Mer receptor tyrosine kinase signaling: prevention of apoptosis and alteration of cytoskeletal architecture without stimulation or proliferation

Mer is a member of the Axl/Mer/Tyro3 receptor tyrosine kinase family, a family whose physiological function is not well defined. We constructed a Mer chimera using the epidermal growth factor receptor (EGFR) extracellular and transmembrane domains and the Mer cytoplasmic domain. Stable transfection of the Mer chimera into interleukin 3 (IL-3)-dependent murine 32D cells resulted in ligand-activable surface receptor that tyrosine autophosphorylated, stimulated intracellular signaling, and dramatically reduced apoptosis initiated by IL-3 withdrawal. However, unlike multiple other ectopically expressed receptor tyrosine kinases including full-length EGFR or an EGFR/Axl chimera, the Mer chimera did not stimulate proliferation. Moreover, and in contrast to EGFR, Mer chimera activation induced adherence and cell flattening in the normally suspension-growing 32D cells. The Mer chimera signal also blocked IL-3-dependent proliferation leading to G(1)/S arrest, dephosphorylation of retinoblastoma protein, and elongation of cellular processes. Unlike other agonists that lead to a slow (4-8 days) ligand-dependent differentiation of 32D cells, the combined Mer and IL-3 signal resulted in differentiated morphology and growth cessation in the first 24 h. Thus the Mer chimera blocks apoptosis without stimulating growth and produces cytoskeletal alterations; this outcome is clearly separable from the proliferative signal produced by most receptor tyrosine kinases.     

Identification of signalling pathways activated by Tyro3 that promote cell survival,proliferation and invasiveness in human cancer cells

Tyro3 is a member of the TAM subfamily of receptor tyrosine kinases alongside Axl and MerTK, which are activated by homologous ligands Gas6 and protein S. The TAMs activate signalling pathways that mediate diverse functions including cell survival, proliferation, phagocytosis and immune regulation, and defects in TAM-dependent processes are associated with the development of human autoimmune diseases and numerous cancers. In this study, we have focused on the signalling and functional roles of Tyro3, about which much remains unknown. For this purpose, we used cultured human cancer cell lines with different levels of TAM expression to reveal the relative significance of Tyro3 amongst the TAMs. Knockdown of Tyro3 expression by siRNA in MGH-U3 cells, which express Tyro3 as sole TAM, caused a reduction in cell viability, which could not be rescued by TAM ligand, demonstrating the dependence of these cells solely on Tyro3. In contrast, the reduced viability of SCC-25 cells upon Tyro3 knockdown could be rescued by Gas6 as these cells express both Tyro3 and Axl and hence Axl expression was preserved. An increase in the fraction of Tyro3 knockdown cells in the early apoptotic phase was observed in four different cell lines each with a different TAM expression profile, revealing a broad anti-apoptotic function of Tyro3. Furthermore, in the Tyro3-dependent cells, Tyro3 depletion caused a significant increase in cells in the G0/G1 phase of the cell cycle concomitant with decreases in the G2/M and S phases. In addition, a cancer pathway gene discovery array revealed distinct sets of genes that were altered in expression in cancer cells upon Tyro3 knockdown. Together, these results have elucidated further a role of Tyro3 in promoting multiple tumour-supporting pathways in human cancer cells, which differs in extent depending on the presence of other TAMs in the same cells.     

Ligand recognition and homophilic interactions in Tyro3: structural insights into the Axl/Tyro3 receptor tyrosine kinase family

The receptor Tyro3 together with Axl and Mer form the Axl/Tyro3 family of receptor tyrosine kinases. Members of this family play essential roles in spermatogenesis, immunoregulation, and phagocytosis. Gas6, the product of growth arrest-specific gene, activates the kinase activity of all three receptors. Here, we report the first biochemical and structural characterization of a member of this family, namely of a fragment spanning the two N-terminal Ig domains of the extracellular part of human Tyro3. Its ligand binding specificity profile is identical to the activation profile of the native receptor. The 1.95-A crystal structure suggests a common ligand-binding site in this receptor family located at the interface of the Ig domains and unusually rich in cis-prolines. Furthermore, both in the crystal and in solution we observed the ligand-independent dimerization of the receptor fragment. This homophilic interaction emphasizes previous functional reports, which hinted that in addition to signal transduction, members of this family of receptors might participate in cell adhesion.     

Axl and Tyro3 modulate female reproduction by influencing gonadotropin-releasing hormone neuron survival and migration

GnRH neurons must undergo a complex and precise pattern of neuronal migration to appropriately target their projections to the median eminence to trigger gonadotropin secretion and thereby control reproduction. Using NLT GnRH cells as a model of early GnRH neuronal development, we identified the potential importance of Axl and Tyro3, members of the TAM (Tyro3, Axl, and Mer) family of receptor tyrosine kinases in GnRH neuronal cell survival and migration. Silencing studies evaluated the role of Tyro3 and Axl in NLT GnRH neuronal cells and suggest that both play a role in Gas6 stimulation of GnRH neuronal survival and migration. Analysis of mice null for both Axl and Tyro3 showed normal onset of vaginal opening but delayed first estrus and persistently abnormal estrous cyclicity compared with wild-type controls. Analysis of GnRH neuronal numbers and positioning in the adult revealed a total loss of 24% of the neuronal network that was more striking (34%) when considered within specific anatomical compartments, with the largest deficit surrounding the organum vasculosum of the lamina terminalis. Analysis of GnRH neurons during embryogenesis identified a striking loss of immunoreactive cells within the context of the ventral forebrain compartment (36%) and not more rostrally. Studies using caspase 3 cleavage as a marker of apoptosis showed that Axl(-/-), Tyro3(-/-) double-knockout mice had increased cell death in the nose and dorsal forebrain, supporting the underlying mechanism of cell loss. Together these data suggest that Axl and Tyro3 mediate the survival and appropriate targeting of GnRH neurons to the ventral forebrain, thereby contributing to normal reproductive function and cyclicity in the female.     

Increased plasma levels of the soluble Mer tyrosine kinase receptor in systemic lupus erythematosus relate to disease activity and nephritis

Introduction  

Mer and Tyro3 are receptor tyrosine kinases important for the phagocytosis of apoptotic cells. Together with Axl, they constitute the TAM receptor family. These receptors can be shed from the cell membrane and their soluble extracellular regions can be found in plasma. The objective of this study was to elucidate whether the plasma levels of soluble Mer (sMer) and Tyro3 (sTyro3) were increased in systemic lupus erythematosis (SLE), rheumatoid arthritis (RA), or critical limb ischemia (CLI).     

Gas6 negatively regulates the Staphylococcus aureus-induced inflammatory response via TLR signaling in the mouse mammary gland

Staphylococcus aureus (S. aureus)-induced mastitis is the most frequent, pathogenic, and prevalent infection of the mammary gland. The ligand growth arrest-specific 6 (Gas6) is a secretory protein that binds to and activates Tyro3, Axl, and MerTK receptors. This study explored the role of Gas6 in S. aureus-induced mastitis. Our results revealed that TLR receptors initiate the innate immune response in mammary gland tissues and epithelial cells and that introducing S. aureus activates TLR2 and TLR6 to drive multiple intracellular mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) pathways. Moreover, S. aureus also induces Gas6, which then activates the TAM receptor kinase pathway, which is related to the inhibition of TLR2- and TLR6-mediated inflammatory pathways through SOCS1 and SOCS3 induction. Gas6 absence alone was found to be involved in the downregulation of TAM receptor-mediated anti-inflammatory effects by inducing significantly prominent expression of TRAF6 and low protein and messenger RNA expression of SOCS1 and SOCS3. S. aureus-induced MAPK and NF-ĸB p65 phosphorylation were also dependent on Gas6, which negatively regulated the production of Pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) in S. aureus-treated mammary tissues and mammary epithelial cells. Our in vivo and in vitro study uncovered the Gas6-mediated negative feedback mechanism, which inhibits TLR2- and TLR6-mediated MAPK and NF-ĸB signaling by activating TAM receptor kinase (MerTK, Axl, and Tyro3) through the induction of SOCS1/SOCS3 proteins.     

TAMpering with toll-like receptor signaling

Toll-like receptors (TLRs) provoke a profound inflammatory response during host defense and must be controlled in order to avoid autoimmune and inflammatory diseases. In this issue, Rothlin et al. (2007) uncover a complex negative feedback mechanism to limit TLR signaling involving the Tyro3/Axl/Mer (TAM) family of receptor tyrosine kinases, which induce expression of the inhibitory proteins SOCS1 and SOCS3.     

The first laminin G-like domain of protein S is essential for binding and activation of Tyro3 receptor and intracellular signalling

The homologous proteins Gas6 and protein S (ProS1) are both natural ligands for the TAM (Tyro3, Axl, MerTK) receptor tyrosine kinases. ProS1 selectively activates Tyro3; however, the precise molecular interface of the ProS1-Tyro3 contact has not been characterised. We used a set of chimeric proteins in which each of the C-terminal laminin G-like (LG) domains of ProS1 were swapped with those of Gas6, as well as a set of ProS1 mutants with novel added glycosylations within LG1. Alongside wildtype ProS1, only the chimera containing ProS1 LG1 domain stimulated Tyro3 and Erk phosphorylation in human cancer cells, as determined by Western blot. In contrast, Gas6 and chimeras containing minimally the Gas6 LG1 domain stimulated Axl and Akt phosphorylation. We performed in silico homology modelling and molecular docking analysis to construct and evaluate structural models of both ProS1-Tyro3 and Gas6-Axl ligand-receptor interactions. These analyses revealed a contact between the ProS1 LG1 domain and the first immunoglobulin domain of Tyro3, which was similar to the Gas6-Axl interaction, and involved long-range electrostatic interactions that were further stabilised by hydrophobic and polar contacts. The mutant ProS1 proteins, which had added glycosylations within LG1 but which were all outside of the modelled contact region, all activated Tyro3 in cells with no hindrance. In conclusion, we show that the LG1 domain of ProS1 is necessary for activation of the Tyro3 receptor, involving protein-protein interaction interfaces that are homologous to those of the Gas6-Axl interaction.     

Normalization of TAM post-receptor signaling reveals a cell invasive signature for Axl tyrosine kinase

Background

Tyro3, Axl, and Mertk (TAMs) are a family of three conserved receptor tyrosine kinases that have pleiotropic roles in innate immunity and homeostasis and when overexpressed in cancer cells can drive tumorigenesis.

Methods

In the present study, we engineered EGFR/TAM chimeric receptors (EGFR/Tyro3, EGFR/Axl, and EGF/Mertk) with the goals to interrogate post-receptor functions of TAMs, and query whether TAMs have unique or overlapping post-receptor activation profiles. Stable expression of EGFR/TAMs in EGFR-deficient CHO cells afforded robust EGF inducible TAM receptor phosphorylation and activation of downstream signaling.

Results

Using a series of unbiased screening approaches, that include kinome-view analysis, phosphor-arrays, RNAseq/GSEA analysis, as well as cell biological and in vivo readouts, we provide evidence that each TAM has unique post-receptor signaling platforms and identify an intrinsic role for Axl that impinges on cell motility and invasion compared to Tyro3 and Mertk.

Conclusion

These studies demonstrate that TAM show unique post-receptor signatures that impinge on distinct gene expression profiles and tumorigenic outcomes.

     

Gas6 induces proliferation in prostate carcinoma cell lines expressing the Axl receptor

Axl is a tyrosine kinase receptor and although it is expressed in malignancy such as leukemia, colon cancer, melanoma, endometrial, prostate and thyroid cancers, its role has not been completely elucidated yet and appears to be complex. The ligand of Axl, Gas6, is a 75 KDa multimodular protein with an N-terminal gamma-carboxy-glutamic acid that is essential for binding. Gas6 has a mitogenic effect on several normal cell lines. The receptor Axl is expressed in primary prostate carcinoma and in prostate cancer cell lines as such as PC-3 and DU 145. We demonstrated a mitogenic activity determined by Gas6/Axl interaction in these undifferentiated metastatic human prostatic cancer cell lines. This effect is proportional to Axl expression, not due to inhibition of apoptosis, and induces AKT and MAPK phosphorylation. However, only MEK phosphorylation seems to be essential for growth signaling. Our results suggest that Axl overexpression and activation by Gas6 could be involved in progression of prostate neoplastic disease.     

High incidence of distal vaginal atresia in mice lacking Tyro3 RTK subfamily

Vaginal atresia is a congenital abnormality of the female genitourinary system, and the specific molecular mechanism leading to failure of vaginal development remains to be elucidated. Here, we report that the female mice lacking Tyro3 RTK subfamily (Tyro3, Axl, and Mer) exhibit a high incidence of distal vaginal atresia. The ratios of the vaginal atresia in Tyro3 RTKs mutant female mice are as follows: 2.5% for Mer(-/-) mice, 4.0% for Axl(-/-)Mer(-/-), 3.7% for Mer(-/-)Tyro3(-/-), 16.06% for Tyro(-/-)Axl(-/-)Mer(-/-) mice. We did not find the vaginal atresia in Axl(-/-), Tyro3(-/-), Axl(-/-) Tyro(-/-), and wild-type mice. These observations suggest that Tyro3 RTKs play roles collaboratively in vaginal development, and Mer is more critical, Axl and Tyro3 support the function of Mer. The phenotype of mice with the vaginal atresia was characterized in this study. Tyro3 RTKs mutant mouse could be a useful model to study the mechanism of vaginal atresia formation.     

An SH2 domain-dependent,phosphotyrosine-independent interaction between Vav1 and the Mer receptor tyrosine kinase: a mechanism for localizing guanine nucleotide-exchange factor action

Mer belongs to the Mer/Axl/Tyro3 receptor tyrosine kinase family, which regulates immune homeostasis in part by triggering monocyte ingestion of apoptotic cells. Mutations in Mer can also cause retinitis pigmentosa, again due to defective phagocytosis of apoptotic material. Although, some functional aspects of Mer have been deciphered, how receptor activation lead to the physiological consequences is not understood. By using yeast two-hybrid assays, we identified the carboxyl-terminal region of the guanine nucleotide-exchange factor (GEF) Vav1 as a Mer-binding partner. Unlike similar (related) receptors, Mer interacted with Vav1 constitutively and independently of phosphotyrosine, yet the site of binding localized to the Vav1 SH2 domain. Mer activation resulted in tyrosine phosphorylation of Vav1 and release from Mer, whereas Vav1 was neither phosphorylated nor released from kinase-dead Mer. Mutation of the Vav1 SH2 domain phosphotyrosine coordinating Arg-696 did not alter Mer/Vav1 constitutive binding or Vav1 tyrosine phosphorylation but did retard Vav1 release from autophosphorylated Mer. Ligand-dependent activation of Mer in human monocytes led to Vav1 release and stimulated GDP replacement by GTP on RhoA family members. This unusual constitutive, SH2 domain-dependent, but phosphotyrosine-independent, interaction and its regulated local release and subsequent activation of Rac1, Cdc42, and RhoA may explain how Mer coordinates precise cytoskeletal changes governing the ingestion of apoptotic material by macrophages and pigmented retinal epithelial cells.     

Vitamin K, an emerging nutrient in brain function

Historically discovered for its role in blood coagulation, there is now convincing evidence that vitamin K has important actions in the nervous system. As a unique cofactor to the γ-glutamyl carboxylase enzyme, vitamin K contributes to the biological activation of proteins Gas6 and protein S, ligands for the receptor tyrosine kinases of the TAM family (Tyro3, Axl, and Mer). Functionally, Gas6 has been involved in a wide range of cellular processes that include cell growth, survival, and apoptosis. In brain, vitamin K also participates in the synthesis of sphingolipids, an important class of lipids present in high concentrations in brain cell membranes. In addition to their structural role, sphingolipids are now known to partake in important cellular events such as proliferation, differentiation, senescence and cell-cell interactions. In recent years, studies have linked alterations in sphingolipid metabolism to age-related cognitive decline and neurodegenerative diseases such as Alzheimer's disease (AD). Emerging data also point to unique actions of the K vitamer menaquinone-4 (MK-4) against oxidative stress and inflammation. Finally, there is now data to suggest that vitamin K has the potential to influence psychomotor behavior and cognition. This review presents an overview of what is known of the role of vitamin K in brain function.     

Tyro3, Axl,and Mertk receptors differentially participate in platelet activation and thrombus formation

Background

Previously, several studies have shown that Tyro3, Axl, and Mertk (TAM) receptors participate in platelet activation and thrombosis. However, the role of individual receptors is not fully understood.

Methods

Using single receptor-deficient platelets from TAM knockout mice in the C57BL/6?J strain, we performed a knockout study using single TAM-deficient mice. We treated platelets isolated from TAM knockout mice with the Glycoprotein VI (GPVI) agonists convulxin, poly(PHG), and collagen-related triple-helical peptide (CRP), as well as thrombin for in-vitro experiments. We used a laser-induced cremaster arterial injury model for thrombosis experiments in vivo.

Results

Deficiency of the tyrosine kinase receptors, Axl or Tyro3, but not Mertk, inhibited aggregation, spreading, JON/A binding, and P-selectin expression of platelets in vitro. In vivo, platelet thrombus formation was significantly decreased in Axl^?/? and Tyro3^?/? mice, but not in Mertk^?/? mice. Upon stimulation with glycoprotein VI (GPVI) agonists, tyrosine phosphorylation of signaling molecules, including spleen tyrosine kinase (Syk) and phospholipase C-γ2 (PLCγ2), was decreased in Axl^?/? and Tyro3^?/? platelets, but not in Mertk^?/? platelets. While platelet aggregation induced by agonists did not differ in the presence or absence of the Gas6 neutralizing antibody, the platelet aggregation was inhibited by anti-Axl or anti-Tyro3 neutralizing antibodies antibody, but not the anti-Mertk antibody. Additionally, the recombinant extracellular domain of Axl or Tyro3, but not that of Mertk, also inhibited platelet aggregation.

Conclusions

These data suggest that Axl and Tyro3, but not Mertk, have an important role in platelet activation and thrombus formation, and mechanistically may do so by a pathway that regulates inside to outside signaling and heterotypic interactions via the extracellular domains of TAMs.

     

Structural basis for Gas6-Axl signalling

Receptor tyrosine kinases of the Axl family are activated by the vitamin K-dependent protein Gas6. Axl signalling plays important roles in cancer, spermatogenesis, immunity, and platelet function. The crystal structure at 3.3 A resolution of a minimal human Gas6/Axl complex reveals an assembly of 2:2 stoichiometry, in which the two immunoglobulin-like domains of the Axl ectodomain are crosslinked by the first laminin G-like domain of Gas6, with no direct Axl/Axl or Gas6/Gas6 contacts. There are two distinct Gas6/Axl contacts of very different size, both featuring interactions between edge beta-strands. Structure-based mutagenesis, protein binding assays and receptor activation experiments demonstrate that both the major and minor Gas6 binding sites are required for productive transmembrane signalling. Gas6-mediated Axl dimerisation is likely to occur in two steps, with a high-affinity 1:1 Gas6/Axl complex forming first. Only the minor Gas6 binding site is highly conserved in the other Axl family receptors, Sky/Tyro3 and Mer. Specificity at the major contact is suggested to result from the segregation of charged and apolar residues to opposite faces of the newly formed beta-sheet.     

TAM kinase signaling is indispensable for proper skeletal muscle regeneration in mice

Skeletal muscle regeneration following injury results from the proliferation and differentiation of myogenic stem cells, called satellite cells, located beneath the basal lamina of the muscle fibers. Infiltrating macrophages play an essential role in the process partly by clearing the necrotic cell debris, partly by producing cytokines that guide myogenesis. Infiltrating macrophages are at the beginning pro-inflammatory, but phagocytosis of dead cells induces a phenotypic change to become healing macrophages that regulate inflammation, myoblast fusion and growth, fibrosis, vascularization and return to homeostasis. The TAM receptor kinases Mer and Axl are known efferocytosis receptors in macrophages functioning in tolerogenic or inflammatory conditions, respectively. Here we investigated their involvement in the muscle regeneration process by studying the muscle repair following cardiotoxin-induced injury in Mer^−/− mice. We found that Axl was the only TAM kinase receptor expressed on the protein level by skeletal muscle and C2C12 myoblast cells, while Mer was the dominant TAM kinase receptor in the CD45⁺ cells, and its expression significantly increased during repair. Mer ablation did not affect the skeletal muscle weight or structure, but following injury it resulted in a delay in the clearance of necrotic muscle cell debris, in the healing phenotype conversion of macrophages and consequently in a significant delay in the full muscle regeneration. Administration of the TAM kinase inhibitor BMS-777607 to wild type mice mimicked the effect of Mer ablation on the muscle regeneration process, but in addition, it resulted in a long-persisting necrotic area. Finally, in vitro inhibition of TAM kinase signaling in C2C12 myoblasts resulted in decreased viability and in impaired myotube growth. Our work identifies Axl as a survival and growth receptor in the mouse myoblasts, and reveals the contribution of TAM kinase-mediated signaling to the skeletal muscle regeneration both in macrophages and in myoblasts.Subject terms: Mechanisms of disease, Immunological disorders     

Identification of cell surface molecules involved in dystroglycan-independent Lassa virus cell entry

Although O-mannosylated dystroglycan is a receptor for Lassa virus, a causative agent of Lassa fever, recent findings suggest the existence of an alternative receptor(s). Here we identified four molecules as receptors for Lassa virus: Axl and Tyro3, from the TAM family, and dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and liver and lymph node sinusoidal endothelial calcium-dependent lectin (LSECtin), from the C-type lectin family. These molecules enhanced the binding of Lassa virus to cells and mediated infection independently of dystroglycan. Axl- or Tyro3-mediated infection required intracellular signaling via the tyrosine kinase activity of Axl or Tyro3, whereas DC-SIGN- or LSECtin-mediated infection and binding were dependent on a specific carbohydrate and on ions. The identification of these four molecules as Lassa virus receptors advances our understanding of Lassa virus cell entry.