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.

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.

Galectin-3 Stimulates Tyro3 Receptor Tyrosine Kinase and Erk Signalling, Cell Survival and Migration in Human Cancer Cells.

The TAM (Tyro3, Axl, MerTK) subfamily of receptor tyrosine kinases (RTKs) and their ligands, Gas6 and protein S (ProS1), are implicated in tumorigenesis and chemoresistance in various cancers. The ß-galactoside binding protein galectin-3 (Gal-3), which is also implicated in oncogenesis, has previously been shown to be a ligand for MerTK. However, the selectivity of Gal-3 for the other TAM receptors, and its TAM-mediated signalling and functional properties in cancer cells, remain to be explored. The present study was aimed at determining these, including through direct comparison of Gal-3 with the two canonical TAM ligands. Exogenous Gal-3 rapidly stimulated Tyro3 receptor phosphorylation to the same extent as the Tyro3 ligand ProS1, but not Axl, in the cultured human cancer cell lines SCC-25 (express both Tyro3 and Axl) and MGH-U3 (express Tyro3 only). Gal-3 also activated intracellular Erk and Akt kinases in both cell lines and furthermore protected cells from acute apoptosis induced by staurosporine but not from serum-starvation induced apoptosis. In addition, Gal-3 significantly stimulated cancer cell migration rate in the presence of the Axl blocker BGB324. Therefore, these results have shown Gal-3 to be a novel agonist for Tyro3 RTK, activating a Tyro3-Erk signalling axis, as well as Akt signalling, in cancer cells that promotes cell survival, cell cycle progression and cell migration. These data therefore reveal a novel mechanism of Tyro3 RTK activation through the action of Gal-3 that contrasts with those of the known TAM ligands Gas6 and ProS1.

Tumour-Secreted Protein S (ProS1) Activates a Tyro3-Erk Signalling Axis and Protects Cancer Cells from Apoptosis.

The TAM subfamily (Tyro3, Axl, MerTK) of receptor tyrosine kinases are implicated in several cancers, where they have been shown to support primary tumorigenesis as well as secondary resistance to cancer therapies. Relatively little is known about the oncogenic role of Tyro3, including its ligand selectivity and signalling in cancer cells. Tyro3 showed widespread protein and mRNA expression in a variety of human cancer cell lines. In SCC-25 head and neck cancer cells expressing both Tyro3 and Axl, Western blotting showed that both natural TAM ligands ProS1 and Gas6 rapidly stimulated Tyro3 and Erk kinase phosphorylation, with ProS1 eliciting a greater effect. In contrast, Gas6 was the sole stimulator of Axl and Akt kinase phosphorylation. In MGH-U3 bladder cancer cells, which express Tyro3 alone, ProS1 was again the stronger stimulator of Tyro3 and Erk stimulation but additionally stimulated Akt phosphorylation. Conditioned medium from ProS1-secreting 786-0 kidney cancer cells replicated the kinase activation effects of recombinant ProS1 in SCC-25 cells, with specificity confirmed by ProS1 ligand traps and warfarin. In addition, ProS1 protected cancer cells from acute apoptosis induced by staurosporine, as well as additionally, long-term serum starvation-induced apoptosis in MGH-U3 cells (Tyro3 only), which reflects its additional coupling to Akt signalling in these cells. In conclusion, we have shown that ProS1 is a tumour-derived functional ligand for Tyro3 that supports cancer cell survival. Furthermore, the ProS1-Tyro3 interaction is primarily coupled to Erk signalling although it displays signalling diversity dependent upon its representative expression as a TAM receptor in tumour cells.