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. ? ?
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