Andersen Carsten B, Sakaue Hiroshi, Nedachi Taku, Kovacina Kristina S, Clayberger Carol, Conti Marco, Roth Richard A
Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, CA 94305, U.S.A.
Biochem J. 2003 Jan 15;369(Pt 2):227-38. doi: 10.1042/BJ20021243.
In the present study, we have characterized the Xenopus Akt expressed in oocytes from the African clawed frog Xenopus laevis and tested whether its activity is required for the insulin- and progesterone-stimulated resumption of meiosis. A cDNA encoding the Xenopus Akt was isolated and sequenced, and its expression in the Xenopus oocyte was confirmed by reverse transcription PCR and Northern blotting. Using phosphospecific antibodies and enzyme assays, a large and rapid activation of the Xenopus Akt was observed upon insulin stimulation of the oocytes. In contrast, progesterone caused a modest activation of this kinase with a slower time course. To test whether the activation of Akt was required in the stimulation of the resumption of meiosis, we have utilized two independent approaches: a functional dominant negative Akt mutant and an inhibitory monoclonal antibody. Both the mutant Akt, as well as the inhibitory monoclonal antibody, completely blocked the insulin-stimulated resumption of meiosis. In contrast, both treatments only partially inhibited (by approx. 30%) the progesterone-stimulated resumption of meiosis when submaximal doses of this hormone were utilized. These data demonstrate a crucial role for Akt in the insulin-stimulated cell cycle progression of Xenopus oocytes, whereas Akt may have an ancillary function in progesterone signalling.
在本研究中，我们对非洲爪蟾（非洲爪蟾）卵母细胞中表达的非洲爪蟾Akt进行了特性分析，并测试了其活性对于胰岛素和孕酮刺激的减数分裂恢复是否必要。分离并测序了编码非洲爪蟾Akt的cDNA，并通过逆转录PCR和Northern印迹法证实了其在非洲爪蟾卵母细胞中的表达。使用磷酸特异性抗体和酶分析，在胰岛素刺激卵母细胞后观察到非洲爪蟾Akt的大量快速激活。相比之下，孕酮导致该激酶适度激活，且时间进程较慢。为了测试Akt的激活在刺激减数分裂恢复中是否必要，我们采用了两种独立的方法：一种功能性显性负性Akt突变体和一种抑制性单克隆抗体。突变型Akt以及抑制性单克隆抗体均完全阻断了胰岛素刺激的减数分裂恢复。相比之下，当使用次最大剂量的这种激素时，两种处理仅部分抑制（约30%）孕酮刺激的减数分裂恢复。这些数据表明Akt在非洲爪蟾卵母细胞的胰岛素刺激的细胞周期进程中起关键作用，而Akt可能在孕酮信号传导中具有辅助功能。
