Webb Rachel J, Tinworth Lorna, Thomas Geraint M H, Zaccolo Manuela, Carroll John
Department of Physiology, University College London, Gower Street, London, WC1E 6BT, UK.
Dev Biol. 2008 May 1;317(1):36-45. doi: 10.1016/j.ydbio.2008.01.045. Epub 2008 Feb 14.
Localisation of Protein Kinase A (PKA) by A-Kinase Anchoring Proteins (AKAPs) is known to coordinate localised signalling complexes that target cAMP-mediated signalling to specific cellular sub-domains. The cAMP PKA signalling pathway is implicated in both meiotic arrest and meiotic resumption, thus spatio-temporal changes in PKA localisation during development may determine the oocytes response to changes in cAMP. In this study we aim to establish whether changes in PKA localisation occur during oocyte and early embryo development. Using fluorescently-labelled PKA constructs we show that in meiotically incompetent oocytes PKA is distributed throughout the cytoplasm and shows no punctuate localisation. As meiotic competence is acquired, PKA associates with mitochondria. Immature germinal vesicle (GV) stage oocytes show an aggregation of PKA around the GV and PKA remains co-localised with mitochondria throughout oocyte maturation. After fertilisation, the punctuate, mitochondrial distribution was lost, such that by the 2-cell stage there was no evidence of PKA localisation. RT-PCR and Western blotting revealed two candidate AKAPs that are known to be targeted to mitochondria, AKAP1 and D-AKAP2. In summary these data show a dynamic regulation of PKA localisation during oocyte and early embryo development.
已知A激酶锚定蛋白（AKAPs）对蛋白激酶A（PKA）的定位可协调局部信号复合物，使cAMP介导的信号传导靶向特定的细胞亚结构域。cAMP-PKA信号通路与减数分裂停滞和减数分裂恢复均有关联，因此发育过程中PKA定位的时空变化可能决定卵母细胞对cAMP变化的反应。在本研究中，我们旨在确定PKA定位在卵母细胞和早期胚胎发育过程中是否发生变化。使用荧光标记的PKA构建体，我们发现，在减数分裂能力不足的卵母细胞中，PKA分布于整个细胞质中，没有点状定位。随着减数分裂能力的获得，PKA与线粒体结合。未成熟的生发泡（GV）期卵母细胞显示PKA在GV周围聚集，并且在整个卵母细胞成熟过程中PKA一直与线粒体共定位。受精后，点状的线粒体分布消失，以至于到2细胞期时没有PKA定位的证据。逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法显示有两种已知靶向线粒体的候选AKAPs，即AKAP1和D-AKAP2。总之，这些数据表明在卵母细胞和早期胚胎发育过程中PKA定位存在动态调节。
