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Absence of Reciprocal Feedback Between MPF and ERK2 MAP Kinase in Mitotic Xenopus laevis Embryo Cell-Free Extract

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Absence of Reciprocal Feedback Between MPF and ERK2 MAP Kinase in Mitotic Xenopus laevis Embryo Cell-Free Extract
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     ©    2    0    0    7     L   A    N    D    E    S     B    I   O    S   C    I    E    N   C    E .     D   O     N   O    T     D    I    S    T    R    I    B    U    T    E . [Cell Cycle 6:4, 489-496, 15 February 2007]; ©2007 Landes Bioscience Franck Bazile 1  Aude Pascal 1  Anthi Karaiskou 2 Franck Chesnel 1 Jacek Z. Kubiak  1, * 1 Biology and Genetics of Development; Mitosis and Meiosis Group; Institute of Genetics and Development of Rennes; University Rennes; Rennes Cedex, France 2 Biology of Development; Biology of the Oocyte Group; University Paris VI; Paris, France*Correspondence to: Jacek Z. Kubiak; Biology and Genetics of Development, Mitosis and Meiosis Group; 1 UMR 6061-CNRS; University Rennes 1; IFR140 GFAS, Faculty of Medicine; 2 Avenue Prof. Léon Bernard; CS 34317; Rennes cedex 35043 France; Tel.: 33.02.23.23.46.98; Fax: 33.02.23.23.44.78; Email: jacek.kubiak@univ-rennes1.frOriginal manuscript submitted: 01/09/07Manuscript accepted: 01/18/07Previously published online as a Cell Cycle   E-publication:http://www.landesbioscience.com/journals/cc/abstract.php?id=3860 KEY WORDS cell cycle, cell-free extract, cyclin B, embryo, ERK2 MAP kinase, mitosis, MG132, MPF, proteasome/ubiquitin pathway, protein degra-dation,  Xenopus laevis   ACKNOWLEDGEMENTS  We thank Marcel Méchali and Thierry Lorca for generous gifts of antibodies, Denis Michel and Yannick Arlot for the gift of the ubiquitin-containing plasmid and Evelyn Houliston for valuable discussions and English correction of the final version of this article. This work  was supported by grant from Ligue Contre le Cancer (Comité d’Ille-et-Vilaine) and ARC (4298) to J.Z.K. Report Absence of Reciprocal Feedback Between MPF and ERK2 MAP Kinase in Mitotic Xenopus laevis   Embryo Cell-Free Extract    ABSTRACT MPF and MAP kinase ERK2 are two major M-phase kinases. They interact with each other in a complex way during meiotic maturation of Xenopus laevis  oocytes. Here we study their interrelationship during first mitosis in X. laevis  embryo cell-free extract perturbing the polyubiquitination pathway as a tool. Recombinant ubiquitin K48R (Ub-K48R) mutant protein arrests mitotic cyclin B degradation in the extract. This results in both increased accumulation of phosphorylated form of cyclin B2 and MPF activity as well as mitotic phosphorylation of its substrates. Ub-K48R also increased the mitotic phosphorylation of ERK2. Simultaneous addition of Ub-K48R and the proteasome inhibitor MG 132 strength-ened and further prolonged MPF activity, MCM4 phosphorylation and accumulation of phosphorylated forms of cyclin B2. ERK2 phosphorylation levels increased and persisted longer than upon action of Ub-K48R alone. This shows a synergistic effect of inhibi-tion of two different steps of ubiquitin-proteasome pathway on MPF activity and mitotic phosphorylation and ubiquitination of specific M-phase proteins. On the other hand, complete inhibition of ERK2 activation using U0126 had no effect either on MPF activity or on MCM4 phosphorylation either in control or in Ub-K48R-supplemented extracts. Experimental reduction of MPF activity by addition of recombinant p21 Cip  protein resulted in significant reduction of ERK2 phosphorylation. Thus, the reciprocal feedback observed between MPF and ERK2 in meiosis is not observed during mitotic M-phase in cell-free Xenopus  embryo extracts. ERK2 phosphorylation is regulated by the levels of MPF activity, however no influence of ERK2 on MPF activity could be detected. These results show a fundamental difference in the relationship between the two major M-phase kinases in meiotic and mitotic cell cycle. INTRODUCTION Meiotic and mitotic phases are driven by activation and inactivation of MPF (M-phase promoting factor), a complex of cyclin-dependent kinase 1 (CDK1) and its regulatory subunit cyclin B. 1-4  For a long time cyclin B degradation was considered as a major mechanism of MPF inactivation. 5-7  It is indeed one of the key events of the cell cycle. However, MPF inactivation both during meiosis 8  and mitosis 9  was shown to depend primarily on proteasome-dependent dissociation of CDK1 from cyclin B and not on cyclin B proteolysis. Polyubiquitination of cyclin B is necessary for targeting the MPF complex to proteasomes as well as for the subsequent separation of cyclin B and CDK1. 8  Following this dissociation cyclin B is degraded within the proteasome while free, inacti-vated CDK1 is released into the cytoplasm. 8,9  Accordingly, MPF inactivation occurs in the presence of proteasome inhibitors arresting cyclin B degradation in meiotic and mitotic  Xenopus   cell-free extracts. 8,9  In contrast, interference with the polyubiquitination process up-stream of proteasome-dependent degradation stabilises MPF activity during mitosis in cytoplasmic extracts. 10 Ubiquitin is a highly conserved, small (76 amino acids) polypeptide whose COOH terminus is ligated to lysine residues of various substrates (e.g., cyclin B upon mitosis exit) through an isopeptide bond (reviewed in ref. 11). Mitotic polyubiquitination of cyclin B takes place within the MPF complex, i.e., when cyclin B is still associated with CDK1. 8  It is achieved through a common action of three sequential enzymes: E1-ubiquitin-activating enzyme (UBA), E2-ubiquitin-conjugating enzyme (UBC) and E3-ubiquitin-protein ligase. 12,13  Polyubiquitination is achieved via formation of multiple rounds of ubiquitina-tion during which COOH terminus of a new ubiquitin molecule forms an isopeptide bond with lysine residue of ubiquitin previously attached to the substrate. 11  Lysine 48 of ubiquitin is one of the major residues used during polyubiquitination, even if all seven  www.landesbioscience.com Cell Cycle 489     ©    2    0    0    7     L   A    N    D    E    S     B    I   O    S   C    I    E    N   C    E .     D   O     N   O    T     D    I    S    T    R    I    B    U    T    E . 490 Cell Cycle 2007; Vol. 6 Issue 4   lysine residues can form such isopeptide bonds. 14-17  ItsmutationtoIts mutation to arginine in K48R mutant perturbes polyubiquination. 18,19 In  Xenopus laevis   the MAP kinase ERK2 (extracellularly regu-lated kinase 2), like MPF, is activated both during meiosis and mitosis in early embryo, the two of them fulfilling partially overlap-ping roles. ERK2 function during mitosis has been implicated in spindle-assembly checkpoint control, 20-23  however, this role could not be demonstrated in mouse embryos. 24-26  Other functions of ERK2 include keeping microtubules relatively short by increasing assembly/disassembly turnover rates, 27  and maintaining nuclear lamins in a disassembled form. 28  During female meiosis in oocytes ERK2 is responsible for maintaining an M-phase phenotype while MPF is transiently inactivated during the M 1 /M 2  transition. 24,29,30  It then becomes essential for the maintaining of M 2  arrest of oocytes. 29,31,32 During each early embryonic mitosis in  Xenopus laevis  , the periods of ERK2 and MPF activity do not coincide. ERK2 becomes activated  while MPF activity is beginning to decline. 33,34  The confined ERK2 activity following MPF inactivation is essential for the prolongation of certain M-phase events, including chromatin condensation and high MT turnover. 34 ERK2 is activated by a dual phosphorylation at specific threonine and tyrosine residues. 35,36  The upstream kinase cascade includes Raf and/or Mos (MAPK kinase kinase) and MEK (MAPK kinase). 29,37-39  During both meiosis and the first embryonic mitosis it is Mos which acts as the MAPK kinase kinase. 40  Phosphorylation and kinase activity of ERK2 during meiosis is, however, much higher than during embryonic mitosis. 41 In meiosis, MPF and ERK2 are linked by numerous interactions described in  Xenopus   oocytes. A full positive feedback activating ERK2 via MPF and vice versa was described upon meiotic matura-tion (reviewed in ref. 42). During the two first embryonic mitoses, the decreasing levels of ERK2 activity seem proportional to also decreasing levels of MPF rising a hypothesis that the MPF activity determines the degree of ERK2 activation later in the same mitosis. 41  The interactions between MPF and ERK2 were however not well characterized during this period.In this paper we investigate the mutual interactions between MPF and ERK2 during the first mitosis in cell-free extracts using K48R ubiquitin mutant to perturb polyubiquitination/proteasome pathway as well as inhibitors of MEK/ERK2 activating pathway and CDKs. MATERIAL AND METHODS Frogs.  Xenopus laevis   females were purchased from NASCO (Fort  Atkinson, WI, USA). Drugs.  MG132 was purchased from Biomol (Pennsylvania, USA) and U0126 from Promega Corp. (Madison, USA). Other chemicals  were obtained either from Sigma or ICN (Irvine, CA, USA) unless otherwise stated. Egg collection and activation.  Femaleswereinjectedsubcutane-Females were injected subcutane-ously 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 (100 mM KCl, 1 mM MgCl 2 , 50 m M CaCl 2 , 10 mM HEPES, 50 mM sucrose pH 7.6), 43   washed in XB, treated for 1.5 minutes with 0.5 m g/ml calcium iono-phore A23187 and then extensively washed in XB. Activated eggs  were then incubated in XB at 21˚C. Cell-free extracts.  Cytoplasmicextractsfromcalciumionophore-Cytoplasmic extracts from calcium ionophore-activated embryos before the first embryonic mitosis were prepared according to a classical protocol 43  modified as previously described. 41  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 m g/ml each) and 25 m g/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 10 minutes at 4˚C and a clarification spin of the supernatant 10,000 g for 10 minutes at 4˚C in which cytochalasin D, AEBSF, aprotinin, leupeptin, pepstatin and chymostatin were again added. The resulting low-speed super-natants were then reincubated at 21˚C for 60 min and every 5 min, 2 m l 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, 44  heated at 85˚C for 5 minutes and stored at -20˚C (for Western blot analyses). Recombinant proteins.  Monomeric  Xenopus   wild-type ubiq-uitin coding sequence (76 amino acids) and Shc partial sequence (AA 236-431) 45  were subcloned into pQE30 expression vector (Qiagen, Courtaboeuf, France). The K48R ubiquin was prepared by site directed mutagenesis as recommended by the manufacturer (Stratagene Europe, The Netherlands) and the mutation was verified by DNA sequencing. His-tagged Shc and K48R ubiquitin proteins  were produced in the bacterial strain M15 by inducing protein expression with isopropyl-1-thio- b -D-galactoside (1 mM) for 3 h at 37˚C. After centrifugation, the bacterial pellets were incubated  with lysis buffer (6 M guanidine-HCl, 0.1M NaH 2 PO 4 , 10 mM Tris- HCl pH 8) overnight at room temperature to solubilize fusion proteins. Bacterial extracts were centrifuged at 10,000 g and 15˚C for 20 min. The supernatants were then loaded on Ni-NTA agarose (Qiagen) equilibrated columns. After the supernatants have flown through the resin, a series of washings was performed using the following buffers: 0.1 M NaH 2 PO 4 , 10 mM Tris-HCl with pH decreasing from 8 to 5.9 and urea concentrations varying from 8M to 0 to eliminate aspecific binding and progressively renature the fusion protein, respectively. Fusion proteins bound to the resin were eluted with a pH 4.5 buffer (0.1 M NaH 2 PO 4 , 10 mM tris-HCl, 0.5 mM AEBSF; pH 4,5) and recovered as 1 ml aliquots. The three most concentrated fractions were then centrifuged at 10,000 g through a Nanosep filter (3-KDa cut-off; Millipore, Guyancourt, France) to concentrate the proteins (10–20x) and for buffer exchange (elution buffer replaced by XB). Protein concentrations were estimated by the Bio-Rad protein assay. Proteins were aliquoted and stored at -70˚C until use. GST-p21 Cip  was produced as described. 46  The stock solu-tion was at 0.1 mg/ml and the final concentration in extracts equal to 6 m g/ml. Electrophoresis, antibodies and western blotting.  Extracts were subjected to electrophoresis on 8 to 12.5% SDS-PAGE gels. 44  Separated proteins were transferred to nitrocellulose membranes (Hybond C, Amersham Biosciences) according to standard proce-dures and probed either with antibodies against cyclin B2 (gift from Thierry Lorca), MCM4 (gift from Marcel Méchali), ERK2 and Phospho-ERK2 (Santa Cruz Biotechnology, CA, USA) or pentaHis (Qiagen). 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). MPF and ERK2 MAP Kinase in First Embryonic Mitosis     ©    2    0    0    7     L   A    N    D    E    S     B    I   O    S   C    I    E    N   C    E .     D   O     N   O    T     D    I    S    T    R    I    B    U    T    E .   In vitro assay for histone H1 kinase activity.  MPF activity in embryos or in cell-free extracts was measured as previously described 41   with minor modifications: extracts (1 m l) were diluted in 25 m l MPF buffer (80 mM b -glycerophosphate, 50 mM sodium fluoride, 20 mM EGTA, 15 mM MgCl 2 , 1 mM DTT, 20 mM HEPES, pH 7.4) supplemented with 0.5 mM sodium orthovanadate and 5 m g/ m l of leupeptin, aprotinin, pepstatin and chymostatin and containing 0.4 mg/ml H1 histone (type III-S), 1 m Ci [ g  32 P] 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 5 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. RESULTS K48R ubiquitin prolongs mitotic M-phase.  Inhibition of the proteolytic activity of the proteasome using MG-132 does not prevent MPF inactivation and hence does not interfere with mitotic progression in  Xenopus   cell-free extracts. 9  Therefore, we studied the effects of increasing amounts of recombinant K48R ubiquitin (Ub-K48R) added to extracts entering the first embryonic M-phase in vitro. Ub-K48R retarded cyclin B degradation in the extract in a dose-dependent manner (Fig. 1A), concomitantly enhancing histone H1 kinase activity and significantly prolonging its duration (Fig. 1B). Detailed analysis of cyclin B2 Western blots revealed that the uppermost, phosphorylated, band of cyclin B2 was more intense in Ub-K48R treated than in control extracts (compare the intensity of the upper band of cyclin B2 in control extract (XB) and extracts  with 45, 90, 135, 180 m M Ub-K48R in Fig. 1A). Ub-K48R addition triggered formation of a typical ladder of polyubiquitinated proteins  which increased in intensity with time (Fig. 1C). This shows that in contrast to MG-132 Ub-K48R interferes with MPF inactivation and perturbs deeply the mitotic progression in embryonic extracts.To verify the specificity of the effects of Ub-K48R we added the same amount of another recombinant His-tagged polypeptide of similar molecular weight, corresponding to a part of  Xenopus   Shc, which has no role in mitosis. 47  Shc did not influence neither cyclin B2 stability, nor MCM4 phosphorylation nor histone H1 kinase activation/inacti-vation (Fig. 2A and B). The ladder formation was not observed upon His-tagged Shc addition to the extract (data not shown). Figure 1. Ub-K48R inhibits cyclin B2 degradation, strengthens histone H1 kinase activity and induces a ladder of ubiquitinated proteins during the first embryonic M-phase in cell-free extract. Cytosolic extract was incubated at 21˚C for 1 hr with recombinant His-tagged Ub-K48R at 45, 90, 135 or 180 m M or with XB buffer added at the volume equivalent ( ≤  10%) to the highest concentration of Ub-K48R. Samples were collected every 5 min. for Western blotting with anti-cyclin B2 antibody (A), for histone H1 kinase assay (B) and for Western blotting with anti-pentaHis antibody (C; separate experiment from A and B).Figure 2. Xenopus  Shc protein (AA 236-431 of the 472 amino acids com-plete sequence), irrelevant to mitosis has minor effect on mitotic progression in cell-free extract. Cell-free extract was supplemented either with XB buffer or His-tagged Shc (200 m g in 100 m l of extract) before entry into M-phase and incubated at 21˚C for 70 min. Samples were collected every 5 min. for Western blotting with anti-cyclin B2 and anti-MCM4 antibodies (A) and for histone H1 kinase assay (B). MPF and ERK2 MAP Kinase in First Embryonic Mitosis  www.landesbioscience.com Cell Cycle 491     ©    2    0    0    7     L   A    N    D    E    S     B    I   O    S   C    I    E    N   C    E .     D   O     N   O    T     D    I    S    T    R    I    B    U    T    E .   Ub-K48R combined with proteasome inhibitor MG132 further enhances mitotic events.  Visualization of endogenous ubiquitin substrates by Western blotting often requires proteasome inhibition enabling higher accumulation of these substrates. 48,49  We reasoned therefore that the simultaneous addition of Ub-K48R and MG132 could enhance the effects of Ub-K48R alone on M-phase events. Cyclin B2 abundance and MCM4 phosphorylation/dephosphoryla-tion patterns were analyzed by Western blotting (Fig. 3A) and the MPF activity profile was assessed by histone H1 kinase assay (Fig. 3B) in mitotic extracts supplemented with both Ub-K48R/MG132. Cyclin B2 was present for a longer time and in higher quantities in Ub-K48R/MG132-containing samples, MCM4 phosphorylation and histone H1 kinase activity were also prolonged. In a similar experiment we compared cyclin B2 abundance in control extract and in extracts supplemented with MG132 alone, Ub-K48R alone and Ub-K48R/MG132 (Fig. 4A). Clearly, cyclin B2, and particularly its phosphorylated forms accumulate to higher amounts in the pres-ence of Ub-K48R/MG132 than Ub-K48R alone following MPF inactivation (Fig. 4B). This suggests that double interference with ubiquitin-proteasome pathway via Ub-K48R/MG132 addition may exert a synergistic effect on MPF activity via increased cyclin B2 stability and phosphorylation. Modulation of MPF activity modulates ERK2 phosphoryla-tion, while levels of ERK2 phosphorylation do not influence MPF activity.  Since Ub-K48R appeared as a useful tool to increase the mitotic histone H1 kinase and the combination of Ub-K48R+MG132 increased this activity even further, we asked how ERK2 MAP kinase behaved in these experimental conditions. ERK2 phosphorylation  was followed by Western blotting using anti-phospho-ERK anti-body in the experiment shown in Figure 3. The intensity of ERK2 phosphorylation was clearly increased by Ub-K48R addition to the control extract and increased further by Ub-K48R/MG132 addition (Fig. 5A and 2B) in line with the effect on MPF (Fig. 3B). MG132 alone does not change either the MPF 9  or ERK2 phosphorylation (data not shown). This showed that MPF, directly or indirectly, increases ERK2 phosphorylation.To determine directly whether the observed increase in ERK2 phosphorylation was a result of MPF activation rather than an independent effect on other unknown ubiquitin-dependent event  we partially inhibited MPF activity in a control extract by addition of a CDK-specific inhibitor protein p21 Cip and monitored ERK2 phosphorylation. Since our goal was to diminish and not to abolish MPF activity, we used 6 m g/ml p21 Cip . This dose is seven times less Figure 3. Concomitant presence of Ub-K48R and the proteasome inhibitor MG132 in mitotic extract extends MCM4 phosphorylation, further inhibits cyclin B2 degradation and enhances histone H1 kinase activity in compari-son to the presence of Ub-K48R alone. Cell-free extract was supplemented with XB buffer, 200 m M His-tagged Ub-K48R or with 200 m M His-tagged Ub-K48R + 100 m M MG132 and incubated for 60 min at 21˚C. Samples were collected every 5 min. for Western blotting with anti-MCM4 and anti-cyclin B2 antibodies (A) and histone H1 kinase assay (B).Figure 4. Effect of the concomitant presence of Ub-K48R and MG132 and Ub-K48R or MG132 alone on cyclin B2 in mitotic extract. Cell-free extract was supplemented with XB buffer, 100 m M MG132, 200 m M His-tagged Ub-K48R or concomitantly with 200 m M His-tagged Ub-K48R + 100 m M MG132 and incubated for 60 min at 21˚C. Samples were collected every 5 min. for Western blotting with anti-MCM4 and anti-cyclin B2 antibodies (A). The Western blots with anti-cyclin B2 were quantified densitometrically for the phosphorylated forms of cyclin B2 (B), the results being expressed as a fold-increase of cyclin B2 amount compared to that at the beginning of the 1-hour incubation. MPF and ERK2 MAP Kinase in First Embryonic Mitosis 492 Cell Cycle 2007; Vol. 6 Issue 4     ©    2    0    0    7     L   A    N    D    E    S     B    I   O    S   C    I    E    N   C    E .     D   O     N   O    T     D    I    S    T    R    I    B    U    T    E .   than used previously to inhibit CDK1 totally in prophase oocytes. 50   As expected, in such extracts activation of MPF was delayed and its activity reduced (Fig. 6A), while cyclin B2 was degraded 10 min later. The period of maximal mitotic phosphorylation of MCM4 was correspondingly shortened indicating that the M-phase conditions  were shortened similarly as happens during the second embryonic M-phase 41  (Fig. 6B; double arrows). ERK2 phosphorylation was clearly reduced to approximately half, in line with MPF activity (Fig. 6C). Again, this resembles ERK2 behaviour during the second embryonic mitosis. 41 To determine whether conversely ERK2 influences MPF activity,  we added the inhibitor of MEK/ERK2 pathway U0126 to the extract supplemented with Ub-K48R. MCM4 and cyclin B2 in the extracts containing Ub-K48R was not affected by U0126 (Fig. 7A), nor was histone H1 kinase activation/inactivation (Fig. 7B), despite the total abolition of ERK2 phosphorylation (Fig. 7C and D). Altogether, these data show that MPF triggers ERK2 phosphorylation in mitotic extracts, but that unlike in meiosis, feedback between ERK2 and MPF activity is not detectable. DISCUSSION Polyubiquitination-proteasome pathway and MPF inactivation.  Little is known about the critical step of MPF inactivation, i.e., the proteasome-dependent dissociation of cyclin B from CDK. This role is, however, attributed to the lid of the 19S regulatory subunit of the proteasome during meiotic M-phase exit. 8  We have shown that this mechanism is also responsible for MPF inactivation not only upon proteasome proteolytic activity inhibition, but also during unper-turbed embryonic mitoses in  Xenopus    laevis   extract. 9  MPF activity is not arrested at high mitotic levels in the presence of the proteasome inhibitors MG132 or ALLN because the dissociation of cyclins B from CDK1 is not inhibited by these drugs. Therefore, these drugs efficiently slow down cyclin B degradation but do not interfere with MPF inactivation. 9  The effects of Ub-K48R on biochemical mitotic events in cell-free extracts show clearly that this recombinant protein interferes with both cyclin B2 degradation and MPF inactivation, in contrast to MG132 or ALLN. Ub-K48R therefore acts up-stream from cyclin B dissociation from CDK1. Figure 5. ERK2 MAP kinase phosphorylation augments upon Ub-K48R addition and this effect is further enhanced by the concomitant presence of Ub-K48R and MG132. Cell-free extract was supplemented with XB buffer, 200 m M His-tagged Ub-K48R or concomitantly with 200 m M His-tagged Ub-K48R and 100 m M MG132 and incubated for 60 min at 21˚C. Samples were collected every 5 min. for Western blotting with anti-ERK2 and anti-Phospho-ERK2 (P-ERK2) antibodies (A). The Western blots with anti-ERK2 and anti-P-ERK2 were quantified densitometrically and the phosphorylated ERK2 / total ERK2 ratios were plotted (B).Figure 6. Diminution of CDK1 activity is correlated with a decrease in mitotic ERK2 phosphorylation. Mitotic cell-free extract was supplemented with puri-fied p21 Cip  at a final concentration of 6 m g/ml, or with the buffer in which recombinant p21 Cip  was obtained (6 m l of the buffer alone or p21 Cip were added to 100 m l of the extract respectively) and incubated for 60 min at 21˚C. Samples were collected every 5 min. for histone H1 kinase assay (A), Western blotting with anti-cyclin B2 and MCM4 (B) as well as anti-ERK2 and anti-Phospho-ERK2 (P-ERK2) antibodies. The two latter were quantified and the values representing phosphorylated ERK2 / total ERK2 ratios plotted (C). Double-headed arrows in MCM4 blot shows the period of the maximal phosphorylation of this protein indicating that p21 Cip  treatment shortens this period. MPF and ERK2 MAP Kinase in First Embryonic Mitosis  www.landesbioscience.com Cell Cycle 493
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