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.
Yu
Bingzhi
B
Department of Biochemical and Molecular Biology, China Medical University, Shenyang, Liaoning, People's Republic of China. ybzbiochem@yeah.net
Wang
Yajie
Y
Liu
Ying
Y
Liu
Yi
Y
Li
Xinna
X
Wu
Didi
D
Zong
Zhihong
Z
Zhang
Jie
J
Yu
Dahai
D
D000705:Anaphase; D000818:Animals; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D002453:Cell Cycle; D002455:Cell Division; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism / Q000502:physiology*; D019926:Cyclin B / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004622:Embryo, Mammalian / Q000166:cytology*; D004625:Embryo, Nonmammalian; D047108:Embryonic Development; D005306:Fertilization; D016193:G1 Phase; D016195:G2 Phase; D018507:Gene Expression Regulation, Developmental; D006657:Histones / Q000378:metabolism; D007118:Immunoassay; D015151:Immunoblotting; D016200:Maturation-Promoting Factor / Q000037:antagonists & inhibitors / Q000502:physiology*; D000090204:Mesothelin; D008677:Metaphase; D051379:Mice; D008938:Mitosis; D010766:Phosphorylation; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry; D014981:Xenopus
D019926:Cyclin B; D006657:Histones; C000715528:Msln protein, mouse; D014443:Tyrosine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D016203:CDC2 Protein Kinase; D016200:Maturation-Promoting Factor; D000090204:Mesothelin