Cyclic AMP Mediation of Epithelial Cell Function

项目摘要 第二信使依赖性机制的亚细胞隔离提供了 信号传导到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 位置特定的信号机制。一种激酶锚定蛋白（AKAP） 最大的一组多功能支架蛋白，不仅锚定II型cAMP， 依赖性蛋白激酶（PKA），而且还有各种蛋白激酶，磷酸酶， 磷酸二酯酶和第二信使调节信号传导的靶点。AKAP 350/450 基因编码许多不同的剪接变体，范围从250至450 kDa。 AKAP 350/450剪接变体可能支架PKA、蛋白激酶C、PKN、酪蛋白激酶 1、磷酸二酯酶4D 3、蛋白磷酸酶1和2a和钙调蛋白以及a 推定的下游效应物的数量。AKAP 350定位于两个中心体， 我们已经证明，用siRNA去除AKAP 350 A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，虽然以前的调查将注意力集中在AKAP 350上， 中心体和高尔基体，我们最近的研究已经导致认识到， 在大多数细胞中，AKAP 350的大量胞质池与CCAR 1和山羊蛋白结合， mRNA运输并参与微管依赖性应激颗粒的形成。而 我们和其他人已经证明，AKAP 350可以潜在地支架广泛的 蛋白质，支架复合物的实际组成可能是细胞特异性的， 亚细胞器特异性。研究这些大型锚定多蛋白的挑战在于 复合物的作用是识别特定的协调复合物在调节 胞内过程我们假设细胞内AKAP 350-协调 复合体调节通过高尔基体的运输和离散的 胞质结构域内的RNA种类。为了检验这些假设，我们将探讨两个 具体目标。首先，我们将确定AKAP 350 A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 AKAP 350 A及其相关蛋白在调节RNA中的作用 贩运和翻译。这些研究将确定特定多蛋白的作用 在细胞的局部区域中由AKAP 350支撑的复合物。

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.