Requirement of Gamma-Carboxyglutamic Acid Modification and Phosphatidylserine Binding for the Activation of Tyro3, Axl, and Mertk Receptors by Growth Arrest-Specific 6

Abstract

The Tyro3, Axl, and Mertk (TAM) receptors are homologous type I receptor tyrosine kinases that have critical functions in the clearance of apoptotic cells in multicellular organisms. TAMs are activated by their endogenous ligands, growth arrest-specific 6 (Gas6), and protein S (Pros1), that function as bridging molecules between externalized phosphatidylserine (PS) on apoptotic cells and the TAM ectodomains. However, the molecular mechanisms by which Gas6/Pros1 promote TAM activation remains elusive. Using TAM/IFNγR1 reporter cell lines to monitor functional TAM activity, we found that Gas6 activity was exquisitely dependent on vitamin K-mediated γ-carboxylation, whereby replacing vitamin K with anticoagulant warfarin, or by substituting glutamic acid residues involved in PS binding, completely abrogated Gas6 activity as a TAM ligand. Furthermore, using domain and point mutagenesis, Gas6 activity also required both an intact Gla domain and intact EGF-like domains, suggesting these domains function cooperatively in order to achieve TAM activation. Despite the requirement of γ-carboxylation and the functional Gla domain, non-γ-carboxylated Gas6 and Gla deletion/EGF-like domain deletion mutants still retained their ability to bind TAMs and acted as blocking decoy ligands. Finally, we found that distinct sources of PS-positive cells/vesicles (including apoptotic cells, calcium-induced stressed cells, and exosomes) bound Gas6 and acted as cell-derived or exosome-derived ligands to activate TAMs. Taken together, our findings indicate that PS is indispensable for TAM activation by Gas6, and by inference, provides new perspectives on how PS, regulates TAM receptors and efferocytosis.

Gamma‐glutamyl–carboxylated Gas6 mediates positive role of vitamin K on lowering hyperglycemia in type 2 diabetes

“…VK‐mediated gamma‐glutamyl carboxylation of Gas6 (cGas6) shows the highest affinity binding with AXL, a member of the TAM receptor tyrosine kinase protein family . The binding of cGas6 with AXL causes the tyrosine phosphorylation of AXL, followed by the activation of PI3K .…”

“…VK-mediated gamma-glutamyl carboxylation of Gas6 (cGas6) shows the highest affinity binding with AXL, a member of the TAM receptor tyrosine kinase protein family. 8,10 The binding of cGas6 with AXL causes the tyrosine phosphorylation of AXL, followed by the activation of PI3K. 6,7 After the activation, PI3K stimulates the formation of PIP3, which causes the phosphorylation of PKB (or AKT), followed by GLUT4 translocation from the intracellular pool to the plasma membrane and glucose uptake.…”

“…The gamma‐glutamyl carboxylase (GGCX)–mediated VK‐dependent posttranslational conversion of protein‐bound glutamate residues into gamma‐carboxyglutamate produces the carboxylated form of Gas6, namely, cGas6. Interestingly, Gas6–AXL interaction was markedly attenuated by inhibiting the gamma‐glutamyl carboxylation of Gas6, which suggests the requirement of gamma‐carboxyglutamic acid residues for the biological activities of Gas6 . There is so far no study in the literature that has examined the plasma levels of cGas6 and its relationship with glycemic parameters and VK in individuals with and without T2D.…”

“…Interestingly, Gas6-AXL interaction was markedly attenuated by inhibiting the gamma-glutamyl carboxylation of Gas6, which suggests the requirement of gamma-carboxyglutamic acid residues for the biological activities of Gas6. [8][9][10] There is so far no study in the literature that has examined the plasma levels of cGas6 and its relationship with glycemic parameters and VK in individuals with and without T2D. Moreover, VK supplementation has been found to play a beneficial role in regulating glucose metabolism.…”

AXL as a Target in Breast Cancer Therapy

“…Up until recently protein S was thought to exclusively be a ligand for TYRO3 and MER, but recently it has been shown to be capable of binding to and activating AXL in glioblastoma cells [9]. Activation of AXL is not complete until a further interaction with the phospholipid phosphatidyl serine (PS) occurs, mediated by the gamma-carboxyglutamic acid (Gla) domain on GAS6 following its posttranslational modification [10]. PS is a phospholipid that is normally restricted to the intracellular portion of the phospholipid bilayer but is externalized in apoptotic cells or cells that are otherwise stressed, such as in virally infected cells.…”