Cyclin B Dissociation from CDK1 Precedes its Degradation Upon MPF Inactivation in Mitotic Extracts...

©2006 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.

www.landesbioscience.com Cell Cycle 1687

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

Franck ChesnelFranck BazileAude Pascal Jacek Z. Kubiak *

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

*Correspondence to: Jacek Z. Kubiak; CNRS-UMR 6061; Biology and Genetics ofDevelopment; 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.: +33.0.2.23.23.46.98; Fax: +33.0.2.23.23.44.78; Email: [email protected]

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

Previously published online as a Cell Cycle E-publication:http://www.landesbioscience.com/journals/cc/abstract.php?id=3123

KEY WORDS

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

ACKNOWLEDGEMENTS

We thank Marcel Méchali, Tim Hunt, JohnGannon, Jean-Pierre Tassan, Daniel Fisher,and Thierry Lorca for generous gifts of anti-bodies, Marie-Anne Felix for ∆90 cyclin B andLénaïck Detivaud and Laurent Meijer for p9beads. We thank Yannick Arlot-Bonnemains,Laurent Richard-Parpaillon, Daniel Fisher,Olivier Haccard and Jacek Gaertig for discus-sions and critical reading of the manuscript.This work was supported by grant 4298 fromARC and from Ligue Contre le Cancer(Comité d'Ille-et-Vilaine) to J.Z.K.

Report

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

ABSTRACTCyclin 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 meioticmetaphase II-arrest in Xenopus cell-free extract revealed that the proteasome-dependentdissociation of cyclin B from CDK1 is sufficient to inactivate MPF without cyclin B degra-dation. In this study we analyze whether the same mechanism operates during the exitfrom mitotic M-phase. We show in Xenopus cell-free extract undergoing the first or thesecond embryonic mitosis that CDK1 oscillations are not affected by proteasome inhi-bition with MG132 or ALLN despite effective inhibition of cyclins B degradation. Themajority of cyclins B1 and B2 surviving CDK1 inactivation is CDK-free and cyclin B2becomes resistant to phosphatase λ dephosphorylation. The pool of cyclins B remainingafter CDK1 inactivation in the presence of MG132 is mitotically inert, while exogenousor newly synthesized cyclin B activates CDK1. This suggests that cyclins B remainsequestered 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-boundcyclins B diminish clearly faster. Our results thus show that cyclin B dissociation fromCDK1 precedes cyclins B degradation upon CDK1 inactivation in mitotic embryo extractsand that proteasome proteolytic activity is dispensable for both activation and inactivationof CDK1 in such extracts.

INTRODUCTIONM-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 inactivationwas considered to depend rigorously on cyclin B degradation via ubiquitin/proteasomepathway.1,2 However, Nishiyama and colleagues3 have shown that MPF inactivation isuncoupled from cyclin B degradation. The proteolysis of mitotic cyclins is coordinated bythe E3 ligase complex or Anaphase Promoting Complex/Cyclosome (APC/C; for a reviewsee ref. 4). It polyubiquitinates substrates for degradation, thereby targeting them to 26Sproteasome. Sequential recognition of mitotic substrates determines their sequentialdegradation. Cyclin A is degraded first shortly after M-phase entry and B-type cyclins followduring later M-phase stages.5,6 In calcium-activated Xenopus laevis CSF extract all cyclinsB are degraded within ten minutes; cyclin B1 first, then cyclins B4, B2 and B5 whereascyclin B3 protein is not expressed in oocytes and early embryos.7 Sequential degradationof 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 bytwo 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 recognitiondepends on degradation signals within the amino-acid sequence of the protein to bedegraded (e.g., D-box in A- and B-type cyclins2,9 KEN-box in Cdc2010 or GxEN-box inXkid;11 double degradation motifs present in Aurora A: a D-box and a “D-box ActivatingDomain” or A-box12). Cyclins degradation starts via APC/C-Cdc20-dependent polyubiq-uitination and propagates via APC/C-Cdh1-dependent mechanism13 (for a review seeref. 8).

B-type cyclins in Xenopus laevis oocytes and early embryos are associated with CDK1and not with CDK2. B1 cyclin is very similar to B4 and the two of them are degraded thefastest. B2 cyclin resembles B5 and their degradation occurs the latest.7 B1, B2 and B4

cyclins are the most abundant during oocyte maturation. Theirquantities rise following fertilization, while B5 decreases.7 Inparthenogenetic embryos 60 min post-activation (at 22˚C, thusbefore the first mitosis) cyclin B1 is in roughly equal mixture withcyclin B2.14 In fully-grown stage VI Xenopus oocyte, there are10-100 times more molecules of CDK1 than cyclin B (the majorityof it is cyclin B2;14 and a pool of free CDK1 is present.15 Taking intoaccount a strong affinity of CDK1 to cyclin B, no free cyclin Bshould be present in prophase oocytes. During the first embryoniccell cycle the levels of cyclin B1, B2 and B4 are similar as in prophaseoocyte.7,14 The quantity of cyclin B5 was not precisely measured atthat time, however, judging by Western blot analysis it is present inclearly lower levels than B1, B2 and B4 cyclins respectively.7

Therefore, during the first mitosis the levels of all cyclins B increasenot more than three and half times in comparison to prophaseoocyte. Since cyclin A is degraded early in mitosis and the levels ofCDK1 does not change during this period, it means that the levelsof CDK1 are at least three times higher than the level of all B-typecyclins.7,14 Therefore, all four cyclins B should be associated withCDK1 at that time.

Association of CDK1 with a specific cyclin most likely determinesthe enzymatic properties of the complex, as shown for cyclin A- orcyclin B-CDK1 in relation to the regulation of microtubule dynamicsand nucleation.16,17 Probably, more subtle differences concernCDK1 associated with different B-type cyclins. Phosphorylation ofcyclins B on multiple N-terminal residues is associated with MPFactivation.18,19,20,21,22 Cyclins B are phosphorylated by CDK1itself.20 They can be, however, phosphorylated also by Mos,23 Plk124

or MAP kinase.25 Differential phosphorylation of a cyclin componentcould 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 eukaryoticsomatic cells in the mitotic M-phase.26,27,28,29 It blocks also themeiotic MI/MII transition in mouse oocytes.30 This M-phase arrestwas thought to be due to the inhibition of cyclins B degradationwhich stabilizes high MPF activity. However, exceptions to this rulewere also described. The first embryonic mitosis of the rat embryo isarrested by MG132 with high levels of non-degraded cyclin B andrelatively low MPF activity.31 Similarly, human colon carcinomaHct-116 cells with induced MAD2 haplo-insufficiency escape cyclinB 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 isseparated from cyclin B degradation upon activation of CSF extractof Xenopus laevis in the presence of proteasome proteolytic activityinhibitor MG115 and that proteasomes possess non-proteolyticactivity dissociating CDK1 from cyclin B. During the exit frommeiotic metaphase II (MII) in CSF extract, inactivation of MPFrequires only the physical separation of cyclin B and CDK1 and notcyclin B degradation. However, as mentioned above, cyclin B degra-dation appears necessary for the transition between two meioticM-phases in mammalian oocytes.30,34,35 Despite that the role of theproteasome in this meiotic transition was not questioned in Xenopuslaevis oocytes, it was postulated that APC/C is dispensable for thisprocess, suggesting modified targeting of cyclin B to the proteasomesduring meiotic M-phase,36 while in mouse oocytes it is certainlyinvolved.30,35,37 Thus, different mechanisms could be involved inMPF inactivation during somatic or embryonic mitoses, as well asupon the first or second meiotic division in different species.

In oocytes, numerous meiosis-specific proteins are abundant oractivated to high degree (e.g., Mos, MAP kinases ERK, Rsk proteins)and stabilize cyclins B both in Xenopus as well as in mouseoocytes.38,39,40,41 In contrast to meiosis, the early mitotic cell cyclesare controlled by short and regular MPF oscillations guided by cyclesof cyclins B synthesis and degradation.42 ERK2 MAP kinase, themajor meiotic player in cyclin B stabilization, is also activated duringearly embryonic mitoses in Xenopus, however relatively late and tomuch lower levels than during meiosis.43 Such a natural activationof ERK2 during mitosis has no effect on MPF stability (see Fig. 7 inref. 44). Experimental activation of ERK2 to high levels duringmitosis, however, stabilizes cyclin B and MPF activity arrestingembryos and cell-free extracts in M-phase.45,46,47 In addition, theexit from the M II-arrest depends on external signal, while theembryonic cycles are driven by internal calcium-dependent clock.48

Thus, the mechanisms regulating cyclin B stability and MPF inacti-vation differ substantially between the meiotic and mitotic M-phases.They could influence proteasome-dependent dissociation of CDK1and cyclin B during MPF inactivation. For these reasons we examinedeffects of proteasome inhibitors on progression of mitotic M-phasesin cell-free embryo extract.

MATERIAL AND METHODSFrogs. 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 subcutaneouslyinjected with human chorionic gonadotropin (500-600 IU perfemale; Organon, Puteaux, France) and kept overnight at 21˚C in110 mM NaCl. Unfertilized eggs collected from “overnight lay” weredejellied 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 pH7.6), washed in XB, treated for 1.5 minute with 0.5 µg/ml calciumionophore A23187 and then extensively washed in XB. Activatedeggs were then incubated in XB at 21˚C.

Cell-free extracts. Cytoplasmic cell-free cycling extracts fromcalcium ionophore-activated embryos were prepared as previouslydescribed by Murray49 with slight modifications. Briefly, embryoswere cultured at 21˚C in XB for 60 minutes post-activation. Theywere transferred into appropriate tubes (5 mL ultra-clear™ centrifugetubes; Beckman Coulter, Roissy, France) containing 0.5 mL XB with0.1 mM AEBSF, aprotinin, leupeptin, pepstatin, chymostatin (10 µg/mLeach) and 25 µg/mL cytochalasin D and packed through a short spinat 700 rpm. After removal of any excess XB medium, embryos weresubjected to two consecutive centrifugations: a crushing spin,10,000 g for ten minutes at 4˚C and a clarification spin of the super-natant 10,000 g for ten minutes at 4˚C in which cytochalasin D,AEBSF, aprotinin, leupeptin, pepstatin and chymostatin were againadded. The resulting low-speed supernatants were then reincubatedat 21˚C for 60 to 120 minutes and every five minutes, 2-µl aliquotswere taken out and either frozen in liquid nitrogen and stored at-70˚C (for subsequent H1 kinase activity assays) or mixed withLaemmli sample buffer, heated at 85˚C for five minutes and storedat -20˚C (for Western blot analyses).50

Electrophoresis, antibodies and Western blotting. Extracts weresubjected to electrophoresis on 8 to 12.5% SDS-PAGE gels.50

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

1688 Cell Cycle 2006; Vol. 5 Issue 15


Separated proteins were transferred to nitrocellulose membranes(Hybond C, Amersham Biosciences) according to standard proce-dures and probed either with antibodies against cyclin B1 (gift fromTim Hunt), cyclin B2 (gift from Thierry Lorca), cyclin B4 (gift fromJohn Gannon), MCM4 (gift from Marcel Méchali) or Eg3 (giftfrom Jean-Pierre Tassan). Antigen-antibody complexes were revealedusing alkaline phosphatase conjugated anti-rabbit or anti-mousesecondary antibody (diluted 1:20,000) in combination with EnhancedChemifluorescence reagent (ECF; Amersham Biosciences). Signalquantification was performed using ImageQuant 5.2 software(Amersham Biosciences).

In vitro assay for histone H1 kinase activity. MPF activity inembryos or in cell-free extracts was measured as previously described,51

with minor modifications: extracts (1 µl) were diluted in 25 µl MPFbuffer (80 mM β-glycerophosphate, 50 mM sodium fluoride, 20 mMEGTA, 15 mM MgCl2, 1 mM DTT, 20 mM HEPES, pH 7.4)supplemented with 0.5 mM sodium orthovanadate and 5 µg/µl ofleupeptin, aprotinin, pepstatin and chymostatin and containing0.4 mg/ml H1 histone (type III-S), 1 µCi [γ32P] ATP (specific activity:3000 Ci/mmol; Amersham Biosciences) and 0.8 mM ATP. Afterincubation for 30 minutes at 30˚C, phosphorylation reactions werestopped by adding Laemmli sample buffer and heating for fiveminutes at 85˚C. Histone H1 was separated by SDS-PAGE andincorporated radioactivity was measured by autoradiography of the gelusing a STORM phosphorimager (Amersham Biosciences) followedby a data analysis with ImageQuant 5.2 software.

Sepharose p9 beads precipitation. The p9 sepharose beads usedfor affinity precipitation were kindly provided by Lenaïck Detivaudand Laurent Meijer. Ten µl of extracts were added to 10 µl p9 beadspreequilibrated with homogeneizing buffer (MOPS pH 7.2, 60 mMβ-glycerophosphate, 15 mM EGTA, 15 mM MgCl2, 2 mM dithio-threitol, 1 mM sodium fluoride, 1 mM sodium orthovanadate and1 mM disodium phenyl phosphate) extemporaneously supplementedwith 1% (w/v) BSA, 1mM AEBSF and aprotinin, leupeptin, pep-statin, chymostatin (10 µg/ml each). The mixtures were agitated for2.5 h at 4˚C. After a brief centrifugation (5,000 g, 1 min, 4˚C), thesupernatant was collected to be analyzed by Western blotting whilethe pelleted p9 beads were washed four times with 1 ml of washingbuffer (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, chymo-statin (10 µg/ml each). The beads were then resuspended in 12 µl of2× Laemmli sample buffer and heated at 85˚C for 5 min. Samples(whole extracts, p9 supernatant, p9 eluate) were then subjected to12% SDS-PAGE, and cyclins B and CDK1 were detected byWestern blotting.

Phage λ protein phosphatase treatment. Cell-free extract wassampled at appropriate time points and 1 µl was treated with 200units of λ protein phosphatase (New England Biolabs) in a 15-µlreaction mixture consisting of phosphatase buffer (50 mM HEPESpH 7.5, 0.1 mM Na2EDTA, 5 mM dithiothreitol and 0.01%Brij35) supplemented with 2 mM MnCl2. When indicated, 20 mMsodium orthovanadate and 50 mM sodium fluoride were furtheradded to the mixtures to inactivate phosphatase. Following a 15-minincubation at 30˚C, phosphatase reactions were terminated by addi-tion of an equal volume of 2× Laemmli sample buffer and heatingfor five minutes at 85˚C. Samples were then subjected to 9% SDS-PAGE, and cyclins B1 and B2 were detected by Western blotting.

Recombinant cyclins B. Sea urchin ∆90 cyclin B was kindlyprovided by Marie-Anne Felix.

RESULTSInhibition of cyclin B degradation does not modify progression

of MPF activity in mitotic extract. MG132 is a potent inhibitor ofchymotrypsin-like proteolytic activity of proteasomes (for a reviewsee ref. 52). To characterize the effects of this inhibitor on the mitoticextracts we followed cyclin B2 degradation pattern (Fig. 1A) andhistone H1 kinase activity (Fig. 1B) in presence of increasingconcentrations of MG132. Cyclin B2 degradation was completed at30 min time point in the control extract (Fig. 1A). It was only slightlyslowed down by 20 µM MG132 in comparison to the control andattained a maximal level, similar to the control, at 35 min time-point(Fig. 1A, upper and lower panels), while 100 µM MG132 enabled


Figure 1. Dose-dependent effect of MG132 on cyclin B2 degradation. Thelow-speed cytoplasmic embryo extract was prepared 60 min. post ionophore-activation of metaphase II (M II) oocytes. MG132 was added at 20, 100and 200 µM final concentration or 1% DMSO (equivalent of the concen-tration of DMSO in the extract with 200µM MG132), incubated at 21˚Cand sampled every 5 min for 60 min. (A) Samples (10 µg of cytoplasmicproteins) were analyzed by 10% SDS-PAGE followed by Western blottingwith anti-cyclin B2 antibody (upper panel). Signals were detected usingECF 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 usingImageQuantTM software.

to preserve significant amounts of cyclin B throughout the one-hourincubation (Fig. 1A). Further increase of the drug concentration till200 µM did not provoke any increase in the efficiency of the inhi-bition of cyclin B2 degradation (Fig. 1A). Strikingly, the profiles ofhistone H1 kinase activity examined in parallel with the WesternBlot analysis of cyclin B2 were very similar in the control as well asin MG132-treated extracts for each concentration of the drug tested(Fig. 1B).

To further study the effects of proteasome inhibition on embryonicM-phase in vitro we used the drug in the final concentration of100 µM. Almost identical profiles of histone H1 kinase activityobtained in mitotic control extracts and upon proteasome inhibition(Figs. 1B and 2A) suggested that at least some mitotic events in theextract remained unchanged despite the potent inhibition of cyclinsB1, B2 and B4 proteolysis (Fig. 2B). To verify this, we followedchanges of chosen mitotic markers previously described in the labo-ratory as useful indicators of the timing of mitotic events.43,53 Tothis end, we studied the pattern of Eg3 kinase undergoing charac-teristic mitotic phosphorylation reflected by a decreased velocity inSDS/PAGE and MCM4, a DNA replication regulator undergoingeven more clear-cut mitotic changes than Eg3 (Fig. 2B). The resultsshowed that upon MG132 treatment cyclins B1 and B4 degradationwas greatly reduced as observed with cyclin B2. Despite the massiveinhibition of B-type cyclins degradation, the profiles of electro-phoretic migration of Eg3 and MCM4 in MG132-treated extractsare indistinguishable from the control ones (Fig. 2B, double-headedarrows show the periods of the maximum up-shift of these proteinson SDS/PAGE). This shows that both the timing and the efficiencyof phosphorylation and dephosphorylation of Eg3 and MCM4proteins are not affected (directly or indirectly) by proteasomeinhibition, and that the duration of the mitotic events studied hereare unchanged in all extracts despite permanent cyclins B high levelsin the MG-treated extracts.

To confirm the selectivity of MG132 action as chymotrypsin-likeproteasome inhibitor in the mitotic extract we tested another drugALLN and studied histone H1 kinase activity in parallel withWestern Blot analysis of cyclin B2 and Eg3 (Fig. 3A). 40 µM ALLNdid not perturb histone H1 kinase activity profile (Fig. 3A) whileinhibiting cyclin B2 degradation quite similarly as MG132 (Fig. 3Band C). Further increase in ALLN concentration (tested up to 100 µM)only slightly decreased the dynamics of cyclin B2 degradation, againwithout affecting histone H1 kinase activity profile (data notshown). Also the period of mitotic phosphorylation of Eg3 remainedthe same in ALLN-treated and control extracts (Fig. 3B). This showsthat the two proteasome inhibitors have similar effects on M-phaseevents in Xenopus embryo cell-free extract. Together, these data providecompelling evidence that inhibition of proteasome proteolyticactivity during embryonic mitosis does not prevent MPF inactivation asshown for activated CSF meiotic extract by Nishiyama and colleagues.3

Cyclins B1 and B2 remaining after MPF inactivation in MG132-treated extract are not associated with CDK1. Since cyclins Bdegradation and MPF inactivation appeared as separated events duringthe mitotic exit in the above experiments, we verified the quantitativerelationship between B1 and B2 cyclins and CDK1 during this periodas done previously by Nishiyama et al.3 during the exit from meiot-ic arrest. To this end we compared the behavior of CDK1 and cyclinsB1 and B2 in samples collected at ten minutes (before M-phaseentry) and 60 min (following the mitotic exit; see Fig. 4A) ofincubation (Figs. 4A, B), and examined whether these cyclins Bcoprecipitate with p9-coated beads specific to CDKs;54 (Fig. 4B).

Cyclin B1 migrates as a double band due to the presence of twodistinct form of alternatively spliced cyclin B1.7 Accordingly, degra-dation of the two forms of cyclin B1 is concomitant (see Figs. 2Band 4B in ref. 7). Cyclin B2 migrates as a double band (see Figs. 1A,2B and 3B), where the upper band corresponds to the phosphory-lated form of cyclin B2, which appears upon cyclin association withCDK.22 Upon mitotic exit, the non-phosphorylated form of cyclinB2 disappears first, and the phosphorylated one remains as a majorband during a short period (see for instance Figs. 1A, 2B). In thepresence of MG132 or ALLN, the majority of cyclin B2 remainingin the extract following the inactivation of histone H1 kinase activitywas found in this phosphorylated form (see Figs. 1A, 2B and 3B).

In the experiment presented in Figure 4, CDK1 in whole extractwas detected in all samples at a similar level (see Fig. 4B), upper leftpanel, CDK1 in whole extract at 10 and 60 min). Cyclin B1, whenpresent, migrated always as a double band. Cyclin B2 was present asa double band at ten minutes both in the control and MG132-treated


Figure 2. MG132 (100 µM) inhibits cyclin B1, B2 and B4 degradation butdoes not change the timing neither of histone H1 kinase activity nor of Eg3and MCM4 proteins phosphorylation during embryonic M-phase in thecell-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 followedby immunoblotting with anti-cyclin B1, cyclin B2, cyclin B4, Eg3 and MCM4antibodies. The double headed arrows show the period of the maximalup-shift of Eg3 and MCM4 proteins due to their mitotic phosphorylation.



extracts, absent since degraded at 60 min in the control extract andpresent in high quantity as well as up-shifted in 60 min time-pointin the presence of MG132. The p9 beads efficiently removed CDK1from all samples of the MG132-treated and untreated extracts, asexpected (Fig. 4B, compare the middle and the rightmost panels).Also cyclins B1 and B2 were coprecipitated by the beads at ten minutesin the control extract and were absent at 60 min since alreadydegraded (Fig. 4B, rightmost panel). In the MG132-treated extractcyclins B1 and B2 were efficiently coprecipitated by p9 beads onlyat ten minutes, while weak signals were found at 60 min (Fig. 4B,rightmost panel). However, cyclins B1 and B2 are absent from thep9 supernatant in all samples but at 60 min in the presence ofMG132 showing that at that particular time point the two cyclins B

in this extract are free of CDK1 (Fig. 4B, the middle panel). Theweak signals of cyclins B1 and B2 associated with the beads observedat 60 min in the MG132-treated extract were most probably non-specific contamination of the pellet by cyclins B present in thesupernatant. It is noteworthy that the two cyclins B studied in thisexperiment behaved in the same way, as expected. This experimentwas repeated twice with identical results. It shows that followingMPF inactivation in the presence of MG132 the majority of remain-ing cyclins B1 and B2 are free of CDK1.

Cyclin B2 remaining after MPF inactivation in MG132-treatedextract is protected against dephosphorylation. Since the majorityof cyclins B1 and B2 are not associated with CDK1 inMG132-treated extracts following mitotic exit and in additioncyclin B2 remains in a phosphorylated state as if it were still associ-ated with CDK1, we wanted to characterize better this particularform of cyclin B2. To this end, we tested whether such cyclin B2could be dephosphorylated by an exogenous phosphatase. CyclinB1, whose dephosphorylation cannot be visualized by modificationof the electrophoretic mobility (our unpublished observations andsee Fig. 4C), was used as a control to distinguish between dephos-phorylation of cyclin B2 and its potential degradation duringincubation since both cyclins B have very similar characteristics withregard to proteolysis.

Phage λ phosphatase was added for 15 min. to the control andMG132-treated extracts coming from the same experiment as shownabove (Figs. 4A, B). Samples at the 20 min time point (the verybeginning of the M-phase) for the control extract as well as theMG132-treated counterpart and 50 min sample following MPFinactivation in the presence of MG132 were analyzed (10 and 60 minneighbor samples were used for p9 beads precipitation; (Fig. 4C). Asexpected, cyclin B1 did not react to the presence of either λ phos-phatase or the phosphatase buffer (Fig. 4C, upper panel). Thisdemonstrates that cyclin B1 remains stable in our experimentalconditions at least for 15 min. Indeed, cyclin B2 behaved in a clearlydifferent way than cyclin B1. In the control extract it was efficientlydephosphorylated by λ phosphatase as indicated by the disappear-ance of the upper band (correlated with increased intensity of thelower one) already after 15 min incubation and followed furtheruntil 45 min (Fig. 4C, leftmost, 15 min and 45 min). However, evenincubation of the extract in the phosphatase buffer followed by incu-bation at 30˚C provoked similar, but slightly less rapid reaction.Note that traces of phosphorylated form of cyclin B2 are still presentafter 15 min incubation and they disappear at 45 min (Fig. 4CDMSO t20 second lane), while they have totally disappeared after15 min incubation upon λ phosphatase treatment (Fig. 4C DMSOt20 third lane). Disappearance of the phosphorylated form of cyclinB2 was however, inhibited by sodium vanadate and fluoride presentin the reaction mixture (Fig. 4C DMSO t20 fourth lane). Thedisappearance of the phosphorylated band of cyclin B2 is due todephosphorylation since cyclin B1 was not degraded in the samesample and we observed a parallel increase in the intensity of thenon-phosphorylated, lower band of this cyclin on Western blot.Identical results were obtained for the 20 min sample from theMG132-treated extract (Fig. 4C, middle panels, 15 and 45 min).However, neither buffer alone nor phage λ phosphatase were able toefficiently dephosphorylate cyclin B2 from the 50 min sample in theMG132-treated extract (Fig. 4C, rightmost panels, MG t50). Theseresults indicate that cyclin B2 undergoes some qualitative changesafter the exit from the M-phase upon MG132 treatment whichmakes it less sensitive to dephosphorylation. We believe that phos-


Figure 3. ALLN (40 µM) has a similar effect on biochemical events duringembryonic M-phase in the cell-free extract as MG132. (A) Histone H1kinase assay of the extract prepared 60 min. post-activation and supple-mented either with 40 µM ALLN or 0.25% DMSO as a control. (B) Westernblots with anti-cyclin B2 (upper panel) and Eg3 (lower panel) antibodiesshow that cyclin B2 degradation is inhibited by ALLN and that the period ofEg3 up-shift (double headed arrows) is identical in the control and inALLN-treated extracts. C) Specific signals for cyclin B2 shown in (Fig. 3B)(upper panel) were quantified using ImageQuantTM software.

phorylated cyclin B2 was efficiently protected against λ phosphatasebecause sequestered within the proteasome. Moreover, we observedclear diminution of the intensity of all bands specifically in threesamples collected at 50 min time point in the presence of MG132and incubated at 30˚C (Fig. 4C MG t50, rightmost panel lanes two,

three and four in cyclin B1 and B2). Significantly, the decreasedintensity of the two bands of both cyclin B1 and B2 in these threesamples was slightly more significant in 45 min time point than in15 min. It suggests progressive degradation of all forms of thesecyclins only in those samples. This was most likely due to thediminution of the concentration of MG 132 in the reaction mixturewhich activated to some extent the proteolytic activity of protea-somes. This latter observation favors our hypothesis that cyclins B1and B2 are indeed sequestered within the proteasome in the presenceof MG 132 after MPF inactivation.

Cyclins B pool remaining after mitotic exit in the presence ofMG132 is mitotically inert, but newly synthesized cyclin B activateshistone H1 kinase in the presence of MG132. If the pool of cyclinsB remaining in the extract following histone H1 kinase inactivationin the presence of MG132 is indeed sequestered within the proteasomeas suggested by Nishiyama et al.3 and our above experiments, itshould be inaccessible for CDK1 and thus mitotically inert. If thispool of cyclin B is able to leave the proteasome it should be capableto associate with CDK1 and thus induce precocious subsequentM-phase in cycling extracts. To test which hypothesis is correct weassayed histone H1 kinase activity as well as cyclin B2 levels byWestern blotting in control and MG132-treated extracts during twohours to allow the second M-phase to occur in a control cyclingextract. We observed that the second peak of histone H1 kinaseactivity in MG132-treated extract was not accelerated but delayed incomparison to the control extract (Fig. 5A). Cyclin B2 was degradedupon mitotic exit and then resynthesized in the control extract,while it was only slightly diminished upon M-phase exit in MG132-treated extract, and further accumulated to sensitively higher levelsthan in the control extract as shown by Western blot analysis (Fig. 5B).While the maximal quantity of cyclin B2 during the second M-phasewas present at 110 min. time-point (Fig. 5B), a comparable amountof this protein in the MG132-treated extract was already present at70 min. time-point (Fig. 5B). Despite faster accumulation of cyclinB2 in this extract the increase in histone H1 kinase activity wasobserved only at 110-120 min. (Fig. 5B). The absence of accelerationof the second M-phase in MG132-treated extract suggested that thepool of cyclin B remaining after mitotic exit is inaccessible toCDK1. This again argued for cyclin B sequestration within theproteasome. The second wave of activation of histone H1 kinase inthe extract shows however, that newly synthesized cyclin B indeedassociates with CDK1 in the continuous presence of MG132. Weconclude that two different pools of cyclin B are therefore present inthe extract following mitotic exit in the presence of MG132: themitotically inert one, which was synthesized before the exit from thefirst embryonic M-phase, and the mitotically active, which isneo-synthesized following this mitotic exit. This experiment alsoshows that even prolonged presence of MG132 does not prevent theextract to enter the second M-phase.

Exogenous sea urchin cyclin B also activates histone H1 kinasein the presence of MG132. To verify experimentally the capacity ofMG132-treated extract to activate MPF, we added exogenous non-degradable ∆90 cyclin B of sea urchin during the mitotic exit andcompared evolution of histone H1 kinase activity (Fig. 6A), endogenouscyclin B2 levels and MCM 4 phosphorylation pattern (Fig. 6B).Non-degradable cyclin B addition provokes gradual histone H1kinase increase in MG132-treated extract (Fig. 6A; dotted line).Western blot analysis confirmed that endogenous cyclin B2 wasdegraded in the control extract without MG132 (data not shown)and preserved in MG132-treated extract upon histone H1 kinase


Figure 4. Cyclin B2 remaining after MPF inactivation in the presence of 100 µMMG132 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 H1kinase was assayed. (B) Coprecipitation of cyclins B1 and B2 with CDKproteins on p9 beads. The presence or absence of CDK1 and cyclins B weredetected by Western blot in 10 min and 60 min samples of the wholecell-free mitotic extract (left panel). Sibling samples of these extracts wereincubated with p9 beads for 2.5 h at 4˚C under constant agitation. CDK1was absent in the supernatant (middle panel) and precipitated entirely withthe beads (right panel). In the supernatant, cyclins B1 and B2 are detectedonly 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 thecontrol as well as in the MG132-treated extract, absent at 60 min time pointin the control and present in a very weak amount at 60 min time point inthe presence of MG132 (right panel). (C) One µl of the extracts from 20and 50 min samples of the same experiment was treated with 200 Units ofλ protein phosphatase in the presence of buffer (in a 15 µl final volume)supplemented or not with 20 mM sodium orthovanadate and 50 mM sodiumfluoride (PPase inh.). The mixtures were incubated for 15 to 45 min at 30˚Cand the samples were then analyzed by Western blotting for the presenceof cyclins B1 and/or B2.



inactivation (Fig. 6B, upper panel, time points 60-120 min) as in allpreviously described experiments. Anti-MCM4 Western blotshowed that the phosphorylation of this protein reincreased steadilyin MG132-containing extract only when supplemented with exoge-nous ∆90 cyclin B (Fig. 6B, lower panel, time points 60-120 min).This clearly indicates that exogenous cyclin B forms active complexeswith CDK1 and provokes early M-phase events in the presence ofMG132 despite sequestration of the endogenous cyclin pool.

MG132 only slightly delays mitotic activation of MPF. Theslight delay in histone H1 kinase activation during the entry into thesecond embryonic M-phase in cycling extracts upon prolongedexposure to MG132 (Fig. 5A) could suggest that requirements for aproteasome-dependent degradation differ between the first andsecond embryonic M-phase. Indeed, numerous differences in theregulation of these two M-phases in vitro were shown recently byour laboratory.43 Alternatively, proteolysis of some “early” substratesof the proteasome could be required for a correct entry into the

M-phase during both, the first or the second, embryonic mitoses. Inthe latter case, troubles with histone H1 kinase activation in cyclingextracts could be due to the prolonged incubation of extracts withMG132 affecting such an early proteolysis.

To verify these possibilities, we first added MG132 to the extractsprepared 108-110 min after M II oocytes activation, i.e., just beforethe entry into the second embryonic M-phase. The profiles of histoneH1 kinase activity were almost identical in the presence and absenceof MG132 (Fig. 7A). However, we noticed that when the mitoticentry in the untreated control was delayed, most likely due to thedifferences 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 embryonicM-phase in vitro is not inhibited by MG132 similarly as the first


Figure 5. Cyclin B2 remaining in the extract after MPF inactivation in thepresence of 100 µM MG132 does not accelerate subsequent M-phase.(A) Histone H1 kinase activity was assayed every five mintutes for two hoursin the extracts containing either 100 µM MG132 or 0.5 % DMSO (control).Second mitosis is already completed in the control extract at 120 min timepoint, while the mitotic increase of histone H1 kinase activity only begins inthe presence of MG132. (B) Cyclin B2 was detected in parallel to histoneH1 kinase assay in the two extracts. Note the complete degradation ofcyclin B2 following each mitosis (35 and 120 min time points) in the controlextract and much less important diminution of cyclin B2 following the firstmitosis in the MG132-treated extract. Despite higher amount of cyclin B2detected by Western blot in the MG132-treated extract, the second M-phaseentry is not accelerated. Note also a discrete difference between the ratioof two bands of cyclin B2 during the first and the second M-phase in cyclingextract as visualized by Western blotting. Cyclin B2 is present as a cleardouble band during the first M-phase and mostly as a single, phosphorylatedupper band during the second. This is an artefact observed in cyclingextracts, while in extracts dedicated specifically to the second M-phase thetwo bands of cyclin B2 are present also during the second cell cycle(compare with Fig. 1B) in Chesnel et al. 2005a).

Figure 6. Recombinant sea urchin ∆ 90 cyclin B added by the end of theM-phase to the extract forms active complex with CDK1 and allows histoneH1 kinase reactivation. The low-speed extract was incubated at 21˚C fortwo hours with 100 µM MG132. At 50 min of incubation (expected time ofthe mitotic exit), ∆ 90 cyclin B was added to the half of the extract at thefinal concentration of 15 µM, while another half containing only MG132was sampled as a control. (A) Histone H1 kinase activity was assayed everyten minutes for two hours in the extract containing 100 µM MG132 and sup-plemented or not with ∆ 90 cyclin B. Note the reincrease of histone H1kinase activity following full inactivation of the mitotic histone H1 kinaseactivity in the extract supplemented with ∆ 90 cyclin B (dotted line) in con-trast to the control without exogenous cyclin B. (B) Western blot analysis ofcyclin B2 (upper panels) and MCM4 (lower panels) was performed on sib-ling samples. Cyclin B2 Western blot confirms that MG132 prevented cyclinB2 degradation in this experiment (double arrows in the upper panels).MCM4 is dephosphorylated following histone H1 kinase inactivation inMG132-treated control extract and rephosphorylated starting from t = 80min following ∆90 cyclin B addition only (double arrows in the lower panel).

one, and argued rather in favor of our second hypothesis thatincreased exposure to the presence of MG132 delays slightly histoneH1 kinase activation.

To better characterize this possibility, we returned to the analysisof the first embryonic M-phase. This time, extracts were preparedearlier (50-58 min post-activation) to extend MG132 incubationbefore the mitotic entry. We added the drug at 0 and 15 min timepoints after the beginning of incubation at 21˚C. In control extractsfrom six independent experiments, the peak of histone H1 kinaseactivity has never been observed before 35 min time point followingthe beginning of incubation (average 40 min). MG132 eitherdelayed the peak of histone H1 kinase activity or did not influencethe timing of its activation in all experiments (Fig. 8A). Usually, alonger incubation (MG132 added at t = 0) resulted in longer delayand lower amplitudes of histone H1 kinase activity in parallel withdelayed pattern of MCM4 phosphorylation/dephosphorylation

(Fig. 8A and upper panel in 8B). The efficiency of inhibition ofcyclin B2 proteolysis was, however, comparable in each case (Fig. 8B;lower panel). The delay caused by MG132 was not always strictlycorrelated with the increased time of incubation. In two out of sixexperiments, shorter incubation with the drug (MG132 added att = 15 min) resulted in histone H1 kinase activity peak slightly delayedbeyond the longer incubation (starting at t = 0; data not shown).

These results confirm that even prolonged presence of MG132does not prevent mitotic entry in the extract and only slightly delaysthis process.

Levels of cyclins B1, B2 and B4 associated with CDK1 diminishfaster than the decline of total pools of these cyclins upon mitoticexit in CHX-treated mitotic extract. Proteasome inhibitors couldmodify numerous activities in the extract. Therefore, the mechanism


Figure 8. MG132 does not block histone H1 kinase activation during thefirst embryonic M-phase in vitro even upon longer incubation before thebeginning of mitotic events. (A), Histone H1 kinase assay of the extract pre-pared 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 blotanalyses of MCM4 (upper panels) and cyclin B2 (lower panels) were per-formed on sibling samples. MCM4 Western blot confirms that MG132 pro-vokes delay in mitotic exit and dephosphorylation of this protein in longerincubation (MG132 added at t0). Cyclin B2 degradation is prevented ineach extract containing MG132.

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



of MPF inactivation described by Nishiyama et al.3

in meiotic extract and in the current paper in themitotic ones could differ in intact extracts. To veri-fy whether the dissociation of cyclins B fromCDK1 upon MPF inactivation precedes cyclins Bdegradation in embryo extract we compared thedynamics of diminutions of CDK1-coupled cyclinsB1, B2 and B4 relative to the dynamics of thedecline of the total levels of the same cyclins indi-vidually without proteasome inhibition. CHX wasadded to the mitotic extracts to eliminate the newlysynthesized pool of cyclins potentially not yet asso-ciated with CDK1. We reasoned that if our hypoth-esis is valid we should observe earlier decline ofCDK1-associated cyclins B and later of total poolsof each cyclin B.

CHX was added 18 min following the begin-ning of extract incubation at 21˚C and MCM4,cyclin B1, B2 and B4 samples were analyzed byWestern blotting every three minutes. MCM4down-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), corre-sponding to the period when all analyzed cyclins Bwere degraded, were incubated with p9 beads. Thetotal levels of each cyclin B (Fig. 9B–D; input forcyclins B1, B2 and B4) were compared to the lev-els of p9-bound cyclins (Figs. 9B–D; p9-bound forcyclins B1, B2 and B4). In parallel, CDK1 levelswere followed in both input and p9-bound materi-al by Western blotting (Figs. 9B–D, left, CDK1).As expected, in the case of each of three cyclins Bstudied we found significant advance of the declineof CDK1-associated pool compared to the total oneof the same cyclin (Figs. 9B–D, Western blot andquantification on rightmost curves).

Altogether, the present results enable us to pos-tulate that dissociation of cyclins B (at least B1, B2and B4) from CDK1 and not their degradation perse is responsible for MPF inactivation duringmitotic exit in Xenopus embryo cell-free extract.

DISCUSSIONIn this study, we have investigated the role of the

proteasome proteolytic activity in the regulation ofthe mitotic activity of MPF as well as the timing ofphosphorylation and dephosphorylation of chosenmitotic marker proteins (Eg3 and MCM4;43,53) during the first twoembryonic M-phases in vitro. Our results have shown that (1) cyclinB degradation is not necessary for MPF inactivation upon mitoticexit during the first and the second embryonic M-phases in cell-freeextracts, as shown before for the meiotic CSF extract by Nishiyamaet al.,3 (2) the pool of cyclin B remaining in the extract followingmitotic exit in the presence of MG132 is free of CDK1 and mitoti-cally inert, while the extract maintains the capacity to activate MPFwhen exogenous or newly-synthesized cyclin is present, (3) inhibi-tion of the proteolytic activity of the proteasome does not prevent

mitotic M-phase entry in Xenopus embryo extracts, even if the pro-longed presence of MG132 can perturb the correct timing of MPFactivation during the two mitoses studied. These results prompted usto investigate whether the dissociation of B-type cyclins from theirCDK1 partner precedes their degradation in mitotic cell-freeextracts. Accordingly, we observed differences between the dynamicsof decline in CDK1-bound cyclins B1, B2 and B4 vs. their total lev-els in CHX-treated mitotic extracts, which directly confirmed ourhypothesis. These results, together with those from Nishiyama andcolleagues3 concerning activated CSF extract, enable us to postulate


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

the sequence of events accompanying MPF inactivation duringmitotic exit in Xenopus embryo cell-free extract (Fig. 10).

MPF inactivation and cyclin B degradation are separate eventsduring meiotic and mitotic exits. It is well established that cyclin Bdegradation is a key event of the mitotic exit and enables the cellcycle progression beyond M-phase.1,2,55 However, it does not play adirect role in MPF inactivation neither during meiosis3 nor duringmitosis (this paper). Nishiyama and colleagues3 have clearly demon-strated 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 viaubiquitin-proteasome pathway while cyclin B is polyubiquitinatedand targeted to the proteasome while still complexed with CDK1.They have also shown that the pool of cyclin B remaining in the extractafter M-phase exit is found in high molecular weight complexesassociated with the 26S proteasome, which enabled to postulate thatCDK1-free cyclin B remains sequestered inside the proteasomesupon the inhibition of the proteolytic activity of the proteasome.Our results showing that during mitosis in cell-free Xenopus embryoextract under similar conditions (i.e., in presence of MG132 insteadof MG115 used by Nishiyama et al.3) cyclin B accessibility to cytoso-lic enzymes, such as phosphatases and CDK1, is greatly reducedreinforce this hypothesis (this paper). Similar uncoupling of a sin-gle subunit of a multisubunit complexes prior proteasome-depen-dent degradation was reported in the case of ubiquitinated Sic1(from CDK-cyclin complex56,57) and IκB (from NF-κB58). It wasproposed that either ubiquitination acts as “proteinaceous detergent”destabilizing the folded state of proteins,59 or the ATPases residentin the 26S proteasome have an “unfoldase” activity that dissociatesubiquitinated subunits.60 Our demonstration that three B-typecyclins (B1, B2 and B4) dissociate from CDK1 before their degra-dation upon mitotic exit in extracts in which protein synthesis wasinhibited strongly suggests that the mechanism of MPF inactivationthrough proteasome-dependent separation of B-type cyclins discov-ered in meiotic extract by Nishiyama and colleagues3 could be uni-versal. Josefsberg et al.31 have shown that in rat zygotes MPF inacti-vation could proceed without cyclin B degradation in the presenceof MG132. This is in agreement with our results in Xenopus embryoextracts described in this paper and demonstrates that the phenomenonof MPF inactivation without cyclin B degradation is not restrictedto cell-free extracts.

The lack of inhibition of MPF inactivation by MG132, MG115or ALLN contrasts with the M-phase arrest triggered in XenopusCaCl2-treated CSF extract and cycling embryo extracts by N-terminalfragment of cyclin B2 addition (B2Nt).61 The way B2Nt stabilizesMPF and arrests mitotic exit seems to be provoked, however, by thesaturation of the proteolytic machinery. B2Nt competes for degra-dation of other substrates and inhibits proteolysis of endogenouscyclin B. The latter remains associated with CDK1 and maintainhigh MPF activity. Moreover, their continuous synthesis results in asteady increase in histone H1 kinase activity while oscillations areobserved in controls where BSA or other proteins were added (seeFigs. 3 and 4 in ref. 61). Since B2Nt contains sequences necessaryfor its ubiquitination it seems that it saturates APC/C, and thereforeprovoke 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 arrestingthe M-phase upon CSF extract activation.3 One cannot exclude,however, that wild-type B2Nt inhibits cyclinB/CDK1 dissociatingactivity of proteasome by association with the proteasome lid blocking

entry of other substrates into the proteasome cylinder. The differencebetween the effects of proteasome inhibitors and B2Nt on mitoticextracts suggests that the inhibition of cyclin B degradation at differentsteps of the ubiquitin-proteasome pathway provokes extremely differentresults regarding MPF activity.

Our observations that cyclins B dissociation from CDK1 precedestheir degradation in extracts without MG132 or ALLN suggestsstrongly that this mechanism is indeed a direct cause of MPF inac-tivation also upon mitotic exits in Xenopus cell-free extract. Thedegradation of cyclins B, already present within the proteasome andinaccessible from the cytoplasm, follows (as summarized in Fig. 10).

Why somatic cells are arrested in M-phase by proteasome inhi-bition? In Xenopus, the ability to inactivate MPF without cyclin Bdegradation is observed in cell-free extracts derived of MII oocytes3

and early embryos (this paper). This contrasts with Xenopus XL2somatic cells obtained from later embryonic stages, which are arrestedin M-phase upon ALLN treatment.28 This could argue for anevolution of mechanisms inactivating MPF during later Xenopusdevelopment. However, such a switch could have quantitative ratherthan qualitative character. Oocytes and embryos, contrarily tosomatic cells, contain much higher ratio of proteasomes per unit ofcytoplasm due to accumulation during oogenesis, which couldincrease the efficiency of dissociation of cyclin B from CDK1. Wediminished the quantity of proteasome in premitotic extracts byimmunodepletion (using a polyclonal antibody against 20S protea-some kindly provided by Toshinobu Tokumoto).62 The most efficientdepletion in our hands—up to 64% (as visualized by Western blottingusing two different monoclonal antibodies directed against protea-some subunits α2 and α4;1-4D5 and GC3β)62 did not change thedynamics of histone H1 kinase inactivation upon mitotic exit (ourunpublished results). Thus, the quantity of proteasome in cytoplasmof one-cell embryo indeed exceeds the quantity necessary to promoteefficient MPF inactivation. Supposedly lower concentration ofproteasomes in the cytoplasm of somatic cells might favor saturation ofproteasome with other than cyclins B substrates upon the presenceof a proteasome inhibitor. This could facilitate in turn, the retentionof active CDK1/cyclin B complexes in the cytoplasm of these cells.Alternatively, in somatic cells other substrates than in oocytes orearly embryos could be degraded by the proteasome before theinitiation 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 cellswith a peak in M-phase.63,64 It is also degraded in cell-free extract of


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



G1 synchronized XL2 cells65 and upon MII oocytes activation.66

However, its level remains constant in maturing oocytes duringMI/MII meiotic transition66 as well as in mitotic extract of one-cellXenopus embryos (our unpublished data). In addition, ALLNinhibits degradation of this protein in Xenopus XL2 cells and theircell-free extract.63,65 However, Aurora A is not a good candidate fora trigger of MPF inactivation since its proteolysis occurs late inmitosis. In contrast, Nek2A from NIMA kinases family is an earlysubstrate of proteasome (for a review see ref. 67) and it seems abetter candidate for this role. Nek2A is present in somatic cells and isabsent in oocytes and early Xenopus embryos (ref. 68 and our unpub-lished results). Its downregulation in HeLa cells results in prolongationof mitosis, which could suggest a potential role of this protein as anaccelerator of MPF inactivation during mitosis in these cells.69 Onthe other hand, in HeLa cells overexpressing GFP-Nek2A anaphaseis delayed,70 which shows that Nek2A is a more complex player ofmitotic regulation. However, in calcium-activated CSF extract ofXenopus, where endogenous Nek2A is absent, non-degradablemutant of this protein prevents cyclins degradation.70 The latterstrongly suggests that Nek2A proteolysis could indeed be a signal formitotic cyclins degradation. Finally, inhibition of degradation ofmitotic forms of Erp/Emi proteins (known to be involved in meioticCSF arrest; for a review see e.g., ref. 71) could also arrest somaticcells in the M-phase upon proteasome inhibition upstream fromboth Nek2A and cyclin B proteolysis. Recently, Giménez-Abián etal.72 provided evidence that in mammalian cells anaphase couldoccur in the presence of non-degraded cyclin B and securin. Moreinterestingly, they showed that some non-identified substrates of pro-teasome which regulate mitotic progression are degraded in mam-malian cells in APC/C-independent manner.73 Inhibition of prote-olysis of these proteins, which could include Nek2A and/orErp/Emi-like proteins, seems indeed to arrest cells upstream fromcyclin B polyubiquitination. However, since their identity remainsunknown, the question whether different mechanisms of MPF inac-tivation operate during development remains still open.

Mitotically inert and active cyclin B pools. Nishiyama andcoworkers3 have demonstrated by gel filtration that the cyclin B poolremaining in activated CSF extract in the presence of MG115 is inhigh molecular weight complexes and by coimmunoprecipitationthat it is associated with the 26S proteasome. They postulated thatCDK1-free cyclin B remains sequestered within the proteasomes.Our experiments showing that this pool of cyclin B remains inac-cessible both for endogenous phosphatases and exogenous phage λphosphatase and that it is incapable to accelerate the next M-phaseentry provides the first evidence for functional masking of cyclin Bwithin such a cytoplasm. This further strengthens the hypothesisthat this pool of cyclin B is sequestered within the proteasomesfollowing meiotic3 as well as mitotic (this paper) MPF inactivationin the presence of the proteasome inhibitors.

The behavior of cycling extracts in which MPF is activated (andMCM4 phosphorylated) for the second time in the presence ofMG132 (Fig. 5) suggests that newly synthesized pool of cyclin B ismitotically active because remaining free in cytoplasm. Similarly,non-degradable ∆90 cyclin B added to the extract following mitoticexit in the presence of MG132 activates MPF, too (Fig. 6). Since thewindow of polyubiquitination is restricted to the M-phase2 whenAPC/C-Cdc20 becomes phosphorylated and active74,75 it is notsurprising that following MPF inactivation newly synthesized orexogenous cyclin B remains free in the cytoplasm and avoid seques-tration within the proteasomes. Thus, two different pools of cyclin B

(free and sequestered within the proteasome) are present in theextract following the M-phase exit in the presence of the drug.

Proteasome inhibition and the mitotic entry. We show here thatthe extracts enter the first or the second mitotic M-phase in the pres-ence of MG132 (and ALLN for the first M-phase). It demonstratesthat the mitotic entry in the extract does not require proteolyticactivity of the proteasome. However, upon prolonged incubationwith MG132 the peak of histone H1 kinase is slightly delayed. Thiscould suggest that proteasome inhibition interferes either with someprocesses determining the timing of the mitotic entry (but not withthe machinery of MPF activation itself ), or that the drug has someunspecific effects. These results are in contrast to the data obtainedby Kawahara et al.76 showing that proteasome inhibitors preventmitotic entry in sea urchin embryos. On the other hand, interferencewith polyubiquitination, a process upstream fromproteasome-dependent proteins degradation, in Xenopus embryoextract delays MPF activation77 similarly as MG132 in our study.This supports our hypothesis that the ubiquitin-proteasome path-way is involved rather in the control of the timing of MPF activationthan in its activation itself. Species-specific differences in proteasomemachinery regulating mitotic entry cannot be, however, excluded.

References1. 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 dissociationof 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 Cell2002; 9:931-43.

5. Minshull J, Golsteyn R, Hill CS, Hunt T. The A- and B-type cyclin associated cdc2 kinasesin 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-typecyclins of Drosophila are accumulated and destroyed in temporally distinct events thatdefine 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 dur-ing Xenopus oocyte maturation. Development 2001; 128:3795-807.

8. Castro A, Bernis C, Vigneron S, Labbé JC, Lorca T. The anaphase-promoting complex: Akey 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-Cys41does 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 theD 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-activatingdomain (DAD) is a new proteolysis signal that stimulates the silent D-Box sequence ofAurora-A. EMBO Rep 2002; 3:1209-14.

13. Raff JW, Jeffers K, Huang JY. The roles of Fzy/Cdc20 and Fzr/Cdh1 in regulating thedestruction 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 anddestruction of A- and B-type cyclins during oogenesis and meiotic maturation in Xenopuslaevis. J Cell Biol 1999; 114:755-65.

15. De Smedt V, Poulhe R, Cayla X, Dessauge F, Karaiskou A, Jessus C, Ozon R. Thr161 phos-phorylation 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 dynamicsand length by cyclin A- and cyclin B-dependent kinases in Xenopus egg extracts. J Cell Biol1992; 118:1097-108.

17. Buendia B, Draetta G, Karsenti E. Regulation of the microtubule nucleating activity ofcentrosomes in Xenopus egg extracts: Role of cyclin A-associated protein kinase. J Cell Biol1992; 116:1431-42.

18. Meijer L, Arion D, Golsteyn R, Pines J, Brizuela L, Hunt T, Beach D. Cyclin is a componentof 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 ofpre-MPF activation. EMBO J 1991; 10:177–82.

20. Izumi T, Maller JL. Phosphorylation of Xenopus cyclins B1 and B2 is not required for cellcycle transitions. Mol Cell Biol 1991; 11:3860-7.


21. Li J, Meyer AN, Donoghue DJ. Requirement for phosphorylation of cyclin B1 for Xenopusoocyte maturation. Mol Biol Cell 1995; 6:1111–24.

22. Peter M, Le Peuch C, Labbé JC, Meyer AN, Donoghue DJ, Dorée M. Initial activation ofcyclin-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 componentof 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 kinase1 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 retentionsequence 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 ofcyclin B degradation and induction of cell-cycle arrest in mammalian cells by the neutralcysteine 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 proteolysisin plants. Identification of the destruction box pathway and metaphase arrest produced bythe proteasome inhibitor MG132. Plant Cell 1998; 10:2063-76.

28. Uzbekov R, Chartrain I, Philippe M, Arlot-Bonnemains Y. Cell cycle analysis and synchro-nization 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 ofp53 and p21Waf1 expression on G2/M phase arrest of colorectal carcinoma HCT116 cellsto proteasome inhibitors. Int J Oncol 2004; 24:935-41.

30. Terret ME, Wassmann K, Waizenegger I, Maro B, Peters JM, Verlhac MH. The meiosisI-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-phasepromoting factor at exit from first embryonic mitosis in the rat is independent of cyclin B1degradation. Biol Reprod 2001; 64:871-8.

32. Michel LS, Liberal V, Chatterjee A, Kirchwegger R, Pasche B, Gerald W, Dobles M, SorgerPK, Murty VV, Benezra R. MAD2 haplo-insufficiency causes premature anaphase andchromosome instability in mammalian cells. Nature 2001; 409:355-9.

33. Michel L, Diaz-Rodriguez E, Narayan G, Hernando E, Murty VV, Benezra R. Completeloss of the tumor suppressor MAD2 causes premature cyclin B degradation and mitoticfailure 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 involvedin 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. Homologuedisjunction in mouse oocytes requires proteolysis of securin and cyclin B1. Nat Cell Biol2003; 5:1023-5.

36. Peter M, Castro A, Lorca T, Le Peuch C, Magnaghi-Jaulin L, Doreé M, Labbé JC. The APCis 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 checkpointis 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 isa 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 inmouse oocytes is controlled through microtubule-dependent destruction of cyclin B in thepresence of CSF. EMBO J 1993; 12:3773-8.

40. Verlhac MH, Kubiak JZ, Weber M, Géraud G, Colledge WH, Evans MJ, Maro B. Mos isrequired for MAP kinase activation and is involved in microtubule organization duringmeiotic maturation in the mouse. Development 1996; 122:815-22.

41. Kalab P, Kubiak JZ, Verlhac MH, Colledge WH, Maro B. Activation of p90rsk duringmeiotic maturation and first mitosis in mouse oocytes and eggs: MAP kinase-independentand -dependent activation. Development 1996; 122:1957-64.

42. Murray AW, Kirschner MW. Cyclin synthesis drives the early embryonic cell cycle. Nature1989; 339:275-80.

43. Chesnel F, Vignaux F, Richard-Parpaillon L, Huguet A, Kubiak JZ. Differences in regulationof 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 incycling 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 stabilizesM-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 forexit 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 earlyembryonic 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 bacterio-

phage 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 postvitellogenicXenopus oocytes. Dev Biol 1997; 188:122-33.

52. Lee DH, Goldberg AL. Proteasome inhibitors: Valuable new tools for cell biologists. TrendsCell Biol 1998; 8:397-403.

53. Chesnel F, Gautier I, Richard-Parpaillon L, Kubiak JZ. Each mitosis can be different: Howthe cell cycle machinery modulates early embryonic M-phases. In: Tokumoto T, ed. Newimpact 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 char-acterization 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 bymaternal 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 inhibitorp40SIC1 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. Phosphorylationof 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, andHedgehog signaling pathways. Genes Dev 1999; 13:505-10.

59. Johnson ES, Ma PC, Ota IM, Varshavsky A. A proteolytic pathway that recognizes ubiq-uitin 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 Xenopuscyclin 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 duringXenopus oocyte maturation. Exp Cell Res 1999; 247:313-9.

63. Klotzbucher A, Pascreau G, Prigent C, Arlot-Bonnemains Y. A method for analyzing theubiquitination 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 cycle-dependent 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, JessusC. Progesterone regulates the accumulation and the activation of Eg2 kinase in Xenopusoocytes. J Cell Sci 2000; 113:1127-38.

67. Hayward DG, Fry AM. Nek2 kinase in chromosome instability and cancer. Cancer Lett2005:1-12.

68. Hames RS, Wattam SL, Yamano H, Bacchieri R, Fry AM. APC/C-mediated destruction ofthe centrosomal kinase Nek2A occurs in early mitosis and depends upon a cyclin A-typeD-box. The EMBO J 2001; 20:7117–27.

69. Fletcher L, Cerniglia GJ, Yen TJ, Muschel RJ. Live cell imaging reveals distinct roles in cellcycle 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 mitoticdegradation of Nek2A depends on Cdc20-independent interaction with the APC/C. NatCell Biol 2006, (in press).

71. Schmidt A, Rauh NR, Nigg EA, Mayer TU. Cytostatic factor: An activity that puts the cellcycle 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 assemblycheckpoint 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. Proteasomeactivity is required for centromere separation independently of securin degradation inhuman 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 ofthe 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. Inhibitingproteasome activity causes overreplication of DNA and blocks entry into mitosis in seaurchin embryos. J Cell Sci 2000; 113:2659-70.

77. Mahaffey DT, Gorbea C, Rechsteiner M. Evidence that DNA replication is not regulatedby ubiquitin-dependent proteolysis in Xenopus egg extract. Exp Cell Res 2003; 288:225-34.



Cyclin B Dissociation from CDK1 Precedes its Degradation Upon MPF Inactivation in Mitotic Extracts...

Documents

Transcript of Cyclin B Dissociation from CDK1 Precedes its Degradation Upon MPF Inactivation in Mitotic Extracts...