Bingzhi Yu

Protein kinase a modulates Cdc25B activity during meiotic resumption of mouse oocytes

Protein kinase A (PKA) play a critical role in maintaining the meiotic arrest. However, the steps downstream of PKA remain largely unknown. In this study, we investigated the regulation of meiotic resumption by PKA/Cdc25B pathway in mouse oocytes. Injection of mRNA coding for Cdc25b‐S321A had a more potent maturation‐inducing ability than Cdc25b‐WT. When co‐injected with PKA inhibitor, Cdc25B‐WT had similar activities with Cdc25B‐S321A. Meanwhile, the phosphorylation of Cdc25B‐S321 was detected in germinal vesicle (GV) oocytes by Western blotting with a phospho‐Ser321‐specific antibody and the band disappeared when oocytes reenter into the meiotic cell cycle. Furthermore, Cdc25B‐WT translocated to the nucleus shortly before GV breakdown (GVBD), whereas phosphorylated Cdc25B‐S321 expressed exclusively in the cytoplasm and the signal could not be detected in GVBD oocytes. Taken together, these data indicate that Cdc25B‐Serine321 is the potential PKA target and Cdc25B subcellular localization determines its function during the process of maintaining GV arrest in mouse oocytes. Developmental Dynamics 237:3777–3786, 2008.

Protein kinase A regulates cell cycle progression of mouse fertilized eggs by means of MPF

Cell cycle of one‐cell stage mouse fertilized eggs was accompanied by fluctuation in the concentration of adenosine 3′5′‐monophosphate (cAMP) and in the activity of free catalytic subunit of cAMP‐dependent protein kinase (PKA). The concentration of cAMP and the activity of free catalytic subunit of PKA decreased at the onset of mitosis and increased at the transition between mitosis and G1 phase. Stimulation of PKA by microinjection of cAMP into one‐cell stage mouse embryos at G2 phase induced interphase arrest and prevented the activation of M‐phase promoting factor (MPF). Upon blockage of the activation of PKA by microinjecting a thermostable PKA inhibitor (PKI) into one‐cell stage mouse embryos at G2 phase, the increase in the MPF activity occurred 30 min earlier than in control group. When a high dose of PKI was microinjected, a transition into interphase was prevented, and the activity of MPF remained high. Western blot analysis showed that Cdc2 remained phosphorylated in cAMP microinjected embryos by the time when control embryos were at metaphase and showed dephosphorylated Cdc2; conversely, Cdc2 dephosphorylation was accelerated in PKI‐microinjected embryos. At the same time, Cdc2 was phosphorylated at Tyr15 at G2 phase and even at M phase when cAMP was microinjected but was dephosphorylated when PKI was microinjected. PKI microinjection also prevented cyclin B degradation and sustained MPF activity, thus delaying the transition from metaphase to anaphase. Our results show that PKA, by inhibiting MPF, regulates cell cycle progression of fertilized eggs. Developmental Dynamics 232:98–105, 2005.

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

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.

CDC25B Acts as a Potential Target of PRKACA in Fertilized Mouse Eggs

Abstract Protein kinase A (PRKACA) has been documented as a pivotal regulator in meiosis and mitosis arrest. Although our previous work has established that PRKACA regulates cell cycle progression of mouse fertilized eggs by inhibiting M-phase promoting factor (MPF), little is known about the intermediate factor between PRKACA and MPF in the mitotic cell cycle. In this study, we investigated the role of the PRKACA/CDC25B pathway on the early development of mouse fertilized eggs. Overexpression of unphosphorylatable CDC25B mutant (Cdc25b-S321A or Cdc25b-S229A/S321A) rapidly caused G2-phase eggs to enter mitosis. Microinjection of either Cdc25b-WT or Cdc25b-S229A mRNA also promoted G2/M transition, but much less efficiently than Cdc25b-S321A and Cdc25b-S229A/S321A. Moreover, mouse fertilized eggs overrode the G2 arrest by microinjection of either Cdc25b-S321A or Cdc25b-S229A/S321A mRNA, which efficiently resulted in MPF activation by directly dephosphorylating CDC2A-Tyr15, despite culture under conditions that maintained exogenous dibutyryl cAMP. Using a highly specific antibody against phospho-Ser321 of CDC25B in Western blotting, we showed that CDC25B-Ser321 was phosphorylated at the G1 and S phases, whereas Ser321 was dephosphorylated at the G2 and M phases in vivo. Our findings identify CDC25B as a potential target of PRKACA and show that PRKACA regulates G2/M transition by phosphorylating CDC25B-Ser321 but not CDC25B-Ser229 on the first mitotic division of mouse fertilized eggs..

The Role of 14-3-3ε Interaction with Phosphorylated Cdc25B at Its Ser321 in the Release of the Mouse Oocyte from Prophase I Arrest

The protein kinase A (PKA)/Cdc25B pathway plays a critical role in maintaining meiotic arrest in mouse oocytes. However, the molecular mechanism underlying this interchange is not known. In this study, we assessed the role of 14-3-3ε interaction with phosphorylated Cdc25B at its Ser321 as the mouse oocyte is released from prophase I arrest. The 14-3-3ε isoform is a highly conserved protein with various regulatory roles, including maintenance of meiotic arrest. Cdc25B phosphatase is also a key cell cycle regulator. 14-3-3ε binds to Cdc25B-WT, which was abrogated when Ser321 of Cdc25B was mutated to Ala. In addition, we found that 14-3-3ε and Cdc25B were co-localized. Cdc25B was translocated from the cytoplasm to the nucleus shortly before germinal vesicle breakdown (GVBD) during the primary oocyte stage of oogenesis. However, mutation of Ser321 to Ala completely abolished the cytoplasmic localization of Cdc25B. Furthermore, oocytes co-expressing of Cdc25B-WT or Cdc25B-Ser321D and 14-3-3ε were unable to undergo GVBD. In contrast, co-expression of 14-3-3ε and Cdc25B-Ser321A induced GVBD and allowed the process to continue. Down-regulation of 14-3-3ε caused partial meiotic resumption. Taken together, these data indicate that Ser321 of Cdc25B is the specific binding site for 14-3-3ε binding, and that 14-3-3ε is the significant factor in Cdc25B regulation during meiotic resumption of GV stage.

mTOR-rictor is the Ser473 kinase for AKT1 in mouse one-cell stage embryos

Mammalian target of rapamycin (mTOR) controls cell growth and proliferation via the raptor-mTOR (TORC1) and rictor-mTOR (TORC2) protein complexes. The mTORC2 containing mTOR and rictor is thought to be rapamycin insensitive and it is recently shown that both rictor and mTORC2 are essential for the development of both embryonic and extra embryonic tissues. To explore rictor function in the early development of mouse embryos, we disrupted the expression of rictor, a specific component of mTORC2, in mouse fertilized eggs by using rictor shRNA. Our results showed that one-cell stage eggs that were lack of rictor could not enter into the two-cell stage normally. Recent biochemical studies suggests that TORC2 is the elusive PDK2 (3′-phosphoinositide-dependent kinase 2) for AKT/PKB Ser473 phosphorylation, which is deemed necessary for AKT function, so we microinjected AKT-S473A into mouse fertilized eggs to investigate whether AKT-S473A is downstream effector of mTOR.rictor to regulate the mitotic division. Our findings revealed that the rictor induced phosphorylation of AKT in Ser473 is required for TORC2 function in early development of mouse embryos.

Ser149 is another potential PKA phosphorylation target of Cdc25B in G2/M transition of fertilized mouse eggs

It is well documented that protein kinase A (PKA) acts as a negative regulator of M phase promoting factor (MPF) by phosphorylating cell division cycle 25 homolog B (Cdc25B) in mammals. However, the molecular mechanism remains unclear. In this study, we identified PKA phosphorylation sites in vitro by LC-MS/MS analysis, including Ser149, Ser229, and Ser321 of Cdc25B, and explored the role of Ser149 in G2/M transition of fertilized mouse eggs. The results showed that the overexpressed Cdc25B-S149A mutant initiated efficient MPF activation by direct dephosphorylation of Cdc2-Tyr15, resulting in triggering mitosis prior to Cdc25B-WT. Conversely, overexpression of the phosphomimic Cdc25B-S149D mutant showed no significant difference in comparison with the control groups. Furthermore, we found that Cdc25B-Ser149 was phosphorylated at G1 and S phases, whereas dephosphorylated at G2 and M phases, and the phosphorylation of Cdc25B-Ser149 was modulated by PKA in vivo. In addition, we examined endogenous and exogenous Cdc25B, which were expressed mostly in the cytoplasm at the G1 and S phases and translocated to the nucleus at the G2 phase. Collectively, our findings provide evidence that Ser149 may be another potential PKA phosphorylation target of Cdc25B in G2/M transition of fertilized mouse eggs and Cdc25B as a direct downstream substrate of PKA in mammals, which plays important roles in the regulation of early development of mouse embryos.

Effect of LHRH-PE40 on target cells via LHRH receptors.

Objective. To detect the effect and cytotoxicity of luteinizing hormone-releasing hormone-Pseudomonas aeruginosa exotoxin 40 (LHRH-PE40) on target cells using LHRH receptors (LHRHR). Methods. The affinity of LHRH-PE40 and LHRH binding to LHRHR on the membrane surface of target cells were measured by enzyme linked immunosorbent assay. Morphological observations with light microscope were used to analyze its receptor pathway, with Spiegelmer, and cytotoxicity. IC50 values of LHRH-PE40, which caused 50% inhibition of tumor cell growth were evaluated by MTT assay. The target cells were exposed to LHRH-PE40 and its cytotoxicity was analyzed by scanning and transmission electron microscopies, agarose gel electrophoresis, and flow cytometry. Results. LHRH-PE40 killed target cells by LHRHR pathway. The morphological changes in these cells showed decreased cell size, cytoplasmic membrane blebbing, and chromatin condensation and margination. At a certain concentration and time point, HeLa cells were also induced to undergo programmed cell death. Conclusion. LHRH-PE40 induced target cells apoptosis via LHRHR.

Ser 15 of WEE1B is a potential PKA phosphorylation target in G2/M transition in one-cell stage mouse embryos

The WEE1 kinase family has been shown to be the major kinase family responsible for phosphorylating Tyr 15 on cyclin-dependent kinase 1 (CDK1). WEE1 homolog 2 (WEE2, also known as WEE1B) was first identified in Xenopus laevis and more recently in humans and mice, and is responsible for phosphorylating the CDK1 inhibitory site and maintaining meiotic arrest in oocytes. However, the mechanism by which WEE1B is regulated in one-cell stage mouse embryos remains to be elucidated. In the present study, we examined the role of WEE1B-Ser 15 in G2/M transition of one-cell stage mouse embryos. WEE1B-Ser 15 was phosphorylated during the G1 and S phases, whereas Ser 15 was dephosphorylated during G2 and M phases in vivo. Overexpression of the phosphor-mimic Wee1B-S15D mutant delayed the re-entry of embryos into mitosis more efficiently than Wee1B-wild type (Wee1B-WT) by direct phosphorylation of CDK1-Tyr 15. The results of the present study suggested that WEE1B acts as a direct downstream substrate of protein kinase A (PKA) and that Ser 15 of WEE1B is a potential PKA phosphorylation target in the G2/M transition of mouse embryos. In addition, WEE1B inhibits mitosis by negatively regulating M phase promoting factor activity in one-cell stage mouse embryos.

Protein kinase B/Akt may regulate G2/M transition in the fertilized mouse egg by changing the localization of p21Cip1/WAF1

Protein kinase B (PKB, also called Akt) is known as a serine/threonine protein kinase. Some studies indicate that the Akt signalling pathway strongly promotes G2/M transition in mammalian cell cycle progression, but the mechanism remains to be clarified, especially in the fertilized mouse egg. Here, we report that the expression of Akt at both the protein and mRNA level was highest in G2 phase, accompanied by a peak of Akt activity. In addition, the subcellular localization of p21Cip1/WAF1 has been proposed to be critical in the cell cycle. Hence, we detected the expression and localization of p21Cip1/WAF1 after injecting fertilized mouse eggs with Akt mRNA. In one‐cell stage fertilized embryos microinjected with mRNA coding for a constitutively active myristoylated Akt (myr‐Akt), p21Cip1/WAF1 was retained in the cytoplasm. Microinjection of mRNA of kinase‐deficient Akt(Akt‐KD) resulted in nuclear localization of p21Cip1/WAF1. Meanwhile, microinjection of different types of Akt mRNA affected the phosphorylation status of p21Cip1/WAF1. However, there was no obvious difference in the protein expression of p21Cip1/WAF1. Therefore, Akt controls the cell cycle by changing the subcellular localization of p21Cip1/WAF1, most likely by affecting the phosphorylation status of p21Cip1/WAF1. Copyright