Ceramide‐1‐Phosphate: Characterizing a Fluorescent Lipid and Discovering New Binding Proteins

Abstract

Sphingolipids are a class of biomolecules that play key roles in cellular signaling and membrane trafficking, with main players including sphingosine, sphingosine‐1‐phosphate (S1P), ceramide, and ceramide‐1‐phosphate (C1P). Specifically, C1P has been shown to play a role in important intra‐ and extracellular events such as cell proliferation, phagocytosis, inflammation, tumor metastasis, and migration of macrophages. Despite the identification of several C1P binding proteins such as cytosolic phospholipase A2α (cPLA2α), Annexin A2, diacylglycerol kinase γ, tumor necrosis factor‐α converting enzyme (TACE), and prostaglandin D synthase, there are still many gaps to fill in our understanding of C1P binding proteins and how these cellular interactions are facilitated. Therefore, we characterized a fluorescently labeled C1P for its in vitro and cellular activity, and sought to identify more C1P binding proteins. Here, we investigated a new fluorescently labeled C1P, called TopFluor‐C1P, to see if it could mimic C1P naturally found in mammalian cells. TopFluor‐C1P proved to be non‐toxic to cells, localize to the trans‐Golgi Network and plasma membrane, and successfully recruit the translocation of cPLA2α, as previous work has shown with native C1P. We also used fluorescence recovery after photobleaching (FRAP) to determine the mobile fraction and diffusion coefficient of TopFluor‐C1P at the plasma membrane to understand C1P membrane dynamics. This revealed the diffusion coefficient of C1P is less than other signaling lipids such as phosphatidylserine and cholesterol, and more similar to that of other sphingolipids. From our studies we determined TopFluor‐C1P is a valuable mimetic that can be further utilized to study many important biophysical and cellular questions regarding C1P properties in membranes and its role in lipid‐lipid and lipid‐protein interactions. In addition we aimed to discover the C1P binding proteome. Therefore, we performed an IP assay from A549 lung adenocarcinoma cells using lipid‐coated beads. The mass spectrometry analysis of lipid phosphomonoesters identified 212 C1P, 280 ceramide, 588 S1P, and 449 phosphatidic acid binding proteins. From this data we were were able to identify proteins that were found among all four structurally similar lipids, or were specific to only one lipid. We also used the Gene Ontology consortium and MetaCore pathways analysis software to further identify the molecular function, cellular localization and pathways involved in C1P binding proteins. Identifying these direct effectors of C1P will aid us in discovering C1P lipid‐protein binding characteristics, and help us better understand the role of C1P within the cell and in disease and cancer metabolism.Support or Funding InformationThis material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE‐1313583.