Anchored Protein Kinase A in Signal Transduction

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)，并具有独特的结构域，靶向细胞器中的连接位点。锚定的PKA与底物/效应蛋白的共定位使cAMP信号的有效接收和精确聚焦传递成为可能。细胞生理学的关键方面是由激活的PKA和嵌入或并列在皮质肌动蛋白细胞骨架中的效应器之间的相遇来控制的。人们对直接与肌动蛋白细胞骨架的特定区域结合的AKAP的性质知之甚少。AKAPKL亚型适用于将PKAII偶联到皮质F-肌动蛋白网络的性质和蛋白质上。AKAPKL蛋白有一个RII锚定位点，锚定结构域连接和交联F-肌动蛋白，还有一个与NSF结合的结构域。然而，AKAP-KL4将PKAII传递到极化细胞的侧面，而AKAP-KL2则在根尖表面富集。因此，锚蛋白将PKAII传递到不同的微环境和效应器星座。AKAP范式预测，内在靶向域和同源对接分子的独特组合解释了AKAP-PKAII复合体的特定位置和功能。主要研究人员和他的团队将描述a)控制AKAP-KL2和AKAP-KL4在极化的MDCK细胞中不对称分布的靶向结构域，以及b)将不同的AKAP-PKAII复合体引导到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。一个中心目标是阐明史诗导向的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转位以及b)局部F-肌动蛋白网络的组织，这一命题将得到系统的研究。将描述介导细胞骨架重塑的下游靶点。主要研究人员和他的团队将在体内研究线虫体内锚定的类似PKAI的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，这些结构特征赋予了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缺失的线虫中。生化和生理后果将在体内进行测试。

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.