CpnA's role in cAMP signaling and the actin cytoskeleton in Dictyostelium

描述(申请人提供)：Copine是一种高度保守的钙依赖膜结合蛋白，存在于许多不同的真核生物中。从单细胞生物到人类的各种生物体中都发现了铜绿素，这表明铜绿素在大多数真核细胞中发挥着重要的基本功能。越来越多的证据表明，铜绿素在信号通路中扮演着调节蛋白质的角色。模式生物盘基网柄菌具6个Copine基因(cpnA-cpnF)，为研究Copine的功能提供了理想的系统。鞭毛虫可以作为单细胞阿米巴虫独立生活，但当被置于饥饿条件下时，单个阿米巴虫相互发出信号，首先聚集，然后分化成细胞，形成多细胞子实体。这一简单的发育程序不仅被广泛用于研究发育，还被广泛用于研究几种基本的细胞过程，包括趋化作用介导的细胞运动、信号转导、细胞程序性死亡和细胞分化。以往的研究表明，网柄苔藓中的一种Copine蛋白CpnA是正常发育所必需的。上一次拨款的总体目标是将我们在发育过程中对cpnA细胞的发现(目标1)与CpnA靶蛋白的鉴定(目标2)联系起来，以产生关于CpnA功能的更具体的假设。在这两个具体目标方面都取得了重大进展，在前一次赠款发放期间收集的数据导致了关于CpnA功能的两个新假设。这两个假设构成了这一更新建议的两个具体目的：1)确定CpnA是否是cAMP磷酸二酯酶的负调节因子，RegA；2)确定CpnA是否是肌动蛋白细丝聚合的负调节因子。这项拨款提案描述了使用生化、遗传学和显微镜方法来检验这些假说的实验。这些实验的结果将对这一领域产生重大影响，因为它是第一个定义Copine蛋白质的机械作用的实验。人类铜绿素通过调节ErB2受体的信号通路在癌症中发挥作用，并通过调节肿瘤坏死因子受体的信号通路在免疫反应中发挥作用。增加我们对铜绿素如何发挥作用的科学知识可以使我们更好地了解癌症和免疫细胞相关疾病。

DESCRIPTION (provided by applicant): Copines are highly conserved calcium-dependent membrane-binding proteins found in numerous diverse eukaryotic organisms. The wide array of organisms ranging from single-celled organisms to humans in which copines are found suggest that copines carry out fundamental functions important in most eukaryotic cells. A growing body of evidence indicates that copines act as regulatory proteins in signaling pathways. The model organism Dictyostelium discoideum has six copine genes (cpnA-cpnF) and provides an ideal system for studying copine function. Dictyostelium can live independently as single-celled amoebae, but when placed in starvation conditions, single amoeba signal each other to first aggregate and then differentiate into cells that form a multicellular fruiting body. This simple developmental program in Dictyostelium is widely used to study not only development, but also several basic cell processes including chemotaxis-mediated cell motility, signal transduction, programmed cell death, and cell differentiation. Previous studies showed that one of the copine proteins in Dictyostelium, CpnA, is required for normal development. The overall goal of the previous grant award was to correlate our findings on cpnA- cells during developmental processes (aim 1) and the identification of the target proteins of CpnA (aim 2) to generate more specific hypotheses about the function of CpnA. Significant progress was made on both of these specific aims and data collected during the previous grant award period has led to two new hypotheses about the function of CpnA. These two hypotheses form the two specific aims of this renewal proposal: 1) Determine if CpnA is a negative regulator of the cAMP phosphodiesterase, RegA and 2) Determine if CpnA is a negative regulator of actin filament polymerization. This grant proposal describes experiments using biochemical, genetics, and microscopy approaches to test these hypotheses. The results of these experiments will make a significant impact on this field by being the first to define a mechanistic role for a copine protein. Human copines have been implicated to play a role in cancer by regulating signaling pathways involving the ErB2 receptor and in immune responses by regulating signaling pathways involving the TNF (tumor necrosis factor) receptor. Increasing our scientific knowledge of how copines function could lead to a better understanding of cancer and immune cell-related diseases.