SPATIAL REGULATION OF PKA BY AKAP-RI INTERACTIONS

描述（由申请人提供）：这项工作的广泛，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。着床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机制尚不清楚。PKA的空间调控可以由a激酶锚定蛋白（AKAPs）提供，这些蛋白通过I型（RI）或II型（RII）调控亚基锚定激酶。对基因敲除小鼠的研究表明，RI是发育过程中防止PKA活性不受调节的主要代偿亚基，而RII不是必需的。然而，关于RI在胚胎中调节PKA作用的机制尚无信息。我们发现在胚胎中多个akap存在于不同的位置并结合RI，这表明AKAP-RI相互作用对PKA的空间调控很重要。与这一观点一致的是，AKAP-RI关联的扰动破坏了着床前胚胎的发育。其中一个akap， AKAP7gamma，其核位置与RI相似（但与RII不同），这表明AKAP7gamma/拴住了参与基因调控的PKA。第二种与RI结合的AKAP， PAP7，在胚胎皮层区域发现，那里也存在RI。为了研究rα在着床前胚胎中的作用以及AKAPs对PKA作用的空间调控，我们建议：(1)确定rα在着床前胚胎发育中的作用。将通过RNA干扰方法消除RIalpha，并检测胚胎的发育潜力。(2)确定着床前胚胎发育是否依赖于AKAPs对PKA的适当定位。PKA-AKAP的相互作用将被特异性干扰RI或rii结合的肽抑制剂破坏。(3)确定AKAP7gamma和PAP7在着床前胚胎发育中的具体作用。通过RNA干扰法去除AKAP7gamma和PAP7，并检测其对胚胎发育的影响。这些研究将促进我们对早期哺乳动物发育的认识，并与不孕不育的临床问题和辅助生殖程序有关。

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.