Cyclic AMP Mediation of Epithelial Cell Function

项目总结 第二信使依赖机制的亚细胞隔离提供了 信号对离散细胞器系统的特异性，或者在极化上皮细胞的情况下， 质膜结构域。多蛋白支架协调细胞特异性和细胞内 特定于位置的信号机制。A激酶锚定蛋白(AKAPs)折衷 最大的一组多功能支架蛋白，不仅锚定II型cAMP- 依赖蛋白激酶(PKA)，还包括各种蛋白激酶、磷酸酶、 磷酸二酯酶和第二信使调节信号的靶标。AKAP350/450 基因编码了从250 kDa到450 kDa的许多不同的剪接变体。 AKAP350/450剪接变异体可能作为PKA、蛋白激酶C、PKN、酪蛋白激酶的支架 1、磷酸二酯酶4D3、蛋白磷酸酶1和2a、钙调蛋白以及 假定的下游效应器的数量。AKAP350定位于着丝粒和 Golgi装置，我们已经证明了AKAP350A与siRNA一起耗尽会导致 高尔基体结构的破坏以及聚合微管的改变。 重要的是，虽然之前的调查都将注意力集中在AKAP350上 中心体和高尔基体，我们最近的研究使人们认识到在 大多数细胞是一个大的AKAP350胞浆池，与CCAR1和Caprin结合并调节 并参与微管依赖的应力颗粒的形成。而当 我们和其他人已经证明，AKAP350可以潜在地搭建起广泛的 蛋白质，支架复合体的实际组成可能既是细胞特有的，也是 亚细胞器特异型。研究这些大的锚定多蛋白的挑战 识别特定的配位复合体在调节细胞周期中的作用 细胞内过程。我们假设细胞内AKAP350协调 复合体既调节通过高尔基体的贩运，也调节离散的 胞质结构域中的RNA物种。为了检验这些假设，我们将探讨两个问题 明确的目标。首先，我们将确定AKAP350A及其相关蛋白在 调节高尔基体的结构和功能。第二，我们将调查 AKAP350A及其相关蛋白的胞液池在调节RNA中的作用 贩卖和翻译。这些研究将确定特定多蛋白的作用。 以AKAP350为支架的复合体位于细胞的局部区域。

PROJECT SUMMARY 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.