Ceramide Activated Protein Phosphatases

神经酰胺是一类密切相关的分子，具有生物效应的功能。 脂类在调节各种人的应激反应和生长/死亡中的作用 癌细胞。我们对神经酰胺的理解中的关键缺失元素源于 缺乏对神经酰胺隔室特异性功能的定义，以及缺乏分子- 明确的行动目标。由该项目支持的我们实验室的研究已经确定 神经酰胺激活的丝氨酸苏氨酸磷酸酶(CAPP)，特别是PP1和PP2A作为直接的 神经酰胺体外激活的靶点。最近的结果为我们在以下方面提供了突破 确定在质膜(PM)产生神经酰胺的特定途径。这里, 我们将研究PM产生的神经酰胺会迅速激活的假说 Pp1α导致不同蛋白质的去磷酸化，启动了一个不同的程序 对细胞黏附和迁移的调节。我们还在开发一种新的检测方法来 具体定量PM神经酰胺。通过这一突破，我们将实现以下目标： 目的1.建立一种定量分析方法来测量PM和 确定神经酰胺代谢的关键酶在这个隔室(稳定状态)。目标2. 定义刺激诱导PM神经酰胺的形成及其机制。目标3. 明确PM神经酰胺调节PP1a的生化机制。加在一起， 这些方法应该第一次导致明确定义一个具体、直接和 神经酰胺作用的相关靶点(PP1)具有特定的调节作用 PM神经酰胺对细胞黏附和迁移的影响。

Ceramide constitutes a family of closely related molecules that function as bioeffector lipids with roles in the regulation of stress responses and growth/death of various human cancer cells. Critical missing elements in our understanding of ceramide stem from the lack of defining compartment-specific functions of ceramides and from lack of molecularly- defined targets of action. Studies in our lab supported by this project have identified ceramide-activated Ser–Thr phosphatases (CAPPs), specifically PP1 and PP2A as direct targets activated by ceramide in vitro. Recent results have provided us a breakthrough in defining a specific pathway of ceramide generation at the plasma membrane (PM). Here, we will investigate the hypothesis that ceramide generated at the PM acutely activates PP1α that leads to the dephosphorylation of distinct proteins, launching a distinct program of regulation of cell adhesion and migration. We are also developing a novel assay to quantitate PM ceramide specifically. With this breakthrough, we will address these aims: Aim 1. Develop a quantitative assay to measure ceramide generation at the PM and define key enzymes of ceramide metabolism in this compartment (steady state). Aim 2. Define stimulus-induced formation of PM ceramide and the mechanisms involved. Aim 3. Define biochemical mechanisms of regulation of PP1a by PM ceramide. Taken together, these approaches should result, for the first time, in clearly defining a specific, direct, and relevant target for ceramide action (PP1) with a specific function in mediating the effects of PM ceramide on cell adhesion and migration.