Involvement Of Protein Kinase B In Early Development Of Mouse Fertilized Eggs

IntroductionEarly embryonic division is characterized by a rapid succession of inter-phase and mitotic state in many species. The progression through M phase is controlled by maturation/M phase - promoting factor ( MPF) , a highly conserved complex consisting of a kinase, Cdc2, and an activating subunit, cyclin B. MPF undergoes tight regulation during the G2/M transition by phosphoryla-tion and dephosphorylation of Cdc2. In the G2 phase of vertebrate cells, Cdc2 is kept inactivated by inhibitory phosphorylation on tyrosine 15 ( Tyr - 15 ) and threonine 14 (Thr -14);these phophorylation are catalyzed by Weel/Mytl kinase. And if in this state, the Cdc2/cyclin B complex remains enzymatically inactive (pre - MPF). On entry into M phase, Cdc25, a dual - specificity phos-phatase family, dephosphorylates Cdc2 on both Tyr - 15 and Thr - 14, causing the activation of MPF. Thus, in principle, the G2/M transition can be induced by inactivation of Weel/Mytl kinases or activation of Cdc25 phosphatases or both. However, it is unknown how the dominance between the activities of the Weel/Mytl kinases and Cdc25 phosphatases is initially reversed to activate Cdc2/cyclin B at entry into M phase.Protein kinase B (PKB, also named kinase AKT) is known as a serine/ threonine protein kinase and a downstream molecular of PI3K. It's well established PKB play an important role in cellular processes such as glucose metabolism, cell proliferation, apoptosis, transcription and cell migration. There are recent indications that PKB could have a wide spread function in cell cycle regulation, since it involved in the regulation both of G1 phase progession and of G2/M transition. Despite the continuing discovery of new Akt members and ourincreasing knowledge of their basic biochemistry and molecular biology, relatively little is known about the effect of PKB in the regulation of mammalian development of early embryo, especially the mouse fertilized eggs. The mouse fertilized egg is the most simple and natural cell cycle model in vertebrate which is closer to human. There are few reports about the mechanism on the regulation of early development of mouse fertilized eggs especially the events of entry into M- phase. Here we reported that PKB causes the activation of MPF and strongly promotes the development of 1 - cell stage mouse fertilized eggs by inducing Akt- dependent phosphorylation of Cdc25B, a number of Cdc25 phosphatase family. Our findings identify Cdc25B as a target of PKB/Akt and provide new insight into the effect of PKB/Akt in the regulation of the development of mouse early embryos.Materials and methods1. Materials and reagentsFemales of 3 - 4 week - old Kunming strain mice were supplied by the Department of Laboratory of Animals, China Medical University. The constructs encoding the wild type Akt (pCIS2 - WT -Akt) and myristoylated Akt (pCIS2- myr - Akt) were gifts from Prof. Michael J. Quon at National Institutes of Health. The full - length cdc25B cDNA clone ( pBSK - cdc25B) was kindly provided by Dr. Tony Hunter at Salk Institute. pGEM - T vector was purchased from Promega. pBluescript II/SK vector and E. coli JM109 were previous kept in our lab. Restriction endonuclease Hind HI A Xhol ^ BamHI and Xbal were purchased from Fermentas. mMESSAGE mMACHINE kit from Ambion. Quick-Change Site - directed Mutagenesis Kit from STRATAGENE. Anti - Cdc2 -pTyrl5 monoclonal antibody and anti - cyclin B antibody from Santa - Cruz. 0- dianidine ? (3 - naphthyl acid phosphate and reagents for M - phase promoting factor ( MPF) and PKB activity assay from Sigma.2. Superovulation and eggs collectionFemale mice 4-6 weeks old were abdominal injected with 10IU of pregnant mares serum gonadotropin (PMSG) , and 48h later with 10IU human chori-onic gonadotropin (hCG). A single female was placed with a single male for fertilization. One - cell embryos were collected with M2 medium the next day (24h after hCG injection) from oviduct of females possessing a vaginal plug. After injected with different kinds of mRNA, embryos were transferred to culture in a drop of M16 medium, at 37 C, in a humidified atmosphere of 5% C02 in air, under paraffin oil.3. Construction of mRNA expression vectorsThe pCIS2 - WT - Akt and pCIS2 - myr - Akt were subcloned into pBlue-script II/SK vector using Hind HI and BamHI. These two recombinants were named pBSK - PKB - WT and pBSK - myr - PKB. The coding sequence of cdc25b was amplified by PCR using the pBSK - cdc25B as template and introduced the enzyme - incision site of Xhol and BamHI into 5' and 3' end respectively. The product was cloned into the vector pBluescript II/SK and named pB-SK-cdc25B-WT.Using Site - directed Mutagenesis Kit to mutate Lysine 179 to Alanine of PKB and to mutate Serine 351 to nonphosphorylatable alanine of Cdc25B, and these two mutants were called pBSK - PKB - KD and pBSK - Cdc25B - S351A respectively.All the recombinant plasmids were sequenced to verify the inserted gene correct and mutation successful. 4. In vitro transcriptionAll the constructs in pBluescript were cut singly with Xbal and in vitro transcribed into 5'- capped mRNA for microinjection by using mMESSAGE mMA-CHINE kit. After a transcription reaction was done, the reaction mixture was treated with DNase I to remove the DNA template. Then the mixture was extracted with phenol/chloroform, and RNA was precipitated with IiCl.5. mRNA microinjectionMouse fertilized eggs were microinjected using a micropipette and Eppen-dorf transferman manipulators mounted on a Olympus IX - 70 inverted microscope with DIC optics. Eggs were placed in a drop of M2 medium under paraffin oil in a lid of 3cm Falcon culture dish. Typical injection volume was 5% of total cell volume or lOpl per egg. mRNA was diluted to various concentrations in 5mmol/L Tris and 0.5mmol/L EDTA ( pH7.4) 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 30hr and 35hr after injection of hCG, and the results were analyzed statistically.6. MPF activity assayMPF kinase activity was measured using histone HI kinase assay. Five oo-cytes cultured in M16 medium were collected and subjected to freezing and thawing three times. A total of 25ul of MPF buffer was then added to the disrupted cells. Hie mixture was incubated at 30°C for 7min. After that, 25ul aliquots were spotted on Whatman p81 paper. The radioactivity on the filter paper was counted with a Beckman scintillation counter.7. PKB activity assay10 one - cell stage fertilized eggs cultured in Ml6 medium were cultured and lysed as described above, assayed for 30 min in PKB reaction buffer, the follow reaction was as same as the assay of Histone HI describe above.8. Western blottingProtein extracts of mouse fertilized eggs were prepared by adding appropriate number of oocytes in a minimal volume of collection medium to 20ul of protein extraction buffer. The extracts were briefly vortexed, quickly frozen on dry ice, and stored at -20^ until used. Laemmli sample buffer was added to the protein extracts, and the mixture was boiled for 5min and resolved on a 12% SDS - PAGE gel. For immunoblotting, the fractionated proteins were transferred to a nitro - cellulose membrane. The primary antibody against pTyrl5 of Cdc2 or cyclinB was used and alkaline phosphatase - conjugated IgG was as secondary antibody. The proteins were detected by using 0 - dianidine and |3 - naphthyl acid phosphate as the substrates of alkaline phosphatase.Result1. Microinjection of mRNA of PKB interferes cell division of 1 - cell stage mouse fertilized eggsTo examine whether PKB affects the mitotic cell cycle, 1 - cell stage mousefertilized eggs were isolated and cultured in M16 medium after microinjection of 0.03ng mRNA of PKB - WI\myr - PKB and PKB - KD, respectively. The control groups were in two situations: no microinjection or be given microinjection of TE buffer. In the control groups, embryos remained at the 1 - cell stage 26hr after hCG injection(G2 phase) , but over 63% of embryos reached 2 -cell stage 4hr later(30hr after the injection of hCG) , and there was no significant difference between the two control groups. Embryos microinjected with mRNA of PKB - WT were in 1 - cell stage 26hr after the hCG injection as control, but over 75.21% of embryos reached 2 - cell stage 30hr after the hCG injection. 15. 39% of embryos were died 35hr after the hCG injection. When embryos micro-injected with mRNA of myr - PKB, a constitutively activated, myristylated form of PKB, the percentage of cleavage was up to 91. 26% , and the percentage of death was down to 10.32% , even 34 of total 126 embryos reached 2 - cell stage at 26.5hr after the injection of hCG. Irregular division occurred 30hr after the hCG injection. On the other hand, only 12. 84% of embryos that microinjected with mRNA of PKB - KD reached 2 - cell stage 30hr after the hCG injection. These results demonstrated that microinjection of different kinds of mRNA of PKB can interfered the division of 1 - cell stage fertilized eggs and suggested that PKB may have the biological function of promoting the cell division of mouse early embryo.2. Effect of PKB mRNA microinjection on MPF activity and phosphoryla-tion status of Cdc2 - Tyrl5The promotion of the development of 2 - cell stage by PKB mRNA microinjection into fertilized eggs suggests a role in regulating mouse embryonic cleavage. Therefore we measured MPF activity at different time points when the first division of the fertilized eggs occurred. In PKB - WT group, MPF activity reached its peak value at 27.5hr after the hCG injection, about 30 minutes former than in control groups. In myr - PKB group, the peak of MPF activity occurred at 26. 5hr after the injection of hCG, about 1. 5hr former than the control group. In PKB - KD group, MPF activity reached its peak value even at 30hr, and the peak value was lower than that of control group. The phosphorylation status of Cdc2 - Tyrl5 in each group was coincident with the MPF activity.Meanwhile, we examined the expression of cyclinB at the point of G2/M transition, and there were no significant difference among the groups. These results suggests that PKB can promote the cleavage of 1 - cell stage fertilized eggs by means of affecting the phosphorylation status of Cdc2 - Tyrl5, directly or indirectly , and then increasing the activity of MPF.3. The PKB/Cdc25B pathway can operate in 1 -cell stage fertilized eggs Cdc25B acts as an initiator of the early mitotic events. It could well play a role in the activation of a centrosomal sub - population of Cdc2 - cyclin Bl that is next translocated to the nucleus where activation of Cdc25C will initiate an amplification loop driving the cell into mitosis. It has been reported that in Hela cells, PKB/Akt phosphorylates CDC25B on serine 353, resulting in a nuclear export - dependent cytoplasmic accumulation of the phosphatase. We then tested whether PKB can directly act on and promote Cdc25B (a potential substrate for PKB) in mouse fertilized eggs. We utilized Scansite software and Clustal W multiple sequence alignment program to predicted the relevant target sites of PKB on mouse Cdc25B. We finally got that mouse Cdc25B has a serine residue at position 351 (Ser351) , which corresponds to Ser353 of human Cdc25B, a residue phosphorylated by human PKB. We therefore injected 1 - cell stage eggs first with PKB - WT mRNA or myr - PKB mRNA as above and then with mRNA encoding either wild - type Cdc25B ( Cdc25B - WT) or a PKB - nonphosphory-latable Ser351 to Ala Cdc25B mutant (Cdc25B -S351A). The results showed that Cdc25B - S351A can strongly inhibit the effect of PKB. Embryos coex-pressed with PKB and Cdc25B - S351A remained at 1 - cell stage 28 hr after hCG injection, and only 9. 64% of total embryos reached 2 - cell stage 30hr after hCG. Even in the myr - PKB and Cdc25B - S351A coexpression group, Cdc25B - S351A can also decrease the percentage of division to 17. 72%. Meanwhile the Cdc25B - S351A mutant can inhibit the activation of MPF and affect the phosphorylation status of Cdc2 - Tyrl5. These results suggest strongly that PKB should directly phosphorylate Cdc25B on Ser351 and can promote its function in mouse fertilized eggs. Taken together, the present results indicate that the PKB/Cdc25B pathway can operate in 1 -cell stage fertilized eggs.DiscussionPKB/Akt is a signaling molecular that is known to enhance cell proliferation, survival as well as glycogen and protein synthesis. Here we provide evidence for an additional role of this Akt kinase in the control of the first round mitosis of mouse fertilized eggs. PKB overexpression in the 1 - cell mouse fertilized eggs promotes the cell division of early embryos, and exogenous expression of constitutively active PKB (myr-PKB) can enhance this effect. However, over-expression of kinase - dead PKB ( PKB - KD ) can inhibit the development of mouse fertilized eggs. So, we can say that PKB was a promoter of the first round mitosis of mouse fertilized eggs. Although the wild type PKB can partially promote early embryos division, it does much less efficiently than the myr -PKB. Because the only difference between WT - PKB and myr - PKB is the ability to interaction with the lipid bilayer, we can conclude that PKB interaction with lip-ids is essential for the signaling early embryo division.The role of PKB/Akt in promoting progression through Gl and into S -phase has been well characterized. It was reported to promote Gl progression through activation of the cyclin Dl/ Cdk4 complex. By contrast, PKB is known to phosphorylates p21Wafl/Ciplon Thrl45 and on p27Kipl Thrl57 and Thr 198, resulting in cytoplasmic translocation and suppression of the function. These results indicate that the Akt promotes cell cycle progression in Gl phase by activating positive regulators and inactivating negative ones. Otherwise, PKB/Akt is suggested to fuction as a G2/M initiator. Akt activation by overexpression of a constitutively active form or by the loss of PTEN could overcome the G2/M arrest that was induced by gamma irradiation. In addition, PTEN null ES cells were reported to transit faster from the G2/M to the Gl phase of the cell cycle than wild - type ES cells, and LY294002, an inhibitor of PI3K, elicited G2/M arrest in HEK 293 cells. Although these results suggest that the PKB strongly promotes G2/M transition in a mammalian cell cycle progression, the direct relationship between the PKB and regulation of the G2/M transition is not fully understood. In this study, we show that PKB, especial the myr - PKB, can pro-mote the initiation of MPF activation by means of affecting the phosphorylation status of Cdc2 - Tyrl5. These results suggest that PKB may be an upstream regulator of MPF activation, similar to a role in maturation of starfish Asterina pectinifera, Xenopus and mouse oocyte. Meanwhile, we also noticed that in the overexpression PKB - KD group, more than 45% of the eggs reached 2 - cell stage accompanied with the activation of MPF when extended the time to 35hr after hCG injection. In support of our findings, others have also reported that inhibiting PDK - PKB signaling in mouse oocytes with LY -294002 total prevents resumption of meiosis at 55min when control oocytes have already undergone GVBD and more than 50% of these oocytes undergo GVBD following 2h of culture. These datas suggest that alternative pathways leading to MPF activation exit and act synergistically with PKB pathway.In primary oocytes from the starfish Asterina pectinifera, Akt was reported to inhibit Mytl through Akt - dependent phosphorylation and down - regulation at the G2/M transition. Moreover, in HEK293T cells, Akt promotes G2/M cell cycle progression by inducing phophorylation - dependent 14 -3 - 39 binding and cytoplasmic localization of Weel kinase. Thus, PKB may shift the balance during G2/M transition in favour of dephosphorylated and activated Cdc2 required for mitotic entry.It is known that Cdc2 activity is regulated by phosphorylation. Cdc2 activity is inhibited by phosphorylation on Serl4 and Tyrl5 catalyzed by Weel/Mytl kinase. Tyrl5 is a target for Cdc25 phosphatase. Activated Cdc25 then dephos-phorylates Cdc2 on Tyrl5, which activates Cdc2. In humans, three Cdc25 iso-forms have been identified, and two of these, Cdc25B and Cdc25C phosphatase, have roles in regulating G2/M progression. In vivo, Cdc25B is active during G2 phase, before Cdc25C is activated at the G2/M transition, and Cdc25B is more efficient than Cdc25C at promoting mitosis. These data point to Cdc25B as the initiator of mitosis, whereas the role of Cdc25C is to ensure full activation of MPF and thereby rapid entry into mitosis. Cdc25B has been shown to be phosphorylated by a number of protein kinase including CK2, Cdk/cyclins, CHK kinases, p38, MAPKAP, Aurora - A kinase, and pEg3 kinase, and these events have been shown to be responsible for changes in the localization and/orthe activity of the phosphatase. Recently, it was reported that in Hela cells, PKB can phosphorylate Cdc25B on Serine 353, resulting in a nuclear export -dependent cytoplasmic accumulation of the phosphatase. Our data extend these observations and investigate the effect of PKB/Cdc25B pathway during the first round mitosis of mouse fertilized eggs. Our results suggest strongly that PKB should directly phosphorylate Cdc25B on Ser -351 and can promote its function during the first round 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 ( NES ) and nuclear localization signal ( NLS) sequences and to be regulated through its interaction with 14-3 -3 proteins. Cdc25B accumulation in the cytoplasm has been correlated with prophase 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. Recently it has been shown that active MPF first appears at centrosomes in prophase, and Cdc25B can specifically activate MPF on centrosomes. These results shown that an important mitotic function of Cdc25B occurs in the centrosome of cytoplasm. In somatic cells, the centrosome is established as an integrator of positive and negative pathways for entry to mitosis and essential moleculars such as Cdc25B, Cdc2, Aurora - A, Cdc2/cyclinB and active PKB are associated with the centrosome at G2/M transition. These suggests that PKB - dependent phosphoryla-tion of Cdc25B may involve in the initiation of MPF activation in the centrosome. Indeed, we have predicted that there are NES between 52 -65 residues and NLS between 343 - 352 residues on the mouse Cdc25B sequence. According our data, the most possible phosphorylation - site of PKB on mouse Cdc25B sequence, Ser351, was just located in the NLS. So we deduced that Cdc25B -S351 phosphorylation by PKB may masked the NLS and selectively reduced the rate of Cdc25B nuclear import while leaving its rate of export unaffected, presumably resulting in more efficient exclusion of Cdc25B from the nucleus, and initiate the activation of MPF on the centrosome, then promote the cell division of 1 - cell mouse fertilized eggs.In conclusion, we have reported for the first time that PKB can phosphoryl-ate Cdc25B -S351 and mask the NLS, which causes more efficient exclusion of Cdc25B. Thus Cdc25B can activate MPF on centrosome in the cytoplasm and improve the cell division of mammalian early embryos. Further analysis of direct phosphorylation of PKB on Cdc25B and the experiments about the subcellular localization of pCdc25B - S351 will be proceeded and will help us understand better the mechanism of the regulation of cell cycle in early embryo development.Conclusion1. PKB can promote the division of mouse 1 - cell stage fertilized eggs. The activation of this kinase is necessary for the first round mitosis of mouse embryos.2. PKB can promote the cleavage of 1 - cell stage fertilized eggs by means of affecting the phosphorylation status of Cdc2 - Tyrl5, directly or indirectly, and then increasing the activity of MPF.3. PKB can phosphorylate Cdc25B -S351 and mask the NLS, which causes more efficient exclusion of Cdc25B. Thus Cdc25B can activate MPF on centrosome in the cytoplasm and improve the cell division of mammalian early embryos. PKB/Cdc25B pathway can operate in 1 - cell stage fertilized eggs.