Anchored Protein Kinase A in Signal Transduction

描述（由申请人提供）：A-激酶锚蛋白（AKAP）介导cAMP携带的信号靶向。AKAP结合蛋白激酶All（PKAII），并具有将栓系激酶靶向细胞器中的对接位点的独特结构域。锚定PKA与底物/效应蛋白的共定位使得cAMP信号的有效接收和精确聚焦传输成为可能。细胞生理学的关键方面受到激活的PKA和嵌入或并列于皮质肌动蛋白细胞骨架的效应器之间的相遇的控制。AKAP与肌动蛋白细胞骨架的特化区域直接结合的特性知之甚少。AKAPKL同种型适于将PKAII偶联至皮质F-肌动蛋白网络的性质和蛋白质。AKAPKL蛋白具有RII拴系位点、双链连接和交联F-肌动蛋白的锚定结构域和结合NSF的结构域。然而，AKAP-KL 4将PKAII路由到极化细胞的侧表面，而AKAP-KL 2在顶表面富集。因此，锚蛋白将PKAII递送到不同的微环境和效应物的星座。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。主要研究者和他的小组将表征a）控制AKAP-KL 2和AKAP-KL 4在极化MDCK细胞中的不对称分布的靶向结构域和B）引导不同AKAP-PKA II复合物在肌动蛋白细胞骨架中的结构和功能上不同的目的地的对接蛋白。将确定特定对接相互作用的分子基础，并开发工具以选择性地破坏AKAP-KL 2或AKAP-KL 4对PKAII的锚定。一个中心目标是阐明epically-oriented AKAPKL 2-PKAII复合物的生理作用。这个锚定的PKA Ⅱ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的命题将被系统地研究。介导细胞骨架重塑的下游靶点将被表征。主要研究者和他的小组将研究锚定PKAI样PKA在C. elegans in vivo.他们在AKAPce和Rce中发现了赋予亚型选择性高亲和力结合活性的独特结构特征。将野生型和突变型转基因引入AKAPce或Rce无效C。优雅将在体内测定生物化学和生理学结果。

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.