Tang et al., 2008b;Isoda et al., 2011). Further functional analysis of the OSD1 putative phosphorylation sites is required to establish the role of this CYCA1;2/TAM-CDKA;1-mediated phosphorylation in meiotic cell cycle progression. ...
Meiosis is a specialized type of cell division that generates haploid gametes. At meiosis, two divisions follow a single DNA replication event leading to ploidy halving. A stepwise sister chromatids cohesion release also occurs to allow the two successive balanced rounds of chromosome segregation. In addition to general cell-cycle regulators, meiosis requires specific proteins. The aim of this thesis was to understand the molecular mechanisms leading to two successive balanced chromosome segregations. We show that OSD1 promotes meiotic progression through APC/C inhibition and we identified a functional network between OSD1, CYCA1;2/TAM and TDM in Arabidopsis. This functional network controls three key steps of meiotic progression; the prophase-meiosis I transition, the meiosis I-meiosis II transition and the meiosis exit. In addition, we characterized the two Arabidopsis thaliana Shugoshin paralogs, which are conserved proteins involved in sister chromatid cohesion protection. We also identified Patronus, an uncharacterized protein, as a novel protector of meiotic centromeric cohesion. We suggest that Patronus is a novel APC/C regulator that prevents cohesins release during meiotic interkinesis. This work identified two APC/C regulators essential for meiosis in Arabidopsis thaliana.
... We do not know how Cyclin B3 stimulates anaphase. However, cyclin:Cdk kinase activity phosphorylates certain APC/C subunits, such as Cdc27, as well as phosphorylating and inactivating an inhibitor of the APC/C, Emi2 [30,31]. We suggest that Cyclin B3:Cdk1 has unique substrate specificity [32] and spatial-temporal distribution that make it an effective activator of APC/C. ...
The timing mechanism for mitotic progression is still poorly understood. The spindle assembly checkpoint (SAC), whose reversal upon chromosome alignment is thought to time anaphase [1-3], is functional during the rapid mitotic cycles of the Drosophila embryo; but its genetic inactivation had no consequence on the timing of the early mitoses. Mitotic cyclins-Cyclin A, Cyclin B, and Cyclin B3-influence mitotic progression and are degraded in a stereotyped sequence [4-11]. RNAi knockdown of Cyclins A and B resulted in a Cyclin B3-only mitosis in which anaphase initiated prior to chromosome alignment. Furthermore, in such a Cyclin B3-only mitosis, colchicine-induced SAC activation failed to block Cyclin B3 destruction, chromosome decondensation, or nuclear membrane re-assembly. Injection of Cyclin B proteins restored the ability of SAC to prevent Cyclin B3 destruction. Thus, SAC function depends on particular cyclin types. Changing Cyclin B3 levels showed that it accelerated progress to anaphase, even in the absence of SAC function. The impact of Cyclin B3 on anaphase initiation appeared to decline with developmental progress. Our results show that different cyclin types affect anaphase timing differently in the early embryonic divisions. The early-destroyed cyclins-Cyclins A and B-restrain anaphase-promoting complex/cyclosome (APC/C) function, whereas the late-destroyed cyclin, Cyclin B3, stimulates function. We propose that the destruction schedule of cyclin types guides mitotic exit by affecting both Cdk1 and APC/C, whose activities change as each cyclin type is lost.
Copyright © 2015 Elsevier Ltd. All rights reserved.
... To study redundant functions of B56 subunits, we performed a combinatorial knockdown of all five B56 subunits in the "Aα exchange" cell line. Generated from the PC6-3 subline of PC12 cells cycle regulation (Foley et al., 2011;Isoda et al., 2011;Chambon et al., 2013;Kruse et al., 2013;Porter et al., 2013;Xu et al., 2013), apoptosis (Li et al., 2002;Silverstein et al., 2002;Holland et al., 2007;Ruvolo et al., 2008;Jin et al., 2010), embryonic development (Hannus et al., 2002;Yang et al., 2003;Rorick et al., 2007;Jin et al., 2009Jin et al., , 2011Varadkar et al., 2014), tumorigenesis (Li et al., 2007;Shouse et al., 2008Shouse et al., , 2010Nobumori et al., 2013), and the longevity of worms (Padmanabhan et al., 2009). The crystal structure of B56containing PP2A has been determined. ...
Recent GWASs reveal that the FAM13A gene is associated with human lung function and a variety of lung diseases, including COPD, asthma, lung cancer, and pulmonary fibrosis. The biological functions of Fam13a, however, have not been studied. In an effort to identify novel substrates of B56-containing PP2As, we found that B56-containing PP2As and Akt act antagonistically to control reversible phosphorylation of Fam13a on Serine322 (Ser322). We show that Ser322 phosphorylation acts as a molecular switch to control the subcellular distribution of Fam13a. Fam13a shuttles between the nucleus and cytoplasm. When Ser322 is phosphorylated by Akt, the binding between Fam13a and 14-3-3 is enhanced, leading to cytoplasmic sequestration of Fam13a. B56-containing PP2As dephosphorylate phospho-Ser322 and promote nuclear localization of Fam13a. We generated Fam13a knockout mice. Fam13a mutant mice are viable and healthy, indicating that Fam13a is dispensable for embryonic development and physiological functions in adult animals. Intriguingly, Fam13a has the ability to activate the Wnt pathway. Although Wnt signaling remains largely normal in Fam13a knockout lungs, depletion of Fam13a in human lung cancer cells causes an obvious reduction in Wnt signaling activity. Our work provides important clues to elucidate the mechanism by which Fam13a may contribute to human lung diseases.
© 2015 by The American Society for Cell Biology.
... Emi1 and Emi2 share the F-box and DB motifs, the zinc-binding region (ZBR) with the in-between-RING-fingers (IBR) domain topology and the C6HC-type zinc-binding motif [22,41], and the C-terminal region with a conserved 14-residue sequence ending in the RL residues, termed the RL tail [42]. The phosphorylation status of the C-terminal region controls the interaction between Emi2 and APC/C [43]. The F-box motif (named after cyclin F) interacts with Skp1, and the F-box proteins have been characterized as components of the Skp1-cullin-F-box (SCF) ubiquitin-ligase complexes [44]. ...
Anaphase-promoting complex or cyclosome (APC/C) is a multisubunit ubiquitin ligase E3 that targets cell-cycle regulators. Cdc20 is required for full activation of APC/C in M phase, and mediates substrate recognition. In vertebrates, Emi2/Erp1/FBXO43 inhibits APC/C-Cdc20, and functions as a cytostatic factor that causes long-term M phase arrest of mature oocytes. In this study, we found that a fragment corresponding to the zinc-binding region (ZBR) domain of Emi2 inhibits cell-cycle progression, and impairs the association of Cdc20 with the APC/C core complex in HEK293T cells. Furthermore, we revealed that the ZBR fragment of Emi2 inhibits in vitro ubiquitin chain elongation catalyzed by the APC/C cullin-RING ligase module, the ANAPC2-ANAPC11 subcomplex, in combination with the ubiquitin chain-initiating E2, E2C/UBE2C/UbcH10. Structural analyses revealed that the Emi2 ZBR domain uses different faces for the two mechanisms. Thus, the double-faced ZBR domain of Emi2 antagonizes the APC/C function by inhibiting both the binding with the coactivator Cdc20 and ubiquitylation mediated by the cullin-RING ligase module and E2C. In addition, the tail region between the ZBR domain and the C-terminal RL residues [the post-ZBR (PZ) region] interacts with the cullin subunit, ANAPC2. In the case of the ZBR fragment of the somatic paralogue of Emi2, Emi1/FBXO5, these inhibitory activities against cell division and ubiquitylation were not observed. Finally, we identified two sets of key residues in the Emi2 ZBR domain that selectively exert each of the dual Emi2-specific modes of APC/C inhibition, by their mutation in the Emi2 ZBR domain and their transplantation into the Emi1 ZBR domain.
... 59 Finetuning of CDK1 activity during metaphase II arrest is achieved through a feedback loop with combined action of CDK1 and PP2A (in Xenopus eggs, with a B56β/ε regulatory subunit 60 ) on EMI2, an inhibitor of the anaphase-promoting complex/cyclosome. [60][61][62] Data from studies using Okadaic acid to inhibit PP2A and FTY720 to activate PP2A provide evidence that this same mechanism functions in metaphase II mouse eggs. 63 In summary, this study demonstrated that exit from prophase I arrest is impaired in ENSA-deficient oocytes. ...
Mammalian oocytes in ovarian follicles are arrested in meiosis at prophase I. This arrest is maintained until ovulation, upon which the oocyte exits from this arrest, progresses through meiosis I and to metaphase of meiosis II. The progression from prophase I to metaphase II, known as meiotic maturation, is mediated by signals that coordinate these transitions in the life of the oocyte. ENSA (α-endosulfine) and ARPP19 (cAMP-regulated phosphoprotein-19) have emerged as regulators of M phase, with function in inhibition of protein phosphatase 2A (PP2A) activity. Inhibition of PP2A maintains the phosphorylated state of CDK1 substrates, thus allowing progression into and/or maintenance of an M-phase state. We show here ENSA in mouse oocytes plays a key role in the progression from prophase I arrest into M phase of meiosis I. The majority of ENSA-deficient oocytes fail to exit from prophase I arrest. This function of ENSA in oocytes is dependent on PP2A, and specifically on the regulatory subunit PPP2R2D (also known as B55δ). Treatment of ENSA-deficient oocytes with okadaic acid to inhibit PP2A rescues the defect in meiotic progression, with okadaic acid-treated, ENSA-deficient oocytes being able to exit from prophase I arrest. Similarly, oocytes deficient in both ENSA and PPP2R2D are able to exit from prophase I arrest to an extent similar to wild-type oocytes. These data are evidence of a role for ENSA in regulating meiotic maturation in mammalian oocytes, and also have potential relevance to human oocyte biology, as mouse and human have genes encoding both Arpp19 and Ensa.
... The B-type subunit is crucial for discriminating between each PP2A holoenzyme, as it dictates substrate specificity, subcellular localization and catalytic activity of the heterotrimer (Sents et al, 2012). Despite the well-recognized importance of PP2A, very few cellular functions, including intracellular signalling (Bengtsson et al, 2009;Yan et al, 2010;Rodgers et al, 2011), cell division (Yan et al, 2000;Chan and Amon, 2009;Jayadeva et al, 2010;Manchado et al, 2010;Brownlee et al, 2011;Foley et al, 2011;Isoda et al, 2011;Song et al, 2011;Wang et al, 2011) and apoptosis (Merrill et al, 2012;Yan et al, 2012), have been undisputedly attributed to specific PP2A holoenzymes, as defined by their regulatory subunit. To date, the putative roles of PP2A regulatory subunits in development remain largely undisclosed. ...
To supply tissues with nutrients and oxygen, the cardiovascular system forms a seamless, hierarchically branched, network of lumenized tubes. Here, we show that maintenance of patent vessel lumens requires the Bα regulatory subunit of protein phosphatase 2A (PP2A). Deficiency of Bα in zebrafish precludes vascular lumen stabilization resulting in perfusion defects. Similarly, inactivation of PP2A-Bα in cultured ECs induces tubulogenesis failure due to alteration of cytoskeleton dynamics, actomyosin contractility and maturation of cell-extracellular matrix (ECM) contacts. Mechanistically, we show that PP2A-Bα controls the activity of HDAC7, an essential transcriptional regulator of vascular stability. In the absence of PP2A-Bα, transcriptional repression by HDAC7 is abrogated leading to enhanced expression of the cytoskeleton adaptor protein ArgBP2. ArgBP2 hyperactivates RhoA causing inadequate rearrangements of the EC actomyosin cytoskeleton. This study unravels the first specific role for a PP2A holoenzyme in development: the PP2A-Bα/HDAC7/ArgBP2 axis maintains vascular lumens by balancing endothelial cytoskeletal dynamics and cell-matrix adhesion.
... Unfertilized eggs of vertebrates are arrested at metaphase of meiosis II by Emi2, an inhibitor of the anaphase promoting complex/cyclosome (APC/C) that targets the Cdk1 co-activator cyclin B for destruction [37]. The stability and inhibitory potency of Emi2 are antagonistically regulated by Cdk1 and PP2A-B56. ...
... p53 protein was also reported to be a substrate of PP2A-B56c1 and -B56c3 [48,49]. PP2A-B56b1 dephosphorylates Pim-1 [50], TrkA [31], tyrosine hydroxylase [51], Akt1 [52,53] and Emi2 [54]. In addition, B56a targets cMyc, APC, ankyrin B [55] and SK1 [56], and PP2A-B56c3 dephosphorylates ChK2 [57]. ...
Extracellular signal-regulated kinase (ERK) signalling plays a central role in various biological processes, including cell migration, but it remains unknown what factors directly regulate the strength and duration of ERK activation. We found that, among the B56 family of protein phosphatase 2A (PP2A) regulatory subunits, B56γ1 suppressed EGF-induced cell migration on collagen, bound to phosphorylated-ERK, and dephosphorylated ERK, whereas B56α1 and B56β1 did not. B56γ1 was immunolocalized in nuclei. The IER3 protein was immediately highly expressed in response to costimulation of cells with EGF and collagen. Knockdown of IER3 inhibited cell migration and enhanced dephosphorylation of ERK. Analysis of the time course of PP2A-B56γ1 activity following the costimulation showed an immediate loss of phosphatase activity, followed by a rapid increase in activity, and this activity then remained at a stable level that was lower than the original level. Our results indicate that the strength and duration of the nuclear ERK activation signal that is initially induced by ERK kinase (MEK) are determined at least in part by modulation of the phosphatase activity of PP2A-B56γ1 through two independent pathways.
... Interestingly, phosphorylation of Emi2 by Rsk recruits the phosphatase PP2A/B56 β/ε, which continuously antagonizes the inhibitory phosphorylations performed by Cdk1/Plx1/CK1. 39 Inhibited APC maintains cyclin B-Cdk1 activity, which in turn will permit the activation of the Gwl-Arpp19 pathway and the subsequent inhibition of PP2A-B55, preventing this massive protein dephosphorylation. It is worth noticing that recruitment and phosphorylation of Emi2 by Plx1 and CK1 depend on the phosphorylation of this protein by cyclin B-Cdk1 in a cluster of consensus sites (S/T-P) and that these sites are recognized by PP2A-B56β/ε; however, this phosphate does not massively dephosphorylate cyclin B-Cdk1 substrates. ...
Mitotic division is induced by protein phosphorylation. For a long time the supported hypothesis was that mitotic entry and exit were the exclusive result of cyclin B-Cdk1 kinase activation and inactivation, whereas the phosphatase activity required to dephosphorylate mitotic substrates was thought to be constant during mitosis. Recent data demonstrate that phosphatase activity must also be tightly regulated to promote correct cell division. Here we describe the new pathway involved in phosphatase regulation and the questions that this discovery raises concerning the classic view of cell cycle regulation.
... This suggests that TAM could regulate OSD1 to prevent precocious meiotic exit. Alternatively, phosphorylation by TAM could inactivate OSD1 and thus allow exit from meiosis I. Interestingly, the activity and stability of Emi2/Erp1-the vertebrate meiotic APC/C inhibitoris regulated by phosphorylation [33,34]. Further functional analysis of the OSD1 putative phosphorylation sites is required to establish the role of this CYCA1;2/TAM-CDKA;1-mediated phosphorylation in meiotic cell cycle progression. ...
Cell cycle control is modified at meiosis compared to mitosis, because two divisions follow a single DNA replication event. Cyclin-dependent kinases (CDKs) promote progression through both meiosis and mitosis, and a central regulator of their activity is the APC/C (Anaphase Promoting Complex/Cyclosome) that is especially required for exit from mitosis. We have shown previously that OSD1 is involved in entry into both meiosis I and meiosis II in Arabidopsis thaliana; however, the molecular mechanism by which OSD1 controls these transitions has remained unclear. Here we show that OSD1 promotes meiotic progression through APC/C inhibition. Next, we explored the functional relationships between OSD1 and the genes known to control meiotic cell cycle transitions in Arabidopsis. Like osd1, cyca1;2/tam mutation leads to a premature exit from meiosis after the first division, while tdm mutants perform an aberrant third meiotic division after normal meiosis I and II. Remarkably, while tdm is epistatic to tam, osd1 is epistatic to tdm. We further show that the expression of a non-destructible CYCA1;2/TAM provokes, like tdm, the entry into a third meiotic division. Finally, we show that CYCA1;2/TAM forms an active complex with CDKA;1 that can phosphorylate OSD1 in vitro. We thus propose that a functional network composed of OSD1, CYCA1;2/TAM, and TDM controls three key steps of meiotic progression, in which OSD1 is a meiotic APC/C inhibitor.
Developmental arrest in preimplantation embryos is one of the major causes of assisted reproduction failure. It is briefly defined as a delay or a failure of embryonic development in producing viable embryos during ART cycles. Permanent or partial developmental arrest can be observed in the human embryos from 1-cell to blastocyst stages. These arrests mainly arise from different molecular biological defects, including epigenetic disturbances, ART processes, and genetic variants. Embryonic arrests were found to be associated with a number of variants in the genes playing key roles in embryonic genome activation, mitotic divisions, subcortical maternal complex formation, maternal mRNA clearance, repairing DNA damage, transcriptional and translational controls. In this review, the biological impacts of these variants are comprehensively evaluated in the light of existing studies. The creation of diagnostic gene panels and potential ways of preventing developmental arrests to obtain competent embryos are also discussed.
The anaphase-promoting complex/cyclosome (APC/C), a multi-subunit ubiquitin ligase essential for cell cycle control, is regulated by reversible phosphorylation. APC/C phosphorylation by cyclin-dependent kinase 1 (Cdk1) promotes Cdc20 co-activator loading in mitosis to form active APC/C-Cdc20. However, detailed phospho-regulation of APC/C dynamics through other kinases and phosphatases is still poorly understood. Here, we show that an interplay between polo-like kinase (Plx1) and PP2A-B56 phosphatase on a flexible loop domain of the subunit Apc1 (Apc1-loop500 ) controls APC/C activity and mitotic progression. Plx1 directly binds to the Apc1-loop500 in a phosphorylation-dependent manner and promotes the formation of APC/C-Cdc20 via Apc3 phosphorylation. Upon phosphorylation of loop residue T532, PP2A-B56 is recruited to the Apc1-loop500 and differentially promotes dissociation of Plx1 and PP2A-B56 through dephosphorylation of Plx1-binding sites. Stable Plx1 binding, which prevents PP2A-B56 recruitment, prematurely activates the APC/C and delays APC/C dephosphorylation during mitotic exit. Furthermore, the phosphorylation status of the Apc1-loop500 is controlled by distant Apc3-loop phosphorylation. Our study suggests that phosphorylation-dependent feedback regulation through flexible loop domains within a macromolecular complex coordinates the activity and dynamics of the APC/C during the cell cycle.
Members of the highly conserved pleiotropic CK1 family of serine/threonine-specific kinases are tightly regulated in the cell and play crucial regulatory roles in multiple cellular processes from protozoa to human. Since their dysregulation as well as mutations within their coding regions contribute to the development of various different pathologies, including cancer and neurodegenerative diseases, they have become interesting new drug targets within the last decade. However, to develop optimized CK1 isoform-specific therapeutics in personalized therapy concepts, a detailed knowledge of the regulation and functions of the different CK1 isoforms, their various splice variants and orthologs is mandatory. In this review we will focus on the stress-induced CK1 isoform delta (CK1δ), thereby addressing its regulation, physiological functions, the consequences of its deregulation for the development and progression of diseases, and its potential as therapeutic drug target.
In metazoans that undergo sexual reproduction, genomic inheritance is ensured by two distinct types of cell cycle, mitosis and meiosis. Mitosis maintains the genomic ploidy in somatic cells reproducing within a generation, whereas meiosis reduces by half the ploidy in germ cells to prepare for successive generations. The meiotic cell cycle is believed to be a derived form of the mitotic cell cycle; however, the molecular mechanisms underlying both of these processes remain elusive. My laboratory has long studied the meiotic cell cycle in starfish oocytes, particularly the control of meiotic M-phase by maturation- or M phase-promoting factor (MPF) and the kinase cyclin B-associated Cdk1 (cyclin B-Cdk1). Using this system, we have unraveled the molecular principles conserved in metazoans that modify M-phase progression from the mitotic type to the meiotic type needed to produce a haploid genome. Furthermore, we have solved a long-standing enigma concerning the molecular identity of MPF, a universal inducer of M-phase both in mitosis and meiosis of eukaryotic cells.
Cell signaling is governed by dynamic changes in kinase and phosphatase activities, which are difficult to assess with discontinuous readout methods. Here, we introduce an NMR-based reporter approach to directly identify active kinases and phosphatases in complex physiological environments such as cell lysates and to measure their indi-vidual activities in a semi-continuous fashion. Multiplexed NMR profiling of reporter phosphorylation states provides unique advantages for kinase inhibitor studies and reveals reversible modulations of cellular enzyme activities under different metabolic conditions.
Members of the highly conserved and ubiquitously expressed pleiotropic CK1 family play major regulatory roles in many cellular processes including DNA-processing and repair, proliferation, cytoskeleton dynamics, vesicular trafficking, apoptosis, and cell differentiation. As a consequence of cellular stress conditions, interaction of CK1 with the mitotic spindle is manifold increased pointing to regulatory functions at the mitotic checkpoint. Furthermore, CK1 is able to alter the activity of key proteins in signal transduction and signal integration molecules. In line with this notion, CK1 is tightly connected to the regulation and degradation of β-catenin, p53, and MDM2. Considering the importance of CK1 for accurate cell division and regulation of tumor suppressor functions, it is not surprising that mutations and alterations in the expression and/or activity of CK1 isoforms are often detected in various tumor entities including cancer of the kidney, choriocarcinomas, breast carcinomas, oral cancer, adenocarcinomas of the pancreas, and ovarian cancer. Therefore, scientific effort has enormously increased (i) to understand the regulation of CK1 and its involvement in tumorigenesis- and tumor progression-related signal transduction pathways and (ii) to develop CK1-specific inhibitors for the use in personalized therapy concepts. In this review, we summarize the current knowledge regarding CK1 regulation, function, and interaction with cellular proteins playing central roles in cellular stress-responses and carcinogenesis.
Mitotic divisions result from the oscillating activity of cyclin-dependent kinase 1 (Cdk1). Cdk1 activity is terminated by
the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets cyclin B for destruction. In somatic divisions,
the early mitotic inhibitor 1 (Emi1) and the spindle assembly checkpoint (SAC) regulate cell cycle progression by inhibiting
the APC/C. Early embryonic divisions lack these APC/C-inhibitory components, which raises the question of how those cycles
are controlled. We found that the APC/C-inhibitory activity of XErp1 (also known as Emi2) was essential for early divisions
in Xenopus embryos. Loss of XErp1 resulted in untimely destruction of APC/C substrates and embryonic lethality. XErp1’s APC/C-inhibitory
function was negatively regulated by Cdk1 and positively by protein phosphatase 2A (PP2A). Thus, Cdk1 and PP2A operate at
the core of early mitotic cell cycles by antagonistically controlling XErp1 activity, which results in oscillating APC/C activity.
Vertebrate eggs arrest at second meiotic metaphase. The fertilizing sperm causes meiotic exit through Ca(2+)-mediated activation of the anaphase-promoting complex/cyclosome (APC/C). Although the loss in activity of the M-phase kinase CDK1 is known to be an essential downstream event of this process, the contribution of phosphatases to arrest and meiotic resumption is less apparent, especially in mammals. Therefore, we explored the role of protein phosphatase 2A (PP2A) in mouse eggs using pharmacological inhibition and activation as well as a functionally dominant-negative catalytic PP2A subunit (dn-PP2Ac-L199P) coupled with live cell imaging. We observed that PP2A inhibition using okadaic acid induced events normally observed at fertilization: degradation of the APC/C substrates cyclin B1 and securin resulting from loss of the APC/C inhibitor Emi2. Although sister chromatids separated, chromatin remained condensed, and polar body extrusion was blocked as a result of a rapid spindle disruption, which could be ameliorated by non-degradable cyclin B1, suggesting that spindle integrity was affected by CDK1 loss. Similar cell cycle effects to okadaic acid were also observed using dominant-negative PP2Ac. Preincubation of eggs with the PP2A activator FTY720 could block many of the actions of okadaic acid, including Emi2, cyclin B1, and securin degradation and sister chromatid separation. Therefore, in conclusion, we used okadaic acid, dn-PP2Ac-L199P, and FTY720 on mouse eggs to demonstrate that PP2A is needed to for both continued metaphase arrest and successful exit from meiosis.
Entry into mitosis in eukaryotes requires the activity of cyclin-dependent kinase 1 (Cdk1). Cdk1 is opposed by protein phosphatases
in two ways: They inhibit activation of Cdk1 by dephosphorylating the protein kinases Wee1 and Myt1 and the protein phosphatase
Cdc25 (key regulators of Cdk1), and they also antagonize Cdk1’s own phosphorylation of downstream targets. A particular form
of protein phosphatase 2A (PP2A) containing a B55δ subunit (PP2A- B55δ) is the major protein phosphatase that acts on model
CDK substrates in Xenopus egg extracts and has antimitotic activity. The activity of PP2A-B55δ is high in interphase and low in mitosis, exactly opposite
that of Cdk1. We report that inhibition of PP2A-B55δ results from a small protein, known as α-endosulfine (Ensa), that is
phosphorylated in mitosis by the protein kinase Greatwall (Gwl). This converts Ensa into a potent and specific inhibitor of
PP2A-B55δ. This pathway represents a previously unknown element in the control of mitosis.
The cyclin dependent kinase Cdk1 controls the cell cycle, which is best understood in the model organism S. cerevisiae. Research performed during the past decade has significantly improved our understanding of the molecular machinery of the cell cycle. Approximately 75 targets of Cdk1 have been identified that control critical cell cycle events, such as DNA replication and segregation, transcriptional programs and cell morphogenesis. In this review we discuss currently known targets of Cdk1 in the budding yeast S. cerevisiae and highlight the role of Cdk1 in several crucial processes including maintenance of genome stability.
Members of the polo subfamily of protein kinases have emerged as important regulators in diverse aspects of the cell cycle and cell proliferation. A large body of evidence suggests that a highly conserved polo-box domain (PBD) present in the C-terminal non-catalytic region of polo kinases plays a pivotal role in the function of these enzymes. Recent advances in our comprehension of the mechanisms underlying mammalian polo-like kinase 1 (Plk1)-dependent protein-protein interactions revealed that the PBD serves as an essential molecular mediator that brings the kinase domain of Plk1 into proximity with its substrates, mainly through phospho-dependent interactions with its target proteins. In this review, current understanding of the structure and functions of PBD, mode of PBD-dependent interactions and substrate phosphorylation, and other phospho-independent functions of PBD are discussed.
Emi2 (also called Erp1) inhibits the anaphase-promoting complex/cyclosome (APC/C) and thereby causes metaphase II arrest in unfertilized vertebrate eggs. Both the D-box and the zinc-binding region (ZBR) of Emi2 have been implicated in APC/C inhibition. However, it is not well known how Emi2 interacts with and hence inhibits the APC/C. Here we show that Emi2 binds the APC/C via the C-terminal tail, termed here the RL tail. When expressed in Xenopus oocytes and egg extracts, Emi2 lacking the RL tail fails to interact with and inhibit the APC/C. The RL tail itself can directly bind to the APC/C, and, when added to egg extracts, either an excess of RL tail peptides or anti-RL tail peptide antibody can dissociate endogenous Emi2 from the APC/C, thus allowing APC/C activation. Furthermore, and importantly, the RL tail-mediated binding apparently promotes the inhibitory interactions of the D-box and the ZBR (of Emi2) with the APC/C. Finally, Emi1, a somatic paralog of Emi2, also has a functionally similar RL tail. We propose that the RL tail of Emi1/Emi2 serves as a docking site for the APC/C, thereby promoting the interaction and inhibition of the APC/C by the D-box and the ZBR.
The entry into mitosis is controlled by Cdc2/cyclin B, also known as maturation or M-phase promoting factor (MPF). In Xenopus egg extracts, the inhibitory phosphorylations of Cdc2 on Tyr-15 and Thr-14 are controlled by the phosphatase Cdc25 and the
kinases Myt1 and Wee1. At mitosis, Cdc25 is activated and Myt1 and Wee1 are inactivated through phosphorylation by multiple
kinases, including Cdc2 itself. The Cdc2-associated Suc1/Cks1 protein (p9) is also essential for entry of egg extracts into
mitosis, but the molecular basis of this requirement has been unknown. We find that p9 strongly stimulates the regulatory
phosphorylations of Cdc25, Myt1, and Wee1 that are carried out by the Cdc2/cyclin B complex. Overexpression of the prolyl
isomerase Pin1, which binds to the hyperphosphorylated forms of Cdc25, Myt1, and Wee1 found at M-phase, is known to block
the initiation of mitosis in egg extracts. We have observed that Pin1 specifically antagonizes the stimulatory effect of p9
on phosphorylation of Cdc25 by Cdc2/cyclin B. This observation could explain why overexpression of Pin1 inhibits mitotic initiation.
These findings suggest that p9 promotes the entry into mitosis by facilitating phosphorylation of the key upstream regulators
of Cdc2.
We have previously shown that Greatwall kinase (Gwl) is required for M phase entry and maintenance in Xenopus egg extracts. Here, we demonstrate that Gwl plays a crucial role in a novel biochemical pathway that inactivates, specifically during M phase, "antimitotic" phosphatases directed against phosphorylations catalyzed by cyclin-dependent kinases (CDKs). A major component of this phosphatase activity is heterotrimeric PP2A containing the B55delta regulatory subunit. Gwl is activated during M phase by Cdk1/cyclin B (MPF), but once activated, Gwl promotes PP2A/B55delta inhibition with no further requirement for MPF. In the absence of Gwl, PP2A/B55delta remains active even when MPF levels are high. The removal of PP2A/B55delta corrects the inability of Gwl-depleted extracts to enter M phase. These findings support the hypothesis that M phase requires not only high levels of MPF function, but also the suppression, through a Gwl-dependent mechanism, of phosphatase(s) that would otherwise remove MPF-driven phosphorylations.
To explore the mechanisms and evolution of cell-cycle control, we analyzed the position and conservation of large numbers
of phosphorylation sites for the cyclin-dependent kinase Cdk1 in the budding yeast Saccharomyces cerevisiae. We combined specific chemical inhibition of Cdk1 with quantitative mass spectrometry to identify the positions of 547 phosphorylation
sites on 308 Cdk1 substrates in vivo. Comparisons of these substrates with orthologs throughout the ascomycete lineage revealed
that the position of most phosphorylation sites is not conserved in evolution; instead, clusters of sites shift position in
rapidly evolving disordered regions. We propose that the regulation of protein function by phosphorylation often depends on
simple nonspecific mechanisms that disrupt or enhance protein-protein interactions. The gain or loss of phosphorylation sites
in rapidly evolving regions could facilitate the evolution of kinase-signaling circuits.
The eukaryotic cell division cycle is characterized by a sequence of orderly and highly regulated events resulting in the duplication and separation of all cellular material into two newly formed daughter cells. Protein phosphorylation by cyclin-dependent kinases (CDKs) drives this cycle. To gain further insight into how phosphorylation regulates the cell cycle, we sought to identify proteins whose phosphorylation is cell cycle regulated. Using stable isotope labeling along with a two-step strategy for phosphopeptide enrichment and high mass accuracy mass spectrometry, we examined protein phosphorylation in a human cell line arrested in the G1 and mitotic phases of the cell cycle. We report the identification of >14,000 different phosphorylation events, more than half of which, to our knowledge, have not been described in the literature, along with relative quantitative data for the majority of these sites. We observed >1,000 proteins with increased phosphorylation in mitosis including many known cell cycle regulators. The majority of sites on regulated phosphopeptides lie in [S/T]P motifs, the minimum required sequence for CDKs, suggesting that many of the proteins may be CDK substrates. Analysis of non-proline site-containing phosphopeptides identified two unique motifs that suggest there are at least two undiscovered mitotic kinases.
• cell cycle
• cyclin-dependent kinase
• mass spectrometry
• proteomics
Inhibition of okadaic acid-sensitive phosphatases released the cyclin degradation pathway from its inhibited state in extracts
prepared from unfertilized Xenopus eggs arrested at the second meiotic metaphase. It also switched on cyclin protease activity
in a permanent fashion in interphase extracts prepared from activated eggs. Even after cdc2 kinase inactivation, microinjection
of okadaic acid-treated interphase extracts pushed G2-arrested recipient oocytes into the M phase, suggesting that the phosphatase
inhibitor stabilizes the activity of an unidentified factor which shares in common with cdc2 kinase the maturation-promoting
factor activity.
Exit from mitosis requires the inactivation of mitotic cyclin-dependent kinases (CDKs). In the budding yeast, Saccharomyces cerevisiae, inactivation of CDKs during late mitosis involves degradation of B-type cyclins as well as direct inhibition of cyclin-CDK complexes by the CDK-inhibitor protein Sic1 (refs 1,2,3). Several striking similarities exist between Sic1 and Cdc6, a DNA replication factor essential for the formation of pre-replicative complexes at origins of DNA replication. Transcription of both genes is activated during late mitosis by a process dependent on Swi5 (ref. 10). Like Sic1, Cdc6 binds CDK complexes in vivo and downregulates them in vitro. Here we show that Cdc6, like Sic1, also contributes to inactivation of CDKs during late mitosis in S. cerevisiae. Deletion of the CDK-interacting domain of Cdc6 does not inhibit the function of origins of DNA replication during S phase, but instead causes a delay in mitotic exit; this delay is accentuated in the absence of Sic1 or of cyclin degradation. By contributing to mitotic exit and inactivation of CDKs, Cdc6 helps to create the conditions that are required for its subsequent role in the formation of pre-replicative complexes at origins of DNA replication.
Metaphase-to-anaphase transition is a fundamental step in cell cycle progression where duplicated sister-chromatids segregate to the future daughter cells. The anaphase-promoting complex/cyclosome (APC/C) is a highly regulated ubiquitin-ligase that triggers anaphase onset and mitotic exit by targeting securin and mitotic cyclins for destruction. It was previously shown that the Xenopus polo-like kinase Plx1 is essential to activate APC/C upon release from cytostatic factor (CSF) arrest in Xenopus egg extract. Although the mechanism by which Plx1 regulates APC/C activation remained unclear, the existence of a putative APC/C inhibitor was postulated whose activity would be neutralized by Plx1 upon CSF release. Here we identify XErp1, a novel Plx1-regulated inhibitor of APC/C activity, and we demonstrate that XErp1 is required to prevent anaphase onset in CSF-arrested Xenopus egg extract. Inactivation of XErp1 leads to premature APC/C activation. Conversely, addition of excess XErp1 to Xenopus egg extract prevents APC/C activation. Plx1 phosphorylates XErp1 in vitro at a site that targets XErp1 for degradation upon CSF release. Thus, our data lead to a model of APC/C activation in Xenopus egg extract in which Plx1 targets the APC/C inhibitor XErp1 for degradation.
Unfertilized vertebrate eggs are arrested in metaphase of meiosis II with high cyclin B/Cdc2 activity to prevent parthenogenesis. Until fertilization, exit from metaphase is blocked by an activity called cytostatic factor (CSF), which stabilizes cyclin B by inhibiting the anaphase-promoting complex (APC) ubiquitin ligase. The APC inhibitor early mitotic inhibitor 1 (Emi1) was recently found to be required for maintenance of CSF arrest. We show here that exogenous Emi1 is unstable in CSF-arrested Xenopus eggs and is destroyed by the SCFβTrCP ubiquitin ligase, suggesting that endogenous Emi1, an apparent 44-kDa protein, requires a stabilizing factor. However, anti-Emi1 antibodies crossreact with native Emi2/Erp1/FBXO43, a homolog of Emi1 and conserved APC inhibitor. Emi2 is stable in CSF-arrested eggs, is sufficient to prevent CSF release, and is rapidly degraded in a Polo-like kinase 1-dependent manner in response to calcium-mediated egg activation. These results identify Emi2 as a candidate CSF maintenance protein.
• cyclin B
• meiosis
• maturation-promoting factor
• oocyte maturation
The serine/threonine-specific casein kinase I delta (CKIdelta) is ubiquitously expressed in all tissues, is p53 dependently induced in stress situations and plays an important role in various cellular processes. Our immunohistochemical analysis of the human placenta revealed strongest expression of CKIdelta in extravillous trophoblast cells and in choriocarcinomas. Investigation of the functional role of CKIdelta in an extravillous trophoblast hybrid cell line revealed that CKIdelta was constitutively localized at the centrosomes and the mitotic spindle. Inhibition of CKIdelta with the CKI-specific inhibitor IC261 led to structural alterations of the centrosomes, the formation of multipolar spindles, the inhibition of mitosis and, in contrast to other cell lines, the induction of apoptosis. Our findings indicate that CKIdelta plays an important role in the mitotic progression and in the survival of cells of trophoblast origin. Therefore, IC261 could provide a new tool in treating choriocarcinomas.
Vertebrate eggs awaiting fertilization are arrested at metaphase of meiosis II by a biochemical activity termed cytostatic factor (CSF). This activity inhibits the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that triggers anaphase onset and mitotic/meiotic exit by targeting securin and M-phase cyclins for destruction. On fertilization a transient rise in free intracellular calcium causes release from CSF arrest and thus APC/C activation. Although it has previously been shown that calcium induces the release of APC/C from CSF inhibition through calmodulin-dependent protein kinase II (CaMKII), the relevant substrates of this kinase have not been identified. Recently, we characterized XErp1 (Emi2), an inhibitor of the APC/C and key component of CSF activity in Xenopus egg extract. Here we show that calcium-activated CaMKII triggers exit from meiosis II by sensitizing the APC/C inhibitor XErp1 for polo-like kinase 1 (Plx1)-dependent degradation. Phosphorylation of XErp1 by CaMKII leads to the recruitment of Plx1 that in turn triggers the destruction of XErp1 by phosphorylating a site known to serve as a phosphorylation-dependent degradation signal. These results provide a molecular explanation for how the fertilization-induced calcium increase triggers exit from meiosis II.
In vertebrate meiosis, unfertilized eggs are arrested in metaphase II by cytostatic factor (CSF), which is required to maintain mitotic cyclin-dependent kinase activity. Fertilization triggers a transient increase in cytosolic free Ca²⁺, which leads to CSF inactivation and ubiquitin-dependent cyclin destruction through the anaphase promoting complex or cyclosome (APC/C). The Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and the Polo-like kinase Plx1 are essential factors for Ca²⁺-induced meiotic exit, but the critical targets of these kinases were unknown. The APC/C inhibitor Emi2 or XErp1 has recently been characterized as a pivotal CSF component, required to maintain metaphase II arrest and rapidly destroyed in response to Ca²⁺ signaling through phosphorylation by Plx1 and ubiquitination by the SCFβTrCP complex. An important question is how the increase in free Ca²⁺ targets Plx1 activity toward Emi2. Here, we demonstrate that CaMKII is required for Ca²⁺-induced Emi2 destruction, and that CaMKII functions as a “priming kinase,” directly phosphorylating Emi2 at a specific motif to induce a strong interaction with the Polo Box domain of Plx1. We show that the strict requirement for CaMKII to phosphorylate Emi2 is a specific feature of CSF arrest, and we also use phosphatase inhibitors to demonstrate an additional mode of Emi2 inactivation independent of its destruction. We firmly establish the CSF component Emi2 as the first-known critical and direct target of CaMKII in CSF release, providing a detailed molecular mechanism explaining how CaMKII and Plx1 coordinately direct APC/C activation and meiotic exit upon fertilization.
• fertilization
• meiosis
• ubiquitin ligases
Fertilizable mammalian oocytes are arrested at the second meiotic metaphase (mII) by the cyclinB-Cdc2 heterodimer, maturation promoting factor (MPF). MPF is stabilized via the activity of an unidentified cytostatic factor (CSF), thereby suspending meiotic progression until fertilization. We here present evidence that a conserved 71 kDa mammalian orthologue of Xenopus XErp1/Emi2, which we term endogenous meiotic inhibitor 2 (Emi2) is an essential CSF component. Depletion in situ of Emi2 by RNA interference elicited precocious meiotic exit in maturing mouse oocytes. Reduction of Emi2 released mature mII oocytes from cytostatic arrest, frequently inducing cytodegeneration. Mos levels autonomously declined to undetectable levels in mII oocytes. Recombinant Emi2 reduced the propensity of mII oocytes to exit meiosis in response to activating stimuli. Emi2 and Cdc20 proteins mutually interact and Cdc20 ablation negated the ability of Emi2 removal to induce metaphase release. Consistent with this, Cdc20 removal prevented parthenogenetic or sperm-induced meiotic exit. These studies show in intact oocytes that the interaction of Emi2 with Cdc20 links activating stimuli to meiotic resumption at fertilization and during parthenogenesis in mammals.
The periodic destruction of mitotic cyclins is triggered by the activation of the anaphase-promoting complex/cyclosome (APC/C) in mitosis. Although the ability of the APC/C to recognize destruction box (D-box) substrates oscillates throughout the cell cycle, the mechanism regulating APC/C binding to D-box substrates remains unclear. Here, we show that the APC/C inhibitor Emi1 tightly binds both the APC/C and its Cdh1 activator, binds to the D-box receptor site on the APC/C(Cdh1), and competes with APC/C substrates for D-box binding. Emi1 itself contains a conserved C-terminal D-box, which provides APC/C-binding affinity, and a conserved zinc-binding region (ZBR), which antagonizes APC/C E3 ligase activity independent of tight APC binding. Mutation of the ZBR converts Emi1 into a D-box-dependent APC/C substrate. The identification of a direct Emi1-APC/C complex further explains how Emi1 functions as a stabilizing factor for cyclin accumulation and the need to destroy Emi1 for APC/C activation in mitosis. The combination of a degron/E3 recognition site and an anti-ligase function in Emi1 suggests a general model for how E3 substrates evolve to become pseudosubstrate inhibitors.
During interkinesis, a metaphase II (MetII) spindle is built immediately after the completion of meiosis I. Oocytes then remain MetII arrested until fertilization. In mouse, we find that early mitotic inhibitor 2 (Emi2), which is an anaphase-promoting complex inhibitor, is involved in both the establishment and the maintenance of MetII arrest. In MetII oocytes, Emi2 needs to be degraded for oocytes to exit meiosis, and such degradation, as visualized by fluorescent protein tagging, occurred tens of minutes ahead of cyclin B1.
Emi2 antisense morpholino knockdown during oocyte maturation did not affect polar body (PB) extrusion. However, in interkinesis the central spindle microtubules from meiosis I persisted for a short time, and a MetII spindle failed to assemble. The chromatin in the oocyte quickly decondensed and a nucleus formed. All of these effects were caused by the essential role of Emi2 in stabilizing cyclin B1 after the first PB extrusion because in Emi2 knockdown oocytes a MetII spindle was recovered by Emi2 rescue or by expression of nondegradable cyclin B1 after meiosis I.
Vertebrate oocytes awaiting fertilization are arrested at metaphase of meiosis II by cytostatic factor (CSF). This arrest is due to inhibition of the anaphase-promoting complex/cyclosome, in part by a newly identified protein, Emi2 (xErp1). Emi2 is required for maintenance of CSF arrest in egg extracts, but its function in CSF establishment in oocytes and the normal embryonic cell cycle is unknown. Here we show that during oocyte maturation, Emi2 appears only after metaphase I, and its level peaks at CSF arrest (metaphase II). In M phase, Emi2 undergoes a phosphorylation-dependent electrophoretic shift. Microinjection of antisense oligonucleotides against Emi2 into stage VI oocytes blocks progression through meiosis II and the establishment of CSF arrest. Recombinant Emi2 rescues CSF arrest in these oocytes and also causes CSF arrest in egg extracts and in blastomeres of two-cell embryos. Fertilization triggers rapid, complete degradation of Emi2, but it is resynthesized in the first embryonic cell cycle to reach levels 5-fold lower than during CSF arrest. However, depletion of the protein from cycling egg extracts does not prevent mitotic cell cycle progression. Thus, Emi2 plays an essential role in meiotic but not mitotic cell cycles.
Spatial and temporal coordination of polo-like kinase 1 (Plk1) activity is necessary for mitosis and cytokinesis, and this is achieved through binding to phosphorylated docking proteins with distinct subcellular localizations. Although cyclin-dependent kinase 1 (Cdk1) creates these phosphorylated docking sites in metaphase, a general principle that explains how Plk1 activity is controlled in anaphase after Cdk1 inactivation is lacking. Here, we show that the microtubule-associated protein regulating cytokinesis (PRC1) is an anaphase-specific binding partner for Plk1, and that this interaction is required for cytokinesis. In anaphase, Plk1 creates its own docking site on PRC1, whereas in metaphase Cdk1 phosphorylates PRC1 adjacent to this docking site and thereby prevents binding of Plk1. Mutation of these Cdk1-sites results in a form of PRC1 that prematurely recruits Plk1 to the spindle during prometaphase and blocks mitotic progression. The activation state of Cdk1, therefore, controls the switch of Plk1 localization from centrosomes and kinetochores during metaphase, to the central spindle during anaphase.
Vertebrate eggs arrest at metaphase of meiosis II due to an activity known as cytostatic factor (CSF). CSF antagonizes the ubiquitin ligase activity of the anaphase-promoting complex/cyclosome (APC/C), preventing cyclin B destruction and meiotic exit until fertilization occurs. A puzzling feature of CSF arrest is that APC/C inhibition is leaky. Ongoing cyclin B synthesis is counterbalanced by a limited amount of APC/C-mediated cyclin B destruction; thus, cyclin B/Cdc2 activity remains at steady state. How the APC/C can be slightly active toward cyclin B, and yet restrained from ubiquitinating cyclin B altogether, is unknown. Emi2/XErp1 is the critical CSF component directly responsible for APC/C inhibition during CSF arrest. Fertilization triggers the Ca2+-dependent destruction of Emi2, releasing the APC/C to ubiquitinate the full pool of cyclin B and initiate completion of meiosis. Previously, we showed that a phosphatase maintains Emi2's APC/C-inhibitory activity in CSF-arrested Xenopus egg extracts. Here, we demonstrate that phosphatase inhibition permits Emi2 phosphorylation at thr-545 and -551, which inactivates Emi2. Furthermore, we provide evidence that adding excess cyclin B to CSF extracts stimulates Cdc2 phosphorylation of these same residues, antagonizing Emi2-APC/C association. Our findings suggest a model wherein the pool of Emi2 acts analogously to a rheostat by integrating Cdc2 and phosphatase activities to prevent cyclin B overaccumulation and Cdc2 hyperactivity during the indefinite period of time between arrival at metaphase II and eventual fertilization. Finally, we propose that inactivation of Emi2 by Cdc2 permits mitotic progression during early embryonic cleavage cycles.
In vertebrates, unfertilized eggs (or mature oocytes) are arrested at metaphase of meiosis II by a cytoplasmic activity called cytostatic factor (CSF). The classical Mos-MAPK pathway has long been implicated in CSF arrest of vertebrate eggs, but exactly how it exerts CSF activity remains unclear. Recently, Erp1 (also called Emi2), an inhibitor of the anaphase-promoting complex/cyclosome (APC/C) required for degradation of the mitotic regulator cyclin B (ref. 5), has also been shown to be a component of CSF in both Xenopus and mice. Erp1 is destroyed on fertilization or egg activation, like Mos. However, despite these similarities the Mos-MAPK (mitogen-activated protein kinase) pathway and Erp1 are thought to act rather independently in CSF arrest. Here, we show that p90rsk, the kinase immediately downstream from Mos-MAPK, directly targets Erp1 for CSF arrest in Xenopus oocytes. Erp1 is synthesized immediately after meiosis I, and the Mos-MAPK pathway or p90rsk is essential for CSF arrest by Erp1. p90rsk can directly phosphorylate Erp1 on Ser 335/Thr 336 both in vivo and in vitro, and upregulates both Erp1 stability and activity. Erp1 is also present in early embryos, but has little CSF activity owing, at least in part, to the absence of p90rsk activity. These results clarify the direct link of the classical Mos-MAPK pathway to Erp1 in meiotic arrest of vertebrate oocytes.
The mechanisms that control cell growth during the cell cycle are poorly understood. In budding yeast, cyclin dependent kinase 1 (Cdk1) triggers polarization of the actin cytoskeleton and bud emergence in late G1 through activation of the Cdc42 GTPase. However, Cdk1 is not thought to be required for subsequent growth of the bud. Here, we show that Cdk1 has an unexpected role in controlling bud growth after bud emergence. Moreover, we show that G1 cyclin-Cdk1 complexes specifically phosphorylate multiple proteins associated with Cdc24, the guanine nucleotide-exchange factor (GEF) that activates the Cdc42 GTPase. A mutant form of a Cdc24-associated protein that fails to undergo Cdk1-dependent phosphorylation causes defects in bud growth. These results provide a direct link between Cdk1 activity and the control of polarized cell growth.
Polo-like kinase-1 (Plk1) phosphorylates a number of mitotic substrates, but the diversity of Plk1-dependent processes suggests the existence of additional targets. Plk1 contains a specialized phosphoserine-threonine binding domain, the Polo-box domain (PBD), postulated to target the kinase to its substrates. Using the specialized PBD of Plk1 as an affinity capture agent, we performed a screen to define the mitotic Plk1-PBD interactome by mass spectrometry. We identified 622 proteins that showed phosphorylation-dependent mitosis-specific interactions, including proteins involved in well-established Plk1-regulated processes, and in processes not previously linked to Plk1 such as translational control, RNA processing, and vesicle transport. Many proteins identified in our screen play important roles in cytokinesis, where, in mammalian cells, the detailed mechanistic role of Plk1 remains poorly defined. We go on to characterize the mitosis-specific interaction of the Plk1-PBD with the cytokinesis effector kinase Rho-associated coiled-coil domain-containing protein kinase 2 (Rock2), demonstrate that Rock2 is a Plk1 substrate, and show that Rock2 colocalizes with Plk1 during cytokinesis. Finally, we show that Plk1 and RhoA function together to maximally enhance Rock2 kinase activity in vitro and within cells, and implicate Plk1 as a central regulator of multiple pathways that synergistically converge to regulate actomyosin ring contraction during cleavage furrow ingression.
Before fertilization, vertebrate eggs are arrested in meiosis II by cytostatic factor (CSF), which holds the anaphase-promoting complex (APC) in an inactive state. It was recently reported that Mos, an integral component of CSF, acts in part by promoting the Rsk-mediated phosphorylation of the APC inhibitor Emi2/Erp1. We report here that Rsk phosphorylation of Emi2 promotes its interaction with the protein phosphatase PP2A. Emi2 residues adjacent to the Rsk phosphorylation site were important for PP2A binding. An Emi2 mutant that retained Rsk phosphorylation but lacked PP2A binding could not be modulated by Mos. PP2A bound to Emi2 acted on two distinct clusters of sites phosphorylated by Cdc2, one responsible for modulating its stability during CSF arrest and one that controls binding to the APC. These findings provide a molecular mechanism for Mos action in promoting CSF arrest and also define an unusual mechanism, whereby protein phosphorylation recruits a phosphatase for dephosphorylation of distinct sites phosphorylated by another kinase.
• anaphase-promoting complex
• cytostatic factor Cdc2
• cyclin B
• meiosis
• phosphatase
Protein phosphatase 2A (PP2A), a major phospho-serine/threonine phosphatase, is conserved throughout eukaryotes. It dephosphorylates a plethora of cellular proteins, including kinases and other signaling molecules involved in cell division, gene regulation, protein synthesis and cytoskeleton organization. PP2A enzymes typically exist as heterotrimers comprising catalytic C-, structural A- and regulatory B-type subunits. The B-type subunits function as targeting and substrate-specificity factors; hence, holoenzyme assembly with the appropriate B-type subunit is crucial for PP2A specificity and regulation. Recently, several biochemical and structural determinants have been described that affect PP2A holoenzyme assembly. Moreover, the effects of specific post-translational modifications of the C-terminal tail of the catalytic subunit indicate that a 'code' might regulate dynamic exchange of regulatory B-type subunits, thus affecting the specificity of PP2A.
Fully grown oocytes of the frog (Rana pipiens) undergo cytoplasmic and nuclear maturation when treated with progesterone after the follicular envelopes have been removed. The mechanism of this maturation was investigated by injection of cytoplasm from progesterone-treated oocytes at various stages of maturation into fully grown but immature oocytes. The injected cytoplasm becomes effective in inducing maturation by 12 hours after progesterone administration, reaches a maximum effectiveness around 20 hours, and then declines after the donor oocytes complete maturation. However, even cytoplasm from early embryos retains some capacity to induce oocyte maturation. The frequency with which maturation is induced is proportional to the volume of the injected cytoplasm. Progesterone itself is not directly responsible for the maturation-producing effect of injected cytoplasm since injected progesterone does not promote maturation. However, externally applied progesterone does induce the completion of the first meiotic division, presumably by releasing a cytoplasmic “maturation promoting factor.” The production of this cytoplasmic factor was not affected by removal of the nucleus.
Greatwall (GW) is a new kinase that has an important function in the activation and the maintenance of cyclin B-Cdc2 activity. Although the mechanism by which it induces this effect is unknown, it has been suggested that GW could maintain cyclin B-Cdc2 activity by regulating its activation loop. Using Xenopus egg extracts, we show that GW depletion promotes mitotic exit, even in the presence of a high cyclin B-Cdc2 activity by inducing dephosphorylation of mitotic substrates. These results indicate that GW does not maintain the mitotic state by regulating the cyclin B-Cdc2 activation loop but by regulating a phosphatase. This phosphatase is PP2A; we show that (1) PP2A binds GW, (2) the inhibition or the specific depletion of this phosphatase from mitotic extracts rescues the phenotype induced by GW inactivation and (3) the PP2A-dependent dephosphorylation of cyclin B-Cdc2 substrates is increased in GW-depleted Xenopus egg extracts. These results suggest that mitotic entry and maintenance is not only mediated by the activation of cyclin B-Cdc2 but also by the regulation of PP2A by GW.
The c-mos proto-oncogene product, pp39mos, is present in unfertilized Xenopus eggs, and disappears on fertilization. Microinjection of synthetic mos RNA into two-cell embryos induces cleavage arrest at metaphase. By contrast, egg cytosol extracts, when immunodepleted of endogenous pp39mos, lose their cleavage-arresting activity in injected embryos. These results demonstrate that Mos protein is the cytostatic factor CSF, long known as an endogenous meiotic inhibitor in vertebrate eggs.
The casein kinase I family of serine/threonine protein kinases is highly conserved from yeast to humans. Until only recently, both the function and regulation of these enzymes remained poorly uncharacterised in that they appeared to be constitutively active and were capable of phosphorylating an untold number of other proteins. While relatively little was known regarding the exact function of the higher eukaryotic isoforms, the casein kinase I (CKI) isoforms from yeast have been genetically linked to vesicular trafficking, DNA repair, cell cycle progression and cytokinesis. All five S. cerevisiae isoforms are known to be associated with discrete cellular compartments and this localization has been shown to be absolutely essential for their respective functions. New evidence now suggests that the CKI isoforms in more complex systems also exhibit non-homogeneous subcellular distributions that may prove vital to defining the function and regulation of these enzymes. In particular, CKIα, the most-characterized vertebrate isoform, is associated with cytosolic vesicles, the mitotic spindle and structures within the nucleus. Functions associated with these localizations coincide with those previously reported in yeast, suggesting a conservation of function. Other reports have indicated that each of the remaining CKI isoforms have the capacity to make associations with components of several signal transduction pathways, thereby channeling CKI function toward specific regulatory events. This review will examine what is now known about the higher eukaryotic CKI family members from the perspective localization as a means of gaining a better understanding of the function and regulation of these kinases.
The casein kinase I family of serine/threonine protein kinases is highly conserved from yeast to humans. Until only recently, both the function and regulation of these enzymes remained poorly uncharacterised in that they appeared to be constitutively active and were capable of phosphorylating an untold number of other proteins. While relatively little was known regarding the exact function of the higher eukaryotic isoforms, the casein kinase I (CKI) isoforms from yeast have been genetically linked to vesicular trafficking, DNA repair, cell cycle progression and cytokinesis. All five S. cerevisiae isoforms are known to be associated with discrete cellular compartments and this localization has been shown to be absolutely essential for their respective functions. New evidence now suggests that the CKI isoforms in more complex systems also exhibit non-homogeneous subcellular distributions that may prove vital to defining the function and regulation of these enzymes. In particular, CKIalpha, the most-characterized vertebrate isoform, is associated with cytosolic vesicles, the mitotic spindle and structures within the nucleus. Functions associated with these localizations coincide with those previously reported in yeast, suggesting a conservation of function. Other reports have indicated that each of the remaining CKI isoforms have the capacity to make associations with components of several signal transduction pathways, thereby channeling CKI function toward specific regulatory events. This review will examine what is now known about the higher eukaryotic CKI family members from the perspective localization as a means of gaining a better understanding of the function and regulation of these kinases.
Many protein kinases themselves are regulated by reversible phosphorylation. Upon cell stimulation, specific kinases are transiently phosphorylated and activated. Several of these protein kinases are substrates for protein phosphatase 2A (PP2A), and PP2A appears to be the major kinase phosphatase in eukaryotic cells that downregulates activated protein kinases. This idea is substantiated by the observation that some viral proteins and naturally occurring toxins target PP2A and modulate its activity. There is increasing evidence that PP2A activity is regulated by extracellular signals and during the cell cycle. Thus, PP2A is likely to play an important role in determining the activation kinetics of protein kinase cascades.
The mitotic and meiotic cell cycle share many regulators, but there are also important differences between the two processes. The meiotic maturation of Xenopus oocytes has proved useful for understanding the regulation of Cdc2-cyclin-B, a key activator of G2/M progression. New insights have been made recently into the signalling mechanisms that induce G2-arrested oocytes to resume and complete the meiotic cell cycle.
We have developed a proteomic approach for identifying phosphopeptide binding domains that modulate kinase-dependent signaling
pathways. An immobilized library of partially degenerate phosphopeptides biased toward a particular protein kinase phosphorylation
motif is used to isolate phospho-binding domains that bind to proteins phosphorylated by that kinase. Applying this approach
to cyclin-dependent kinases (Cdks), we identified the polo-box domain (PBD) of the mitotic kinase polo-like kinase 1 (Plk1)
as a specific phosphoserine (pSer) or phosphothreonine (pThr) binding domain and determined its optimal binding motif. This
motif is present in known Plk1 substrates such as Cdc25, and an optimal phosphopeptide containing the motif disrupted PBD-substrate
binding and localization of the PBD to centrosomes. This finding reveals how Plk1 can localize to specific sites within cells
in response to Cdk phosphorylation at those sites and provides a structural mechanism for targeting the Plk1 kinase domain
to its substrates.
The meiotic cell cycle, which is comprised of two consecutive M-phases, is crucial for the production of haploid germ cells. Although both mitotic and meiotic M-phases share cyclin-B-Cdc2/CDK1 as a key controller, there are meiosis-specific modulations in the regulation of cyclin-B-Cdc2. Recent insights indicate that a common pattern in these modulations can be found by considering the particular activities of mitogen-activated protein kinase (MAPK) during meiosis. The G(2)-phase arrest of meiosis I is released via specific, MAPK-independent signalling that leads to cyclin-B-Cdc2 activation; thereafter, however, the meiotic process is under the control of interplay between MAPK and cyclin-B-Cdc2.
Metaphase arrest in meiosis I or II before fertilization is a common and unique feature of oogenesis in many animal species. How and why oocytes from many species are arrested at metaphase, rather than after the completion of meiosis, has long remained a mystery. This article reviews recent advances in our understanding of the mechanisms and biological significance of meiotic metaphase arrest in animal oocytes.
Cyclin-dependent kinases (CDKs) limit the activation of DNA replication origins to once per cell cycle by preventing the assembly of pre-replicative complexes (pre-RCs) during S, G2 and M phases of the cell cycle in the budding yeast Saccharomyces cerevisiae. CDKs inhibit each pre-RC component (ORC, Cdc6, Cdt1/Mcm2-7) by different mechanisms. We show here that the mitotic CDK, Clb2/Cdc28, binds tightly to an amino-terminal domain (NTD) of Cdc6, and that Cdc6 in this complex is unable to assemble pre-RCs. We present evidence indicating that this Clb2-dependent mechanism contributes to preventing re-replication in vivo. CDK interaction with the NTD of Cdc6 is mediated by the cyclin subunit Clb2, and could be reconstituted with recombinant Clb2 protein and synthetic NTD peptides. Tight Clb2 binding occurred only when the NTD was phosphorylated on CDK consensus sites. Human CDKs containing cyclins A, B and E also bound specifically to phospho-NTD peptides. We propose that direct binding of cyclins to phosphopeptide motifs may be a widespread phenomenon contributing to the targeting of CDKs to substrates.
Meiosis is the type of cell division that gives rise to eggs and sperm. Errors in the execution of this process can result in the generation of aneuploid gametes, which are associated with birth defects and infertility in humans. Here, we review recent findings on how cell-cycle controls ensure the coordination of meiotic events, with a particular focus on the segregation of chromosomes.
Stable maintenance of genetic information requires chromosome segregation to occur with high accuracy. Anaphase is triggered when ring-shaped cohesin is cleaved by separase, a protease regulated by association with its inhibitor securin. Dispensability of vertebrate securin strongly suggests additional means of separase regulation. Indeed, sister chromatid separation but not securin degradation is inhibited by constitutively active cyclin-dependent kinase 1 (Cdk1) and can be rescued solely by preventing phosphorylation of separase. We demonstrate that Cdk1-dependent phosphorylation of separase is not sufficient for inhibition. In a second step, Cdk1 stably binds phosphorylated separase via its regulatory cyclin B1 subunit. Complex formation results in inhibition of both protease and kinase, and we show that vertebrate separase is a direct inhibitor of Cdk1. This unanticipated function of separase is negatively regulated by securin but independent of separase's proteolytic activity.
Vertebrate oocytes are arrested at second meiotic metaphase by cytostatic factor (CSF) while awaiting fertilization. Accumulating evidence has suggested that inhibition of the anaphase-promoting complex/cyclosome (APC/C) is responsible for this arrest. Xenopus polo-like kinase 1 (Plx1) is required for activation of the APC/C at the metaphase-anaphase transition, and calcium elevation, upon fertilization/activation of eggs, acting through calmodulin-dependent kinase II (CaMKII) is sufficient to activate the APC/C and terminate CSF arrest. However, connections between the Plx1 pathway and the CaMKII pathway have not been identified.
Overexpression of Plx1 causes CSF release in the absence of calcium, and depletion of Plx1 from egg extracts blocks induction of CSF release by calcium and CaMKII. Prior phosphorylation of the APC/C inhibitor XErp1/Emi2 by CaMK II renders it a good substrate for Plx1, and phosphorylation by both kinases together promotes its degradation in egg extracts. The pathway is enhanced by the ability of Plx1 to cause calcium-independent activation of CaMKII. The results identify the targets of CaMKII and Plx1 that promote egg activation and define the first known pathway of CSF release in which an APC/C inhibitor is targeted for degradation only when both CaMKII and Plx1 are active after calcium elevation at fertilization.
Plx1 with an intact polo-box domain is necessary for release of CSF arrest and sufficient when overexpressed. It acts at the same level as CaMKII in the pathway of calcium-induced CSF release by cooperating with CaMKII to regulate APC/C regulator(s), such as XErp1/Emi2, rather than by directly activating the APC/C itself.
The Wee1 kinase phosphorylates and inhibits cyclin-dependent kinase 1 (Cdk1), thereby delaying entry into mitosis until appropriate conditions have been met. An understanding of the mechanisms that regulate Wee1 should provide new insight into how cells make the decision to enter mitosis. We report here that Swe1, the budding-yeast homolog of Wee1, is directly regulated by Cdk1. Phosphorylation of Swe1 by Cdk1 activates Swe1 and is required for formation of a stable Swe1-Cdk1 complex that maintains Cdk1 in the inhibited state. Dephosphorylation of Cdk1 leads to further phosphorylation of Swe1 and release of Cdk1. Thus, Cdk1 both positively and negatively regulates its own inhibitor. Regulation of the Swe1-Cdk1 complex is likely to play a critical role in controlling the transition into mitosis.
The anaphase promoting complex/cyclosome (APC/C) is a ubiquitin ligase that has essential functions in and outside the eukaryotic cell cycle. It is the most complex molecular machine that is known to catalyse ubiquitylation reactions, and it contains more than a dozen subunits that assemble into a large 1.5-MDa complex. Recent discoveries have revealed an unexpected multitude of mechanisms that control APC/C activity, and have provided a first insight into how this unusual ubiquitin ligase recognizes its substrates.
The polo-box domain (PBD) of mammalian polo-like kinase 1 (Plk1) is essential in targeting its catalytic activity to specific subcellular structures critical for mitosis. The mechanism underlying Plk1 recruitment to the kinetochores and the role of Plk1 at this site remain elusive. Here, we demonstrate that a PBD-binding protein, PBIP1, is crucial for recruiting Plk1 to the interphase and mitotic kinetochores. Unprecedentedly, Plk1 phosphorylated PBIP1 at T78, creating a self-tethering site that specifically interacted with the PBD of Plk1, but not Plk2 or Plk3. Later in mitosis, Plk1 also induced PBIP1 degradation in a T78-dependent manner, thereby enabling itself to interact with other components critical for proper kinetochore functions. Absence of the p-T78-dependent Plk1 localization induced a chromosome congression defect and compromised the spindle checkpoint, ultimately leading to aneuploidy. Thus, Plk1 self-regulates the Plk1-PBIP1 interaction to timely localize to the kinetochores and promote proper chromosome segregation.
Erp1 (also called Emi2), an inhibitor of the APC/C ubiquitin ligase, is a key component of cytostatic factor (CSF) responsible for Meta-II arrest in vertebrate eggs. Reportedly, however, Erp1 is expressed even during meiosis I in Xenopus oocytes. If so, it is a puzzle why normally maturing oocytes cannot arrest at Meta-I. Here, we show that actually Erp1 synthesis begins only around the end of meiosis I in Xenopus oocytes, and that specific inhibition of Erp1 synthesis by morpholino oligos prevents entry into meiosis II. Furthermore, we demonstrate that premature, ectopic expression of Erp1 at physiological Meta-II levels can arrest maturing oocytes at Meta-I. Thus, our results show the essential role for Erp1 in the meiosis I/meiosis II transition in Xenopus oocytes and can explain why normally maturing oocytes cannot arrest at Meta-I.
Vertebrate oocytes are arrested in metaphase II of meiosis prior to fertilization by cytostatic factor (CSF). CSF enforces a cell-cycle arrest by inhibiting the anaphase-promoting complex (APC), an E3 ubiquitin ligase that targets Cyclin B for degradation. Although Cyclin B synthesis is ongoing during CSF arrest, constant Cyclin B levels are maintained. To achieve this, oocytes allow continuous slow Cyclin B degradation, without eliminating the bulk of Cyclin B, which would induce release from CSF arrest. However, the mechanism that controls this continuous degradation is not understood.
We report here the molecular details of a negative feedback loop wherein Cyclin B promotes its own destruction through Cdc2/Cyclin B-mediated phosphorylation and inhibition of the APC inhibitor Emi2. Emi2 bound to the core APC, and this binding was disrupted by Cdc2/Cyclin B, without affecting Emi2 protein stability. Cdc2-mediated phosphorylation of Emi2 was antagonized by PP2A, which could bind to Emi2 and promote Emi2-APC interactions.
Constant Cyclin B levels are maintained during a CSF arrest through the regulation of Emi2 activity. A balance between Cdc2 and PP2A controls Emi2 phosphorylation, which in turn controls the ability of Emi2 to bind to and inhibit the APC. This balance allows proper maintenance of Cyclin B levels and Cdc2 kinase activity during CSF arrest.
Until fertilization, the meiotic cell cycle of vertebrate eggs is arrested at metaphase of meiosis II by a cytoplasmic activity termed cytostatic factor (CSF), which causes inhibition of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets mitotic cyclins-regulatory proteins of meiosis and mitosis-for degradation. Recent studies indicate that Erp1/Emi2, an inhibitor protein for the APC/C, has an essential role in establishing and maintaining CSF arrest, but its relationship to Mos, a mitogen-activated protein kinase (MAPK) kinase kinase that also has an essential role in establishing CSF arrest through activation of p90 ribosomal S6 kinase (p90rsk), is unclear. Here we report that in Xenopus eggs Erp1 is a substrate of p90rsk, and that Mos-dependent phosphorylation of Erp1 by p90rsk at Thr 336, Ser 342 and Ser 344 is crucial for both stabilizing Erp1 and establishing CSF arrest in meiosis II oocytes. Semi-quantitative analysis with CSF-arrested egg extracts reveals that the Mos-dependent phosphorylation of Erp1 enhances, but does not generate, the activity of Erp1 that maintains metaphase arrest. Our results also suggest that Erp1 inhibits cyclin B degradation by binding the APC/C at its carboxy-terminal destruction box, and this binding is also enhanced by the Mos-dependent phosphorylation. Thus, Mos and Erp1 collaboratively establish and maintain metaphase II arrest in Xenopus eggs. The link between Mos and Erp1 provides a molecular explanation for the integral mechanism of CSF arrest in unfertilized vertebrate eggs.
Evolutionary change in gene regulation is a key mechanism underlying the genetic component of organismal diversity. Here, we study evolution of regulation at the posttranslational level by examining the evolution of cyclin-dependent kinase (CDK) consensus phosphorylation sites in the protein subunits of the pre-replicative complex (RC). The pre-RC, an assembly of proteins formed during an early stage of DNA replication, is believed to be regulated by CDKs throughout the animals and fungi. Interestingly, although orthologous pre-RC components often contain clusters of CDK consensus sites, the positions and numbers of sites do not seem conserved. By analyzing protein sequences from both distantly and closely related species, we confirm that consensus sites can turn over rapidly even when the local cluster of sites is preserved, consistent with the notion that precise positioning of phosphorylation events is not required for regulation. We also identify evolutionary changes in the clusters of sites and further examine one replication protein, Mcm3, where a cluster of consensus sites near a nucleocytoplasmic transport signal is confined to a specific lineage. We show that the presence or absence of the cluster of sites in different species is associated with differential regulation of the transport signal. These findings suggest that the CDK regulation of MCM nuclear localization was acquired in the lineage leading to Saccharomyces cerevisiae after the divergence with Candida albicans. Our results begin to explore the dynamics of regulatory evolution at the posttranslational level and show interesting similarities to recent observations of regulatory evolution at the level of transcription.
• DNA replication
• MCM3
• phosphorylation