Anchored Protein Kinase A in Signal Transduction

描述（由申请人提供）：a激酶锚定蛋白（AKAPs）介导cAMP携带的信号靶向。AKAPs结合蛋白激酶All (PKAII)，并具有独特的结构域，将栓系激酶靶向到细胞器的对接位点。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是由激活的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接触控制的。对于直接结合肌动蛋白细胞骨架特殊区域的akap的特性，我们所知甚少。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蛋白有一个RII系聚位点，二价连接和交联F-actin的锚定结构域和一个结合NSF的结构域。然而，AKAP-KL4将PKAII传递到极化细胞的外侧表面，而AKAP-KL2则富集于细胞的顶端表面。因此，锚定蛋白将PKAII传递到不同的微环境和效应物群中。AKAP范式预测，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描述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，b)引导不同的AKAP-PKAII复合物到达肌动蛋白细胞骨架中结构和功能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，并开发工具来选择性地破坏AKAP-KL2或AKAP-KL4对PKAII的锚定。中心目的是阐明外皮取向的AKAPKL2-PKAII复合体的生理作用。这个锚定的PKAII复合体同时控制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。首席研究员和他的团队将在线虫体内研究锚定的pkai - like 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，赋予了同型选择性高亲和力结合活性。将野生型和突变型转基因引入AKAPce或Rce无效的秀丽隐杆线虫。生物化学和生理后果将在体内进行分析。

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.