SPATIAL REGULATION OF PKA BY AKAP-RI INTERACTIONS

AKAP-RI 相互作用对 PKA 的空间调节

• 批准号: 6841139
• 负责人: CARMEN J WILLIAMS
• 金额: $ 28.53万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6841139
• 关键词: A kinase anchoring protein; RNA interference; binding proteins; developmental genetics; embryo implantation; embryogenesis; genetic regulation; immunofluorescence technique; inhibitor /antagonist; laboratory mouse; polymerase chain reaction; protein kinase A; protein localization; protein protein interaction; protein structure function; synthetic peptide; western blottings

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.

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。植入前胚胎的卵裂球是非常大的细胞，其中 cAMP 和 PKA 具有关键功能，这一点已得到充分证明。然而，在胚胎不同区域局部激活和抑制 PKA 的机制仍然未知。 PKA 的空间调节可由 A 激酶锚定蛋白 (AKAP) 提供，AKAP 是通过 I 型 (RI) 或 II 型 (RII) 调节亚基锚定激酶的蛋白质。使用基因敲除小鼠的研究表明，RI 是防止发育过程中 PKA 活性失控的主要代偿亚基，而 RII 并不是必需的。然而，目前还没有关于 RI 调节胚胎中 PKA 作用的机制的信息。我们已经证明，在胚胎的不同位置发现了多个 AKAP 并与 RI 结合，这表明 AKAP-RI 相互作用对于 PKA 的空间调节很重要。与这一观点一致，AKAP-RI 关联的扰动会破坏植入前胚胎的发育。其中一个 AKAP，AKAP7gamma，具有与 RI（但不是 RII）相似的核位置，表明 AKAP7gamma/束缚参与基因调控的 PKA。第二种与 RI 结合的 AKAP，即 PAP7，存在于也存在 RI 的胚胎皮质区域。为了研究 RIalpha 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 Rlalpha 在植入前胚胎发育过程中的作用。 RIalpha 将通过 RNA 干扰方法消除，并检测胚胎的发育潜力。 (2) 确定植入前胚胎发育是否取决于 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AKAP7gamma和PAP7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AKAP7gamma和PAP7，并检查对胚胎发育的影响。这些研究将增进我们对早期哺乳动物发育的了解，并与不孕症的临床问题和辅助生殖中使用的程序相关。