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Ser 15 of WEE1B Is a Potential PKA Phosphorylation Target in G2/M Transition in One-Cell Stage Mouse Embryos

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Ser 15 of WEE1B Is a Potential PKA Phosphorylation Target in G2/M Transition in One-Cell Stage Mouse Embryos MOLECULAR MEDICINE REPORTS 7: 1929-1937, 2013 Ser 15 of WEE1B is a potential PKA phosphorylation target in G2/M transition in one-cell stage mouse embryos CHAO LIU1,2, YANCHUN LIU1, YANG LIU2, DIDI WU1, ZHIDONG LUAN2, ENHUA WANG3 and BINGZHI YU1,3 1Department of Biochemistry and Molecular Biology, China Medical University, Shenyang; 2Department of Developmental Biology, Liaoning Medical University, Jinzhou; 3Institute of Pathology and Pathopysiology, China Medical University, Shenyang, Liaoning, P.R. China Received December 17, 2012; Accepted March 28, 2013 DOI: 10.3892/mmr.2013.1437 Abstract. The WEE1 kinase family has been shown to be the Introduction major kinase family responsible for phosphorylating Tyr 15 on cyclin-dependent kinase 1 (CDK1). WEE1 homolog 2 (WEE2, M phase-promoting factor (MPF) is a universal regulator of M also known as WEE1B) was first identified in Xenopus laevis phase in the eukaryotic cell cycle (1,2). Its activation induces and more recently in humans and mice, and is responsible for entry into M phase and its subsequent inactivation is necessary phosphorylating the CDK1 inhibitory site and maintaining to exit from M phase. The activity of MPF in higher eukary- meiotic arrest in oocytes. However, the mechanism by which otic cells is primarily regulated by the formation of a complex WEE1B is regulated in one-cell stage mouse embryos remains between the catalytic cyclin-dependent kinase 1 (CDK1, to be elucidated. In the present study, we examined the role previously known as CDC2) subunit and the regulatory of WEE1B-Ser 15 in G2/M transition of one-cell stage mouse CCNB1 (previously known as cyclin B) subunit, and further embryos. WEE1B-Ser 15 was phosphorylated during the G1 and by phosphorylation/dephosphorylation at Thr 14 and Tyr 15 of S phases, whereas Ser 15 was dephosphorylated during G2 and CDK1 associated with CCNB1 (1-4). Until the end of G2 phase, M phases in vivo. Overexpression of the phosphor-mimic MPF remains inactive through the inhibitory phosphoryla- Wee1B-S15D mutant delayed the re-entry of embryos into tion of these residues by the WEE1 kinase family. WEE1 mitosis more efficiently than Wee1B-wild type (Wee1B-WT) proteins are dual-specificity kinases that uniquely function by direct phosphorylation of CDK1-Tyr 15. The results of the to phosphorylate specific Tyr/Thr residues on CDKs. There present study suggested that WEE1B acts as a direct down- are three WEE1 family members: WEE1 homolog 1 (WEE1, stream substrate of protein kinase A (PKA) and that Ser 15 of also designated as somatic WEE1A); WEE1 homolog 2 WEE1B is a potential PKA phosphorylation target in the G2/M (WEE2, also known as WEE1B) and PKMYT1 (previously transition of mouse embryos. In addition, WEE1B inhibits known as MYT1). Various genetic and biochemical studies mitosis by negatively regulating M phase promoting factor have indicated that WEE1, which was originally identified in activity in one-cell stage mouse embryos. the fission yeast Schizosaccharomyces pombe mutant with a small cell or WEE phenotype, is a Tyr-specific protein kinase conserved in all eukaryotes (5). In eukaryotic cells, WEE1A is a nuclear protein that is capable of phosphorylating Tyr 15 but not Thr 14 of CDK1 and thus inhibiting its kinase activity, thereby preventing entry into mitosis (6-9). PKMYT1, a membrane-associated inhibitory kinase, is present in meta- Correspondence to: Professor Bingzhi Yu, Department of zoans and phosphorylates CDK1 efficiently on Thr 14 and Biochemistry and Molecular Biology, China Medical University, Tyr 15 residues (6,10-15) and the human PKMYT1 kinase 92 Bei Er Road, Heping District, Shenyang, Liaoning 110001, preferentially phosphorylates CDK1 on Thr 14 in a cyclin- P. R. Ch i na dependent manner (13). WEE1B, a newly identified member of E-mail: [email protected] the WEE1 kinase family, is conserved from yeast to humans and has recently been shown to suppress CDK1 activity by Abbreviations: CDK1, cyclin-dependent kinase 1; Wee1B-WT, Wee1B-wild type; Wee1B-KD, Wee1B-kinase dead; PKA, protein phosphorylating its inhibitory sites (16-21). While WEE1A kinase A; MPF, M phase-promoting factor; PMSG, pregnant mare and PKMYT1 are well documented as important regulators serum gonadotropin; hCG, human chorionic gonadotropin of mitosis in multiple cell types in numerous species (22), the role and regulation of WEE1B in zygotes and early embryo Key words: WEE1B, mouse embryo, protein kinase A, M phase- development remain to be elucidated. promoting factor, cyclin-dependent kinase 1 Protein kinase A (PKA), a cAMP-dependent Ser/Thr kinase, is composed of two catalytic (C) subunits held in an inactive state in association with a regulatory (R) subunit 1930 LIU et al: WEE1B ACTS AS A DIRECT SUBSTRATE OF PKA dimer. PKA constitutes one of the most important components of Health. The protocol for animal handling and the treatment of several signal transduction pathways, which are important procedures were approved by the Committee on the Ethics in cell growth, differentiation, proliferation and cell cycle of Animal Experiments of the China Medical University. control. Studies in starfish, Xenopus and mammalian oocytes Rabbit anti-WEE1B (mWEE1B-specific antibodies raised in demonstrate that the maintenance of high levels of cAMP and New Zealand white rabbits against the keyhole limpet hemo- active PKA are important in meiotic arrest (23-26). Previous cyanin-conjugated peptide MADTETDQGLNKK), rabbit studies have suggested that PKA may directly phosphorylate anti-phospho-WEE1B-pSer 15 (phospho-WEE1B-pSer 15 and regulate the activity of CDC25B phosphatase (25,27-29) antibodies raised in New Zealand white rabbits against the and WEE1A kinase (20,30,31) to form a bidirectional regula- keyhole limpet hemocyanin-conjugated phosphor-peptide tory loop for MPF activity. Generally, cAMP levels are high in TDQGLNKKLpSFSF) and rabbit anti-non-phospho-WEE1B- meiosis-arrested oocytes and high levels of cAMP may result pSer 15 (non-phospho-WEE1B-pSer 15 antibodies raised in in high PKA activity, both of which are essential for the main- New Zealand white rabbits against the keyhole limpet hemocy- tenance of meiotic arrest (32-34). In particular, when cAMP anin-conjugated non-phospho-peptide TDQGLNKKLSFSF) levels decrease, PKA is deactivated and unable to phosphory- were purchased from Signalway Antibody Co., Ltd. (Baltimore, late CDC25 phosphatase, which is subsequently activated. MD, USA). Goat anti-pTyr 15 of CDK1, rabbit anti-β-actin and As a result, activated CDC25 phosphatase translocates to the H-89 were provided by Santa Cruz Biotechnology, Inc. (Santa nucleus where it dephosphorylates CDK1 and thus activates Cruz, CA, USA). mMESSAGE mMACHINE T7 Ultra kit was MPF (35,36). The concurrent deactivation of WEE1A kinase purchased from Ambion (Carlsbad, CA, USA) and [γ-32P] ATP allows the inhibitory phosphates to be removed from CDK1 from Peking Yahui Biotechnology Co., Ltd. (Beijing, China). by CDC25 phosphatase (12,36,37). However, the pathway Unless otherwise specified, all other reagents were purchased between high PKA activity and the repression of MPF activity from Sigma (St. Louis, MO, USA). has yet to be fully elucidated. To examine the effects of PKA on WEE1B in mammalian cells, WEE1B has been predicted as Collection and culture of one-cell stage mouse embryos. a potential PKA substrate in mice using the scansite software Mouse embryos at the one-cell stage were collected and (http://scansite.mit.edu/) and two potential PKA phosphoryla- cultured as previously described (38). Female mice were tion sites, including Ser 15 and Thr 170 were predicted. The injected with 10 IU of pregnant mare serum gonadotropin two sites lie within a consensus sequence for PKA (KKLS), (PMSG) and then with 10 IU of human chorionic gonadotropin which includes a positively charged R or K residue upstream (hCG) 46-48 h post-PMSG, and caged with Kunming males. of an S residue. Acting as a candidate target of PKA, it has One-cell stage embryos were obtained from females 18-20 h been demonstrated that Ser 15 is the major phosphorylation post-hCG injection and incubated for 2 min in M2 medium site in vitro and is involved in the regulation of meiotic arrest suspended in 300 µg/ml hyaluronidase to remove cumulus in mouse oocytes by modification of phosphorylation and cells, washed extensively using M2 medium and then the dephosphorylation (21). However, the role of Ser 15 in the embryos were microinjected or fixed. Following microinjec- mitotic cell cycle of one-cell stage mouse embryos remains tion, embryos were cultured to the different cell cycle stages in unknown. In the present study, using a highly specific anti- M16 medium at 37˚C in a humidified atmosphere of 5% CO2. body against phospho-Ser 15 of WEE1B in Western blotting, Embryos pretreated with dibutyryl cyclic AMP (dbcAMP) or we demonstrated that WEE1B-Ser 15 was phosphorylated H-89 were incubated and collected at different time points. during G1 and S phases, whereas Ser 15 was dephosphorylated Mitotic stages (G1, S, G2 and M phases) were defined as previ- during G2 and M phases in vivo. Furthermore, we prelimi- ously described (38,39): G1 phase, 12-21 h post-hCG injection; narily examined the role of Ser 15 of WEE1B as the major S phase, 21-27 h; G2 phase, 27-30 h and M phase, 30-33 h. The PKA phosphorylation site in vivo and the inhibitory effects of rate of cleavage, i.e., the number of two-cell embryos resulting the kinase are strengthened when this residue is phosphory- from the division of a one-cell embryo, was counted in three lated. Collectively, our results suggest that Ser 15 of WEE1B independent experiments under a phase contrast microscope at is a potential PKA phosphorylation target in G2/M transition 31, 35 or 30 h post-hCG injection in the absence or presence of of one-cell stage mouse embryos and WEE1B acts as a direct dbcAMP and/or H-89.
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