Dual inhibition of Cdc2 protein kinase activation during apoptosis in Xenopus egg extracts

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1 Dual inhibition of Cdc2 protein kinase activation during apoptosis in Xenopus egg extracts Yuichi Tsuchiya, Shin Murai and Shigeru Yamashita Department of Biochemistry, Toho University School of Medicine, Ota-ku, Tokyo, Japan Keywords apoptosis; Cdc2; Cdc25C; cyclin B; Xenopus egg extracts Correspondence Y. Tsuchiya, Department of Biochemistry, Toho University School of Medicine, Omori-nishi, Ota-ku, Tokyo, Japan Fax: Tel: ext tsuchiya@med.toho-u.ac.jp (Received 25 July 2014, revised 12 January 2015, accepted 26 January 2015) doi: /febs When intracellular damage accumulates in proliferating somatic cells, the cell cycle usually arrests in G 1 or G 2 in a checkpoint-dependent manner, either to repair the damage or to die by apoptosis. In contrast, early embryonic cells lack checkpoint-mediated cell-cycle arrest, and it is not clear whether apoptosis in early embryonic cells occurs at a specific cell cycle stage or at random points. Here, we examined the functional molecular link between the embryonic cell cycle and apoptosis using Xenopus egg extracts. When apoptosis was induced in egg extracts by addition of exogenous cytochrome c during cellcycle progression, cyclin B accumulation was inhibited, Cdc2 was not activated, and the cell cycle arrested at interphase. However, addition of recombinant cyclin B failed to activate Cdc2 due to the strong inhibitory phosphorylation of Cdc2 Tyr15 in apoptotic egg extracts. We found that endogenous Cdc25C, which activates the Cdc2 cyclin B complex by dephosphorylating Cdc2 Tyr15, was inactivated by caspase-mediated cleavage at two sites in the N-terminal regulatory domain. When the hyperactive Cdc25A catalytic fragment was added together with recombinant cyclin B to artificially dephosphorylate Cdc2 Tyr15, M-phase induction was restored in apoptotic egg extracts, indicating that the blockage of cyclin B accumulation and the caspase-mediated inactivation of Cdc25C dually inhibited Cdc2 activation. Apoptosis induction in cytostatic factor-arrested metaphase egg extracts resulted in inactivation of Cdc2 without cyclin B degradation. These results suggest that apoptotic inactivation of Cdc25C plays an important role in arresting the embryonic cell cycle at interphase during apoptosis. Introduction During the execution phase of apoptosis, various cellular activities are affected by the caspase-mediated cleavage of key regulatory proteins. Despite the significant impact of caspase-mediated cleavage on physiological functions, a relatively small number of substrate proteins have been identified, and many targets remain to be found [1,2]. Therefore, it is important to identify new, physiologically important substrate proteins for caspases, and to examine how their functions are modified by caspase-mediated cleavage. When intracellular damage accumulates in proliferating somatic cells, the cell cycle usually arrests in G 1 or G 2 in a checkpoint-dependent manner to repair the damage. If damage is too severe to repair, cells initiate the cytochrome c-mediated intrinsic apoptosis pathway to cause death. In contrast, early embryonic cells lack checkpoint-mediated cell-cycle arrest. These cells cannot repair the damage, and inevitably die by apoptosis at later stages. It is not clear whether the apoptosis of early embryonic cells occurs at a specific cell cycle stage or at random points. Abbreviations Cdk, cyclin-dependent kinase; CHX, cycloheximide; CSF, cytostatic factor; Cyt c, cytochrome c; HA, hemagglutinin; MBP, maltose-binding protein; MPF, M phase-promoting factor; PARP, poly(adp-ribose) polymerase; PP1, protein phosphatase FEBS Journal 282 (2015) ª 2015 FEBS

2 Y. Tsuchiya et al. Caspase-mediated cleavage of Xenopus Cdc25C To address this issue, used employed a cell-free experimental system using egg extracts from Xenopus laevis (called Xenopus hereafter). This system is an excellent, widely used biochemical model of the embryonic cell cycle. Currently, two types of egg extracts are frequently used to study the embryonic cell cycle [3]. Cytostatic factor (CSF)-arrested metaphase egg extracts are prepared from unfertilized eggs in the absence of calcium and naturally arrested at metaphase. The addition of 0.4 mm CaCl 2 induces degradation of cyclin B and inactivation of the Mos MEK MAPK Rsk kinase cascade, which is the major CSF components, thereby releasing the egg extracts from metaphase arrest. Subsequent synthesis and accumulation of cyclin B drives the egg extracts into the second M phase after ~ min. In most cases, extracts become arrested at this second M phase, most likely due to incomplete inactivation of CSF [3]. In contrast, cycling egg extracts are prepared from calcium ionophore-treated, parthenogenetically activated interphase eggs. CSF is completely inactivated after ionophore treatment, and the synthesis/degradation cycle of endogenous cyclin B induces the first M phase after ~ 40 min and the second M phase after ~ 100 min. In addition, Xenopus egg extracts are used as a model system of the intrinsic apoptotic program [4 10]. When interphase egg extracts are treated with the general translation inhibitor cycloheximide (CHX) and incubated for several hours, cytochrome c (Cyt c) is spontaneously released from the mitochondria to the cytoplasm and activates the intrinsic apoptosis pathway. Alternatively, apoptosis may be rapidly triggered by adding exogenous Cyt c into any type of egg extracts. Therefore, this cell-free system is an ideal tool to study the direct functional molecular link between the cell cycle and apoptosis. Cyclin-dependent kinases (Cdks) play central roles to drive the cell cycle. Cdk requires the synthesis of specific cyclin subunits to be activated. In addition, the kinase activities of Cdks are also regulated by post-translational phosphorylation and dephosphorylation. In Xenopus egg extracts, M-phase induction requires an active complex of Cdc2 (also known as Cdk1) and cyclin B known as M phase-promoting factor (MPF), and rapid cell-cycle progression is driven by the synthesis/degradation wave of cyclin B. In addition, Wee1A kinase inactivates MPF by phosphorylating the Tyr15 of Cdc2. A related kinase, Myt1, also phosphorylates both Thr14 and Tyr15 of Cdc2. In contrast, Cdc25C phosphatase dephosphorylates both Thr14 and Tyr15 of Cdc2 to activate MPF. Thus, the functional balance among Wee1A, Myt1 and Cdc25C controls cell-cycle progression through the regulation of MPF [11]. In this study, we show that the blockage of cyclin B accumulation and the caspase-mediated inactivation of Cdc25C dually inhibit the activation of MPF, thereby ensuring cell-cycle arrest at interphase in apoptotic egg extracts. Results The embryonic cell cycle was arrested at interphase in apoptotic egg extracts To investigate the functional molecular link between the cell cycle and apoptosis, we first added exogenous Cyt c to two types of egg extracts, and examined whether apoptosis affects the embryonic cell cycle. When CSFarrested metaphase egg extracts were supplied with CaCl 2, both cyclin B1 and cyclin B2 were rapidly degraded, and egg extracts were released into interphase within 15 min. In control egg extracts, the subsequent synthesis and accumulation of endogenous cyclin B1 and cyclin B2 drove the egg extracts into M phase after ~ 120 min, as determined by reactivity with the M phase-specific MPM-2 antibody, which specifically recognizes mitotically Ser/Thr-phosphorylated proteins. The entry into M phase was also confirmed by the dephosphorylation of Cdc2 Tyr15. After 120 min, cyclin B1 was degraded again, whereas cyclin B2 remained stable, and egg extracts were arrested at the second M phase. During this normal embryonic cell cycle, apoptotic cleavage of poly(adp)-ribose polymerase (PARP) was not observed (Fig. 1A, control). In the presence of CHX, endogenous cyclin B1 and cyclin B2 were both degraded at M-phase exit but did not accumulate afterwards, and CHX-treated egg extracts were arrested at interphase, as determined by the absence of MPM-2 staining. CHX addition itself did not induce apoptotic PARP cleavage for at least 120 min (Fig. 1A, +CHX). When exogenous Cyt c was added together with CaCl 2, apoptosis was rapidly induced, as indicated by the apoptotic PARP cleavage within 15 min. Neither MPM-2 staining nor accumulation of endogenous cyclin B1 and cyclin B2 were observed after M-phase exit, indicating that Cyt c-treated egg extracts were arrested at interphase, similar to CHX-treated egg extracts (Fig. 1A, +Cyt c). The cell cycle in cycling egg extracts started from interphase, with a modest amount of endogenous cyclin B1 and cyclin B2 at 0 min. A further synthesis/ degradation wave of endogenous cyclin B1 and cyclin B2 induced the first M phase after ~ 40 min and the second M phase after ~ 100 min in control egg extracts (Fig. 1B, control). In CHX-treated egg extracts, the cell cycle remained at interphase, without further synthesis of cyclin B1 and cyclin B2. MPM-2 FEBS Journal 282 (2015) ª 2015 FEBS 1257

3 Caspase-mediated cleavage of Xenopus Cdc25C Y. Tsuchiya et al. A B Fig. 1. The embryonic cell cycle was arrested at interphase in apoptotic egg extracts. (A) Cell-cycle progression of CSF-arrested egg extracts. Control, CHX-treated and Cyt c-treated CSF-arrested metaphase extracts were supplied with 0.4 mm CaCl 2 at 0 min and incubated. Samples were taken every 15 min. (B) Cell-cycle progression of cycling egg extracts. Control, CHX-treated and Cyt c-treated cycling egg extracts were incubated. Samples were taken every 10 min. In (A) and (B), samples were subjected to western blotting against PARP (aparp), M-phase phosphorylated proteins (MPM-2), total Cdc2, Tyr15- phosphorylated Cdc2 (acdc2 py15), cyclin B1 and cyclin B2. The M s under the gels indicate the duration of M phase. Experiments were repeated at least three times using different batches of eggs, and representative images from one experiment are shown. staining was not observed during incubation, despite the dephosphorylation of Cdc2 Tyr15 and the slow degradation of endogenous cyclin B1 and cyclin B2 after 100 min (Fig. 1B, +CHX). In Cyt c-treated egg extracts, the initial accumulation and degradation of endogenous cyclin B1 and cyclin B2 were not affected, and the first M phase was induced correctly after ~ 40 min. However, endogenous cyclin B1 and cyclin B2 did not accumulate afterwards, and the second M phase was not induced (Fig. 1B, +Cyt c). Apoptotic PARP cleavage was observed within 10 min in Cyt c- treated egg extracts but not in control or CHX-treated egg extracts. The results obtained from the two types of egg extracts suggested that the rapid induction of apoptosis blocked accumulation of endogenous cyclin B1 and cyclin B2 and arrested the cell cycle at interphase. Cyclin B translation was blocked in apoptotic egg extracts To examine why cyclin B did not accumulate in apoptotic egg extracts, we first supplied a radioactive 35 S-Cys/Met mixture to egg extracts and directly detected new protein synthesis. In control CSFarrested metaphase egg extracts, proteins of various sizes were gradually radiolabeled after addition of calcium (Fig. 2A, control). Among these, the most strongly radiolabeled proteins of ~ 50 kda gradually increased and then abruptly disappeared after 150 min, just after the transient appearance of highmolecular-weight radiolabeled proteins at ~ min (Fig. 2A, HMW). This behavior was similar to that of endogenous cyclin B1 (Fig. 1A, acycb1), and also of classical cyclin reported by Murray and Kirschner [12]. In contrast, no radiolabeled proteins were observed in CHX-treated egg extracts (Fig. 2A, +CHX). In Cyt c-treated egg extracts, radiolabeled proteins, including the cyclin B1-like 50 kda proteins, were first detected after 30 min but did not increase further (Fig. 2A, +Cyt c). We therefore suspected that protein synthesis was blocked min after addition of Cyt c in apoptotic egg extracts, thereby preventing accumulation of cyclin B1-like 50 kda proteins FEBS Journal 282 (2015) ª 2015 FEBS

4 Y. Tsuchiya et al. Caspase-mediated cleavage of Xenopus Cdc25C A B C Fig. 2. Cyclin B translation was blocked in apoptotic egg extracts. (A) Radiolabeling of newly synthesized endogenous proteins in egg extracts. Control, CHX-treated and Cyt c-treated CSF-arrested metaphase extracts were supplied with 35 S-Cys/Met and CaCl 2 at 0 min and incubated. Samples were taken every 15 min, separated by SDS/PAGE, and analyzed using a BAS-5000 image analyzer. The cyclin B1-like 50 kda proteins and high-molecular-weight proteins (HMW) are indicated. (B) Examination of the amount and poly(a) length of cyclin B1 and cyclin B2 mrnas. CSF-arrested egg extracts were incubated for 1 h in the absence (control) or presence of exogenous Cyt c. Z-VAD-FMK was also added to block caspase activation (+Cyt c +VAD). After incubation, total RNA was prepared and subjected to RT-PCR to amplify the poly(a) regions of cyclin B mrnas. Amplified products were separated by electrophoresis in 1% agarose and stained using ethidium bromide. (C) Accumulation of endogenous cyclin B2 in apoptotic egg extracts. CSF-arrested metaphase extracts were supplied with CaCl 2 and Cyt c at 0 min and incubated. MG-132 was also added at 0 min (left) or 30 min (right). Samples were taken every 30 min, separated by SDS/PAGE, and subjected to western blotting against cyclin B2. Experiments were repeated at least three times using different batches of eggs, and representative images from one experiment are shown. We next examined whether the reduction of cyclin B protein accumulation during apoptosis was due to modification of mrna, such as degradation and deadenylation. RT-PCR was performed to specifically amplify the poly(a) tail of cyclin B1 and cyclin B2 mrnas in egg extracts in the absence or presence of exogenous Cyt c. The pan-caspase inhibitor, Z-VAD- FMK, was also used to block caspase activation during incubation. However, the amount and length of the cyclin B1 mrna poly(a) tail was not significantly different between control and apoptotic egg extracts (Fig. 2B, CycB1), and only a fraction of the cyclin B2 mrna poly(a) tail was reduced in length in apoptotic egg extracts (Fig. 2B, CycB2). These results suggested that mrna availability was not the major reason for the loss of cyclin B protein accumulation in apoptotic egg extracts. At M-phase exit, cyclin B is specifically polyubiquitylated by anaphase-promoting complex and degraded by proteasome over a short time window. Therefore, the lack of cyclin B accumulation observed in apoptotic extracts may be due to prolonged proteasome-mediated degradation. If so, cyclin B should accumulate normally when proteasome is inhibited in apoptotic extracts. To test this hypothesis, we used the proteasome inhibitor MG-132 to determine whether endogenous cyclin B2 accumulates after M-phase exit in Cyt c-treated extracts. When MG-132 was added together with CaCl 2 and Cyt c at 0 min, the degradation of endogenous cyclin B2 at M-phase exit was effectively blocked (Fig. 2C, left). In contrast, if MG- 132 was added 30 min after CaCl 2 and Cyt c, endogenous cyclin B2 was completely degraded at M-phase exit and did not accumulate thereafter (Fig. 2C, right). This result suggested that the lack of cyclin B accumulation was due to translational inhibition rather than prolonged degradation. Altogether, we conclude that the lack of cyclin B accumulation in apoptotic egg extracts was due to the inhibition of protein synthesis rather than mrna modification or prolonged protein degradation. Cdc2 Tyr15 dephosphorylation was blocked in apoptotic egg extracts If the insufficiency of cyclin B were the sole reason for the interphase arrest of apoptotic egg extracts, then addition of excess recombinant cyclin B would induce M phase. To test this hypothesis, we pre-incubated either CHX-treated or Cyt c-treated cycling egg extracts for 90 min to ensure interphase arrest, and then added recombinant maltose-binding protein-fused FEBS Journal 282 (2015) ª 2015 FEBS 1259

5 Caspase-mediated cleavage of Xenopus Cdc25C Y. Tsuchiya et al. cyclin B1 (MBP cyclin B1) purified from bacteria to egg extracts. We found that addition of MBP cyclin B1 induced M-phase arrest in egg extracts, most likely due to its resistance to proteasome-mediated degradation. Consistent with the historical paper by Solomon et al. [13], MBP cyclin B1 drove CHX-treated egg extracts into M phase after a lag time of ~ 40 min, as determined from the appearance of MPM-2 reactivity and the disappearance of Cdc2 Tyr15 phosphorylation (Fig. 3, CycB1 + CHX). In marked contrast, MBP cyclin B1 did not induce M phase in Cyt c-treated egg extracts. Instead, an abnormally high level of Cdc2 Tyr15 phosphorylation was observed (Fig. 3, CycB1 + Cyt c), indicating that the regulatory system of Cdc2 Tyr15 phosphorylation was also affected in apoptotic egg extracts. Xenopus Cdc25C was converted into smaller fragments in apoptotic egg extracts The above results suggested that either the phosphatase dephosphorylating Cdc2 Tyr15 (Cdc25C) was inactivated, or the kinases phosphorylating Cdc2 Tyr15 (Wee1A and Myt1) were activated, or both. We next performed western blotting against these three proteins to examine whether they were modified during apoptosis in egg extracts. These three proteins are functionally regulated by mitotic phosphorylation. At M phase, hyperphosphorylated Cdc25C is activated Fig. 3. Cdc2 Tyr15 dephosphorylation was blocked in apoptotic egg extracts. CHX-treated (CycB1 + CHX) and Cyt c-treated (CycB1 + Cyt c) cycling egg extracts were pre-incubated for 90 min to ensure interphase arrest. After 90 min, egg extracts were supplied with 0.1 mgml 1 recombinant MBP cyclin B1 and further incubated. Samples were taken every 20 min, separated by SDS/PAGE, and subjected to western blotting against M-phase phosphorylated proteins (MPM-2), total Cdc2 and Tyr15- phosphorylated Cdc2 (acdc2 py15). Experiments were repeated at least three times using different batches of eggs, and representative images from one experiment are shown. The M below the gel indicates the duration of M phase. [14 17], whereas hyperphosphorylated Wee1A and Myt1 are inactivated [18 22]. It was necessary to distinguish whether the observed electrophoretic mobility shifts observed in western blotting were due to caspase-mediated cleavage or changes in phosphorylation status. In control extracts, endogenous Cdc25C migrated as a hyperphosphorylated active 90 kda form at M phase, whereas it migrated as an inactive 70 kda form at interphase [14 17] (Fig. 4, control, acdc25c total). Phosphorylation of Ser287, which inhibits the function of Cdc25C, was detected at interphase, but not at M phase (Fig. 4, control, acdc25c ps287). In CHX-treated egg extracts, Cdc25C remained as the Ser287-phosphorylated 70 kda form after exit from M phase (Fig. 4, +CHX, acdc25c total and acdc25c ps287). In Cyt c-treated extracts, both the 90 and 70 kda forms disappeared during incubation. Instead, two new forms of 60 and 50 kda, both phosphorylated at Ser287, gradually appeared (Fig. 4, +Cyt c, acdc25c total and acdc25c ps287). In contrast, the electrophoretic mobility shifts of endogenous Wee1A and Myt1, low at M phase and high at interphase, were similar between CHX-treated and Cyt c-treated extracts (Fig. 4, awee1a and amyt1). Therefore, we suspect that Cdc25C, rather than Wee1A and Myt1, is specifically modified in apoptotic egg extracts. Xenopus Cdc25C was cleaved after Asp108 and/ or Asp179 by caspases in apoptotic egg extracts Caspases, a family of apoptosis-related cysteine proteases, cleave various protein substrates after specific Asp residues during apoptosis. To determine whether the apoptotic conversion of Cdc25C in Cyt c-treated egg extracts was due to caspase-mediated cleavage, we used the pan-caspase inhibitor Z-VAD-FMK. As expected, Z-VAD-FMK, but not the proteasome inhibitor MG-132, effectively inhibited the generation of smaller fragments derived from endogenous Cdc25C (Fig. 5A), indicating that Cdc25C was cleaved by caspases in apoptotic egg extracts. To determine the cleavage sites of Cdc25C, we used 35 S-radiolabeled recombinant 6xHis-tagged Cdc25C translated in rabbit reticulocyte lysates. The antigen for our Cdc25C antibody is the C-terminal catalytic domain, and Ser287 is located in the middle part of Cdc25C. As both Cdc25C antibodies recognized the same fragments, caspase-mediated cleavage most likely occurred at the N-terminal region of Cdc25C. Therefore, we mutated various Asp residues in the N-terminus of recombinant 6xHis-tagged Cdc25C to Asn, and determined whether the resulting mutant proteins were 1260 FEBS Journal 282 (2015) ª 2015 FEBS

6 Y. Tsuchiya et al. Caspase-mediated cleavage of Xenopus Cdc25C Fig. 4. Xenopus Cdc25C was converted into smaller fragments in apoptotic egg extracts. Control, CHX-treated and Cyt c-treated CSFarrested metaphase extracts were supplied with 0.4 mm CaCl 2 at 0 min, and incubated as in Fig. 1A. Samples were taken every 15 min, separated by SDS/PAGE, and subjected to western blotting against total Cdc25C, Ser287-phosphoryalted Cdc25C (acdc25c ps287), Wee1A and Myt1. The hyper-phosphorylated active 90 kda form, inactive 70 kda form, and apoptotic 60 kda and 50 kda forms of Cdc25C are indicated by arrowheads. The asterisk on the left indicates a cross-reacting protein that overlaps with the 60 kda fragment of Cdc25C. Experiments were repeated at least three times using different batches of eggs, and representative images from one experiment are shown. cleaved in apoptotic egg extracts. In control interphase egg extracts, full-length 6xHis-tagged Cdc25C wildtype was expressed as a 75 kda protein in the absence of exogenous Cyt c, but was cleaved into smaller fragments (60, 50, 25 and 15 kda) when Cyt c was added (Fig. 5B). When Asp108 was mutated to a caspaseresistant Asn residue (D108N), the 60 kda/15 kda apoptotic cleavage fragments disappeared, but the 50 kda/25 kda fragments were unaffected. In another mutant in which Asp179 was mutated to Asn (D179N), 50 kda/25 kda fragments were not observed, but 60 kda/15 kda fragments were still generated. Finally, when both Asp108 and Asp179 were mutated to Asn (D108N/D179N), this double mutant was resistant to apoptotic cleavage, indicating that Cdc25C was cleaved at one of these two sites in apoptotic egg extracts (Fig. 5B). As schematically represented in Fig. 5C, either of these cleavages removes the protein phosphatase 1 (PP1) binding site [23, 24], nuclear export signal [25, 26], and some activating phosphorylation sites [27 31] from the catalytic domain. In contrast, Ser287, the binding site phosphorylated by multiple checkpoint kinases [11], the nuclear localization signal [26] and Rsk2 phosphorylation sites [31] are retained. Oocyte maturation-promoting activities of Xenopus Cdc25A and Cdc25C were differently regulated by N-terminal truncations Four Cdc25 family phosphatases (Cdc25A, Cdc25B, Cdc25C and Cdc25D) have been identified in Xenopus [32, 33]. Among these, only Cdc25C is present in oocytes and eggs, and acts as the physiological M- phase inducer in egg extracts. In contrast, Cdc25A is absent in oocytes and eggs due to rapid degradation by the ubiquitin/proteasome pathway, and then gradually accumulates during embryogenesis [34 37]. It was recently reported that human Cdc25A was cleaved by caspases to generate a fragment that activates Cdks during apoptosis [38, 39]. The cleavage site of Cdc25A is evolutionarily conserved (Asp234 in Xenopus Cdc25A). We preliminarily observed that the recombinant Xenopus Cdc25A D234N mutant was not cleaved by caspases in apoptotic egg extracts, although its rapid proteasome-mediated degradation prevented further analysis (data not shown). To test whether the apoptotic N-terminal cleavages affect the functions of Cdc25A and Cdc25C, we used a well-established Xenopus oocyte maturation system. Fully grown Xenopus immature oocytes are arrested at meiotic prophase I, and the inactive Tyr15-phosphorylated Cdc2 cyclin B2 complex is stockpiled, whereas cyclin B1 is absent from immature oocytes. If an injected material dephosphorylates Cdc2 Tyr15 and activates the endogenous Cdc2 cyclin B2 complex, a white spot will appear at the animal pole of the oocyte, known as germinal vesicle breakdown. Although Cdc25C is the physiological activator of the Cdc2 cyclin B2 complex during oocyte maturation, Cdc25A activates the Cdc2 cyclin B2 complex more efficiently than Cdc25C when ectopically over-expressed in immature oocytes [40]. We injected mrnas encoding EF1a (negative control), and N-terminally hemagglutinin (HA)-tagged FEBS Journal 282 (2015) ª 2015 FEBS 1261

7 Caspase-mediated cleavage of Xenopus Cdc25C Y. Tsuchiya et al. A B Fig. 5. Xenopus Cdc25C in apoptotic egg extracts was cleaved by caspases after Asp108 and/or Asp179. (A) Apoptotic Cdc25C conversion by caspase-mediated cleavage. CSF-arrested metaphase extracts (CSF), CHX-treated interphase extracts (+CHX), Cyt c- treated apoptotic extracts, (+Cyt c), Cyt c-treated extracts containing the caspase inhibitor Z-VAD-FMK (Cyt c + VAD), and Cyt c-treated extracts containing the proteasome inhibitor MG-132 (Cyt c + MG) were incubated for 2 h, separated by SDS/PAGE, and subjected to western blotting against total Cdc25C. The hyperphosphorylated active 90 kda form, inactive 70 kda form, and apoptotic 60 kda and 50 kda forms of Cdc25C are indicated by arrowheads. The asterisk indicates a cross-reacting protein that overlaps with the 60 kda fragment of Cdc25C. (B) Identification of the caspase-mediated cleavage sites of Cdc25C as Asp108 and Asp179. Rabbit reticulocyte lysates containing 35 S-radiolabeled recombinant 6xHis-tagged Cdc25C derivatives (WT, D108N, D179N and D108/179N) were mixed with CHX-treated interphase extracts in the absence ( ) or presence (+) of exogenous Cyt c, and incubated for 2 h. Samples were separated by SDS/PAGE and analyzed using a BAS-5000 image analyzer. The 75 kda full-length form and four caspase-generated fragments (60 kda, 50 kda, 25 kda and 15 kda) are indicated by arrowheads. Experiments were repeated at least three times using different batches of eggs, and representative images from one experiment are shown. (C) Schematic presentation of the caspase-mediated cleavage sites and functionally important domains in Xenopus Cdc25C. C recombinant proteins including HA Cdc25AFL (residues 1 525, full-length form of Cdc25A), HA Cdc25ADN (residues , C-terminal fragment of Cdc25A), HA Cdc25CFL (residues 1 550, full-length form of Cdc25C), HA Cdc25CDN1 (residues , C-terminal 60 kda fragment of Cdc25C) and HA Cdc25CDN2 (residues , C-terminal 50 kda fragment of Cdc25C), into immature oocytes to overexpress the recombinant proteins (Fig. 6A). HA Cdc25ADN, which lacks the recognition motifs for the ubiquitin/proteasome pathway, was expressed more efficiently than HA Cdc25AFL, whereas all three derivatives of recombinant HA Cdc25C were expressed at comparative levels. Western blotting revealed that both HA Cdc25AFL and HA Cdc25ADN efficiently dephosphorylated Cdc2 Tyr15 and induced M phase. In contrast, all three Cdc25C derivatives were less efficient at M-phase induction (Fig. 6B). When the numbers of oocytes showing germinal vesicle breakdown were counted, both HA Cdc25AFL and HA Cdc25ADN induced oocyte maturation as efficiently as progesterone, a physiological inducer of oocyte maturation that was used as a positive control (Fig. 6C). In contrast, HA Cdc25CFL induced oocyte maturation with approximately half the efficiency of progesterone, consistent with a previous report [40]. Notably, the oocyte maturation-inducing activity of HA Cdc25CDN1 was much lower than that of HA Cdc25CFL. HA Cdc25CDN2 was almost inactive (Fig. 6D). These results suggest that, in contrast with Cdc25A, Cdc25C is inactivated upon caspase-mediated N-terminal cleavage. M-phase induction was restored when cyclin B and hyperactive Cdc25A were added together in apoptotic egg extracts The above results prompted us to investigate whether supplementation of cyclin B and dephosphorylation of Cdc2 Tyr15 were sufficient to restore M-phase induction in apoptotic egg extracts. To prepare caspase-resistant Cdc25C, we first tried to purify the Cdc25C D108N/D179N double mutant from various expression systems, but failed to obtain a sufficient amount of recombinant protein. Instead, we expressed 1262 FEBS Journal 282 (2015) ª 2015 FEBS

8 Y. Tsuchiya et al. Caspase-mediated cleavage of Xenopus Cdc25C A B C D Fig. 6. Oocyte maturation-promoting activities of Xenopus Cdc25A and Cdc25C were differently regulated by N-terminal truncations. (A) Schematic presentation of recombinant Xenopus Cdc25A and Cdc25C derivatives used in this assay. (B) Expression of recombinant Cdc25A and Cdc25C derivatives in immature Xenopus oocytes. Synthetic mrnas encoding EF1a (control), HA Cdc25AFL, HA Cdc25ADN, HA Cdc25CFL, HA Cdc25CD1 and HA Cdc25CD2 (as defined in the text), were injected into immature oocytes. After incubation for 6 h, oocytes were crushed and solubilized. Extracted samples were separated by SDS/PAGE and subjected to western blotting against the HA tag, M-phase phosphorylated proteins (MPM-2), total Cdc2, Tyr15-phosphorylated Cdc2 (acdc2 py15) and cyclin B2. The sizes of molecular mass markers are indicated on the right. (C) Oocyte maturation induced by over-expression of Cdc25A derivatives. (D) oocyte maturation induced by over-expression of Cdc25C derivatives. Each mrna was injected into ~ 30 oocytes. Values are means SEM (n = 4). Experiments were repeated at least three times using different batches of eggs, and representative images from one experiment are shown. and purified Cdc25ADN, which dephosphorylated Cdc2 Tyr15 efficiently (Fig. 6), as an N-terminally 6xHis-tagged recombinant protein from bacteria. We pre-incubated Cyt c-treated cycling egg extracts for 90 min to ensure interphase arrest and caspase-mediated cleavage of endogenous Cdc25C, and then added the recombinant proteins to egg extracts. As in Fig. 3, addition of MBP CycB1 alone induced strong Cdc2 Tyr15 phosphorylation, but no MPM-2 reactivity was observed in apoptotic egg extracts (Fig. 7, +CycB1). When 6xHis-tagged Cdc25ADN was added to apoptotic egg extracts alone, Cdc2 Tyr15 was efficiently dephosphorylated. However, only a transient MPM-2 reactivity at min was observed, presumably due to activation of residual endogenous Cdc2 cyclin B complex (Fig. 7, +Cdc25A). When MBP CycB1 and 6xHis-tagged Cdc25ADN were added together to apoptotic egg extracts, Cdc2 Tyr15 was dephosphorylated and continuous MPM-2 reactivity was restored (Fig. 7, +CycB1 + Cdc25A). These results indicated that cyclin B supplementation and the artificial dephosphorylation of Cdc2 Tyr15 were sufficient to induce M phase in apoptotic egg extracts. FEBS Journal 282 (2015) ª 2015 FEBS 1263

9 Caspase-mediated cleavage of Xenopus Cdc25C Y. Tsuchiya et al. Fig. 7. M-phase induction was restored when cyclin B and hyperactive Cdc25A were added together to apoptotic egg extracts. Cyt c-treated cycling egg extracts were prepared and preincubated for 90 min as in Fig. 3 to ensure interphase arrest and caspase-mediated cleavage of endogenous Cdc25C. After 90 min, MBP CycB1 and MBP (+CycB1), 6xHis-tagged Cdc25ADN and MBP (+Cdc25A), and MBP CycB1 and 6xHis-tagged Cdc25ADN (+CycB1 + Cdc25A) were added to egg extracts at 0.05 mgml 1 each, and egg extracts were further incubated. Samples were taken every 20 min, separated by SDS/PAGE, and subjected to western blotting against M-phase phosphorylated proteins (MPM- 2), total Cdc2 and Tyr15-phosphorylated Cdc2 (acdc2 py15). The M below the gel indicates the duration of M phase. Experiments were repeated at least three times using different batches of eggs, and representative images from one experiment are shown. Apoptosis induction inactivated MPF without cyclin B degradation in CSF-arrested metaphase egg extracts Finally, we tested whether the caspase-mediated inactivation of Cdc25C also affected the maintenance of active MPF in CSF-arrested metaphase egg extracts. In CSF-arrested egg extracts treated with CHX, MPM-2 reactivity and Cdc25C phosphorylation were observed for up to 8 h. For an unknown reason, Cdc2 Tyr15 was partially phosphorylated at 0 min despite its high MPF activity, but completely dephosphorylated upon further incubation under our experimental conditions. Both cyclin B1 and cyclin B2 remained stable, suggesting that MPF activity was maintained. In addition, Mos was also stable, and MAPK was fully phosphorylated, indicating that CSF activity was also maintained (Fig. 8, +CHX). When Cyt c was added to CSF-arrested egg extracts, Cdc25C was converted to shorter forms within 4 h. Accordingly, MPM-2 reactivity was gradually lost after 4 h, and Cdc2 Tyr15 phosphorylation decreased at 2 h but reappeared after 4 h. Neither cyclin B1 nor cyclin B2 were degraded during this period, indicating that the cell cycle reverted to interphase without cyclin B degradation, rather than exiting M phase with cyclin B degradation. Interestingly, MAPK was also partially dephosphorylated after 6 h despite the presence of Mos, suggesting inter-dependence between MPF and CSF activities. Our results indicate that apoptosis induction inactivated MPF not only in egg extracts Fig. 8. Apoptosis induction inactivated MPF without cyclin B degradation in CSF-arrested metaphase egg extracts. CHX-treated and Cyt c-treated CSF-arrested metaphase egg extracts were incubated without addition of 0.4 mm CaCl 2 for 8 h. Samples were taken at every 2 h, separated by SDS/PAGE, and subjected to western blotting against Cdc25C, M-phase phosphorylated proteins (MPM-2), total Cdc2, Tyr15-phosphorylated Cdc2 (acdc2 py15), cyclin B1, cyclin B2, Mos, total p42 MAPK and Thr/Tyrphosphorylated p42 MAPK (amapk ptpy). The hyperphosphorylated active 90 kda form, inactive 70 kda form, and apoptotic 60 kda and 50 kda forms of Cdc25C are indicated by arrowheads. The asterisk indicates a cross-reacting protein that overlaps with the 60 kda fragment of Cdc25C. The M s below the gel indicate the duration of M phase. Experiments were repeated at least three times using different batches of eggs, and representative images from one experiment are shown. during the cell cycle, but also in metaphase-arrested egg extracts. Discussion In this study, we showed that Cdc2 activation was dually inhibited by two distinct mechanisms during apoptosis in Xenopus egg extracts. One mechanism was translational block of cyclin B, which inhibited accumulation of endogenous cyclin B (Fig. 2). The other mechanism was caspase-mediated N-terminal cleavage of Cdc25C, which inactivated Cdc25C and prevented Cdc2 Tyr15 dephosphorylation (Figs 3 7). Addition of recombinant cyclin B and a hyperactive Cdc25A catalytic fragment together was sufficient to restore M-phase induction in apoptotic egg extracts (Fig. 7). We showed that apoptosis induction in CSF-arrested metaphase egg extracts also inactivated MPF in the absence of cyclin B degradation (Fig. 8), explaining why MPF activity decreases during apoptosis in unfertilized Xenopus eggs [41, 42] FEBS Journal 282 (2015) ª 2015 FEBS

10 Y. Tsuchiya et al. Caspase-mediated cleavage of Xenopus Cdc25C We observed that accumulation of cyclin B1 and cyclin B2 was blocked at the translational level rather than as a result of mrna modification or prolonged post-translational degradation (Fig. 2). In mammalian cells, protein synthesis is mainly down-regulated at the level of translation initiation during apoptosis. This inhibition is accompanied by modification of multiple translation initiation factors, including phosphorylation of eif2a and caspase-mediated cleavage of eif4g, eif4b, eif2a and the p35 subunit of eif3 [43]. These proteins are evolutionarily conserved, and we speculated that translation initiation is blocked in a similar manner during apoptosis in Xenopus egg extracts. We observed that the translation block in apoptotic extracts proceeded gradually, and was completed ~ 60 min after addition of exogenous Cyt c (Figs 1B and 2). This may explain why a sufficient amount of cyclin B was translated during the first M phase but not the second in apoptotic cycling extracts (Fig. 1B). We showed that Cdc25C, the only physiological activator of MPF in Xenopus oocytes and eggs, was cleaved by caspases during apoptosis (Figs 4 and 5). Asp108-cleaved Cdc25C was less active than Cdc25CFL, and Asp179-cleaved Cdc25C was inactive (Fig. 6D). The caspase-mediated cleavage of Cdc25C was slower than that of PARP, taking more than 60 min to complete in apoptotic extracts (Fig. 3). This slow inactivation is also consistent with the result shown in Fig. 1B, where MPF was found to be activated normally during the first M phase in apoptotic cycling egg extracts (Fig. 1B). We found that cleavage at either Asp108 and Asp179 was not complete, and approximately equal amounts of Asp108- and Asp179- cleaved Cdc25C were generated (Figs 4 and 5). Therefore, the cleavages at Asp108 and Asp179 may be mutually exclusive. Interestingly, the sequence around Asp108 also overlaps the PP1 binding site [23, 24]. Thus, association of PP1 may mask Asp108 and induce cleavage at Asp179, whereas the absence of PP1 may make cleavage at Asp108 more preferable. However, we cannot rule out the possibility that Cdc25C itself adopts two structurally distinct conformations. In any case, both cleavages remove functionally important regions from the catalytic domain, such as the PP1 binding site [23, 24], nuclear export signal [25, 26], and several activating phosphorylation sites [27 31] (Fig. 5C). Recent reports suggested that association of PP1 is required for the dephosphorylation of Ser287, removal of and activation of Cdc25C [23, 24], consistent with our observation that both Asp108- and Asp179-cleaved Cdc25C fragments remained phosphorylated at Ser287 in apoptotic extracts (Fig. 4). In addition, Thr138 phosphorylation is reported to be important for the function of Cdc25C [28, 29]. Thr138 is lost from Asp179-cleaved Cdc25C, but not from Asp108-cleaved Cdc25C. This may explain why the activities of the two truncated Cdc25C fragments were different (Fig. 6D). The cleavage sites identified in Xenopus Cdc25C are not conserved in Cdc25Cs in other animal species. Previous studies have suggested that human Cdc25C is degraded by proteasome [44 46] or calpain [47] to arrest the cell cycle at G 2. In addition, transcriptional control is non-functional in the early embryonic cell cycle, but plays important roles in somatic cells. Indeed, human Cdc25C has been reported to be transcriptionally down-regulated upon DNA damage [48, 49]. Thus, inactivation of Cdc25C may be an evolutionarily conserved strategy to prevent M-phase entry, although the regulatory mechanisms appear different. We also observed strong Cdc2 Tyr15 phosphorylation in apoptotic egg extracts (Fig. 3), indicating that Wee1A and Myt1 were active during apoptosis. Xenopus Wee1A is also functionally involved in apoptosis induction in egg extracts [50] and early embryos [51]. In contrast, previous reports identified human somatic Wee1 as a caspase substrate during Fas- and staurosporine-induced apoptosis in Jurkat cells [52] and during T-lymphocyte stimulation [53]. Therefore, apoptotic regulation of Wee1 may also differ between Xenopus and human. Previous studies indicated that caspase-cleaved Cdc25A de-regulated Cdk activation and induced apoptosis [38, 39]. In Xenopus egg extracts, N-terminally truncated Xenopus Cdc25A dephosphorylated Cdc2 Tyr15 effectively (Fig. 7). However, full-length Cdc25A was rapidly degraded by proteasome, and hence did not function in egg extracts [34 37]. Therefore, Cdc25A may be physiologically functional at later developmental stages in Xenopus. In somatic cells, Cdc25A is mainly expressed during G 1 S, and regulates Cdk2 to control DNA replication. Therefore, it is unlikely that caspase-cleaved Cdc25A induces M phase during apoptosis in somatic cells. Our study raises an interesting question: is it necessary to be in interphase to undergo apoptosis? Most cells in the body are differentiated and arrested at G 0 phase without cell-cycle progression. In proliferating cells, the accumulation of cellular injuries such as DNA damage or oxidative stress leads to cell-cycle arrest in G 1 or G 2 phase before apoptosis induction. In contrast, the term mitotic catastrophe has been used for cell death during or after aberrant mitosis. However, this death process may be either apoptotic or necrotic, and does not strictly require the bona fide FEBS Journal 282 (2015) ª 2015 FEBS 1265

11 Caspase-mediated cleavage of Xenopus Cdc25C Y. Tsuchiya et al. cell death executioner mechanism [54]. Thus, there may be a reason why dying cells are arrested at interphase for apoptosis to be executed correctly. The cytoskeleton and organelles are extensively remodeled during M phase, and these changes may prevent the correct execution of apoptosis. Alternatively, mitotic phosphorylation of intracellular proteins may inhibit caspase activation or cleavage of key protein substrates. Indeed, caspase-mediated cleavage of Xenopus Cdc25C appeared to take longer in M phase (not completed after 2 h, Fig. 8) than in interphase (completed after ~ 90 min, Fig. 4). Alternatively, the engulfment of apoptotic cells by macrophages may be affected. Further studies are necessary to address this interesting question. Experimental procedures Biochemical reagents Unless specifically noted, all biochemical reagents were purchased from Sigma-Aldrich (St Louis, MO, USA) or Wako Pure Chemical (Osaka, Japan). Preparation of Xenopus egg extracts and metabolic labeling Animal care and use of Xenopus female frogs was approved by the Animal Research Committee for Animal Experimentation of Toho University. CSF-arrested metaphase egg extracts were prepared as previously described [6 10]. To start the embryonic cell cycle, 0.4 mm CaCl 2 was added to egg extracts to release the metaphase arrest. At the same time, the general translation inhibitor 0.1 mgml 1 CHX was added to egg extracts to prevent cyclin B synthesis. Alternatively, 10 lgml 1 purified horse heart Cyt c was added to egg extracts to activate the mitochondria-mediated intrinsic apoptosis pathway. To prepare cycling extracts, de-jellied eggs were activated by calcium ionophore treatment with 10 lgml 1 A23187 in 0.2 x MMR (20 mm NaCl, 0.4 mm KCl, 0.4 mm CaCl 2, 0.2 mm MgSO 4, 1 mm HEPES/KOH ph 7.7, 0.02 mm EDTA) for 5 min. After washout of A23187, eggs were further incubated for 25 min in XB (0.1 M KCl, 0.1 mm CaCl 2, 1 mm MgCl 2, 10 mm HEPES/KOH ph 7.7, 50 mm sucrose) before centrifugal crushing. CHX or Cyt c was added to cycling egg extracts at the start of incubation. To radiolabel newly synthesized proteins in egg extracts, [ 35 S]-Cys/Met solution (EasyTag EXPRE 35 S 35 S protein labeling mix; Perkin-Elmer, Yokohama, Japan) was mixed with egg extracts at 1 : 9 v/v, and incubated at 23 C for the indicated time periods. After incubation, the samples were resolved by SDS/PAGE, and detected using a BAS image analyzer (GE Healthcare, Tokyo, Japan). Antibodies and western blotting Rabbit polyclonal antisera for Xenopus Cdc2 and Xenopus Myt1 were raised against their respective C-terminal peptides. Rabbit polyclonal antibodies for Xenopus Cdc25C, Xenopus cyclin B1 and Xenopus cyclin B2 were raised against the recombinant C-terminal catalytic domain, the recombinant N-terminal regulatory domain or recombinant full-length protein, and affinity-purified. Rabbit polyclonal antibodies against Tyr15-phosphorylated Cdc2 (py15) and Ser216-phosphorylated human Cdc25C (ps287, which also cross-reacts with Ser287-phosphorylated Xenopus Cdc25C), and mouse monoclonal antibody against HA tag (6E2) were purchased from Cell Signaling Technology (Tokyo, Japan). Rabbit polyclonal antibody against Xenopus Wee1A (XCT; Life Technologies, Tokyo, Japan), rabbit polyclonal antibody against Mos xe (sc-86, Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal antibody MPM-2 (Merck Millipore, Billerica, MA, USA), and mouse monoclonal antibodies against PARP and an MAP kinase activation sampler kit (BD Biosciences, Tokyo, Japan) were also used. Western blotting was performed as previously described [6 10]. All antibodies were used at 1 : 1000 dilution. Each lane contained 1 ll of egg extracts or lysates from two oocytes. RACE-PAT assay of cyclin B1 and cyclin B2 mrna polyadenylation status The RACE-PAT assay (rapid amplification of cdna ends/ poly(a) tail) was performed as described by Salles et al. [55]. Briefly, total RNA from Xenopus egg extracts was reverse-transcribed using ReverTra Ace (Toyobo, Tokyo, Japan) and a NotI-(dT) 18 primer (GE Healthcare). The cdna obtained was subsequently amplified by PCR using ExTaq polymerase (TaKaRa, Shiga, Japan) with primers specific for the 3 0 untranslated regions of cyclin B1 and cyclin B2 [56]. Amplified products were separated by electrophoresis in 1% agarose, and stained using ethidium bromide. Recombinant protein expression and purification in E. coli and rabbit reticulocyte lysates Gene modification experiments were approved by the Gene Modification Experiment Safety Committee of Toho University. The open reading frames and truncated fragments of cyclin B1, Cdc25A and Cdc25C were amplified by RT-PCR using the primers listed in Table 1. The amplified fragments were cloned into pgem-t Easy (Promega, Tokyo, Japan), and nucleotide sequences were verified. The cyclin B1 open reading frame was sub-cloned into pmal-cri (New England Biolabs, Tokyo, Japan) to 1266 FEBS Journal 282 (2015) ª 2015 FEBS

12 Y. Tsuchiya et al. Caspase-mediated cleavage of Xenopus Cdc25C Table 1. Primers used in this study. Protein Sequence Open reading frame amplification Cyclin B1 F: 5 0 -CCATGGCTTCGCTACGAGTCACCAGAAAC-3 0 Cdc25A R: 5 0 -CTCGAGTCACATGAGTGGGCGGGCCA-3 0 F: 5 0 -CATATGGAGAGGTTTCGTTCTGCTCC-3 0 Cdc25C R: 5 0 -AAGCTTTCATAGTTTCTTCAGCCGGC-3 0 F: 5 0 -CATATGGCAGAGAGTCACATAATGTC-3 0 R: 5 0 -AGATCTAATGGATCAACGGCATGAGCC-3 0 Site-directed mutagenesis Cdc25C D108N F: 5 0 -GCACAATTTGTCCAGTTTAACGGCCTATTTACTCCTGATC-3 0 R:5 0 -GATCAGGAGTAAATAGGCCGTTAAACTGGACAAATTGTGC-3 0 Cdc25C D179N F: 5 0 -GCTACCACAAGAAGTAGTGAATTCACAATTTTCTCCAACACC-3 0 R: 5 0 -GGTGTTGGAGAAAATTGTGAATTCACTACTTCTTGTGGTAGC-3 0 N-terminally truncated protein expression Cdc25ADN F: 5 0 -CATATGGGAGAGGACCTAGAGAACGAC-3 0 Cdc25CDN1 F: 5 0 -GCGCATATGGGCCTATTTACTCCTGATCTC-3 0 Cdc25CDN2 F: 5 0 -CATATGTCACAATTTTCTCCAACACC-3 0 RT-PCR of cyclin B1 and cyclin B2 mrna poly(a) tails xcycb1 3UTR-F F: 5 0 -CTCATGTGAAGGACTACGTGGCATTCC-3 0 xcycb2 3UTR-F F: 5 0 -TAGAACTGTTAAGTGACCCTTTCAAAGAG-3 0 3UTR-R: R: 5 0 -AACTGGAAGAATTCGCGGCCGCAGG-3 0 NotI-(dT) 18 primer R: 5 0 -AACTGGAAGAATTCGCGGCCGCAGGAAT express MBP-fused cyclin B1 (MBP CycB1). Expression of recombinant protein was induced in Escherichia coli BL21 (DE3) using 0.5 mm isopropylthiogalactoside (Merck-Millipore) overnight at 18 C, and the protein was purified using amylose resin (New England Biolabs) according to the manufacturer s instructions. The Cdc25C open reading frame was sub-cloned into pet-15b (Merck-Millipore) to express N-terminally 6xHistagged recombinant proteins. This vector was used for in vitro translation of 35 S-radiolabeled proteins using TnT T7 Quick (Promega) and EasyTag EXPRE 35 S 35 S protein labeling mix (Perkin-Elmer) according to the manufacturers instructions. The rabbit reticulocyte lysates were mixed with interphase egg extracts (CSF-arrested egg extracts supplied with CaCl 2 and CHX) either in the absence or presence of exogenous Cyt c at 1 : 9 v/v, and incubated for 2 h at room temperature. Where indicated, Z-VAD-FMK (Peptide Institute, Osaka, Japan) or MG-132 (Peptide Institute) were added to extracts at 100 lm. After the reaction, samples were resolved by SDS/PAGE and detected using a BAS-5000 image analyzer. Site-directed mutagenesis was performed using a QuikChange II site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA) and the primers listed in Table 1. N-terminally 6xHis-tagged Cdc25ADN was expressed using pet-15b in E. coli BL21 (DE3). Expression of recombinant protein was induced using 0.5 mm isopropylthiogalactoside overnight at 18 C, and protein was purified using a Ni-loaded HiTrap chelating column (GE Healthcare) according to the manufacturer s instructions. Synthetic mrna injection and recombinant protein expression in Xenopus oocytes We used pha, a modified version of pet-15b, to express N-terminally HA-tagged proteins as previously described [10]. The open reading frames and truncated fragments of Cdc25A and Cdc25C were sub-cloned into pha, and in vitro mrna synthesis was performed using mmessage mmachine T7 (Life Technologies) according to the manufacturer s instructions. Synthetic mrna encoding Xenopus EF1a was produced using ptri-xef, the positive control enclosed in the kit, as the template. Stage VI oocytes were manually dissected from nonprimed Xenopus female ovaries. Using Nanoject II (Drummond Scientific Company, Broomall, PA, USA) and a micromanipulator (Narishige, Tokyo, Japan), 23 nl of 1mgmL 1 mrna dissolved in H 2 O (23 ng RNA) was injected per oocyte at the animal/vegetal border. Injected oocytes were incubated in O-R2 (5 mm HEPES/KOH, ph 7.7, 82.5 mm NaCl, 2.5 mm KCl, 1 mm CaCl 2, 1 mm MgCl 2, 1mM Na 2 HPO 4 ) containing 3% Ficoll 400 (Sigma- Aldrich) at 19 C. After 6 h, 5 ll per oocyte of MEB-TX (20 mm HEPES/KOH, ph 7.7, 15 mm MgCl 2, 80 mm sodium glycerol 2-phosphate, 20 mm EGTA, 1 mm dithiothreitol, 0.2 mm phenylmethanesulfonyl fluoride, 0.1% Triton X-100) was added, and oocytes were crushed using wooden toothpicks. Samples were then centrifuged at g for 10 min, and supernatants (oocyte lysates) were mixed with SDS/PAGE buffer and subjected to western blot analysis. The appearance of a white spot at the top of the animal hemisphere, known as germinal vesicle break- FEBS Journal 282 (2015) ª 2015 FEBS 1267