Anchored Protein Kinase A in Signal Transduction

信号转导中的锚定蛋白激酶 A

• 批准号: 6893709
• 负责人: CHARLES S RUBIN
• 金额: $ 36.07万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6893709
• 关键词: A kinase anchoring protein; Caenorhabditis elegans; MDCK cell; actins; biological signal transduction; cellular polarity; confocal scanning microscopy; cyclic AMP; cytoskeletal proteins; cytoskeleton; protein binding; protein isoforms; protein kinase A; protein localization; protein structure function; second messengers; water channel

DESCRIPTION (provided by applicant): A-kinase anchor proteins (AKAPs) mediate targeting of signals carried by cAMP. AKAPs bind protein kinase All (PKAII) and have unique domains that target the tethered kinase to docking sites in organelles. Colocalization of anchored PKA with substrate/effector proteins enables efficient reception and precisely focused transmission of cAMP signals. Critical aspects of cell physiology are controlled by encounters between activated PKA and effectors that are embedded in or juxtaposed to cortical actin cytoskeleton. Little is known about properties of AKAPs that directly bind with specialized regions of actin cytoskeleton. AKAPKL isoforms are adapted for coupling PKAII to properties and proteins of the cortical F-actin network. AKAPKL proteins have an RII tethering site, anchoring domains that bivalently ligate and cross-link F-actin and a domain that binds NSF. However, AKAP-KL4 routes PKAII to the lateral surface of polarized cells, whereas AKAP-KL2 is enriched at the apical surface. Thus the anchor proteins deliver PKAII to different microenvironments and constellations of effectors. An AKAP paradigm predicts that unique combinations of intrinsic targeting domains and cognate docking molecules account for specific locations and functions of AKAP-PKAII complexes. The principal investigator and his group will characterize a) targeting domains that control asymmetric distribution of AKAP-KL2 and AKAP-KL4 in polarized MDCK cells and b) docking proteins that guide different AKAP-PKAII complexes to structurally and functionally distinct destinations in actin cytoskeleton. A molecular basis for specific docking interactions will be determined and tools developed to selectively disrupt anchoring of PKAII by AKAP-KL2 or AKAP-KL4. A central aim is to elucidate a physiological role for the epically-oriented AKAPKL2-PKAII complex. The proposition that this anchored PKAII complex simultaneously controls a) phosphorylation and translocation of a channel protein and b) organization of the local F-actin network, will be systematically investigated. Downstream targets that mediate cytoskeleton remodeling will be characterized. The principal investigator and his group will investigate the structural basis and physiological functions for anchored PKAI-Iike PKA in C. elegans in vivo. They have discovered unique structural features in the AKAPce and Rce that confer isoform-selective high affinity binding activity. Wild type and mutant transgenes will be introduced into AKAPce or Rce null C. elegans. Biochemical and physiological consequences will be assayed in vivo.

描述（由申请人提供）：A-激酶锚定蛋白（AKAP）介导cAMP携带的信号的靶向。 AKAP 结合蛋白激酶 All (PKAII)，并具有将束缚激酶靶向细胞器中对接位点的独特结构域。锚定 PKA 与底物/效应蛋白的共定位可实现 cAMP 信号的有效接收和精确聚焦传输。细胞生理学的关键方面是由激活的 PKA 与嵌入或并置皮质肌动蛋白细胞骨架的效应器之间的相遇控制的。对于直接与肌动蛋白细胞骨架的特殊区域结合的 AKAP 的特性知之甚少。 AKAPKL 亚型适用于将 PKAII 与皮质 F-肌动蛋白网络的特性和蛋白质偶联。 AKAPKL 蛋白具有 RII 束缚位点、二价连接和交联 F-肌动蛋白的锚定域以及结合 NSF 的域。然而，AKAP-KL4 将 PKAII 引导至极化细胞的侧面，而 AKAP-KL2 则富集于顶端表面。因此，锚定蛋白将 PKAII 传递到不同的微环境和效应器群。 AKAP 范例预测，内在靶向结构域和同源对接分子的独特组合解释了 AKAP-PKAII 复合物的特定位置和功能。首席研究员和他的团队将表征 a) 控制极化 MDCK 细胞中 AKAP-KL2 和 AKAP-KL4 不对称分布的靶向结构域，以及 b) 引导不同 AKAP-PKAII 复合物到达肌动蛋白细胞骨架中结构和功能不同目的地的对接蛋白。将确定特定对接相互作用的分子基础，并开发工具来选择性破坏 AKAP-KL2 或 AKAP-KL4 对 PKAII 的锚定。中心目标是阐明以史诗为导向的 AKAPKL2-PKAII 复合物的生理作用。这种锚定的 PKAII 复合物同时控制 a) 通道蛋白的磷酸化和易位以及 b) 局部 F-肌动蛋白网络的组织，这一主张将得到系统的研究。将表征介导细胞骨架重塑的下游靶标。首席研究员和他的团队将研究在线虫体内锚定的 PKAI-Iike PKA 的结构基础和生理功能。他们在 AKAPce 和 Rce 中发现了独特的结构特征，这些特征赋予异构体选择性高亲和力结合活性。野生型和突变型转基因将被引入AKAPce或Rce null线虫中。生化和生理学后果将在体内进行测定。