SPATIAL REGULATION OF PKA BY AKAP-RI INTERACTIONS

描述（由申请人提供）：这项工作的长期目标是确定哺乳动物胚胎如何调节cAMP依赖性蛋白激酶（PKA）的功能。植入前胚胎的卵裂球是非常大的细胞，其中cAMP和PKA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机制仍不清楚。PKA的空间调节可由A-激酶锚蛋白（AKAP）提供，A-激酶锚蛋白通过I型（RI）或II型（RII）调节亚基来锚定激酶。使用基因敲除小鼠的研究表明，RI是主要的补偿亚基，防止在发育过程中不受调节的PKA活性，而RII不是必不可少的。然而，没有关于RI调节PKA在胚胎中作用的机制的信息。我们已经发现，多个AKAP被发现在胚胎中的不同位置，并结合RI，这表明AKAP-RI相互作用是重要的PKA的空间调节。与这一观点相一致，AKAP-RI联合的干扰破坏了植入前胚胎的发育。其中一种AKAP，AKAP 7 γ，具有与RI相似的核定位（但不是RII），表明AKAP 7 γ/tethers PKA参与基因调控。第二个RI结合AKAP，PAP 7，被发现在胚胎的皮质区域，RI也存在。为了研究RI α在植入前胚胎发育中的作用以及AKAP对PKA作用的空间调控，我们拟：（1）确定RI α在植入前胚胎发育中的作用。将通过RNA干扰方法消除RI α，并测定胚胎的发育潜力。(2)确定植入前胚胎发育是否依赖于AKAP对PKA的正确定位。PKA-AKAP相互作用将使用特异性干扰RI或RII结合的肽抑制剂来破坏。(3)确定AKAP 7 γ和PAP 7在植入前胚胎发育过程中的具体作用。将通过RNA干扰方法消除AKAP 7 γ和PAP 7，并将检查对胚胎发育的影响。这些研究将推进我们对哺乳动物早期发育的认识，并与不孕症的临床问题和辅助生殖中使用的程序有关。

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.