Ceramide Activated Protein Phosphatases

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 PP1a 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.