Ceramide Activated Protein Phosphatases

神经酰胺是一类具有生物效应功能的分子 脂质在调节各种人类应激反应和生长/死亡中的作用 癌细胞在我们对神经酰胺的理解中，关键的缺失元素来自于 神经酰胺缺乏明确的区室特异性功能， 明确的行动目标。我们实验室的研究得到了这个项目的支持， 神经酰胺激活的丝氨酸-苏氨酸磷酸酶（CAPP），特别是PP 1和PP 2A作为直接 神经酰胺在体外激活的靶点。最近的结果为我们提供了一个突破， 定义了质膜（PM）上神经酰胺产生的特定途径。在这里， 我们将研究PM产生的神经酰胺急性激活 PP 1 α导致不同蛋白质的去磷酸化，启动不同的程序 调节细胞粘附和迁移。我们还在开发一种新的检测方法， 特异性定量PM神经酰胺。通过这一突破，我们将实现以下目标： 目标1.开发一种定量测定方法，以测量PM时的神经酰胺生成， 定义在该室中神经酰胺代谢的关键酶（稳态）。目标2. 定义刺激诱导的PM神经酰胺形成及其机制。目标3. 定义PM神经酰胺调节PP 1a的生化机制。综合起来看， 这些方法应该导致，第一次，明确界定一个具体的，直接的， 神经酰胺作用的相关靶点（PP 1），具有介导效应的特定功能 PM神经酰胺对细胞粘附和迁移的影响。

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.