Involvement of Lipid Rafts and Caveolae in gas6-Axl Signaling in Endothelial Cells

Abstract 2211

Lipid rafts are localized regions of plasma membranes that contain primarily cholesterol and glycosphingolipids. Many receptor tyrosine kinases including the epidermal growth factor receptor, the platelet derived growth factor receptor, and the insulin receptor, localize specifically to these structures. Lipid rafts have been shown to play a role in intracellular signaling events. More specifically, diverse processes such as dimer formation, autophosphorylation, and intracellular adaptor protein recruitment/activation have been specifically involved in receptor tyrosine kinase/lipid raft interactions. Caveolae are defined as a subclass of lipid rafts and form well-shape invaginations on the cell surface. Caveolin-1 is the major constitutive protein of caveolae. Axl is a receptor tyrosine kinase whose association with lipid rafts and more specifically with caveolae has not been elucidated. Gas6, the ligand for Axl, is a vitamin K dependent protein that is post-translationally modified by an enzymatic process called g-carboxylation. We hypothesize that gas6/Axl mediated intracellular signaling is dependent on its localization to lipid rafts/caveolae in endothelial cells. First, we show that gas6-induced c-Src, Akt and ERK1/2 phosphorylation is mediated via Axl by using Axl siRNA in human umbilical vein endothelial cells (HUVECs). Then, lipid rafts were isolated from HUVECs by a detergent-free lysis method followed by a sucrose gradient centrifugation and western blot analysis. We found that Axl moved into the lipid raft-enriched fractions after 5 and 10 min of gas6 treatment as shown by its colocalization with caveolin-1. Interestingly, after gas6 treatment, c-Src, which is known to act as a transient docking platform for signaling molecules in lipid rafts, follow the same localization pattern to lipid raft-enriched fractions. The requirement of caveolae was then evaluated by transfecting HUVECs with caveolin-1 siRNA. Caveolin-1 knock down abolished completely gas6-induced phosphorylation of c-Src, Akt and ERK1/2 thus highlighting the role of caveolae in gas6-Axl signaling. Taken together these results demonstrate that activation of gas6-dependent signaling pathways involves Axl trafficking to lipid rafts/caveolae. These novel findings identify lipid rafts/caveolae as dynamic scaffolding systems for gas6/Axl interactions in endothelial cells.