TY - JOUR
T1 - Requirement of gamma-carboxyglutamic acid modification and phosphatidylserine binding for the activation of Tyro3, Axl, and Mertk receptors by growth arrest-specific 6
AU - Geng, Ke
AU - Kumar, Sushil
AU - Kimani, Stanley G.
AU - Kholodovych, Vladyslav
AU - Kasikara, Canan
AU - Mizuno, Kensaku
AU - Sandiford, Oleta
AU - Rameshwar, Pranela
AU - Kotenko, Sergei V.
AU - Birge, Raymond B.
N1 - Funding Information:
We would like to thank David Calianese and Viralkumar Davra for helpful discussions, as well as Anita Antes for maintaining the clonally-derived TAM-IFN reporter lines, and Cyril Empig and Bruce Freimark at Peregrine Pharmaceuticals for providing the PS targeting antibody PGN632. We would like to also acknowledge the mass spectrometry and advanced research computing core facilities. The mass spectrometry data were obtained from a Orbitrap instrument funded in part by an NIH grant NS046593, for the support of the UMDNJ Neuroproteomics Core Facility. RBB was supported in part form NIH CA 165077 as well as a grant New Jersey Research Scholars award to Sushil Kumar.
Publisher Copyright:
© 2017 Geng, Kumar, Kimani, Kholodovych, Kasikara, Mizuno, Sandiford, Rameshwar, Kotenko and Birge.
PY - 2017/11/10
Y1 - 2017/11/10
N2 - 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.
AB - 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.
KW - And Mertk receptors
KW - Axl
KW - Growth arrest-specific 6
KW - Phosphatidylserine
KW - Tumor exosomes
KW - Tyro3
KW - Vitamin K
KW - γ-carboxylation
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U2 - 10.3389/fimmu.2017.01521
DO - 10.3389/fimmu.2017.01521
M3 - Article
AN - SCOPUS:85034014771
VL - 8
JO - Frontiers in Immunology
JF - Frontiers in Immunology
SN - 1664-3224
IS - NOV
M1 - 1521
ER -