Cyclic AMP Mediation of Epithelial Cell Function

描述(由申请人提供)：第二信使依赖机制的亚细胞隔离提供了信号到离散细胞器系统或在极化上皮细胞情况下的质膜域的特异性。多蛋白支架协调细胞特定和细胞内特定位置的信号机制。激酶锚定蛋白(AKAPs)是一类最大的多功能支架蛋白，它不仅锚定II型cAMP依赖的蛋白激酶(PKA)，还锚定多种蛋白激酶、磷酸酶、磷酸二酯酶和第二信使调节的信号转导靶标。AKAP350/450基因编码从250 kDa到450 kDa的许多不同的剪接变体。AKAP350/450剪接变异体可能是PKA、蛋白激酶C？、PKN、酪蛋白激酶1、磷酸二酯酶4D3、蛋白磷酸酶1和2a、钙调蛋白以及一些可能的下游效应因子的支架。AKAP350定位于中心体和高尔基体，我们已经证明，AKAP350A的siRNA缺失会导致高尔基体结构的破坏和聚合微管的改变。重要的是，虽然以前的研究集中在中心体和高尔基体上的AKAP350，但我们最近的研究导致人们认识到，在大多数细胞中，大量的AKAP350与CCAR1和Caprin结合，并调节mRNA的运输，并参与微管依赖的应激颗粒的形成。虽然我们和其他人已经证明了AKAP350可以潜在地构建广泛的蛋白质支架，但支架复合体的实际组成可能既是细胞特有的，也是亚细胞器官特有的。研究这些大的锚定多蛋白复合体的挑战是辨别特定的配位复合体在调节细胞内过程中的作用。我们假设，细胞内AKAP350-协调复合体既调节通过高尔基体的转运，也调节胞液结构域中离散RNA物种的加工。为了检验这些假设，我们将追求两个具体目标。首先，我们将确定AKAP350A及其相关蛋白在调节高尔基体结构和功能中的作用。其次，我们将研究AKAP350A及其相关蛋白的胞液池在调节RNA运输和翻译中的作用。这些研究将确定AKAP350支架的特定多蛋白复合体在细胞局部区域的作用。公共卫生相关性：AKAP350是一种大蛋白，可能在细胞内组装大型调节压缩。这些复合体似乎协调细胞内蛋白质和信使RNA的运动，这些信使RNA负责细胞内的远距离过程。这些过程可以对细胞的正常功能产生不同的影响，包括蛋白质的产生和定位到膜表面，以及在正常和病理条件下细胞对各种诱导应激的反应。

DESCRIPTION (provided by applicant): Subcellular sequestration of second messenger-dependent mechanisms provides for specificity of signaling to discrete organelle systems or, in the case of polarized epithelial cells, plasma membrane domains. Multiprotein scaffolds coordinate cell-specific and intracellular location-specific signaling mechanisms. A kinase anchoring proteins (AKAPs) compromise the largest group of multifunctional scaffolding proteins, which anchor not only Type II cAMP- dependent protein kinase (PKA), but also a variety of protein kinases, phosphatases, phosphodiesterases and targets of second messenger regulated signaling. The AKAP350/450 gene codes for a number of different splice variants ranging from 250 to 450 kDa. AKAP350/450 splice variants potentially scaffold PKA, protein kinase C?, PKN, casein kinase 1, phosphodiesterase 4D3, protein phosphatases 1 and 2a and calmodulin as well as a number of putative downstream effectors. AKAP350 is localized to both centrosomes and the Golgi apparatus, and we have demonstrated that depletion of AKAP350A with siRNA leads to disruption of the Golgi structure as well as alteration of polymerizing microtubules. Importantly, while previous investigations have focused attention on AKAP350 at the centrosome and the Golgi apparatus, our recent studies have led to the recognition that in most cells a large cytosolic pool of AKAP350 associates with CCAR1 and caprin and regulates mRNA trafficking and participates in microtubule-dependent stress granule formation. While we and others have demonstrated that AKAP350 can potentially scaffold a wide range of proteins, the actual composition of scaffolded complexes is likely both cell specific as well as subcellular organelle specific. The challenge of studying these large anchored multiprotein complexes is to discern the role of specific coordinated complexes in the regulation of intracellular processes. We have hypothesized that intracellular AKAP350-coordinated complexes regulate both trafficking through the Golgi apparatus and processing of discrete RNA species within cytosolic domains. To examine these hypotheses, we will pursue two specific aims. First, we will determine the role of AKAP350A and its associated proteins in regulating the structure and function of the Golgi apparatus. Second, we will investigate the role of the cytosolic pool of AKAP350A and its associated proteins in regulating RNA trafficking and translation. These studies will establish the roles of specific multiprotein complexes scaffolded by AKAP350 in localized regions of the cell. PUBLIC HEALTH RELEVANCE: AKAP350 is a large protein that can potentially assemble large regulatory compress within the cell. These complexes appear to coordinate the movement intracellular proteins and messenger RNAs responsible for far ranging processes within cells. These processes can have diverse influences on the proper functioning of cells including the production and targeting of proteins to membrane surfaces and the response of cells to various induced stresses in normal and pathological conditions.