The First Laminin G Domain of Protein S (ProS1) is Involved in Activation of Tyro3 Receptor Tyrosine Kinase and Downstream Signaling in Human Cancer Cells

The homologous vitamin K‐dependent proteins Gas6 and protein S (ProS1) are both composed of an N‐terminal phosphatidylserine‐interacting region, a central region of four EGF domains and a C‐terminal globular region composed of two laminin G domains, here termed LG1 and LG2. Both Gas6 and ProS1 are natural ligands for the related TAM (Tyro3, Axl, Mer) receptor tyrosine kinases (RTKs). The Gas6/ProS1‐TAM signaling axis functions to negatively regulate immune cell function as well as stimulate cell survival. However, overexpression of the TAM RTKs in cancers has been shown to promote oncogenesis through stimulation of cancer cell survival, proliferation, invasion and epithelial‐mesenchymal transition. Gas6 activates all 3 TAM receptors, whereas ProS1 is generally believed to be a ligand for Tyro3 and Mer only. The aim of this study is to determine the the structure‐function relationships that confer TAM ligand properties to ProS1 as compared to Gas6, using a chimeric construct approach. The human head and neck squamous cell carcinoma cell line SCC‐25 was used in culture, expressing both Tyro3 and Axl RTKs. Recombinant human Gas6 and ProS1 proteins were prepared, as well as a set of ProS1 chimeras with LG domains swapped with those of Gas6 as follows: Chimera I (Gas6 LG1/ProS1 LG2), Chimera II (ProS1 LG1/Gas6 LG2), Chimera III (Gas6 LG1 & LG2). The proteins were added to cells over a time period of minutes to determine rapid RTK and intracellular signal pathway activation. Cells were lysed and SDS‐PAGE and western blotting performed on extracts using specific antibodies to detect activated, phosphorylated forms of Tyro3, ERK and Akt, as well as GAPDH as a protein loading control. Western blot band intensities were measured by densitometry and relative protein amounts calculated through normalization against the loading control. ProS1 rapidly stimulated Tyro3 in cells, resulting in a two‐fold increase in phospho‐Tyro3, whereas Gas6 had no significant effect on Tyro3. Of the chimeras, only Chimera II significantly activated Tyro3, and did so to the same extent as ProS1. The ProS1/Chimera II effect was mirrored in their two‐fold activation of ERK, which was not significantly stimulated by Gas6 and the other chimeras. In contrast, phospho‐Akt was increased significantly only in response to Gas6 and Chimera III. Furthermore, whilst both Gas6 and ProS1 protected cells from apoptosis induced by staurosporine over 24h, only Gas6 significantly protected cells from long‐term serum‐starvation‐induced apoptosis. These results show that the C‐terminal LG1 domain of ProS1 is necessary for its activation of Tyro3, which links to ERK signaling downstream. In contrast, the Gas6 C‐terminal globular region does not activate Tyro3, but instead is linked to Akt signaling, likely through activation of Axl. Therefore, the specific TAM ligand and receptor expression status of a particular tumor determines activation of a discrete signaling pathway that subsequently governs the tumour cell biological behavior. Support or Funding Information This research is funded by the University of Portsmouth and by the Council for At‐Risk Academics (Cara) . This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .