SPATIAL REGULATION OF PKA BY AKAP-RI INTERACTIONS

描述(申请人提供)：这项工作的广泛的、长期的目标是确定哺乳动物胚胎如何调节cAMP依赖的蛋白激酶(PKA)的功能。植入前胚胎的卵裂球是非常大的细胞，cAMP和PKA在其中具有重要的功能。然而，PKA在胚胎不同区域的局部激活和抑制的机制尚不清楚。PKA的空间调节可以由A-激酶锚定蛋白(AKAPs)提供，AKAPs是通过I型(RI)或II型(RII)调节亚单位锚定PKA的蛋白质。使用基因敲除小鼠的研究表明，RI是主要的代偿亚单位，在发育过程中阻止不受调控的PKA活动，而RII不是必需的。然而，目前还没有关于RI调节胚胎中PKA活动的机制的信息。我们发现在不同位置的胚胎中发现了多个AKAP并与RI结合，这表明AKAP-RI相互作用对PKA的空间调控是重要的。与这一想法一致的是，AKAP-RI关联的扰动扰乱了植入前胚胎的发育。其中AKAP7Gamma具有与RI相似的核位置(而不是RII)，这表明AKAP7Gamma/Tees对参与基因调控的PKA起作用。第二个RI结合的AKAP，PAP7，在胚胎的皮质区域被发现，那里也存在RI。为了研究RIα在植入前胚胎中的作用以及AKAP对PKA作用的空间调节，我们建议：(1)确定RIα在植入前胚胎发育中的作用(S)。RIpha将通过RNA干扰方法被消除，并将检测胚胎的发育潜力。(2)确定植入前胚胎的发育是否依赖于AKAP对PKA的正确定位。PKA-AKAP的相互作用将被特异性干扰RI或RII结合的多肽抑制剂破坏。(3)明确AKAP7Gamma和PAP7在植入前胚胎发育中的具体作用。AKAP7Gamma和PAP7将通过RNA干扰方法消除，并将检测其对胚胎发育的影响。这些研究将促进我们对哺乳动物早期发育的了解，并与不孕不育的临床问题和辅助生殖中使用的程序相关。

DESCRIPTION (provided by applicant): The broad, long term goal of this work is to determine how mammalian embryos regulate the function of cAMP-dependent protein kinase (PKA). Blastomeres of the preimplantation embryo are extremely large cells in which it is well documented that cAMP and PKA have critical functions. However, the mechanisms responsible for localized activation and inhibition of PKA in distinct regions of embryos remain unknown. Spatial regulation of PKA can be provided by A-Kinase Anchor Proteins (AKAPs), proteins that anchor the kinase via either the type I (RI) or the type II (RII) regulatory subunit. Studies using knockout mice have demonstrated that RI is the major compensatory subunit that prevents unregulated PKA activity during development, while RII is not essential. Yet there is no information available regarding the mechanisms by which RI regulates PKA action in embryos. We have shown that multiple AKAPs are found in embryos in distinct locations and bind RI, suggesting that AKAP-RI interactions are important for the spatial regulation of PKA. Consistent with this idea, perturbation of AKAP-RI associations disrupts preimplantation embryo development. One of these AKAPs, AKAP7gamma, has a nuclear location similar to RI (but not RII), suggesting that AKAP7gamma/tethers PKA that is involved in gene regulation. A second RI-binding AKAP, PAP7, is found in the cortical region of embryos where RI also is present. To examine the role of RIalpha in preimplantation embryos and the spatial regulation of PKA action by AKAPs, we propose to: (1) Determine the role(s) of Rlalpha during preimplantation embryo development. RIalpha will be eliminated by RNA interference methods and the developmental potential of the embryos will be assayed. (2) Determine if preimplantation embryo development depends on the proper localization of PKA by AKAPs. PKA-AKAP interactions will be disrupted using peptide inhibitors that specifically interfere with RI or RII-binding. (3) Determine the specific roles of AKAP7gamma and PAP7 during preimplantation embryo development. AKAP7gamma and PAP7 will be eliminated by RNA interference methods, and the effects on embryo development will be examined. These studies will advance our knowledge of early mammalian development, and are relevant to clinical problems of infertility and the procedures used in assisted reproduction.