Plasma Membrane Targeting and Retargeting of Polarity Proteins

总结 将极性蛋白质靶向到相互排斥的质膜（PM）结构域是一个重要的 建立和维持细胞极性的过程--一个由不断扩大的 极性蛋白及其调节因子/效应因子之间的相互作用网络。相比之下， 关于极性蛋白质和PM之间的物理相互作用如何促成这种极化过程。 PI实验室最近的研究表明，多个核心极性蛋白（“多元极性蛋白”） 如Lg 1、aPKC和Dlg也是固有膜结合蛋白。它们含有所谓的多元 通过结合PM磷脂如PI 4P特异性和静电靶向PM的结构域 PIP2我们已经表征了控制极化静电PM靶向的多种机制 多元极性蛋白的功能。我们还发现了一个意想不到的后果， 细胞极性中现有的静电PM靶向：多元极性蛋白质的PM定位是高度依赖的。 易受缺氧和ATP抑制引发的能量应激的影响，这会诱导 PM PI 4P和PIP 2。在这个建议中，我们的目标是进一步建立静电PM靶向作为一个基本的 在正常和能量应激条件下调节细胞极性的机制。具体来说，我们将 调查： 1)调节细胞极性中静电PM靶向的不同机制。重点抓好 调节基底外侧极性蛋白Dlg的静电PM靶向， 磷酸化，其多碱基结构域的变构调节，以及与其他基底外侧极性的相互作用 proteins.我们将阐明如何变构调节静电PM靶向作为一个核心机制， 基底外侧极性蛋白控制彼此的定位和功能。 2)通过静电PM靶向控制aPKC磷酸化。aPKC是关键的激酶驱动细胞 极化及其伪基底区（PSr）也是一种多元结构域， PM靶向和激酶活性的Par-6。使用一种新型的诱导偏振系统，我们将研究 假设Par-6依赖性的aPKC的静电PM靶向控制底物特异性 通过调节aPKC/Par-6的磷酸化，提供了一种时空控制aPKC激酶活性的机制， 细胞极化 3)多元极性蛋白质的静电PM重靶向调节。尽管首相已经彻底下台 由于PM PI 4 P和PIP 2的耗尽，缺氧条件下Lgl和aPKC的靶向功能，我们发现两者都 一旦再氧合开始，蛋白质总是直接重定向其原始基底外侧或顶端PM结构域。 我们将研究脂质和蛋白质为基础的机制，恢复和控制这种极化PM 多元极性蛋白的重定向。这些机制对于我们理解细胞如何 极性在诸如缺氧和局部缺血的能量应激条件下存活。 我们建议的研究将描绘必要的分子细节，整合静电PM 靶向极性蛋白和磷脂的调节网络。发现新机制 调节静电PM靶向将是我们在正常和非正常条件下对细胞极性的认识的基础。 和压力条件。

SUMMARY Targeting polarity proteins to mutually exclusive plasma membrane (PM) domains is an essential process for establishing and maintaining cell polarity – a process that is regulated by an ever-expanding interaction network among polarity proteins and their regulators/effectors. In contrast, much less is known about how physical interactions between polarity proteins and the PM contribute to this polarization process. Recent research from the PI’s lab showed that multiple core polarity proteins (“polybasic polarity proteins”) such as Lgl, aPKC, and Dlg are also inherently membrane-binding proteins. They contain so-called polybasic domains that specifically and electrostatically target to the PM by binding to PM phospholipids such as PI4P and PIP2. We have characterized multiple mechanisms that control the polarized electrostatic PM targeting function of polybasic polarity proteins. We have also discovered an unexpected consequence of such widely existing electrostatic PM targeting in cell polarity: PM localization of polybasic polarity proteins is highly susceptible to energetic stresses triggered by hypoxia and ATP inhibition, which induce dynamic turnover of PM PI4P and PIP2. In this proposal, our goal is to further establish electrostatic PM-targeting as a fundamental mechanism regulating cell polarity under both normal and energetic stressed conditions. Specifically, we will investigate: 1) Diverse mechanisms regulating electrostatic PM targeting in cell polarity. We will focus on the regulation of electrostatic PM targeting of basolateral polarity protein Dlg which is controlled by phosphorylation, allosteric regulation of its polybasic domain, and interactions with other basolateral polarity proteins. We will elucidate how allosteric regulation of electrostatic PM targeting acts as a core mechanism by which basolateral polarity proteins control each other’s localization and functions. 2) Control of aPKC phosphorylation by electrostatic PM targeting. aPKC is the key kinase driving cell polarization and its pseudo-substrate region (PSr) is also a polybasic domain that is allosterically controlled by Par-6 for both PM targeting and kinase activity. Using a novel inducible polarization system, we will investigate the hypothesis that Par-6-dependent electrostatic PM targeting of aPKC controls the substrate-specific phosphorylation of aPKC/Par-6, providing a mechanism of spatiotemporal control of aPKC kinase activity in cell polarization. 3) Regulation of electrostatic PM retargeting of polybasic polarity proteins. Despite total loss of the PM targeting function of Lgl and aPKC under hypoxia due to depletion of PM PI4P and PIP2, we found that both proteins always directly retargeted their original basolateral or apical PM domains once reoxygenation started. We will investigate both lipid- and protein-based mechanisms that restore and control such polarized PM retargeting of polybasic polarity proteins. These mechanisms will be critical to our understanding of how cell polarity survives energetic stress conditions such as hypoxia and ischemia. Our proposed research will delineate essential molecular details that integrate electrostatic PM targeting into the regulatory network of polarity proteins and phospholipids. Discovering novel mechanisms regulating electrostatic PM targeting will be fundamental to our knowledge on cell polarity under both normal and stress conditions.