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BOR - Papers in Press, published online ahead of print June 6, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.060269
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BIOLOGY OF REPRODUCTION 77, 560–568 (2007)
DOI: 10.1095/biolreprod.107.060269
© 2007 by the Society for the Study of Reproduction, Inc.

Involvement of Protein Kinase B/AKT in Early Development of Mouse Fertilized Eggs1

Chen Feng 3, Aiming Yu 3, Ying Liu 3, Jie Zhang 3, Zhihong Zong 3, Wenhui Su 3, Zhe Zhang 3, Dahai Yu 3, Qing-Yuan Sun 4, and Bingzhi Yu 2 3

Department of Biochemical and Molecular Biology,3 China Medical University, Shenyang 110001, Liaoning, China Institute of Zoology,4 Chinese Academy of Sciences, Beijing 100080, China

ABSTRACT

The activation of AKT (also called protein kinase B) is thought to be a critical step in the phosphoinositide 3-kinase pathway that regulates cell growth and differentiation. In this report, we investigated the role of AKT in the regulation of mouse early embryo development. Injection of mRNA coding for a constitutively active myristoylated AKT (myr-Akt1) into one-cell stage fertilized eggs induced cell division more effectively than injection of wild-type AKT (Akt1-WT) mRNA, whereas microinjection of mRNA of kinase-deficient AKT (Akt1-KD) delayed the first mitotic division. Meanwhile, microinjection of different kinds of mRNA of AKT affected the phosphorylation status of CDC2A-Tyr15 and the activation of M-phase promoting factor (MPF). To investigate the intermediate factor between AKT and MPF, we then injected one-cell stage eggs first with Akt1-WT mRNA or myr-Akt1 mRNA and then with mRNA encoding either wild-type CDC25B (Cdc25b-WT) or a AKT-nonphosphorylatable Ser351 to Ala CDC25B mutant (Cdc25b-S351A). Cdc25b-S351A strongly inhibited the effect of AKT. Therefore, AKT causes the activation of MPF and strongly promotes the development of one-cell stage mouse fertilized eggs by inducing AKT-dependent phosphorylation of CDC25B, a member of the CDC25 phosphatase family. Our finding that CDC25B acts as a potential target of AKT provides new insight into the effect of AKT in the regulation of early development of mouse embryos.

AKT, CDC25B, early development, embryo, kinases, mouse, M-phase promoting factor, signal transduction

INTRODUCTION

Early embryonic division is characterized by a rapid succession of the interphase and mitotic state in many species. The progression through M phase is controlled by M-phase promoting factor (MPF) [1], a highly conserved complex consisting of a kinase, CDC2A [2, 3], and an activating subunit, CCNB1 [1, 4–6]. During the G2/M transition, MPF undergoes tight regulation by phosphorylation and dephosphorylation of CDC2A. In the G2 phase of vertebrate cells, CDC2A is kept inactivated by inhibitory phosphorylation on tyrosine 15 (Tyr15) and threonine 14 (Thr14), which are catalyzed by WEE 1/MYT 1 kinase [7–9], and the CDC2A/CCNB1 complex remains enzymatically inactive (pre-MPF) [10]. On entry into M phase, CDC25, a dual-specific phosphatase family, dephosphorylates CDC2A on both Tyr15 and Thr14, causing the activation of MPF [11, 12]. Thus, in principle, the G2/M transition can be induced by inactivation of WEE 1/MYT 1 kinase or activation of CDC25 phosphatase or both [13, 14]. However, it is unknown how the dominance between the activities of the WEE 1/MYT 1 kinase and CDC25 phosphatase is initially reversed to activate CDC2A/CCNB1 at entry into M phase.

AKT (also called protein kinase B) is known as a serine/threonine protein kinase and a downstream factor of phosphoinositide 3-kinase (PI3K). It is well established that AKT plays an important role in cellular processes such as glucose metabolism, cell proliferation, apoptosis, transcription, and cell migration. The PI3K-AKT signaling pathway was reported to promote G1 progression through activation of the CDK4/CCND1 complex [15]. By contrast, AKT is known to inactivate the CDK inhibitors CDKN1A and CDKN1B (previously known as p21 and p27). AKT phosphorylates CDKN1A on Thr145 and CDKN1B on Thr157 and Thr198, resulting in their cytoplasmic translocation and functional suppression [16–21]. These results indicate that AKT promotes cell-cycle progression in G1 phase by activating positive regulators and inactivating negative ones. Otherwise, AKT is suggested to function as a G2/M initiator. AKT activation by overexpression of a constitutively active form or by the loss of PTEN (phosphatase and tensin homologue deleted in chromosome 10) could overcome the G2/M arrest that was induced by gamma irradiation [22]. In addition, PTEN-null embryonic stem (ES) cells were reported to transit faster from the G2/M to the G1 phase of the cell cycle than wild-type ES cells, and LY294002, an inhibitor of PI3K, elicited G2/M arrest in human embryonic kidney (HEK) 293 cells [22]. Although these results suggest that the PI3K-AKT signaling pathway strongly promotes G2/M transition in mammalian cell-cycle progression, the direct relationship between the PI3K-AKT signaling pathway and regulation of the G2/M transition is not fully understood. However, in primary oocytes from the starfish Asterina pectinifera, AKT was reported to inhibit MYT 1 through AKT-dependent phosphorylation and down-regulation at the G2/M transition [23]; in HEK293T cells, AKT promotes G2/M cell-cycle progression by inducing phophorylation-dependent YWHAQ (previously known as 14-3-3{theta}) binding and cytoplasmic localization of WEE 1 kinase [24]. Moreover, as a result, AKT indirectly causes the activation of CDC2A and promotes the cell cycle progression at the G2/M transition. These findings suggest the possibility that the AKT functions as an M-phase initiator in mammals.

Despite the continuing discovery of new substrates of AKT and our increasing knowledge of their basic biochemistry and molecular biology, relatively little is known about the effect of AKT in the regulation of mammalian early embryo development. The mouse fertilized egg is the most simple and natural cell-cycle model in vertebrates that are close to human. There are few reports about the mechanism of the regulation of early development of mouse fertilized eggs, especially the events of entry into M-phase. Here we report that AKT causes the activation of MPF and strongly promotes the development of one-cell stage mouse fertilized eggs by inducing AKT-dependent phosphorylation of CDC25B, a member of CDC25 phosphatase family. Our findings identify CDC25B as a potential target of AKT and provide new insight into the effect of AKT in the regulation of mouse early embryo development.

MATERIALS AND METHODS

Animals and Reagents

Kunming strain mice were obtained from the Department of Laboratory Animals, China Medical University (CMU). All experiments were performed at CMU in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals. The protocol for animal handling and the treatment procedures were reviewed and approved by the CMU Animal Care and Use Committee. Reagents, unless otherwise specified, were from Sigma.

Collection and Culture of Mouse Embryos

One-cell stage mouse embryos were collected and cultured according to the method described by Hogan and Constantini [25]. Female mice at 4–6 wk old were abdominally injected with 10 IU of eCG and 48 h later with 10 IU hCG. A single female was placed with a single male for fertilization. One-cell embryos were collected with M2 medium the next day (20 h after hCG injection) from the oviduct of females possessing a vaginal plug. After injection with mRNA, embryos were cultured in a drop of M16 medium under paraffin oil at 37°C in a humidified atmosphere of 5% CO2 in air.

Construction of mRNA Expression Vectors

The constructs encoding the wild-type AKT (pCIS2-Akt1-WT), myristoylated AKT (pCIS2-myr-Akt1) and kinase-deficient AKT (pCIS2-Akt1-KD) were gifts from Michael J. Quon (National Institutes of Health) [26, 27].

The coding sequences of wild-type AKT (Akt1-WT), kinase-deficient AKT (Akt1-KD), and myristoylated AKT (myr-Akt1) were amplified by PCR using the pCIS2-Akt1-WT, pCIS2-Akt1-KD, and pCIS2-myr-Akt1, respectively, as templates and introduced the enzyme-incision site of HindIII and BamHI into 5' and 3' end, respectively. The products were cloned into the mRNA expression vector pcDNA3.1/myc-His B (Invitrogen) and named pcDNA3.1-Akt1-WT, pcDNA3.1-Akt1-KD, and pcDNA3.1-myr-Akt1. The pBSK-Cdc25b-WT was subcloned into pcDNA3.1/myc-His B using KpnI and BamHI. The recombinant was called pcDNA3.1-Cdc25b-WT.

A Site-Directed Mutagenesis Kit (Stratagene) was used to mutate Ser351 to a nonphosphorylatable alanine of CDC25B, and this mutant was called pcDNA3.1-Cdc25b-S351A.

All the above recombinant plasmids were sequenced to verify the correct gene insertion and successful mutation and were used as templates for in vitro transcription.

In Vitro Transcription

All the constructs in pcDNA3.1/myc-His B were cut singly with AgeI and transcribed in vitro into 5'-capped mRNA for microinjection by using the mMESSAGE mMACHINE kit (Ambion). The in vitro-synthesized mRNA was dissolved in nuclease-free 5 mM Tris and 0.5 mM EDTA (TE; pH 7.4). We determined mRNA yield by measuring absorbance at 260 nm and by carrying out modified nondenaturing gels loaded with RNA.

Messenger RNA Microinjection and Observation of the Mouse Embryos

Various mRNA were injected into one-cell stage embryos at G2 phase using a micropipette and Eppendorf TransferMan manipulators mounted on an Olympus IX-70 inverted microscope with DIC optics. Eggs were placed in a drop of M2 medium under paraffin oil in the lid of a 3-cm Falcon culture dish. Typical injection volume was 5% of total cell volume or 10 pl per egg. Messenger RNA was diluted to various concentrations in TE buffer (pH 7.4) without nuclease contaminant. Eggs in control groups were either not microinjected or microinjected with TE buffer. The percentages of cell division and cell survival were counted under a dissecting microscope 30 h and 35 h after injection of hCG, and the results were analyzed statistically.

Assay of MPF Activity

MPF kinase activity was measured using histone H1 kinase assay [28]. Five eggs cultured in M16 medium were collected, washed in collection buffer (PBS containing 1 mg/ml polyvinyl alcohol, 5 mM EDTA, 10 mM Na3VO4, and 10 mM NaF), and then transferred to an Eppendorf tube containing 5 µl of the collection buffer. The Eppendorf tube was immediately stored at –70°C until the kinase assay was performed.

The frozen eggs were thawed and subjected to freezing and thawing three times. A total of 25 µl of MPF buffer (54 mM ß-glycerophosphate, 14.5 mM p-nitrophenylphosphate, 24 mM 3-(N-morpholino)-propanesulfonic acid [MOPS; pH 7.2], 14.5 mM MgCl2, 14.5 mM ethyleneglycoltetraacetic acid, 0.12 mM EDTA, 1 mM dithiothreitol [DTT], 2.4 µM PKA inhibitor peptide [PKI], 75 mM genistein [a tyrosine kinase inhibitor], 10 µM ML-9 [a myosin light chain kinase inhibitor], 1 mg/ml histone H1 [type III-s], and 1 mg/L each of leupeptin, aprotonin, pepstatin, chymostatin, and trypsin-chymotrypsin inhibitor) was then added to the disrupted cells. The histone H1 kinase reaction was started by adding 25 µl of 20 µCi/ml [{gamma}-32P]ATP (Beijing FuRui Biotechnology) incubated at 30°C for 10 min. Then 25-µl aliquots were spotted on Whatman p81 paper, and the reaction was stopped with 5% H3PO4 solution. After thorough washing, the radioactivity on the filter paper was counted with a BECKMAN scintillation counter.

A parallel incubation was performed to confirm the phosphorylation of histone H1. Protein extract from 10 oocytes was incubated with 50 µl of MPF buffer containing 50 µCi/ml [{gamma}-32P]ATP at 37°C for 30 min, and the reaction was stopped by adding an equal amount of 2x SDS buffer. The reaction was then resolved on a 12% SDS-PAGE gel, and the incorporation of 32P into histone H1 was visualized by autoradiography.

Assay of PKB Activity

Ten eggs cultured in M16 medium were collected and lysed as described above and assayed for 30 min at room temperature in AKT reaction buffer containing 50 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM DTT, 1 µM PKI, 40 µCi/ml [{gamma} -32P]ATP, and 0.2 mg/ml histone H2B used as substrate. AKT activity was also determined by scintillation counting and autoradiography, as in the MPF activity assay.

Western Blotting

Protein extracts of mouse fertilized eggs were prepared by adding approximately 150 eggs in a minimal volume of collection medium to 20 µl of protein extraction buffer (100 mM NaCl, 20 mM Tris-HCL [pH 7.5], 0.5% Triton X-100, 0.5% NP-40) containing 1 mM phenylmethylsulfonyl fluoride and 1 µg/ml leupeptin and pepstatin. Laemmli sample buffer was added to the protein extracts, and the mixture was boiled for 5 min and resolved on a 12% SDS-PAGE gel. For immunoblotting, the fractionated proteins were transferred to a nitrocellulose membrane. The membrane was blocked with 3% BSA in Tris-buffered saline containing 0.05% Tween 20 and probed with primary antibodies in a sealed plastic bag at 4°C overnight. The primary antibody against pTyr15 of CDC2A or CCNB1 (Santa Cruz Biotechnology) was used at 1:400 dilution, or the anti-MYC antibody (Invitrogen) used at 1:1000 dilution.

The membrane was then incubated with alkaline phosphatase-conjugated anti-mouse IgG secondary antibody at 1:3000 (Beijing Zhongshan Biotechnology). The proteins were detected by using O-dianidine and ß-naphthyl acid phosphate as the substrates of alkaline phosphatase.

Statistical Analysis

One-way analysis of variance or Student t-test was used to evaluate the difference between groups, and differences at P < 0.05 were considered to be significant. SPSS 13.0 software was used to perform statistical analyses.

RESULTS

AKT mRNA Microinjection Interferes with Cell Division of One-Cell Stage Mouse Fertilized Eggs

To test whether AKT kinase activity and the membrane targeting are involved in the mitotic cell cycle of fertilized eggs, three AKT constructs encoding Akt1-WT, myr-Akt1, and Akt1-KD were used in our experiment. In the myr-Akt1 construct, the AKT coding region is fused to a myristoylation sequence from pp60 csk. This engineered protein is constitutively active in 3T3L1 cells by being targeted to the membrane [29]. The dominant inhibitory mutant of AKT that we used in this study is a kinase-deficient AKT that results from the substitution of alamine for lysine at position 179 in the canonical ATP-binding domain. This mutant is not only catalytically inactive but has been shown to inhibit the activity and actions of endogenous AKT. Indeed, these catalytically active and inactive mutants have been extensively characterized [30, 31, 32] and have been shown to have dominant effects in other contexts [26, 27, 33, 34].

To examine whether AKT affects the mitotic cell cycle, one-cell stage mouse fertilized eggs were cultured in M16 medium after microinjection of 0.03 ng mRNA of Akt1-WT, myr-Akt1, or Akt1-KD. Two control groups were non-microinjection or microinjection with TE buffer.

At 4 h after mRNA microinjection, 10 one-cell stage fertilized eggs cultured in M16 medium were collected for the assay of AKT activity. The results showed that the AKT activity in Akt1-WT mRNA-injected oocytes was higher than that of control groups, and AKT activity in the myr-Akt1 mRNA-injected group was 20-fold higher than that of control groups (P < 0.01), or about 8- to 10-fold higher than the Akt1-WT mRNA-injected group (P < 0.05). However, AKT activity in the Akt1-KD mRNA-injected group was lower than the control groups (P < 0.05) (Fig. 1A). Western blot analysis showed that the AKT-MYC fusion protein accumulated at high levels in Akt1-WT, myr-Akt1, or Akt1-KD mRNA-injected one-cell stage fertilized eggs (Fig. 1B). These results indicate that the exogenous mRNA of all kinds of AKT could be translated efficiently, and the microinjection of Akt1 constructs could change the activity of AKT in the one-cell stage eggs.


Figure 01
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  		FIG. 1 The activity of AKT and the expression of AKT-myc after Akt1 constructs microinjection. A) 0.03 ng of mRNA coding Akt1-WT, myr- Akt1, or Akt1-KD was injected into each egg; 50–60 eggs were injected. Eggs in control groups were either not microinjected or microinjected with TE buffer. After 4 h, 10 eggs were collected for the assay of AKT activity. AKT activity was analyzed as described in Materials and Methods. Each value was expressed as mean ± SEM from four independent experiments. B) Western blot of AKT-MYC expression in different microinjected groups. Immunostaining was performed using anti-myc. The data shown are representative of at least three independent experiments.

The percentages of cell division and death in each group were calculated after counting under a dissecting microscope 30 h and 35 h after injection of hCG (Tables 1 and 2). In the control groups, embryos remained at the one-cell stage 26 h after hCG injection (G2 phase), but about 63% of embryos reached two-cell stage 4 h later (30 h after the injection of hCG), and there was no significant difference between the two control groups (P > 0.05). Embryos microinjected with mRNA of Akt1-WT were in one-cell stage 26 h after the hCG injection, as in the control; however, about 75.51% of embryos reached two-cell stage 30 h after the hCG injection (P < 0.05), and 16.14% of embryos were dead 35 h after the hCG injection (P < 0.05). For embryos microinjected with mRNA of myr-Akt1, a constitutively activated, myristylated form of AKT, the percentage of cleavage reached 91.19% at 30 h (P < 0.05), and the percentage of dead eggs decreased 11.21% at 35 h (P < 0.05). The cleavage was also accelerated, as indicated by nearly 27% of embryos having reached the two-cell stage at 26 h after the injection of hCG in this group. Irregular division was observed 30 h after the hCG injection. On the other hand, only 12.93% of embryos that microinjected with mRNA of Akt1-KD reached two-cell stage 30 h after the hCG injection (Tables 1 and 2). These results demonstrated that microinjection of different kinds of mRNA of AKT can interfere with the time course of cell division of one-cell stage fertilized eggs, suggesting that AKT may have the biological function of promoting the early mitotic division in mouse early embryos.


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  		TABLE 1 Comparison of percentage of division among control groups and experimental groups.


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  		TABLE 2 Comparison of percentage of embryo death among control groups and experimental groups.

Akt mRNA Microinjection Regulates MPF Activity and CDC2A-Tyr15 Phosphorylation

The promotion of the development of the one-cell stage by Akt1 mRNA microinjection into fertilized eggs suggests a role of AKT in regulating mouse embryonic cleavage.

Firstly, we investigated the time course for the activation of the endogenous AKT and MPF of one-cell stage fertilized eggs in control groups. Mice were primed with eCG for 48 h and then injected with hCG. Eggs were collected at the indicated timepoint after hCG injection for the assay of AKT activity and H1 kinase activity. As anticipated, the change in AKT activity preceded MPF activation (Fig. 2). The activity of AKT reached a maximum at 26 h after hCG injection (Fig. 2A) and preceded MPF activation by 2 h (Fig. 2B). AKT activation was transient, and a substantial decrease was detected between 27 h and 30 h after hCG injection, although the MPF activity peak occurred during this period. These results suggest that endogenous AKT activation occurs prior to MPF activation during the first mitotic division of fertilized eggs and hence should serve as a component of the CDC2A-activation pathway.


Figure 02
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  		FIG. 2 Fluctuation of endogenous AKT and MPF activities in mouse one-cell stage fertilized eggs during mitosis. A) Embryos were collected from female mice that were injected with hCG; the embryos were then bathed in M16 medium and harvested at indicated times after the hCG administration. AKT was assayed by scintillation counting and autoradiography as described in the Materials and Methods section. B) The MPF activity was determined with histone H1 as substrate following microinjection of TE buffer. For each point, eggs were lysed and examined by scintillation counting and autoradiography. At least three independent experiments were performed.

Then, we measured MPF activity at different time points in each experimental group. In the Akt1-WT group, MPF activity reached its peak value at 27.5 h after the hCG injection, about 30 min earlier than in control groups (Fig. 3A). In the myr-Akt1 group, the peak of MPF activity occurred at 26.5 h after the hCG injection, about 1.5 h earlier than the control group (Fig. 3B). In the Akt1-KD group, MPF activity reached its peak value at 30 h after hCG injection, and the peak value was lower than that of the control groups (Fig. 3C). The phosphorylation status of CDC2A-Tyr15 in each group was coincident with the MPF activity (Fig. 3D). Meanwhile, we examined the expression of CCNB1 at the point of G2/M transition in each group, and there was no significant difference among the groups (Fig. 3E). These results suggest that AKT can promote the cleavage of one-cell stage fertilized eggs by affecting the phosphorylation of CDC2A-Tyr15, directly or indirectly, and then increasing the activity of MPF.


Figure 03
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  		FIG. 3 Effect of Akt1 mRNA microinjection on MPF activity and CDC2A-Tyr15 phosphorylation. Histone H1 kinase (MPF) activity was assayed following microinjection of 0.03 ng Akt1-WT mRNA (A), 0.03 ng myr-Akt1 mRNA (B), and 0.03 ng Akt1-KD mRNA (C). For each point, eggs were lysed and MPF activity was examined by scintillation counting and autoradiography. Each value was expressed as mean ± SEM from three independent experiments. D) Western blot analysis of phosphorylation status of CDC2A-Tyr15. One-cell stage eggs injected with TE buffer were collected only 28 h after hCG injection. The eggs injected with Akt1-WT mRNA, myr-Akt1 mRNA, or Akt1-KD mRNA were collected 26.5 h and 28 h after hCG injection. E) The expression of CCNB1 in mouse eggs after Akt1 mRNA microinjection. One-cell stage eggs injected with TE buffer, Akt1-WT mRNA or Akt1-KD mRNA were collected 28 h after hCG injection; and the eggs injected with myr-Akt1 mRNA were collected 26.5 hr after hCG injection. The results of one representative experiment were presented.

The AKT/CDC25B Pathway Operates in Regulating the Division of One-Cell Stage Fertilized Eggs

CDC25B acts as an initiator of early mitotic events [35], and it plays a role in the activation of a centrosomal subpopulation of CDC2A-CCNB1 that is next translocated to the nucleus where activation of CDC25C will initiate an amplification loop driving the cell into mitosis [36, 37]. It has been reported that in Hela cells, AKT phosphorylates CDC25B on serine 353, resulting in a nuclear export-dependent cytoplasmic accumulation of this phosphatase [38]. We then tested whether AKT can directly act on and promote CDC25B activation (a potential substrate for AKT) in mouse fertilized eggs. We used Scansite software (http://scansite.mit.edu) and the Clustal W(1.83) multiple sequence alignment program (http://www.ebi.ac.uk/clustalw/) to predict the relevant target sites of AKT on mouse CDC25B. We finally found that mouse CDC25B has a serine residue at position 351 (Ser351), which corresponds to Ser353 of human CDC25B, a residue phosphorylated by human AKT. We therefore injected one-cell stage eggs first with Akt1-WT mRNA or myr-Akt1 mRNA, as mentioned above, and then with mRNA encoding either wild-type CDC25B (Cdc25b-WT) or an AKT-nonphosphorylatable Ser351 to Ala CDC25B mutant (Cdc25b-S351A). Embryos coexpressed with Akt1 and Cdc25b-S351A remained at one-cell stage 28 h after hCG injection, and only 9.64% of total embryos reached two-cell stage 30 h after hCG. Even in the myr-Akt1 and Cdc25b-S351A coexpression group, Cdc25b-S351A can also decrease the percentage of division to 17.72% (Fig. 4). Meanwhile, the Cdc25b-S351A mutant can inhibit the activation of MPF and affect the phosphorylation status of CDC2A-Tyr15 (Fig. 5, A–C). In these experiments, the AKT-nonphosphorylatable CDC25B mutant (Cdc25b-S351A), but not Cdc25b-WT, was able to block the (maturation-promoting) effect of overexpressed Akt1-WT or myr-Akt1. These results suggest strongly that AKT may directly phosphorylate CDC25B on Ser351 and can promote its function in mouse fertilized eggs. Taken together, the present results indicate that the AKT/CDC25B pathway can operate in regulating mitotic division of one-cell stage fertilized eggs.


Figure 04
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  		FIG. 4 The division rate in cultured mouse embryos after Akt1 and Cdc25b mRNA co-injection. One-cell stage mouse embryos were microinjected first with 0.02 ng Akt1-WT mRNA or myr-Akt1 mRNA as described in the Materials and Methods and then, 1–2 h later, with mRNA encoding either wild-type Cdc25b (Cdc25b-WT) or a AKT-nonphosphorylatable Ser351 to Ala Cdc25b mutant (Cdc25b-S351A). The percentage of division was calculated after counting under a dissecting microscope 30 h after injection of hCG in female mice. The data were pooled from three experiments and are expressed as mean ± SEM.


Figure 05
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  		FIG. 5 Effect of Akt1 and Cdc25b mRNA co-microinjection on MPF activity. A) One-cell stage mouse embryos were microinjected first with 0.02 ng Akt1-WT mRNA and then with mRNA encoding either wild-type Cdc25b (Cdc25b-WT) or a AKT-nonphosphorylatable Ser351 to Ala Cdc25b mutant (Cdc25b-S351A). B) One-cell stage mouse embryos were microinjected first with 0.02 ng myr-Akt1 mRNA and then with mRNA encoding a AKT-nonphosphorylatable Ser351 to Ala Cdc25b mutant (Cdc25b-S351A). The injected eggs were cultured in M16 medium and collected at different time points. For each point, eggs were lysed and MPF activity was examined by scintillation counting and autoradiography. Each value was expressed as mean ± SEM from three independent experiments. C) Western blot analysis of phophorylation status of CDC2A-Tyr15. One-cell stage eggs comicroinjected with Akt1-WT/Cdc25b-WT mRNA, Akt1-WT/Cdc25b-S351A mRNA, and myr-Akt1/Cdc25b-S351A mRNA were collected 28 h after hCG injection. The data shown are representative of three independent experiments.

DISCUSSION

AKT is a signaling molecule that is known to enhance cell proliferation, survival, and glycogen and protein synthesis. Here we provide evidence for an important role of this kinase in the control of the first round of mitosis of mouse fertilized eggs. AKT overexpression in mouse fertilized eggs promoted cell division, and exogenous expression of myr-Akt1 further enhanced this effect. However, overexpression of Akt1-KD inhibited the development of mouse fertilized eggs. So, we propose that AKT is a key promoter of the first round of mitosis in mouse fertilized eggs. Although the Akt1-WT partially promoted early embryo division, its effect was much less efficient than the myr-AKT. Because the only difference between Akt1-WT and myr-AKT is their different abilities to interact with the lipid bilayer [26, 27, 33, 34], we can conclude that AKT interaction with lipids is essential for signaling during early embryo division.

It is known that AKT participates in all physiological functions by phosphorylating a wide array of substrate proteins, including metabolic enzymes, structural proteins, transcription factors, and so on. Among them, GSK-3B was the first physiological target of AKT to be identified and is a key player in AKT signaling [39]. PI-kinase-induced activation of AKT results in AKT phosphorylation of both GSK-3B isoforms (Ser9 of GSK-3ß; Ser21 of GSK-3{alpha}), which inhibits GSK-3B activity. Then, AKT-dependent inactivation of GSK-3B promotes the dephosphorylation and activation of the many substrates of GSK-3B [40]. We examined the phosphorylation status of GSK-3B in mouse eggs of the Akt1-WT and myr-Akt1 injection groups and noticed that overexpression of Akt1 constructs, especially overexpression of myr-Akt1, could facilitate the phosphorylation of both GSK-3B isoforms. But beyond our expectation, when we mutated Ser9 of GSK-3ß and Ser21 of GSK-3{alpha} to nonphosphorylatable alanine, we found that these mutations couldn't interfere with the time course of cell division of one-cell stage fertilized eggs (Bingzhi Yu, unpublished data). Others also reported that phosphorylation of the Drosophila homologue of GSK-3B plays no role in the regulation of development or cell size and number [41], and the finding that both the single and double GSK-3B knockin mice develop normally and are of normal size confirms these observations in a mammalian system [42]. Taken together, all these indicate that PI3K/AKT-mediated GSK-3 phosphorylation does not play a major role in regulating development, cell growth, and proliferation.

Then we further investigated the physiological substrates of AKT and the molecular basis for the regulation of the cell cycle by AKT in mouse fertilized eggs. It is well-known that MPF is a critical regulator of early embryo development in mice [43, 44], and the MPF activity is regulated by phosphorylation. In this study, we showed that endogenous AKT activation occurs prior to MPF activation; moreover, AKT, especially the myr-AKT, promoted the initiation of MPF activation by affecting the phosphorylation status of CDC2A-Tyr15. These results suggest that AKT may be an upstream regulator of MPF activation, similar to its role in the maturation of the starfish Asterina pectinifera, Xenopus, and mouse oocyte [23, 45, 46]. Furthermore, we also noticed that in the AKT-KD overexpression group, more than 45% of the eggs reached two-cell stage, accompanied with the activation of MPF, if the culture time is extended to 35 h after hCG injection (data not shown). In support of our findings, others have also reported that inhibiting PI3K-AKT signaling in mouse oocytes with LY-294002 totally prevents resumption of meiosis at 55 min when control oocytes have already undergone GVBD, and more than 50% of these oocytes undergo GVBD at 2 h of culture [45]. These data suggest that alternative pathways leading to MPF activation exist and act synergistically with the AKT pathway.

In primary oocytes from the starfish Asterina pectinifera, AKT was reported to inhibit MYT 1 through AKT-dependent phosphorylation and down-regulation at the G2/M transition [23]. Moreover, in HEK293T cells, AKT promotes G2/M cell-cycle progression by inducing phophorylation-dependent YWHAQ binding and cytoplasmic localization of WEE 1 kinase [24]. Thus, AKT may shift the balance during G2/M transition in favor of the dephosphorylated and activated CDC2A required for mitotic entry.

It is known that CDC2A activity is regulated by phosphorylation. CDC2A activity is inhibited by phosphorylation on Ser14 and Tyr15 catalyzed by WEE 1/MYT 1 kinase. Tyr15 is a target for CDC25 phosphatase, and activated CDC25 dephosphorylates CDC2A on Tyr15, which activates CDC2A. In humans, three CDC25 isoforms have been identified, and two of them, CDC25B and CDC25C phosphatases, have roles in regulating G2/M progression [35]. In vivo, CDC25B is active during G2 phase before CDC25C is activated at the G2/M transition [47–49], and CDC25B is more efficient than CDC25C in promoting mitosis [50, 51]. These data indicate that CDC25B is the initiator of mitosis, whereas CDC25C acts to ensure full activation of MPF and thereby rapid entry into mitosis. CDC25B has been shown to be phosphorylated by a number of protein kinases including CSNK2A2 (previously known as CK2), CDK/CCNs, CHEK, MAPK, MAPKAP, AURKA, and PEG3 kinase, and these events have been shown to be responsible for changes in the localization and/or the activity of this phosphatase [52–58]. Recently, it was reported that in Hela cells, AKT can phosphorylate CDC25B on Ser353, resulting in a nuclear export-dependent cytoplasmic accumulation of the phosphatase [38]. Our data have extended these observations and explained the effect of the AKT/CDC25B pathway during the first round of mitosis of mouse fertilized eggs. Our results suggest that AKT may directly phosphorylate CDC25B on Ser351 and can promote its function during the first round of mitosis of mouse fertilized eggs.

The functions of proteins are firmly correlated with their subcellular localization. CDC25B localization was shown to depend on nuclear export signal and nuclear localization signal sequences and to be regulated through its interaction with YWHAQ proteins [59–62]. CDC25B accumulation in the cytoplasm has been correlated with spindle formation and it was suggested that this phosphatase pool located in close vicinity of the centrosome was responsible for the activation of the cytoplasmic pool of MPF [48, 55, 63]. Recently, it has been shown that active MPF first appears at centrosomes in prophase, and CDC25B can specifically activate MPF on centrosomes [64, 65]. These results have indicated an important mitotic function of CDC25B in the cytoplasmic centrosome. In somatic cells, the centrosome is established as an integrator of positive and negative pathways for entry to mitosis [66], and essential molecules such as CDC25B, CHEK1, AURKA, CDC2A/CCNB1, and active AKT are associated with the centrosome at G2/M transition [63, 67–69], suggesting that AKT-dependent phosphorylation of CDC25B may be involved in the initiation of MPF activation in the centrosome. Indeed, we have predicted that there are nuclear export signals between 52 and 65 residues and nuclear localization signals between 343 and 352 residues on the mouse CDC25B sequence. According to our data, the most plausible phosphorylation site of AKT on the mouse CDC25B sequence, Ser351, was just located in the nuclear localization signal (Bingzhi Yu, unpublished data). So we deduced that CDC25B-S351 phosphorylation by AKT may have masked the nuclear localization signal and selectively reduced the rate of CDC25B nuclear import. This presumably results in more efficient exclusion of CDC25B from the nucleus, initiates the activation of MPF on the centrosome, and promotes the cell division of one-cell mouse fertilized eggs.

In conclusion, we have reported for the first time that AKT may phosphorylate CDC25B-S351 and then activate MPF to improve the cell division of mammalian early embryos. Further analysis of direct phosphorylation of AKT on CDC25B and the experiments about the subcellular localization of pCDC25B-S351 will be conducted, which will help us better understand the mechanism of the regulation of cell-cycle progression in early embryo development.

ACKNOWLEDGMENTS

We thank Prof. Michael J. Quon for the kind gift of the constructs encoding the wild type AKT (pCIS2-Akt1-WT), myristoylated AKT (pCIS2-myr-Akt1), and the kinase-deficient AKT (pCIS2-Akt1-KD).

FOOTNOTES

1Sponsored by the National Nature Science Foundation of China (30570945). Back

Correspondence: 2Bingzhi Yu, Department of Biochemical and Molecular Biology, China Medical University, Beier Road, Heping District, Shenyang 110001, Liaoning Province, China. FAX: 0086 24 23261253; e-mail address: ybzbiochem{at}yeah.net

Received: 21 January 2007.

First decision: 8 February 2007.

Accepted: 30 May 2007.

REFERENCES

  1. Kim N. Putting the cell cycle in order Science 1996 2741643–1645[Free Full Text]
  2. Dunphy WG, Brizuela L, Beach D, Newport J. The Xenopus Cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis Cell 1988 54424–432
  3. Gautier J, Norbury C, Lohka MJ, Nurse P, Maller J. Purified maturation-promoting factor contains the product of a Xenopus homolog of the fission yeast cell cycle control gene Cdc2 Cell 1988 54433–439[CrossRef][Medline]
  4. Hunt T. Maturation-promoting factor, cyclin, and the control of M-phase Curr Opin Cell Biol 1989 1268–274[CrossRef][Medline]
  5. Labbe JC, Picard A, Peaucellier G, Cavadore JC, Nurse P, Doree M. Purification of MPF from starfish identification as H1 histone kinase p34cdc2 and a possible mechanism for its periodic activation Cell 1989 57253–256[CrossRef][Medline]
  6. Murray AW and Kirschner MW. Cyclin synthesis drives the early embryonic cell cycle Nature 1989 339275–280[CrossRef][Medline]
  7. Coleman TR and Dunphy WG. Cdc2 regulatory factors Curr Opin Cell Biol 1994 6877–882[CrossRef][Medline]
  8. Mueller PR, Coleman TR, Kumagai A, Dunphy WG. Myt1: a membrane-associated inhibitory kinase that phosphorylates Cdc2 on both threonine-14 and tyrosine-15 Science 1995 27086–90[Abstract/Free Full Text]
  9. Mueller PR, Coleman TR, Dunphy WG. Cell cycle regulation of a Xenopus Wee1-like kinase Mol Biol Cell 1995 6119–134[Abstract]
  10. Solomon MJ, Glotzer M, Lee TH, Philippe M, Kirschner MW. Cyclin activation of p34cdc2 Cell 1990 631013–1024[CrossRef][Medline]
  11. Strausfeld U, Labbé JC, Fesquet D, Cavadore JC, Picard A, Sadhu K, Russell P, Dorée M. Dephosphorylation and activation of a p34cdc2/cyclin B complex in vitro by human CDC25 protein Nature 1991 351242–245[CrossRef][Medline]
  12. Millar JBA and Russell P. The cdc25 M-phase inducer: an unconventional protein phosphatase Cell 1992 68407–410[CrossRef][Medline]
  13. King RW, Jackson P K, Kirschner MW. Mitosis in transition Cell 1994 79563–571[CrossRef][Medline]
  14. Dunphy WG. The decision to enter mitosis Trends Cell Biol 1994 4202–207[CrossRef][Medline]
  15. Chang F, Lee JT, Navolanic PM, Steelman LS, Shelton JG, Blalock WL, Franklin RA, McCubrey JA. Involvement of PI3K/Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation: a target for cancer chemotherapy Leukemia 2003 17590–603[CrossRef][Medline]
  16. Fujita N, Sato S, Katayama K, Tsuruo T. Akt-dependent phosphorylation of p27Kip1 promotes binding to 14-3-3 and cytoplasmic localization J Biol Chem 2002 27728706–28713[Abstract/Free Full Text]
  17. Liang J, Petrocelli JT, Kotchetkov R, Connor MK, Han K, Lee JH, Ciarallo S, Catzavelos C, Beniston R, Franssen E, Slingerland JM. PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest Nat Med 2002 81153–1160[CrossRef][Medline]
  18. Sekimoto T, Fukumoto M, Yoneda Y. 14-3-3 suppresses the nuclear localization of threonine 157-phosphorylated p27Kip1 EMBO J 2004 231934–1942[CrossRef][Medline]
  19. Shin I, Yakes FM, Rojo F, Shin NY, Bakin AV, Baselga J, Arteaga CL. PKB/Akt mediates cell-cycle progression by phosphorylation of p27Kip1 at threonine 157 and modulation of its cellular localization Nat Med 2002 81145–1152[CrossRef][Medline]
  20. Viglietto G, Motti ML, Bruni P, Melillo RM, D'Alessio A, Califano D, Vinci F, Chiappetta G, Tsichlis P, Bellacosa A, Fusco A, Santoro M. Cytoplasmic relocalizaiton and inhibition of the cyclin-dependent kinase inhibitor p27Kip1 by PKB/Akt-mediated phosphorylation in breast cancer Nat Med 2002 81136–1144[CrossRef][Medline]
  21. Zhou BP, Liao Y, Xia W, Spohn B, Lee MH, Hung MC. Cytoplasmic localization of p21Cip1/Waf1 by Akt-induced phosphorylation in HER-2/neu-overexpressing cells Nat Cell Biol 2001 3245–252[CrossRef][Medline]
  22. Kandel ES, Skeen J, Majewski N, Cristofano AD, Pandolfi PP, Feliciano CS, Gartel A, Hay N. Activation of Akt/protein kinase B overcomes a G2/M cell cycle checkpoint induced by DNA damage Mol Cell Biol 2002 227831–7841[Abstract/Free Full Text]
  23. Okumura E, Fukuhara T, Yoshida H, Hanada S, Kozutsumi R, Mori M, Tachibana K, Kishimoto T. Akt inhibits Myt1 in the signaling pathway that leads to meiotic G2/M-phase transition Nat Cell Biol 2002 4111–116[CrossRef][Medline]
  24. Kazuhiro K, Naoya F, Takashi T. Akt/protein kinase B-dependent phosphorylation and inactivation of Wee1 Hu promote cell cycle progression at G2/M transition Mol Cell Biol 2005 255725–5737[Abstract/Free Full Text]
  25. Manipulating the mouse embryos: A laboratory manual Hogan B and Constantini LE. 1986New York Cold Harbor Laboratory Press249–256
  26. Alessi DR, Andjelkovic M, Caudwell B, Cron P, Morrice N, Cohen P, Hemmings BA. Mechanism of activation of protein kinase B by insulin and IGF-1 EMBO J 1996 156541–6551[Medline]
  27. Faiyaz A, Li-Na C, Lena SH, Ling-Mei W, Tova RL, Jaclyn HP, Michael JQ, Eva D, Vincent CM. Cyclic nucleotide phosphodiesterase 3B is a downstream target of protein kinase B and may be involved in regulation of effects of protein kinase B on thymidine incorporation in FDCP2 cells J Immunol 2000 1644678–4688[Abstract/Free Full Text]
  28. Gallicano GI, McGaughey RW, Capco DG. Activation of protein kinase C after fertilization is required for remodeling the mouse egg into the zygote Mol Reprod Dev 1997 46587–601[CrossRef][Medline]
  29. Aimee DK, Fumito T, Richard AR. Akt, a Pleckstrin Homology domain containing kinase, is activated primarily by phosphorylation J Biol Chem 1996 27121920–21926[Abstract/Free Full Text]
  30. Datta K, Bellacosa A, Chan TO, Tsichlis PN. Akt is a direct target of the phophatidylinositol 3-kinase: activation by growth factors, v-src and v-Ha-ras, in Sf9 and mammalian cells J Biol Chem 1996 27130835–30839[Abstract/Free Full Text]
  31. Klippel A, Reinhard C, Kavanaugh MW, Apell G, Escobedo MA, Williams LT. Membrane localization of phosphatidylinositol 3-kinase is sufficient to activate multiple signal-transducing kinase pathways Mol Cell Biol 1996 164117–4127[Abstract]
  32. Cong LN, Chen H, Li Y, Zhou L, McGibbon MA, Taylor SI, Quon MJ. Physiological role of Akt in insulin-stimulated translocation of GLUT4 in transfected rat adipose cells Mol Endocrinol 1997 111881–1890[Abstract/Free Full Text]
  33. Lee SR, Park JH, Park EK, Chung CH, Kang SS, Bang OS. Akt-induced promotion of cell-cycle progression at G2/M phase involves upregulation of NF-Y binding activity in PC12 cells J Cell Physiol 2005 205270–277[CrossRef][Medline]
  34. Watanabe S, Umehara H, Murayama K, Okabe M, Kimura T, Nakano T. Activation of Akt signaling is sufficient to maintain pluripotency in mouse and primate embryonic stem cells Oncogene 2006 252697–2707[CrossRef][Medline]
  35. Nilsson I and Hoffmann I. Cell cycle regulation by the Cdc25 phosphatase family Prog Cell Cycle Res 2000 4107–114[Medline]
  36. Hoffmann I, Clarke P, Marcote MJ, Karsenti E, Draetta G. Phosphorylation and activation of human cdc25-C by cdc2-cyclin B and its involvement in the self amplification of MPF at mitosis EMBO J 1993 1253–63[Medline]
  37. Kumagai A and Dunphy W. Regulation of the cdc25 protein during the cell cycle in Xenopus extracts Cell 1992 70139–151[CrossRef][Medline]
  38. Baldin V, Theis-Febvre N, Benne C, Froment C, Cazales M, Burlet-Schiltz O, Ducommun B. PKB/Akt phosphorylates the Cdc25B phosphatase and regulates its intracellular localization Biol Cell 2003 95547–554[CrossRef][Medline]
  39. Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B Nature 1995 378785–789[CrossRef][Medline]
  40. Duble BW and Woodgett JR. GSK-3: tricks of the trade for a multi-tasking kinase J Cell Sci 2003 1161175–1186[Abstract/Free Full Text]
  41. Papadopoulou D, Bianchi MW, Bourouis M. Functional studies of shaggy/glycogen synthase kinase 3 phosphorylation sites in Drosophila melanogaster Mol Cell Biol 2004 244909–4919[Abstract/Free Full Text]
  42. Edward JM, Kei S, Laura JA, Leah R, Natalia S, Rodolfo M, Dario RA. Role that phosphorylation of GSK3 plays in insulin and Wnt signaling defined by knockin analysis EMBO J 2005 241571–1583[CrossRef][Medline]
  43. Yu B, Zheng J, Yu AM, Shi XY, Liu Y, Wu DD, Fu W, Yang J. Effects of protein kinase C on M-phase promoting factor in early development of fertilized mouse eggs Cell Biochem Funct 2004 22291–298[CrossRef][Medline]
  44. Yu B, Wang Y, Liu Y, Liu Y, Li X, Wu DD, Zong Z, Zhang J, Yu D. Protein kinase A regulates cell cycle progression of mouse fertilized eggs by means of MPF Dev Dyn 2005 23298–105[CrossRef][Medline]
  45. Kalous J, Solc P, Baran V, Kubelka M, Schultz RM, Motlik J. PKB/AKT is involved in resumption of meiosis in mouse oocytes Biol Cell 2006 98111–123[CrossRef][Medline]
  46. Patrick S, Magali LB, Julia M, Robert B, Sandrine B, Odile ML, Partrick C. Signal transduction pathways that contribute to CDC2A/cyclin B activation during the first mitotic division in sea urchin embryos Exp Cell Res 2004 296347–357[CrossRef][Medline]
  47. Gabrielli BG, Clark JM, McCormack AK, Ellem KA. Hyperphosphorylation of the N-terminal domain of Cdc25 regulates activity toward cyclin B1/Cdc2 but not cyclin A/Cdk2 J Biol Chem 1997 27228607–28614[Abstract/Free Full Text]
  48. Gabrielli BG, Clark JM, McCormack AK, Ellem KA. Ultraviolet light-induced G2 phase cell cycle checkpoint blocks cdc25-dependent progression into mitosis Oncogene 1997 15749–758[CrossRef][Medline]
  49. Lammer C, Wagerer S, Saffrich R, Mertens D, Ansorge W, Hoffmann I. The cdc25B phosphatase is essential for the G2/M phase transition in human cells J Cell Sci 1998 1112445–2453[Abstract]
  50. Karlsson C, Katich S, Hagting A, Hoffmann I, Pines J. Cdc25B and Cdc25C differ markedly in their properties as initiators of mitosis J Cell Biol 1999 146573–584[Abstract/Free Full Text]
  51. Goldstone S, Pavey S, Forrest A, Sinnamon J, Gabrielli B. Cdc25-dependent activation of cyclin A/cdk2 is blocked in G2 phase arrested cells independently of ATM/ATR Oncogene 2001 20921–932[CrossRef][Medline]
  52. Baldin V, Cans C, Knibiehler M, Ducommun B. Phosphorylation of human CDC25B phosphatase by CDC2A-cyclin A triggers its proteasome dependent degradation J Biol Chem 1997 27232731–32734[Abstract/Free Full Text]
  53. Bulavin DV, Amundson SA, Fornace AJ. p38 and Chk1 kinases: different conductors for the G(2)/M checkpoint symphony Curr Opin Genet Dev 2002 1292–97[CrossRef][Medline]
  54. Cazales M, Schmitt E, Montembault E, Dozier C, Prigent C, Ducommun B. CDC25B phosphorylation by Aurora-A occurs at the G2/M transition and is inhibited by DNA damage Cell Cycle 2005 41233–1238[Medline]
  55. Lindqvist A, Kallstrom H, Karlsson RC. Characterisation of Cdc25B localization and nuclear export during the cell cycle and in response to stress J Cell Sci 2004 1174979–4990[Abstract/Free Full Text]
  56. Manke IA, Nguyen A, Lim D, Stewart MQ, Elia AE, Yaffe MB. MAPKAP kinase-2 is a cell cycle checkpoint kinase that regulates the G2/M transition and S phase progression in response to UV irradiation Mol Cell 2005 1737–48[CrossRef][Medline]
  57. Mirey G, Chartrain I, Froment C, Quaranta M, Bouche JP, Monsarrat B, Tassan JP, Ducommun B. CDC25B phosphorylated by pEg3 localizes to the centrosome and the spindle poles at mitosis Cell Cycle 2005 4806–811[Medline]
  58. Theis FN, Filhol O, Froment C, Cazales M, Cochet C, Monsarrat B, Ducommun B, Baldin V. Protein kinase CK2 regulates CDC25B phosphatase activity Oncogene 2003 22220–232[CrossRef][Medline]
  59. Davezac N, Baldin V, Gabrielli B, Forrest A, Theis-Febvre N, Yashida M, Ducommun B. Regulation of CDC25B phosphatases subcellular localization Oncogene 2000 192179–2185[CrossRef][Medline]
  60. Forrest A and Gabrielli B. Cdc25B activity is regulated by 14-3-3 Oncogene 2001 204393–4401[CrossRef][Medline]
  61. Giles N, Forrest A, Gabrielli B. 14-3-3 acts as an intramolecular bridge to regulate cdc25B localization and activity J Biol Chem 2003 27828580–28587[Abstract/Free Full Text]
  62. Mils V, Baldin V, Pinta I, Goubin F, Papin C, Waye M, Eychene A, Ducommun B. Specific interaction between 14-3-3 isoforms and the human CDC25B phosphatase Oncogene 2000 191257–1265[CrossRef][Medline]
  63. Gabrielli BG, De Souza CP, Tonks ID, Clark JM, Hayward NK, Ellem KA. Cytoplasmic accumulation of cdc25B phosphatase in mitosis triggers centrosomal microtubule nucleation in HeLa cells J Cell Sci 1996 1091081–1093[Abstract]
  64. Jackman M, Lindon C, Nigg EA, Pines J. Active cyclin B1-Cdk1 first appears on centrosomes in prophase Nat Cell Biol 2003 5143–148[CrossRef][Medline]
  65. Lindqvist A, Kallstrom A, Lundgren A, Barsoium E, Rosenthal CK. Cdc25B cooperates with Cdc25A to induce mitosis but has a unique role in activating cyclin B1-Cdk1 at the centrosome J Cell Biol 2005 17135–45[Abstract/Free Full Text]
  66. Kramer A, Lukas J, Bartek J. Checking out the centrosome Cell Cycle 2004 31390–1393[Medline]
  67. Dutertre S, Cazales M, Quaranta M, Froment C, Trabut V, Dozier C, Mirey G, Bouche JP, Theis-Febvre N, Schmitt E, Monsarrat B, Prigent C, Ducommun B. Phosphorylation of CDC25B by Aurora-A at the centrosome contributes to the G2-M transition J Cell Sci 2004 1172523–2531[Abstract/Free Full Text]
  68. Kramer A, Mailand N, Lukas C, Syljuasen RG, Wilkinson CJ, Nigg EA, Bartek J, Lukas J. Centrosome-associated Chk1 prevents premature activation of cyclin-B-Cdk1 kinase Nat Cell Biol 2004 6884–891[CrossRef][Medline]
  69. Wakefield JG, Stephens DJ, Tavare JM. A role for glycogen synthase kinase-3 in mitotic spindle dynamics and chromosome alignment J Cell Sci 2003 116637–646[Abstract/Free Full Text]



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