γ-Carboxylation Is Not Required for gas6-Axl Binding but Is Necessary for gas6-Dependent Endothelial Cell Survival.

Gas6 is a novel member of the vitamin K-dependent family of γ-carboxylated proteins and is a ligand for the receptor tyrosine kinase Axl. Gas6-Axl interactions have been shown to mediate cell survival in vascular endothelium. Although the receptor-binding portion of gas6 lies in the C-terminus, the significance of the N-terminal γ-carboxylated residues (Gla domain) is not clear. To address this question, we recombinantly produced both carboxylated and decarboxylated gas6 and confirmed that the latter is not γ-carboxylated by both Western blotting and fluorescence spectroscopy. Using DAPI staining and flow cytometry, we show that carboxylated gas6 mediates protection of endothelial cells from serum starvation-induced apoptosis whereas decarboxylated gas6 does not. Furthermore, carboxylated gas6, but not decarboxylated gas6, activates Axl and phosphorylates Akt during gas6-Axl-mediated protection of endothelial cells. Interestingly, although decarboxylated gas6 cannot activate Axl and rescue endothelium from serum starvation-induced apoptosis, it can inhibit the survival effect of carboxylated gas6. To further explore the properties of Gla domain of gas6, the binding each form of gas6 to endothelial cells was determined. Both carboxylated and decarboxylated gas6 bind to endothelial cells with an equal affinity of 50 nM. The binding of both forms of gas6 to endothelium is inhibited by an antibody to the extracellular domain of Axl thereby demonstrating that the Gla domain is not required for the direct binding of the C-terminus of gas6 to Axl. These findings support the conclusion that inhibition of gas6-mediated survival by decarboxylated gas6 is not mediated through inhibition of binding of gas6 to its receptor Axl. Rather, the Gla domain of gas6 has a key role by impacting Axl activation via heretofore unknown mechanism(s). Taken together, the results of this study suggest a novel role for γ-carboxylation in gas6 function.