The molecular basis of ceramide-1-phosphate recognition by C2 domains

Abstract

The sphingolipid ceramide‐1‐phosphate (C1P) plays a critical role in the cellular signaling that mediates inflammation, cell proliferation and phagocytosis. C1P has been shown to increase the activity of cytosolic phospholipase A2 α (cPLA2α) as well regulate its translocation to cellular membranes, a process that promotes inflammation through the production of arachidonic acid. In this study we have resolved the first C1P binding site of a peripheral protein. Using NMR we demonstrate the cPLA2α C2 domain interaction site for C1P by mapping chemical shifts. Subsequently, this novel‐binding site is confirmed with in vitro biophysical and biochemical analysis as well as molecular dynamics simulations. To search the human genome for other C1P binding proteins we have performed proteomic studies to began high throughput characterization of the conserved nature of C1P binding. Funding source: American Heart Association SDG0735350N (R.V.S)

The Role of Sphingolipids in Arachidonic Acid Metabolism

“…The mutation of these residues abolishes the ability of the enzyme to be activated and translocated in response to probably C1P formed by A23187 or ATP (8 (an RxRH sequence) have also been shown to be essential for the in vitro affinity of C1P and translocation in response to A23187 (9). These studies demonstrate that C1P is required for the translocation of cPLA 2 a to intracellular membranes in response to stimuli and the subsequent production of AA.…”

Ceramide 1-Phosphate Mediates Endothelial Cell Invasion via the Annexin a2-p11 Heterotetrameric Protein Complex

“…Unlike the binding of cPLA 2 to C-1-P, which has been described as a coincidence detector for C-1-P and zwitterionic membranes (55), annexin a2 binds with very strong affinity (in the low nanomolar range) to membranes containing 10% C-1-P, further sup-FIGURE 5. C-1-P interacts with p11 in endothelial cells.…”

Characterization of eicosanoid synthesis in a genetic ablation model of ceramide kinase

“…In cells, this mutant of cPLA 2 ␣ is not activated in response to infl ammatory agonists demonstrating the requirement of the cPLA 2 ␣ / C1P interaction in eicosanoid biosynthesis ( 15,20 ).…”

“…Plasma was collected and transferred to a clean glass tube. Plasma (50 l) was extracted according to the C1P/sphingolipid analysis protocol described below, and previously described by our laboratory ( 20 ).…”