Cyclin B Dissociation from CDK1 Precedes its Degradation Upon MPF Inactivation in Mitotic Extracts of Xenopus laevis Embryos

Share Embed

Descrição do Produto

[Cell Cycle 5:15, 1687-e12, 1 August 2006]; ©2006 Landes Bioscience

Cyclin B Dissociation from CDK1 Precedes its Degradation Upon MPF Inactivation in Mitotic Extracts of Xenopus laevis Embryos Report


Cyclin B is a regulatory subunit of CDK1 within MPF complex. Degradation of cyclin B via ubiquitin-proteasome pathway seemed to be absolutely required for the M-phase exit. However, inhibition of the proteasome proteolytic activity upon the exit from the meiotic metaphase II-arrest in Xenopus cell-free extract revealed that the proteasome-dependent dissociation of cyclin B from CDK1 is sufficient to inactivate MPF without cyclin B degradation. In this study we analyze whether the same mechanism operates during the exit from mitotic M-phase. We show in Xenopus cell-free extract undergoing the first or the second embryonic mitosis that CDK1 oscillations are not affected by proteasome inhibition with MG132 or ALLN despite effective inhibition of cyclins B degradation. The majority of cyclins B1 and B2 surviving CDK1 inactivation is CDK-free and cyclin B2 becomes resistant to phosphatase λ dephosphorylation. The pool of cyclins B remaining after CDK1 inactivation in the presence of MG132 is mitotically inert, while exogenous or newly synthesized cyclin B activates CDK1. This suggests that cyclins B remain sequestered within the proteasome upon MPF inactivation in the presence of MG132. Comparison of the dynamics of the decline of total and CDK-bound pools of cyclins B1, B2 and B4 upon mitotic exit in absence of protein synthesis reveals that CDK-bound cyclins B diminish clearly faster. Our results thus show that cyclin B dissociation from CDK1 precedes cyclins B degradation upon CDK1 inactivation in mitotic embryo extracts and that proteasome proteolytic activity is dispensable for both activation and inactivation of CDK1 in such extracts.

Original manuscript submitted: 06/05/06 Manuscript accepted: 06/20/06




ALLN, cell cycle, cyclin B, embryo, mitosis, MG132, MPF, proteasome, Xenopus laevis



Previously published online as a Cell Cycle E-publication:


*Correspondence to: Jacek Z. Kubiak; CNRS-UMR 6061; Biology and Genetics of Development; Mitosis and Meiosis Group; IFR140 GFAS; University of Rennes 1; Faculty of Medicine; 2 Ave. Prof. Léon Bernard; CS 34317; 35043 Rennes cedex, France; Tel.: +; Fax: +; Email: jacek. [email protected]


CNRS-UMR 6061; Biology and Genetics of Development; Mitosis and Meiosis Group; IFR140 GFAS; University of Rennes 1; Faculty of Medicine; Rennes, France



Franck Chesnel Franck Bazile Aude Pascal Jacek Z. Kubiak *




M-phase Promoting Factor (MPF) is a universal mitotic regulator composed of an enzymatic subunit—CDK1 (Cyclin-Dependent Kinase 1) and a regulatory one—cyclin B. It is required for both meiotic and mitotic M-phase in all eukaryotic cells. MPF inactivation was considered to depend rigorously on cyclin B degradation via ubiquitin/proteasome pathway.1,2 However, Nishiyama and colleagues3 have shown that MPF inactivation is uncoupled from cyclin B degradation. The proteolysis of mitotic cyclins is coordinated by the E3 ligase complex or Anaphase Promoting Complex/Cyclosome (APC/C; for a review see ref. 4). It polyubiquitinates substrates for degradation, thereby targeting them to 26S proteasome. Sequential recognition of mitotic substrates determines their sequential degradation. Cyclin A is degraded first shortly after M-phase entry and B-type cyclins follow during later M-phase stages.5,6 In calcium-activated Xenopus laevis CSF extract all cyclins B are degraded within ten minutes; cyclin B1 first, then cyclins B4, B2 and B5 whereas cyclin B3 protein is not expressed in oocytes and early embryos.7 Sequential degradation of mitotic cyclins, as well as other mitotic proteins (e.g., Nek2A, Xkid, Emi1, securin, Kip1 and Cin8 kinesins, Cdc20, Prc1, Tome-1, Plk1, Aurora A/Eg2) is orchestrated by two APC/C protein-ubiquitin ligase activators (Cdc20/Fizzy and Cdh1/Fizzy-related) which determine APC/C-substrate affinity (for a review see ref. 8). Substrate recognition depends on degradation signals within the amino-acid sequence of the protein to be degraded (e.g., D-box in A- and B-type cyclins2,9 KEN-box in Cdc2010 or GxEN-box in Xkid;11 double degradation motifs present in Aurora A: a D-box and a “D-box Activating Domain” or A-box12). Cyclins degradation starts via APC/C-Cdc20-dependent polyubiquitination and propagates via APC/C-Cdh1-dependent mechanism13 (for a review see ref. 8). B-type cyclins in Xenopus laevis oocytes and early embryos are associated with CDK1 and not with CDK2. B1 cyclin is very similar to B4 and the two of them are degraded the fastest. B2 cyclin resembles B5 and their degradation occurs the latest.7 B1, B2 and B4









We thank Marcel Méchali, Tim Hunt, John Gannon, Jean-Pierre Tassan, Daniel Fisher, and Thierry Lorca for generous gifts of antibodies, Marie-Anne Felix for ∆90 cyclin B and Lénaïck Detivaud and Laurent Meijer for p9 beads. We thank Yannick Arlot-Bonnemains, Laurent Richard-Parpaillon, Daniel Fisher, Olivier Haccard and Jacek Gaertig for discussions and critical reading of the manuscript. This work was supported by grant 4298 from ARC and from Ligue Contre le Cancer (Comité d'Ille-et-Vilaine) to J.Z.K.

Cell Cycle


Cyclin B Dissociation from CDK1 Precedes its Degradation Upon MPF Inactivation in Mitotic Extracts of Xenopus laevis Embryos

cyclins are the most abundant during oocyte maturation. Their quantities rise following fertilization, while B5 decreases.7 In parthenogenetic embryos 60 min post-activation (at 22˚C, thus before the first mitosis) cyclin B1 is in roughly equal mixture with cyclin B2.14 In fully-grown stage VI Xenopus oocyte, there are 10-100 times more molecules of CDK1 than cyclin B (the majority of it is cyclin B2;14 and a pool of free CDK1 is present.15 Taking into account a strong affinity of CDK1 to cyclin B, no free cyclin B should be present in prophase oocytes. During the first embryonic cell cycle the levels of cyclin B1, B2 and B4 are similar as in prophase oocyte.7,14 The quantity of cyclin B5 was not precisely measured at that time, however, judging by Western blot analysis it is present in clearly lower levels than B1, B2 and B4 cyclins respectively.7 Therefore, during the first mitosis the levels of all cyclins B increase not more than three and half times in comparison to prophase oocyte. Since cyclin A is degraded early in mitosis and the levels of CDK1 does not change during this period, it means that the levels of CDK1 are at least three times higher than the level of all B-type cyclins.7,14 Therefore, all four cyclins B should be associated with CDK1 at that time. Association of CDK1 with a specific cyclin most likely determines the enzymatic properties of the complex, as shown for cyclin A- or cyclin B-CDK1 in relation to the regulation of microtubule dynamics and nucleation.16,17 Probably, more subtle differences concern CDK1 associated with different B-type cyclins. Phosphorylation of cyclins B on multiple N-terminal residues is associated with MPF activation.18,19,20,21,22 Cyclins B are phosphorylated by CDK1 itself.20 They can be, however, phosphorylated also by Mos,23 Plk124 or MAP kinase.25 Differential phosphorylation of a cyclin component could modify the specificity of the complex as well as its metabolism, for example the affinity for ubiquitination and proteolysis. Proteasome inhibition using ALLN or MG132 arrests eukaryotic somatic cells in the mitotic M-phase.26,27,28,29 It blocks also the meiotic MI/MII transition in mouse oocytes.30 This M-phase arrest was thought to be due to the inhibition of cyclins B degradation which stabilizes high MPF activity. However, exceptions to this rule were also described. The first embryonic mitosis of the rat embryo is arrested by MG132 with high levels of non-degraded cyclin B and relatively low MPF activity.31 Similarly, human colon carcinoma Hct-116 cells with induced MAD2 haplo-insufficiency escape cyclin B degradation upon MPF inactivation during mitotic exit.32,33 Therefore, MPF can be inactivated without cyclin B degradation. Nishiyama and colleagues3 have shown that MPF inactivation is separated from cyclin B degradation upon activation of CSF extract of Xenopus laevis in the presence of proteasome proteolytic activity inhibitor MG115 and that proteasomes possess non-proteolytic activity dissociating CDK1 from cyclin B. During the exit from meiotic metaphase II (MII) in CSF extract, inactivation of MPF requires only the physical separation of cyclin B and CDK1 and not cyclin B degradation. However, as mentioned above, cyclin B degradation appears necessary for the transition between two meiotic M-phases in mammalian oocytes.30,34,35 Despite that the role of the proteasome in this meiotic transition was not questioned in Xenopus laevis oocytes, it was postulated that APC/C is dispensable for this process, suggesting modified targeting of cyclin B to the proteasomes during meiotic M-phase,36 while in mouse oocytes it is certainly involved.30,35,37 Thus, different mechanisms could be involved in MPF inactivation during somatic or embryonic mitoses, as well as upon the first or second meiotic division in different species. 1688

In oocytes, numerous meiosis-specific proteins are abundant or activated to high degree (e.g., Mos, MAP kinases ERK, Rsk proteins) and stabilize cyclins B both in Xenopus as well as in mouse oocytes.38,39,40,41 In contrast to meiosis, the early mitotic cell cycles are controlled by short and regular MPF oscillations guided by cycles of cyclins B synthesis and degradation.42 ERK2 MAP kinase, the major meiotic player in cyclin B stabilization, is also activated during early embryonic mitoses in Xenopus, however relatively late and to much lower levels than during meiosis.43 Such a natural activation of ERK2 during mitosis has no effect on MPF stability (see Fig. 7 in ref. 44). Experimental activation of ERK2 to high levels during mitosis, however, stabilizes cyclin B and MPF activity arresting embryos and cell-free extracts in M-phase.45,46,47 In addition, the exit from the M II-arrest depends on external signal, while the embryonic cycles are driven by internal calcium-dependent clock.48 Thus, the mechanisms regulating cyclin B stability and MPF inactivation differ substantially between the meiotic and mitotic M-phases. They could influence proteasome-dependent dissociation of CDK1 and cyclin B during MPF inactivation. For these reasons we examined effects of proteasome inhibitors on progression of mitotic M-phases in cell-free embryo extract.


Frogs. Xenopus laevis females were purchased from NASCO (Fort Atkinson, WI, USA). Drugs. MG132 was purchased from Biomol (Pennsylvania, USA) and ALLN from Sigma (Saint Quentin Fallavier, France). Other chemicals were obtained either from Sigma or ICN (Irvine, CA, USA) unless otherwise stated. Eggs collection and activation. Females were subcutaneously injected with human chorionic gonadotropin (500-600 IU per female; Organon, Puteaux, France) and kept overnight at 21˚C in 110 mM NaCl. Unfertilized eggs collected from “overnight lay” were dejellied with 2% L-cysteine pH 7.81 in XB buffer;49 100 mM KCl, 1 mM MgCl2, 50 µM CaCl2, 10 mM HEPES, 50 mM sucrose pH 7.6), washed in XB, treated for 1.5 minute with 0.5 µg/ml calcium ionophore A23187 and then extensively washed in XB. Activated eggs were then incubated in XB at 21˚C. Cell-free extracts. Cytoplasmic cell-free cycling extracts from calcium ionophore-activated embryos were prepared as previously described by Murray49 with slight modifications. Briefly, embryos were cultured at 21˚C in XB for 60 minutes post-activation. They were transferred into appropriate tubes (5 mL ultra-clear™ centrifuge tubes; Beckman Coulter, Roissy, France) containing 0.5 mL XB with 0.1 mM AEBSF, aprotinin, leupeptin, pepstatin, chymostatin (10 µg/mL each) and 25 µg/mL cytochalasin D and packed through a short spin at 700 rpm. After removal of any excess XB medium, embryos were subjected to two consecutive centrifugations: a crushing spin, 10,000 g for ten minutes at 4˚C and a clarification spin of the supernatant 10,000 g for ten minutes at 4˚C in which cytochalasin D, AEBSF, aprotinin, leupeptin, pepstatin and chymostatin were again added. The resulting low-speed supernatants were then reincubated at 21˚C for 60 to 120 minutes and every five minutes, 2-µl aliquots were taken out and either frozen in liquid nitrogen and stored at -70˚C (for subsequent H1 kinase activity assays) or mixed with Laemmli sample buffer, heated at 85˚C for five minutes and stored at -20˚C (for Western blot analyses).50 Electrophoresis, antibodies and Western blotting. Extracts were subjected to electrophoresis on 8 to 12.5% SDS-PAGE gels.50

Cell Cycle

2006; Vol. 5 Issue 15

Cyclin B Dissociation from CDK1 Precedes its Degradation Upon MPF Inactivation in Mitotic Extracts of Xenopus laevis Embryos

Separated proteins were transferred to nitrocellulose membranes (Hybond C, Amersham Biosciences) according to standard procedures and probed either with antibodies against cyclin B1 (gift from Tim Hunt), cyclin B2 (gift from Thierry Lorca), cyclin B4 (gift from John Gannon), MCM4 (gift from Marcel Méchali) or Eg3 (gift from Jean-Pierre Tassan). Antigen-antibody complexes were revealed using alkaline phosphatase conjugated anti-rabbit or anti-mouse secondary antibody (diluted 1:20,000) in combination with Enhanced Chemifluorescence reagent (ECF; Amersham Biosciences). Signal quantification was performed using ImageQuant 5.2 software (Amersham Biosciences). In vitro assay for histone H1 kinase activity. MPF activity in embryos or in cell-free extracts was measured as previously described,51 with minor modifications: extracts (1 µl) were diluted in 25 µl MPF buffer (80 mM β-glycerophosphate, 50 mM sodium fluoride, 20 mM EGTA, 15 mM MgCl2, 1 mM DTT, 20 mM HEPES, pH 7.4) supplemented with 0.5 mM sodium orthovanadate and 5 µg/µl of leupeptin, aprotinin, pepstatin and chymostatin and containing 0.4 mg/ml H1 histone (type III-S), 1 µCi [γ32P] ATP (specific activity: 3000 Ci/mmol; Amersham Biosciences) and 0.8 mM ATP. After incubation for 30 minutes at 30˚C, phosphorylation reactions were stopped by adding Laemmli sample buffer and heating for five minutes at 85˚C. Histone H1 was separated by SDS-PAGE and incorporated radioactivity was measured by autoradiography of the gel using a STORM phosphorimager (Amersham Biosciences) followed by a data analysis with ImageQuant 5.2 software. Sepharose p9 beads precipitation. The p9 sepharose beads used for affinity precipitation were kindly provided by Lenaïck Detivaud and Laurent Meijer. Ten µl of extracts were added to 10 µl p9 beads preequilibrated with homogeneizing buffer (MOPS pH 7.2, 60 mM β-glycerophosphate, 15 mM EGTA, 15 mM MgCl2, 2 mM dithiothreitol, 1 mM sodium fluoride, 1 mM sodium orthovanadate and 1 mM disodium phenyl phosphate) extemporaneously supplemented with 1% (w/v) BSA, 1mM AEBSF and aprotinin, leupeptin, pepstatin, chymostatin (10 µg/ml each). The mixtures were agitated for 2.5 h at 4˚C. After a brief centrifugation (5,000 g, 1 min, 4˚C), the supernatant was collected to be analyzed by Western blotting while the pelleted p9 beads were washed four times with 1 ml of washing buffer (50 mM Tris-HCl pH 7.4, 250 mM NaCl, 5 mM EDTA, 5 mM EGTA, 5mM sodium fluoride and 0.1% nonidet-P 40) containing 0.5 mM AEBSF, aprotinin, leupeptin, pepstatin, chymostatin (10 µg/ml each). The beads were then resuspended in 12 µl of 2× Laemmli sample buffer and heated at 85˚C for 5 min. Samples (whole extracts, p9 supernatant, p9 eluate) were then subjected to 12% SDS-PAGE, and cyclins B and CDK1 were detected by Western blotting. Phage λ protein phosphatase treatment. Cell-free extract was sampled at appropriate time points and 1 µl was treated with 200 units of λ protein phosphatase (New England Biolabs) in a 15-µl reaction mixture consisting of phosphatase buffer (50 mM HEPES pH 7.5, 0.1 mM Na2EDTA, 5 mM dithiothreitol and 0.01% Brij35) supplemented with 2 mM MnCl2. When indicated, 20 mM sodium orthovanadate and 50 mM sodium fluoride were further added to the mixtures to inactivate phosphatase. Following a 15-min incubation at 30˚C, phosphatase reactions were terminated by addition of an equal volume of 2× Laemmli sample buffer and heating for five minutes at 85˚C. Samples were then subjected to 9% SDSPAGE, and cyclins B1 and B2 were detected by Western blotting. Recombinant cyclins B. Sea urchin ∆90 cyclin B was kindly provided by Marie-Anne Felix.

Figure 1. Dose-dependent effect of MG132 on cyclin B2 degradation. The low-speed cytoplasmic embryo extract was prepared 60 min. post ionophoreactivation of metaphase II (M II) oocytes. MG132 was added at 20, 100 and 200 µM final concentration or 1% DMSO (equivalent of the concentration of DMSO in the extract with 200µM MG132), incubated at 21˚C and sampled every 5 min for 60 min. (A) Samples (10 µg of cytoplasmic proteins) were analyzed by 10% SDS-PAGE followed by Western blotting with anti-cyclin B2 antibody (upper panel). Signals were detected using ECF reagent and quantified using ImageQuantTM software (lower panel). (B) Histone H1 kinase was assayed in parallel to the cyclin B2 Western blot. Following in vitro phosphorylation reaction, samples were analyzed by 10% SDS-PAGE followed by autoradiography and quantification using ImageQuantTM software.

RESULTS Inhibition of cyclin B degradation does not modify progression of MPF activity in mitotic extract. MG132 is a potent inhibitor of chymotrypsin-like proteolytic activity of proteasomes (for a review see ref. 52). To characterize the effects of this inhibitor on the mitotic extracts we followed cyclin B2 degradation pattern (Fig. 1A) and histone H1 kinase activity (Fig. 1B) in presence of increasing concentrations of MG132. Cyclin B2 degradation was completed at 30 min time point in the control extract (Fig. 1A). It was only slightly slowed down by 20 µM MG132 in comparison to the control and attained a maximal level, similar to the control, at 35 min time-point (Fig. 1A, upper and lower panels), while 100 µM MG132 enabled

Cell Cycle


Cyclin B Dissociation from CDK1 Precedes its Degradation Upon MPF Inactivation in Mitotic Extracts of Xenopus laevis Embryos

to preserve significant amounts of cyclin B throughout the one-hour incubation (Fig. 1A). Further increase of the drug concentration till 200 µM did not provoke any increase in the efficiency of the inhibition of cyclin B2 degradation (Fig. 1A). Strikingly, the profiles of histone H1 kinase activity examined in parallel with the Western Blot analysis of cyclin B2 were very similar in the control as well as in MG132-treated extracts for each concentration of the drug tested (Fig. 1B). To further study the effects of proteasome inhibition on embryonic M-phase in vitro we used the drug in the final concentration of 100 µM. Almost identical profiles of histone H1 kinase activity obtained in mitotic control extracts and upon proteasome inhibition (Figs. 1B and 2A) suggested that at least some mitotic events in the extract remained unchanged despite the potent inhibition of cyclins B1, B2 and B4 proteolysis (Fig. 2B). To verify this, we followed changes of chosen mitotic markers previously described in the laboratory as useful indicators of the timing of mitotic events.43,53 To this end, we studied the pattern of Eg3 kinase undergoing characteristic mitotic phosphorylation reflected by a decreased velocity in SDS/PAGE and MCM4, a DNA replication regulator undergoing even more clear-cut mitotic changes than Eg3 (Fig. 2B). The results showed that upon MG132 treatment cyclins B1 and B4 degradation was greatly reduced as observed with cyclin B2. Despite the massive inhibition of B-type cyclins degradation, the profiles of electrophoretic migration of Eg3 and MCM4 in MG132-treated extracts are indistinguishable from the control ones (Fig. 2B, double-headed arrows show the periods of the maximum up-shift of these proteins on SDS/PAGE). This shows that both the timing and the efficiency of phosphorylation and dephosphorylation of Eg3 and MCM4 proteins are not affected (directly or indirectly) by proteasome inhibition, and that the duration of the mitotic events studied here are unchanged in all extracts despite permanent cyclins B high levels in the MG-treated extracts. To confirm the selectivity of MG132 action as chymotrypsin-like proteasome inhibitor in the mitotic extract we tested another drug ALLN and studied histone H1 kinase activity in parallel with Western Blot analysis of cyclin B2 and Eg3 (Fig. 3A). 40 µM ALLN did not perturb histone H1 kinase activity profile (Fig. 3A) while inhibiting cyclin B2 degradation quite similarly as MG132 (Fig. 3B and C). Further increase in ALLN concentration (tested up to 100 µM) only slightly decreased the dynamics of cyclin B2 degradation, again without affecting histone H1 kinase activity profile (data not shown). Also the period of mitotic phosphorylation of Eg3 remained the same in ALLN-treated and control extracts (Fig. 3B). This shows that the two proteasome inhibitors have similar effects on M-phase events in Xenopus embryo cell-free extract. Together, these data provide compelling evidence that inhibition of proteasome proteolytic activity during embryonic mitosis does not prevent MPF inactivation as shown for activated CSF meiotic extract by Nishiyama and colleagues.3 Cyclins B1 and B2 remaining after MPF inactivation in MG132treated extract are not associated with CDK1. Since cyclins B degradation and MPF inactivation appeared as separated events during the mitotic exit in the above experiments, we verified the quantitative relationship between B1 and B2 cyclins and CDK1 during this period as done previously by Nishiyama et al.3 during the exit from meiotic arrest. To this end we compared the behavior of CDK1 and cyclins B1 and B2 in samples collected at ten minutes (before M-phase entry) and 60 min (following the mitotic exit; see Fig. 4A) of incubation (Figs. 4A, B), and examined whether these cyclins B coprecipitate with p9-coated beads specific to CDKs;54 (Fig. 4B). 1690

Figure 2. MG132 (100 µM) inhibits cyclin B1, B2 and B4 degradation but does not change the timing neither of histone H1 kinase activity nor of Eg3 and MCM4 proteins phosphorylation during embryonic M-phase in the cell-free extract. (A) Histone H1 kinase assay of the extract prepared 60 min. post-activation and supplemented either with 100 µM MG132 or 0.5% DMSO as a control. (B) Samples were analyzed by 10% SDS-PAGE followed by immunoblotting with anti-cyclin B1, cyclin B2, cyclin B4, Eg3 and MCM4 antibodies. The double headed arrows show the period of the maximal up-shift of Eg3 and MCM4 proteins due to their mitotic phosphorylation.

Cyclin B1 migrates as a double band due to the presence of two distinct form of alternatively spliced cyclin B1.7 Accordingly, degradation of the two forms of cyclin B1 is concomitant (see Figs. 2B and 4B in ref. 7). Cyclin B2 migrates as a double band (see Figs. 1A, 2B and 3B), where the upper band corresponds to the phosphorylated form of cyclin B2, which appears upon cyclin association with CDK.22 Upon mitotic exit, the non-phosphorylated form of cyclin B2 disappears first, and the phosphorylated one remains as a major band during a short period (see for instance Figs. 1A, 2B). In the presence of MG132 or ALLN, the majority of cyclin B2 remaining in the extract following the inactivation of histone H1 kinase activity was found in this phosphorylated form (see Figs. 1A, 2B and 3B). In the experiment presented in Figure 4, CDK1 in whole extract was detected in all samples at a similar level (see Fig. 4B), upper left panel, CDK1 in whole extract at 10 and 60 min). Cyclin B1, when present, migrated always as a double band. Cyclin B2 was present as a double band at ten minutes both in the control and MG132-treated

Cell Cycle

2006; Vol. 5 Issue 15

Cyclin B Dissociation from CDK1 Precedes its Degradation Upon MPF Inactivation in Mitotic Extracts of Xenopus laevis Embryos

Figure 3. ALLN (40 µM) has a similar effect on biochemical events during embryonic M-phase in the cell-free extract as MG132. (A) Histone H1 kinase assay of the extract prepared 60 min. post-activation and supplemented either with 40 µM ALLN or 0.25% DMSO as a control. (B) Western blots with anti-cyclin B2 (upper panel) and Eg3 (lower panel) antibodies show that cyclin B2 degradation is inhibited by ALLN and that the period of Eg3 up-shift (double headed arrows) is identical in the control and in ALLN-treated extracts. C) Specific signals for cyclin B2 shown in (Fig. 3B) (upper panel) were quantified using ImageQuantTM software.

extracts, absent since degraded at 60 min in the control extract and present in high quantity as well as up-shifted in 60 min time-point in the presence of MG132. The p9 beads efficiently removed CDK1 from all samples of the MG132-treated and untreated extracts, as expected (Fig. 4B, compare the middle and the rightmost panels). Also cyclins B1 and B2 were coprecipitated by the beads at ten minutes in the control extract and were absent at 60 min since already degraded (Fig. 4B, rightmost panel). In the MG132-treated extract cyclins B1 and B2 were efficiently coprecipitated by p9 beads only at ten minutes, while weak signals were found at 60 min (Fig. 4B, rightmost panel). However, cyclins B1 and B2 are absent from the p9 supernatant in all samples but at 60 min in the presence of MG132 showing that at that particular time point the two cyclins B

in this extract are free of CDK1 (Fig. 4B, the middle panel). The weak signals of cyclins B1 and B2 associated with the beads observed at 60 min in the MG132-treated extract were most probably nonspecific contamination of the pellet by cyclins B present in the supernatant. It is noteworthy that the two cyclins B studied in this experiment behaved in the same way, as expected. This experiment was repeated twice with identical results. It shows that following MPF inactivation in the presence of MG132 the majority of remaining cyclins B1 and B2 are free of CDK1. Cyclin B2 remaining after MPF inactivation in MG132-treated extract is protected against dephosphorylation. Since the majority of cyclins B1 and B2 are not associated with CDK1 in MG132-treated extracts following mitotic exit and in addition cyclin B2 remains in a phosphorylated state as if it were still associated with CDK1, we wanted to characterize better this particular form of cyclin B2. To this end, we tested whether such cyclin B2 could be dephosphorylated by an exogenous phosphatase. Cyclin B1, whose dephosphorylation cannot be visualized by modification of the electrophoretic mobility (our unpublished observations and see Fig. 4C), was used as a control to distinguish between dephosphorylation of cyclin B2 and its potential degradation during incubation since both cyclins B have very similar characteristics with regard to proteolysis. Phage λ phosphatase was added for 15 min. to the control and MG132-treated extracts coming from the same experiment as shown above (Figs. 4A, B). Samples at the 20 min time point (the very beginning of the M-phase) for the control extract as well as the MG132-treated counterpart and 50 min sample following MPF inactivation in the presence of MG132 were analyzed (10 and 60 min neighbor samples were used for p9 beads precipitation; (Fig. 4C). As expected, cyclin B1 did not react to the presence of either λ phosphatase or the phosphatase buffer (Fig. 4C, upper panel). This demonstrates that cyclin B1 remains stable in our experimental conditions at least for 15 min. Indeed, cyclin B2 behaved in a clearly different way than cyclin B1. In the control extract it was efficiently dephosphorylated by λ phosphatase as indicated by the disappearance of the upper band (correlated with increased intensity of the lower one) already after 15 min incubation and followed further until 45 min (Fig. 4C, leftmost, 15 min and 45 min). However, even incubation of the extract in the phosphatase buffer followed by incubation at 30˚C provoked similar, but slightly less rapid reaction. Note that traces of phosphorylated form of cyclin B2 are still present after 15 min incubation and they disappear at 45 min (Fig. 4C DMSO t20 second lane), while they have totally disappeared after 15 min incubation upon λ phosphatase treatment (Fig. 4C DMSO t20 third lane). Disappearance of the phosphorylated form of cyclin B2 was however, inhibited by sodium vanadate and fluoride present in the reaction mixture (Fig. 4C DMSO t20 fourth lane). The disappearance of the phosphorylated band of cyclin B2 is due to dephosphorylation since cyclin B1 was not degraded in the same sample and we observed a parallel increase in the intensity of the non-phosphorylated, lower band of this cyclin on Western blot. Identical results were obtained for the 20 min sample from the MG132-treated extract (Fig. 4C, middle panels, 15 and 45 min). However, neither buffer alone nor phage λ phosphatase were able to efficiently dephosphorylate cyclin B2 from the 50 min sample in the MG132-treated extract (Fig. 4C, rightmost panels, MG t50). These results indicate that cyclin B2 undergoes some qualitative changes after the exit from the M-phase upon MG132 treatment which makes it less sensitive to dephosphorylation. We believe that phos-

Cell Cycle


Cyclin B Dissociation from CDK1 Precedes its Degradation Upon MPF Inactivation in Mitotic Extracts of Xenopus laevis Embryos

Figure 4. Cyclin B2 remaining after MPF inactivation in the presence of 100 µM MG132 is free of CDK1 and less sensitive to phage λ phosphatase. (A) Embryo extracts supplemented at the beginning of incubation with MG132 (100 µM) or with DMSO (0.5 %) was sampled every 5 min and histone H1 kinase was assayed. (B) Coprecipitation of cyclins B1 and B2 with CDK proteins on p9 beads. The presence or absence of CDK1 and cyclins B were detected by Western blot in 10 min and 60 min samples of the whole cell-free mitotic extract (left panel). Sibling samples of these extracts were incubated with p9 beads for 2.5 h at 4˚C under constant agitation. CDK1 was absent in the supernatant (middle panel) and precipitated entirely with the beads (right panel). In the supernatant, cyclins B1 and B2 are detected only in the MG132-treated extract at 60 min time point (middle panel), while they are coprecipitated with p9 beads at 10 min time point in the control as well as in the MG132-treated extract, absent at 60 min time point in the control and present in a very weak amount at 60 min time point in the presence of MG132 (right panel). (C) One µl of the extracts from 20 and 50 min samples of the same experiment was treated with 200 Units of λ protein phosphatase in the presence of buffer (in a 15 µl final volume) supplemented or not with 20 mM sodium orthovanadate and 50 mM sodium fluoride (PPase inh.). The mixtures were incubated for 15 to 45 min at 30˚C and the samples were then analyzed by Western blotting for the presence of cyclins B1 and/or B2.

phorylated cyclin B2 was efficiently protected against λ phosphatase because sequestered within the proteasome. Moreover, we observed clear diminution of the intensity of all bands specifically in three samples collected at 50 min time point in the presence of MG132 and incubated at 30˚C (Fig. 4C MG t50, rightmost panel lanes two, 1692

three and four in cyclin B1 and B2). Significantly, the decreased intensity of the two bands of both cyclin B1 and B2 in these three samples was slightly more significant in 45 min time point than in 15 min. It suggests progressive degradation of all forms of these cyclins only in those samples. This was most likely due to the diminution of the concentration of MG 132 in the reaction mixture which activated to some extent the proteolytic activity of proteasomes. This latter observation favors our hypothesis that cyclins B1 and B2 are indeed sequestered within the proteasome in the presence of MG 132 after MPF inactivation. Cyclins B pool remaining after mitotic exit in the presence of MG132 is mitotically inert, but newly synthesized cyclin B activates histone H1 kinase in the presence of MG132. If the pool of cyclins B remaining in the extract following histone H1 kinase inactivation in the presence of MG132 is indeed sequestered within the proteasome as suggested by Nishiyama et al.3 and our above experiments, it should be inaccessible for CDK1 and thus mitotically inert. If this pool of cyclin B is able to leave the proteasome it should be capable to associate with CDK1 and thus induce precocious subsequent M-phase in cycling extracts. To test which hypothesis is correct we assayed histone H1 kinase activity as well as cyclin B2 levels by Western blotting in control and MG132-treated extracts during two hours to allow the second M-phase to occur in a control cycling extract. We observed that the second peak of histone H1 kinase activity in MG132-treated extract was not accelerated but delayed in comparison to the control extract (Fig. 5A). Cyclin B2 was degraded upon mitotic exit and then resynthesized in the control extract, while it was only slightly diminished upon M-phase exit in MG132treated extract, and further accumulated to sensitively higher levels than in the control extract as shown by Western blot analysis (Fig. 5B). While the maximal quantity of cyclin B2 during the second M-phase was present at 110 min. time-point (Fig. 5B), a comparable amount of this protein in the MG132-treated extract was already present at 70 min. time-point (Fig. 5B). Despite faster accumulation of cyclin B2 in this extract the increase in histone H1 kinase activity was observed only at 110-120 min. (Fig. 5B). The absence of acceleration of the second M-phase in MG132-treated extract suggested that the pool of cyclin B remaining after mitotic exit is inaccessible to CDK1. This again argued for cyclin B sequestration within the proteasome. The second wave of activation of histone H1 kinase in the extract shows however, that newly synthesized cyclin B indeed associates with CDK1 in the continuous presence of MG132. We conclude that two different pools of cyclin B are therefore present in the extract following mitotic exit in the presence of MG132: the mitotically inert one, which was synthesized before the exit from the first embryonic M-phase, and the mitotically active, which is neo-synthesized following this mitotic exit. This experiment also shows that even prolonged presence of MG132 does not prevent the extract to enter the second M-phase. Exogenous sea urchin cyclin B also activates histone H1 kinase in the presence of MG132. To verify experimentally the capacity of MG132-treated extract to activate MPF, we added exogenous nondegradable ∆90 cyclin B of sea urchin during the mitotic exit and compared evolution of histone H1 kinase activity (Fig. 6A), endogenous cyclin B2 levels and MCM 4 phosphorylation pattern (Fig. 6B). Non-degradable cyclin B addition provokes gradual histone H1 kinase increase in MG132-treated extract (Fig. 6A; dotted line). Western blot analysis confirmed that endogenous cyclin B2 was degraded in the control extract without MG132 (data not shown) and preserved in MG132-treated extract upon histone H1 kinase

Cell Cycle

2006; Vol. 5 Issue 15

Cyclin B Dissociation from CDK1 Precedes its Degradation Upon MPF Inactivation in Mitotic Extracts of Xenopus laevis Embryos

Figure 5. Cyclin B2 remaining in the extract after MPF inactivation in the presence of 100 µM MG132 does not accelerate subsequent M-phase. (A) Histone H1 kinase activity was assayed every five mintutes for two hours in the extracts containing either 100 µM MG132 or 0.5 % DMSO (control). Second mitosis is already completed in the control extract at 120 min time point, while the mitotic increase of histone H1 kinase activity only begins in the presence of MG132. (B) Cyclin B2 was detected in parallel to histone H1 kinase assay in the two extracts. Note the complete degradation of cyclin B2 following each mitosis (35 and 120 min time points) in the control extract and much less important diminution of cyclin B2 following the first mitosis in the MG132-treated extract. Despite higher amount of cyclin B2 detected by Western blot in the MG132-treated extract, the second M-phase entry is not accelerated. Note also a discrete difference between the ratio of two bands of cyclin B2 during the first and the second M-phase in cycling extract as visualized by Western blotting. Cyclin B2 is present as a clear double band during the first M-phase and mostly as a single, phosphorylated upper band during the second. This is an artefact observed in cycling extracts, while in extracts dedicated specifically to the second M-phase the two bands of cyclin B2 are present also during the second cell cycle (compare with Fig. 1B) in Chesnel et al. 2005a).

inactivation (Fig. 6B, upper panel, time points 60-120 min) as in all previously described experiments. Anti-MCM4 Western blot showed that the phosphorylation of this protein reincreased steadily in MG132-containing extract only when supplemented with exogenous ∆90 cyclin B (Fig. 6B, lower panel, time points 60-120 min). This clearly indicates that exogenous cyclin B forms active complexes with CDK1 and provokes early M-phase events in the presence of MG132 despite sequestration of the endogenous cyclin pool. MG132 only slightly delays mitotic activation of MPF. The slight delay in histone H1 kinase activation during the entry into the second embryonic M-phase in cycling extracts upon prolonged exposure to MG132 (Fig. 5A) could suggest that requirements for a proteasome-dependent degradation differ between the first and second embryonic M-phase. Indeed, numerous differences in the regulation of these two M-phases in vitro were shown recently by our laboratory.43 Alternatively, proteolysis of some “early” substrates of the proteasome could be required for a correct entry into the

Figure 6. Recombinant sea urchin ∆ 90 cyclin B added by the end of the M-phase to the extract forms active complex with CDK1 and allows histone H1 kinase reactivation. The low-speed extract was incubated at 21˚C for two hours with 100 µM MG132. At 50 min of incubation (expected time of the mitotic exit), ∆ 90 cyclin B was added to the half of the extract at the final concentration of 15 µM, while another half containing only MG132 was sampled as a control. (A) Histone H1 kinase activity was assayed every ten minutes for two hours in the extract containing 100 µM MG132 and supplemented or not with ∆ 90 cyclin B. Note the reincrease of histone H1 kinase activity following full inactivation of the mitotic histone H1 kinase activity in the extract supplemented with ∆ 90 cyclin B (dotted line) in contrast to the control without exogenous cyclin B. (B) Western blot analysis of cyclin B2 (upper panels) and MCM4 (lower panels) was performed on sibling samples. Cyclin B2 Western blot confirms that MG132 prevented cyclin B2 degradation in this experiment (double arrows in the upper panels). MCM4 is dephosphorylated following histone H1 kinase inactivation in MG132-treated control extract and rephosphorylated starting from t = 80 min following ∆90 cyclin B addition only (double arrows in the lower panel).

M-phase during both, the first or the second, embryonic mitoses. In the latter case, troubles with histone H1 kinase activation in cycling extracts could be due to the prolonged incubation of extracts with MG132 affecting such an early proteolysis. To verify these possibilities, we first added MG132 to the extracts prepared 108-110 min after M II oocytes activation, i.e., just before the entry into the second embryonic M-phase. The profiles of histone H1 kinase activity were almost identical in the presence and absence of MG132 (Fig. 7A). However, we noticed that when the mitotic entry in the untreated control was delayed, most likely due to the differences in the timing of mitoses in different batches of embryos, the MG132-treated extract exhibited an even longer delay (Fig. 7B). This demonstrates that the entry into the second embryonic M-phase in vitro is not inhibited by MG132 similarly as the first

Cell Cycle


Cyclin B Dissociation from CDK1 Precedes its Degradation Upon MPF Inactivation in Mitotic Extracts of Xenopus laevis Embryos

Figure 7. MG132 does not block histone H1 kinase activation during the second embryonic M-phase in vitro. A) Histone H1 kinase assays were performed on extracts obtained from two different females and prepared 110 min (A) or 108 min (B) post-activation. The low-speed extracts were incubated at 21˚C for 1 hr with 100 µM MG132 or 0.5% DMSO as a control and sampled every 5 min. In B, the cell-free extract was prepared 2 min earlier than in A. While this difference probably participated in the delay in MPF activation in this extract, it certainly cannot explain a 20 min delay in relation to (Fig. 7A). This is likely due to different timing of the second mitosis in the two batches of embryos (from two different females).

one, and argued rather in favor of our second hypothesis that increased exposure to the presence of MG132 delays slightly histone H1 kinase activation. To better characterize this possibility, we returned to the analysis of the first embryonic M-phase. This time, extracts were prepared earlier (50-58 min post-activation) to extend MG132 incubation before the mitotic entry. We added the drug at 0 and 15 min time points after the beginning of incubation at 21˚C. In control extracts from six independent experiments, the peak of histone H1 kinase activity has never been observed before 35 min time point following the beginning of incubation (average 40 min). MG132 either delayed the peak of histone H1 kinase activity or did not influence the timing of its activation in all experiments (Fig. 8A). Usually, a longer incubation (MG132 added at t = 0) resulted in longer delay and lower amplitudes of histone H1 kinase activity in parallel with delayed pattern of MCM4 phosphorylation/dephosphorylation 1694

Figure 8. MG132 does not block histone H1 kinase activation during the first embryonic M-phase in vitro even upon longer incubation before the beginning of mitotic events. (A), Histone H1 kinase assay of the extract prepared 51 min. post-activation and supplemented either with 0.5 % DMSO, or 100 µM MG132 at t0 or t15min of incubation at 21˚C. (B), Western blot analyses of MCM4 (upper panels) and cyclin B2 (lower panels) were performed on sibling samples. MCM4 Western blot confirms that MG132 provokes delay in mitotic exit and dephosphorylation of this protein in longer incubation (MG132 added at t0). Cyclin B2 degradation is prevented in each extract containing MG132.

(Fig. 8A and upper panel in 8B). The efficiency of inhibition of cyclin B2 proteolysis was, however, comparable in each case (Fig. 8B; lower panel). The delay caused by MG132 was not always strictly correlated with the increased time of incubation. In two out of six experiments, shorter incubation with the drug (MG132 added at t = 15 min) resulted in histone H1 kinase activity peak slightly delayed beyond the longer incubation (starting at t = 0; data not shown). These results confirm that even prolonged presence of MG132 does not prevent mitotic entry in the extract and only slightly delays this process. Levels of cyclins B1, B2 and B4 associated with CDK1 diminish faster than the decline of total pools of these cyclins upon mitotic exit in CHX-treated mitotic extract. Proteasome inhibitors could modify numerous activities in the extract. Therefore, the mechanism

Cell Cycle

2006; Vol. 5 Issue 15

Cyclin B Dissociation from CDK1 Precedes its Degradation Upon MPF Inactivation in Mitotic Extracts of Xenopus laevis Embryos

of MPF inactivation described by Nishiyama et al.3 in meiotic extract and in the current paper in the mitotic ones could differ in intact extracts. To verify whether the dissociation of cyclins B from CDK1 upon MPF inactivation precedes cyclins B degradation in embryo extract we compared the dynamics of diminutions of CDK1-coupled cyclins B1, B2 and B4 relative to the dynamics of the decline of the total levels of the same cyclins individually without proteasome inhibition. CHX was added to the mitotic extracts to eliminate the newly synthesized pool of cyclins potentially not yet associated with CDK1. We reasoned that if our hypothesis is valid we should observe earlier decline of CDK1-associated cyclins B and later of total pools of each cyclin B. CHX was added 18 min following the beginning of extract incubation at 21˚C and MCM4, cyclin B1, B2 and B4 samples were analyzed by Western blotting every three minutes. MCM4 down-shift confirmed the exit from the M-phase (Fig. 9A). Cyclin B1 disappeared the first (Fig. 9A, 38 min time point), followed by B2 and B4 (Fig. 9A, 41 min time point) in the whole extract. Samples from six time points (29-44 min), corresponding to the period when all analyzed cyclins B were degraded, were incubated with p9 beads. The total levels of each cyclin B (Fig. 9B–D; input for cyclins B1, B2 and B4) were compared to the levels of p9-bound cyclins (Figs. 9B–D; p9-bound for cyclins B1, B2 and B4). In parallel, CDK1 levels were followed in both input and p9-bound material by Western blotting (Figs. 9B–D, left, CDK1). As expected, in the case of each of three cyclins B studied we found significant advance of the decline of CDK1-associated pool compared to the total one of the same cyclin (Figs. 9B–D, Western blot and quantification on rightmost curves). Altogether, the present results enable us to postulate that dissociation of cyclins B (at least B1, B2 and B4) from CDK1 and not their degradation per se is responsible for MPF inactivation during mitotic exit in Xenopus embryo cell-free extract.


Figure 9. The decline of cyclins B1, B2 and B4 associated with CDK1 is faster than the decline of the total pool of these cyclins in the cell-free mitotic extract in which protein synthesis was inhibited five minutes before the peak of histone H1 kinase activity by 50 µg/ml CHX (added at 18 min. time point). (A) The low-speed extracts were incubated at 21˚C and MCM4 as well as cyclins B1, B2 and B4 levels were examined by Western blotting every three minutes. (B–D) These CDK-associated cyclins B were precipitated on p9-beads and the levels of CDK-associated cyclins and CDK1 (p9-bound) as well as the total levels of cyclins B (Input) were visualized by Western blotting (left panels) and quantified (curves rightmost in B–D). Each of three cyclins B associated with p9 beads (CDK1-associated) analyzed here declines faster than its respective total pool.

In this study, we have investigated the role of the proteasome proteolytic activity in the regulation of the mitotic activity of MPF as well as the timing of phosphorylation and dephosphorylation of chosen mitotic marker proteins (Eg3 and MCM4;43,53) during the first two embryonic M-phases in vitro. Our results have shown that (1) cyclin B degradation is not necessary for MPF inactivation upon mitotic exit during the first and the second embryonic M-phases in cell-free extracts, as shown before for the meiotic CSF extract by Nishiyama et al.,3 (2) the pool of cyclin B remaining in the extract following mitotic exit in the presence of MG132 is free of CDK1 and mitotically inert, while the extract maintains the capacity to activate MPF when exogenous or newly-synthesized cyclin is present, (3) inhibition of the proteolytic activity of the proteasome does not prevent

mitotic M-phase entry in Xenopus embryo extracts, even if the prolonged presence of MG132 can perturb the correct timing of MPF activation during the two mitoses studied. These results prompted us to investigate whether the dissociation of B-type cyclins from their CDK1 partner precedes their degradation in mitotic cell-free extracts. Accordingly, we observed differences between the dynamics of decline in CDK1-bound cyclins B1, B2 and B4 vs. their total levels in CHX-treated mitotic extracts, which directly confirmed our hypothesis. These results, together with those from Nishiyama and colleagues3 concerning activated CSF extract, enable us to postulate

Cell Cycle


Cyclin B Dissociation from CDK1 Precedes its Degradation Upon MPF Inactivation in Mitotic Extracts of Xenopus laevis Embryos

the sequence of events accompanying MPF inactivation during mitotic exit in Xenopus embryo cell-free extract (Fig. 10). MPF inactivation and cyclin B degradation are separate events during meiotic and mitotic exits. It is well established that cyclin B degradation is a key event of the mitotic exit and enables the cell cycle progression beyond M-phase.1,2,55 However, it does not play a direct role in MPF inactivation neither during meiosis3 nor during mitosis (this paper). Nishiyama and colleagues3 have clearly demonstrated the involvement of a non-proteolytic activity of the proteasome (most probably associated with the lid in 19S proteasome subcomplex) in this process. This explained also why CDK1 is not degraded via ubiquitin-proteasome pathway while cyclin B is polyubiquitinated and targeted to the proteasome while still complexed with CDK1. They have also shown that the pool of cyclin B remaining in the extract after M-phase exit is found in high molecular weight complexes associated with the 26S proteasome, which enabled to postulate that CDK1-free cyclin B remains sequestered inside the proteasomes upon the inhibition of the proteolytic activity of the proteasome. Our results showing that during mitosis in cell-free Xenopus embryo extract under similar conditions (i.e., in presence of MG132 instead of MG115 used by Nishiyama et al.3) cyclin B accessibility to cytosolic enzymes, such as phosphatases and CDK1, is greatly reduced reinforce this hypothesis (this paper). Similar uncoupling of a single subunit of a multisubunit complexes prior proteasome-dependent degradation was reported in the case of ubiquitinated Sic1 (from CDK-cyclin complex56,57) and IκB (from NF-κB58). It was proposed that either ubiquitination acts as “proteinaceous detergent” destabilizing the folded state of proteins,59 or the ATPases resident in the 26S proteasome have an “unfoldase” activity that dissociates ubiquitinated subunits.60 Our demonstration that three B-type cyclins (B1, B2 and B4) dissociate from CDK1 before their degradation upon mitotic exit in extracts in which protein synthesis was inhibited strongly suggests that the mechanism of MPF inactivation through proteasome-dependent separation of B-type cyclins discovered in meiotic extract by Nishiyama and colleagues3 could be universal. Josefsberg et al.31 have shown that in rat zygotes MPF inactivation could proceed without cyclin B degradation in the presence of MG132. This is in agreement with our results in Xenopus embryo extracts described in this paper and demonstrates that the phenomenon of MPF inactivation without cyclin B degradation is not restricted to cell-free extracts. The lack of inhibition of MPF inactivation by MG132, MG115 or ALLN contrasts with the M-phase arrest triggered in Xenopus CaCl2-treated CSF extract and cycling embryo extracts by N-terminal fragment of cyclin B2 addition (B2Nt).61 The way B2Nt stabilizes MPF and arrests mitotic exit seems to be provoked, however, by the saturation of the proteolytic machinery. B2Nt competes for degradation of other substrates and inhibits proteolysis of endogenous cyclin B. The latter remains associated with CDK1 and maintain high MPF activity. Moreover, their continuous synthesis results in a steady increase in histone H1 kinase activity while oscillations are observed in controls where BSA or other proteins were added (see Figs. 3 and 4 in ref. 61). Since B2Nt contains sequences necessary for its ubiquitination it seems that it saturates APC/C, and therefore provoke the arrest of the M-phase upstream from the proteasome. Accordingly, N-terminal cyclin B2 fragment with R36S mutation, which is not recognized by destruction system, does not allow arresting the M-phase upon CSF extract activation.3 One cannot exclude, however, that wild-type B2Nt inhibits cyclinB/CDK1 dissociating activity of proteasome by association with the proteasome lid blocking 1696

Figure 10. Schematic sequence of events concerning CDK1 and cyclins B during mitotic exit in Xenopus laevis embryo cell-free extract.

entry of other substrates into the proteasome cylinder. The difference between the effects of proteasome inhibitors and B2Nt on mitotic extracts suggests that the inhibition of cyclin B degradation at different steps of the ubiquitin-proteasome pathway provokes extremely different results regarding MPF activity. Our observations that cyclins B dissociation from CDK1 precedes their degradation in extracts without MG132 or ALLN suggests strongly that this mechanism is indeed a direct cause of MPF inactivation also upon mitotic exits in Xenopus cell-free extract. The degradation of cyclins B, already present within the proteasome and inaccessible from the cytoplasm, follows (as summarized in Fig. 10). Why somatic cells are arrested in M-phase by proteasome inhibition? In Xenopus, the ability to inactivate MPF without cyclin B degradation is observed in cell-free extracts derived of MII oocytes3 and early embryos (this paper). This contrasts with Xenopus XL2 somatic cells obtained from later embryonic stages, which are arrested in M-phase upon ALLN treatment.28 This could argue for an evolution of mechanisms inactivating MPF during later Xenopus development. However, such a switch could have quantitative rather than qualitative character. Oocytes and embryos, contrarily to somatic cells, contain much higher ratio of proteasomes per unit of cytoplasm due to accumulation during oogenesis, which could increase the efficiency of dissociation of cyclin B from CDK1. We diminished the quantity of proteasome in premitotic extracts by immunodepletion (using a polyclonal antibody against 20S proteasome kindly provided by Toshinobu Tokumoto).62 The most efficient depletion in our hands—up to 64% (as visualized by Western blotting using two different monoclonal antibodies directed against proteasome subunits α2 and α4;1-4D5 and GC3β)62 did not change the dynamics of histone H1 kinase inactivation upon mitotic exit (our unpublished results). Thus, the quantity of proteasome in cytoplasm of one-cell embryo indeed exceeds the quantity necessary to promote efficient MPF inactivation. Supposedly lower concentration of proteasomes in the cytoplasm of somatic cells might favor saturation of proteasome with other than cyclins B substrates upon the presence of a proteasome inhibitor. This could facilitate in turn, the retention of active CDK1/cyclin B complexes in the cytoplasm of these cells. Alternatively, in somatic cells other substrates than in oocytes or early embryos could be degraded by the proteasome before the initiation of polyubiquitination of cyclins B associated with CDK1. Such differences include a few proteins described so far. For instance, Aurora A (Eg2) oscillates during the cell cycle in XL2 and HeLa cells with a peak in M-phase.63,64 It is also degraded in cell-free extract of

Cell Cycle

2006; Vol. 5 Issue 15

Cyclin B Dissociation from CDK1 Precedes its Degradation Upon MPF Inactivation in Mitotic Extracts of Xenopus laevis Embryos

G1 synchronized XL2 cells65 and upon MII oocytes activation.66 However, its level remains constant in maturing oocytes during MI/MII meiotic transition66 as well as in mitotic extract of one-cell Xenopus embryos (our unpublished data). In addition, ALLN inhibits degradation of this protein in Xenopus XL2 cells and their cell-free extract.63,65 However, Aurora A is not a good candidate for a trigger of MPF inactivation since its proteolysis occurs late in mitosis. In contrast, Nek2A from NIMA kinases family is an early substrate of proteasome (for a review see ref. 67) and it seems a better candidate for this role. Nek2A is present in somatic cells and is absent in oocytes and early Xenopus embryos (ref. 68 and our unpublished results). Its downregulation in HeLa cells results in prolongation of mitosis, which could suggest a potential role of this protein as an accelerator of MPF inactivation during mitosis in these cells.69 On the other hand, in HeLa cells overexpressing GFP-Nek2A anaphase is delayed,70 which shows that Nek2A is a more complex player of mitotic regulation. However, in calcium-activated CSF extract of Xenopus, where endogenous Nek2A is absent, non-degradable mutant of this protein prevents cyclins degradation.70 The latter strongly suggests that Nek2A proteolysis could indeed be a signal for mitotic cyclins degradation. Finally, inhibition of degradation of mitotic forms of Erp/Emi proteins (known to be involved in meiotic CSF arrest; for a review see e.g., ref. 71) could also arrest somatic cells in the M-phase upon proteasome inhibition upstream from both Nek2A and cyclin B proteolysis. Recently, Giménez-Abián et al.72 provided evidence that in mammalian cells anaphase could occur in the presence of non-degraded cyclin B and securin. More interestingly, they showed that some non-identified substrates of proteasome which regulate mitotic progression are degraded in mammalian cells in APC/C-independent manner.73 Inhibition of proteolysis of these proteins, which could include Nek2A and/or Erp/Emi-like proteins, seems indeed to arrest cells upstream from cyclin B polyubiquitination. However, since their identity remains unknown, the question whether different mechanisms of MPF inactivation operate during development remains still open. Mitotically inert and active cyclin B pools. Nishiyama and coworkers3 have demonstrated by gel filtration that the cyclin B pool remaining in activated CSF extract in the presence of MG115 is in high molecular weight complexes and by coimmunoprecipitation that it is associated with the 26S proteasome. They postulated that CDK1-free cyclin B remains sequestered within the proteasomes. Our experiments showing that this pool of cyclin B remains inaccessible both for endogenous phosphatases and exogenous phage λ phosphatase and that it is incapable to accelerate the next M-phase entry provides the first evidence for functional masking of cyclin B within such a cytoplasm. This further strengthens the hypothesis that this pool of cyclin B is sequestered within the proteasomes following meiotic3 as well as mitotic (this paper) MPF inactivation in the presence of the proteasome inhibitors. The behavior of cycling extracts in which MPF is activated (and MCM4 phosphorylated) for the second time in the presence of MG132 (Fig. 5) suggests that newly synthesized pool of cyclin B is mitotically active because remaining free in cytoplasm. Similarly, non-degradable ∆90 cyclin B added to the extract following mitotic exit in the presence of MG132 activates MPF, too (Fig. 6). Since the window of polyubiquitination is restricted to the M-phase2 when APC/C-Cdc20 becomes phosphorylated and active74,75 it is not surprising that following MPF inactivation newly synthesized or exogenous cyclin B remains free in the cytoplasm and avoid sequestration within the proteasomes. Thus, two different pools of cyclin B

(free and sequestered within the proteasome) are present in the extract following the M-phase exit in the presence of the drug. Proteasome inhibition and the mitotic entry. We show here that the extracts enter the first or the second mitotic M-phase in the presence of MG132 (and ALLN for the first M-phase). It demonstrates that the mitotic entry in the extract does not require proteolytic activity of the proteasome. However, upon prolonged incubation with MG132 the peak of histone H1 kinase is slightly delayed. This could suggest that proteasome inhibition interferes either with some processes determining the timing of the mitotic entry (but not with the machinery of MPF activation itself ), or that the drug has some unspecific effects. These results are in contrast to the data obtained by Kawahara et al.76 showing that proteasome inhibitors prevent mitotic entry in sea urchin embryos. On the other hand, interference with polyubiquitination, a process upstream from proteasome-dependent proteins degradation, in Xenopus embryo extract delays MPF activation77 similarly as MG132 in our study. This supports our hypothesis that the ubiquitin-proteasome pathway is involved rather in the control of the timing of MPF activation than in its activation itself. Species-specific differences in proteasome machinery regulating mitotic entry cannot be, however, excluded. References 1. Murray AW, Solomon MJ, Kirschner MW. The role of cyclin synthesis and degradation in the control of maturation promoting factor activity. Nature 1989; 339:280-6. 2. Glotzer M, Murray AW, Kirschner MW. Cyclin is degraded by the ubiquitin pathway. Nature 1991; 349:132-8. 3. Nishiyama A, Tachibana K, Igarashi Y, Yasuda H, Tanahashi N, Tanaka K, Ohsumi K, Kishimoto T. A non-proteolytic function of the proteasome is required for the dissociation of Cdc2 and cyclin B at the end of M phase. Genes Dev 2000; 14:2344-57. 4. Peters JM. The anaphase-promoting complex: Proteolysis in mitosis and beyond. Mol Cell 2002; 9:931-43. 5. Minshull J, Golsteyn R, Hill CS, Hunt T. The A- and B-type cyclin associated cdc2 kinases in Xenopus turn on and off at different times in the cell cycle. EMBO J 1990; 9:2865-75. 6. Whitfield WG, Gonzalez C, Maldonado-Codina G, Glover DM. The A- and B-type cyclins of Drosophila are accumulated and destroyed in temporally distinct events that define separable phases of the G2-M transition. EMBO J 1990; 9:2563-72. 7. Hochegger H, Klotzbucher A, Kirk J, Howell M, le Guellec K, Fletcher K, Duncan T, Sohail M, Hunt T. New B-type cyclin synthesis is required between meiosis I and II during Xenopus oocyte maturation. Development 2001; 128:3795-807. 8. Castro A, Bernis C, Vigneron S, Labbé JC, Lorca T. The anaphase-promoting complex: A key factor in the regulation of cell cycle. Oncogene 2005; 24:314-25. 9. Lorca T, Devault A, Colas P, Van Loon A, Fesquet D, Lazaro JB, Doreé M. Cyclin A-Cys41 does not undergo cell cycle-dependent degradation in Xenopus extracts. FEBS Lett 1992; 306:90-3. 10. Pfleger CM, Kirschner MW. The KEN box: An APC recognition signal distinct from the D box targeted by Cdh1. Genes Dev 2000; 14:655-65. 11. Castro A, Vigneron S, Bernis C, Labbe JC, Lorca T. Xkid is degraded in a D-box, KEN-box, and A-box-independent pathway. Mol Cell Biol 2003; 23:4126-38. 12. Castro A, Vigneron S, Bernis C, Labbe JC, Prigent C, Lorca T. The D-Box-activating domain (DAD) is a new proteolysis signal that stimulates the silent D-Box sequence of Aurora-A. EMBO Rep 2002; 3:1209-14. 13. Raff JW, Jeffers K, Huang JY. The roles of Fzy/Cdc20 and Fzr/Cdh1 in regulating the destruction of cyclin B in space and time. J Cell Biol 2002; 157:1139-49. 14. Kobayashi H, Minshull J, Ford C, Golsteyn R, Poon R, Hunt T. On the synthesis and destruction of A- and B-type cyclins during oogenesis and meiotic maturation in Xenopus laevis. J Cell Biol 1999; 114:755-65. 15. De Smedt V, Poulhe R, Cayla X, Dessauge F, Karaiskou A, Jessus C, Ozon R. Thr161 phosphorylation of monomeric Cdc2: Regulation by protein phosphatase 2C in Xenopus oocyte. J Biol Chem 2002; 277:28592-600. 16. Verde F, Dogterom M, Stelzer E, Karsenti E, Leibler S. Control of microtubule dynamics and length by cyclin A- and cyclin B-dependent kinases in Xenopus egg extracts. J Cell Biol 1992; 118:1097-108. 17. Buendia B, Draetta G, Karsenti E. Regulation of the microtubule nucleating activity of centrosomes in Xenopus egg extracts: Role of cyclin A-associated protein kinase. J Cell Biol 1992; 116:1431-42. 18. Meijer L, Arion D, Golsteyn R, Pines J, Brizuela L, Hunt T, Beach D. Cyclin is a component of the sea urchin egg M-phase specific histone H1 kinase. EMBO J 1989; 8:2275–82. 19. Gautier J, Maller JL. Cyclin B in Xenopus oocytes: Implications for the mechanism of pre-MPF activation. EMBO J 1991; 10:177–82. 20. Izumi T, Maller JL. Phosphorylation of Xenopus cyclins B1 and B2 is not required for cell cycle transitions. Mol Cell Biol 1991; 11:3860-7.

Cell Cycle


Cyclin B Dissociation from CDK1 Precedes its Degradation Upon MPF Inactivation in Mitotic Extracts of Xenopus laevis Embryos 21. Li J, Meyer AN, Donoghue DJ. Requirement for phosphorylation of cyclin B1 for Xenopus oocyte maturation. Mol Biol Cell 1995; 6:1111–24. 22. Peter M, Le Peuch C, Labbé JC, Meyer AN, Donoghue DJ, Dorée M. Initial activation of cyclin-B1-cdc2 kinase requires phosphorylation of cyclin B1. EMBO Rep 2002; 3:551-6. 23. Roy LM, Singh B, Gautier J, Arlinghaus RB, Nordeen SK, Maller JL. The cyclin B2 component of MPF is a substrate for the c-mosxe proto-oncogene product. Cell 1990; 5:825-31. 24. Toyoshima-Morimoto F, Taniguchi E, Shinya N, Iwamatsu A, Nishida E. Polo-like kinase 1 phosphorylates cyclin B1 and targets it to the nucleus during prophase. Nature 2001; 410:215–20. 25. Walsh S, Margolis SS, Kornbluth S. Phosphorylation of the Cyclin B1 cytoplasmic retention sequence by mitogen-activated protein kinase and Plx. Molec. Cancer Res 2003; 1:280-9. 26. Sherwood SW, Kung AL, Roitelman J, Simoni RD, Schimke RT. In vivo inhibition of cyclin B degradation and induction of cell-cycle arrest in mammalian cells by the neutral cysteine protease inhibitor N-acetylleucylleucylnorleucinal. Proc Natl Acad Sci USA 1993; 90:3353-7. 27. Genschik P, Criqui MC, Parmentier Y, Derevier A, Fleck J. Cell cycle -dependent proteolysis in plants. Identification of the destruction box pathway and metaphase arrest produced by the proteasome inhibitor MG132. Plant Cell 1998; 10:2063-76. 28. Uzbekov R, Chartrain I, Philippe M, Arlot-Bonnemains Y. Cell cycle analysis and synchronization of the Xenopus cell line XL2. Exp Cell Res 1998; 242:60-8. 29. Kim OH, Lim JH, Woo KJ, Kim YH, Jin IN, Han ST, Park JW, Kwon TK. Influence of p53 and p21Waf1 expression on G2/M phase arrest of colorectal carcinoma HCT116 cells to proteasome inhibitors. Int J Oncol 2004; 24:935-41. 30. Terret ME, Wassmann K, Waizenegger I, Maro B, Peters JM, Verlhac MH. The meiosis I-to-meiosis II transition in mouse oocytes requires separase activity. Curr Biol 2003; 13:1797-802. 31. Josefsberg LB, Kaufman O, Galiani D, Kovo M, Dekel N. Inactivation of M-phase promoting factor at exit from first embryonic mitosis in the rat is independent of cyclin B1 degradation. Biol Reprod 2001; 64:871-8. 32. Michel LS, Liberal V, Chatterjee A, Kirchwegger R, Pasche B, Gerald W, Dobles M, Sorger PK, Murty VV, Benezra R. MAD2 haplo-insufficiency causes premature anaphase and chromosome instability in mammalian cells. Nature 2001; 409:355-9. 33. Michel L, Diaz-Rodriguez E, Narayan G, Hernando E, Murty VV, Benezra R. Complete loss of the tumor suppressor MAD2 causes premature cyclin B degradation and mitotic failure in human somatic cells. Proc Natl Acad Sci USA 2004; 101:4459-64. 34. Josefsberg LB, Galiani D, Dantes A, Amsterdam A, Dekel N. The Proteasome is involved in the first metaphase-to-anaphase transition of meiosis in Rat Oocytes. Biol Reprod 2000; 62:1270-7. 35. Herbert M, Levasseur M, Homer H, Yallop K, Murdoch A, McDougall A. Homologue disjunction in mouse oocytes requires proteolysis of securin and cyclin B1. Nat Cell Biol 2003; 5:1023-5. 36. Peter M, Castro A, Lorca T, Le Peuch C, Magnaghi-Jaulin L, Doreé M, Labbé JC. The APC is dispensable for first meiotic anaphase in Xenopus oocytes. Nat Cell Biol 2001; 3:83-7. 37. Tsurumi C, Hoffmann S, Geley S, Graeser R, Polanski Z. The spindle assembly checkpoint is not essential for CSF arrest of mouse oocytes. J Cell Biol 2004; 167:1037-50. 38. Sagata N, Watanabe N, Vande Woude GF, Ikawa Y. The c-mos proto-oncogene product is a cytostatic factor responsible for meiotic arrest in vertebrate eggs. Nature 1989; 342:512-8. 39. Kubiak JZ, Weber M, de Pennart H, Winston NJ, Maro B. The metaphase II arrest in mouse oocytes is controlled through microtubule-dependent destruction of cyclin B in the presence of CSF. EMBO J 1993; 12:3773-8. 40. Verlhac MH, Kubiak JZ, Weber M, Géraud G, Colledge WH, Evans MJ, Maro B. Mos is required for MAP kinase activation and is involved in microtubule organization during meiotic maturation in the mouse. Development 1996; 122:815-22. 41. Kalab P, Kubiak JZ, Verlhac MH, Colledge WH, Maro B. Activation of p90rsk during meiotic maturation and first mitosis in mouse oocytes and eggs: MAP kinase-independent and -dependent activation. Development 1996; 122:1957-64. 42. Murray AW, Kirschner MW. Cyclin synthesis drives the early embryonic cell cycle. Nature 1989; 339:275-80. 43. Chesnel F, Vignaux F, Richard-Parpaillon L, Huguet A, Kubiak JZ. Differences in regulation of the first two M-phases in Xenopus laevis embryo cell-free extracts. Dev Biol 2005; 285:358-75. 44. Walter SA, Guadagno SN, Ferrell Jr JE. Activation of wee1 by p42 MAPK in vitro and in cycling Xenopus egg extracts. Mol Biol Cell 2000; 11:887-96. 45. Haccard O, Sarcevic B, Lewellyn A, Hartley R, Roy L, Izumi T, Erikson E, Maller JL. Induction of metaphase arrest in cleaving Xenopus embryos by MAP kinase. Science 1993; 262:1262-5. 46. Chau AS, Shibuya EK. Mos-induced p42 mitogen-activated protein kinase activation stabilizes M-phase in Xenopus egg extracts after cyclin destruction. Biol Cell 1998; 90:565-72. 47. Chau AS, Shibuya EK. Inactivation of p42 mitogen-activated protein kinase is required for exit from M-phase after cyclin destruction. J Biol Chem 1999; 45:32085-90. 48. Swanson CA, Arkin AP, Ross J. An endogenous calcium oscillator may control early embryonic division. Proc Natl Acad Sci USA 1997; 94:1194-9. 49. Murray AW. Cell cycle extracts. Methods Cell Biol 1991; 36:581-605. 50. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680-5. 51. Chesnel F, Bonnec G, Tardivel A, Boujard D. Comparative effects of insulin on the activation of the Raf/Mos-dependent MAP kinase cascade in vitellogenic versus postvitellogenic Xenopus oocytes. Dev Biol 1997; 188:122-33.


52. Lee DH, Goldberg AL. Proteasome inhibitors: Valuable new tools for cell biologists. Trends Cell Biol 1998; 8:397-403. 53. Chesnel F, Gautier I, Richard-Parpaillon L, Kubiak JZ. Each mitosis can be different: How the cell cycle machinery modulates early embryonic M-phases. In: Tokumoto T, ed. New impact on Proteins Modifications in the Regulation of Reproductive Systems. Trivandrum: Research Signpost, 2005:155-67. 54. Vogel L, Baratte B, Detivaud L, Azzi L, Leopold P, Meijer L. Molecular cloning and characterization of p15(CDK-BP), a novel CDK-binding protein. Biochim Biophys Acta 2002; 1589:219-31. 55. Evans T, Rosenthal ET, Youngblom J, Distel D, Hunt T. Cyclin: A protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division. Cell 1983; 33:389-96. 56. Schwob E, Böhm T, Mendenhall M, Nasmyth K. The B-type cyclin kinase inhibitor p40SIC1 controls the G1/S transition in S. cerevisiae. Cell 1994; 79:233-44. 57. Verma R, Annan RS, Huddleston MJ, Carr SA, Reynard G, Deshaies RJ. Phosphorylation of Sic1p by G1 Cdk required for its degradation and entry into S phase. Science 1997; 278:455-60. 58. Maniatis T. A ubiquitin ligase complex essential for the NF-kappaB, Wnt/Wingless, and Hedgehog signaling pathways. Genes Dev 1999; 13:505-10. 59. Johnson ES, Ma PC, Ota IM, Varshavsky A. A proteolytic pathway that recognizes ubiquitin as a degradation signal. J Biol Chem 1995; 270:17442-56. 60. Larsen CN, Finley D. Protein translocation channels in the proteasome and other proteases. Cell 1997; 91:431-4. 61. van der Velden HM, Lohka MJ. Mitotic arrest caused by the amino terminus of Xenopus cyclin B2. Mol Cell Biol 1993; 13:1480-8. 62. Tokumoto T, Tokumoto M, Seto K, Horiguchi R, Nagahama Y, Yamada S, Ishikawa K, Lohka MJ. Disappearance of a novel protein component of the 26S proteasome during Xenopus oocyte maturation. Exp Cell Res 1999; 247:313-9. 63. Klotzbucher A, Pascreau G, Prigent C, Arlot-Bonnemains Y. A method for analyzing the ubiquitination and degradation of aurora-A. Biol Proced Online 2002; 4:62-9. 64. Kimura M, Kotani S, Hattori T, Sumi N, Yoshioka T, Todokoro K, Okano Y. Cell cycledependent expression and spindle pole localization of a novel human protein kinase, Aik, related to Aurora of Drosophila and yeast Ipl1. J Biol Chem 1997; 272:13766-71. 65. Arlot-Bonnemains Y, Klotzbucher A, Giet R, Uzbekov R, Bihan R, Prigent C. Identification of a functional destruction box in the Xenopus laevis aurora-A kinase pEg2. FEBS Lett 2001; 508:149-52. 66. Frank-Vaillant M, Haccard O, Thibier C, Ozon R, Arlot-Bonnemains Y, Prigent C, Jessus C. Progesterone regulates the accumulation and the activation of Eg2 kinase in Xenopus oocytes. J Cell Sci 2000; 113:1127-38. 67. Hayward DG, Fry AM. Nek2 kinase in chromosome instability and cancer. Cancer Lett 2005:1-12. 68. Hames RS, Wattam SL, Yamano H, Bacchieri R, Fry AM. APC/C-mediated destruction of the centrosomal kinase Nek2A occurs in early mitosis and depends upon a cyclin A-type D-box. The EMBO J 2001; 20:7117–27. 69. Fletcher L, Cerniglia GJ, Yen TJ, Muschel RJ. Live cell imaging reveals distinct roles in cell cycle regulation for Nek2A and Nek2B. Biochim Biophys Acta 2005; 1744:289-92. 70. Hayes MJ, Kimata Y, Wattam SL, Lindon C, Mao G, Yamano H, Fry AM. Early mitotic degradation of Nek2A depends on Cdc20-independent interaction with the APC/C. Nat Cell Biol 2006, (in press). 71. Schmidt A, Rauh NR, Nigg EA, Mayer TU. Cytostatic factor: An activity that puts the cell cycle on hold. J Cell Sci 2006; 119:1213-8. 72. Giménez-Abián JF, Díaz-Martínez LA, Wirth KG, Andrews CA, Giménez-Martín G, Clarke DJ. Regulated separation of sister centromeres depends on the spindle assembly checkpoint but not on the anaphase promoting complex/cyclosome. Cell Cycle 2005a; 4:1561-75. 73. Giménez-Abián JF, Díaz-Martínez LA, Wirth KG, De la Torre C, Clarke DJ. Proteasome activity is required for centromere separation independently of securin degradation in human cells. Cell Cyle 2005; 4:1558-60. 74. Lorca T, Castro A, Martinez AM, Vigneron S, Morin N, Sigrist S, Lehner C, Dorée M, Labbé JC. Fizzy is required for activation of the APC/cyclosome in Xenopus egg extracts. EMBO J 1998; 17:3565-75. 75. Kramer ER, Scheuringer N, Podtelejnikov AV, Mann M, Peters JM. Mitotic regulation of the APC activator proteins CDC20 and CDH1. Mol Biol Cell 2000; 11:1555-69. 76. Kawahara H, Philipova R, Yokosawa H, Patel R, Tanaka K, Whitaker M. Inhibiting proteasome activity causes overreplication of DNA and blocks entry into mitosis in sea urchin embryos. J Cell Sci 2000; 113:2659-70. 77. Mahaffey DT, Gorbea C, Rechsteiner M. Evidence that DNA replication is not regulated by ubiquitin-dependent proteolysis in Xenopus egg extract. Exp Cell Res 2003; 288:225-34.

Cell Cycle

2006; Vol. 5 Issue 15

Lihat lebih banyak...


Copyright © 2017 DADOSPDF Inc.