A pathway map of AXL receptor-mediated signaling network

Introduction

AXL is a transmembrane protein that comes under TAM (TYRO3,AXL and MERTK) family of receptor tyrosine kinases (RTKs) (Myers et al. 2019). It is first identified as a transforming gene isolated from primary human myeloid leukemia cells (O’Bryan et al. 1991). The AXL gene is located in chromosome 19, at 19q13.1(Linz et al. 1993), which encodes a protein of 894 amino acids (Korshunov 2012). AXL receptor has an extracellular, a transmembrane and an intracellular domains. The extracellular domain contains two immunoglobulin-like (IgL) and fibronectin type-III (FN III)-like domains with a single-pass transmembrane domain (Linger et al. 2008). The intracellular tyrosine kinase domain of AXL contains six phosphorylation sites. Among these three phosphorylation sites were in C-terminal domain (Tyr⁷⁷⁹, Tyr⁸²¹ and Tyr⁸⁶⁶) and others in N-terminal domain (Tyr⁶⁹⁸, Tyr⁷⁰², Tyr⁷⁰³). Autophosphorylation of C-terminal phosphorylation sites stimulated by GAS6 has a role in the kinase activity of AXL (Linger et al. 2008, O’Bryan et al. 1991). Phosphorylation of the residues in the N-terminal domain induced by GAS6 has also been shown (Pao-Chun et al. 2009). In particular, phosphorylated Tyr⁷⁰² may stabilize the conformation of the activation loop of AXL, promoting AXL activity (​PMID: 28,724,631​). AXL is known to extensively express in various tissues such as brain, heart, liver, bone marrow, neurons and vascular tissues (Jin et al. 2015, Lemke 2013, Lemke and Rothlin 2008, Nielsen-Preiss et al. 2007).

AXL activation is mediated by a ligand called growth arrest-specific protein 6 (GAS6), which is the product of GAS6 gene. GAS6 is a vitamin-K dependent growth factor mainly express in multiple cells (Sasaki et al. 2006). GAS6 has the highest affinity for AXL among the TAM family members. The stimulation of AXL receptor by binding of GAS6 induces receptor dimerization and subsequently AXL activation, which transfers signals from the extracellular matrix into the cytoplasm and results in various signaling pathways through its downstream substrates. The activation of AXL can also occur through ligand-independent mechanism in response to ROS, which mainly occurs during oxidative stress and overexpression of AXL (Konishi et al. 2004).

The GAS6-mediated AXL pathway regulates multiple physiological processes including cell proliferation, survival, motility, aggregation and anti-inflammation (Linger et al. 2010). Similar to that of the other RTKs, this pathway contributes to different downstream signaling pathways including PI3K/AKT/mTOR, MAPK and JAK/STAT (Brien 1964). The aberrant expression as well as prolonged activation of AXL has been reported in various disease conditions such as cancer, chronic immune disorders and cardiovascular diseases (Linger et al. 2008, O’Bryan et al. 1991). Deregulated levels of GAS6 has also been implicated in diseases including liver fibrosis (Barcena et al. 2015), renal disease (Fiebeler et al. 2004), Alzheimer’s disease (Sainaghi et al. 2017) and multiple sclerosis (Bellan et al. 2016).

Although AXL activation has been reported in many physiological and pathophysiological conditions, the information pertaining to GAS6-mediated AXL signaling network is dispersed throughout the literature. Therefore, we compiled the molecular consequences induced by AXL from the research articles and built a detailed signaling map of GAS6-mediated AXL. We developed a resource of signaling events mediated by AXL similar to the previously published NetPath pathways including FGFR, BDNF, Serotonin, IL-33, IL-18, IL-10 and Oncostatin M (Rex et al. 2020, Sahu et al. 2018, Pinto et al. 2018, Verma et al. 2016, Raju et al. 2014, Sandhya et al. 2013, Dey et al. 2013). The GAS6-mediated AXL signaling pathway described in this study comprises of 216 proteins undergoing six different types of molecular reactions stimulated by AXL. These reactions gathered together through a manual annotation of the scientific literature and depicted as a single pathway map. The GAS6-mediated AXL signaling pathway map is made available through the WikiPathways Database (https://www.wikipathways.org/index.php/Pathway:WP4847).

Methodology

We carried out a literature survey of GAS6-mediated AXL signaling related articles in PubMed to establish a complete pathway map of GAS6/AXL. The scientific articles were fetched from the PubMed using following search terms (“AXL receptor tyrosine kinase” OR “AXL” OR “ARK” OR “JTK11” OR “Tyro7” OR “UFO” OR “GAS6” OR “Growth Arrest Specific Gene 6”) AND (“pathway” OR “signaling” OR “signalling”). The abstract of research articles were screened manually to get only those articles relevant to GAS6/AXL signaling events. We manually curated the signalling reactions based on the previously published NetPath annotation criteria (Kandasamy et al. 2010). We curated only those reactions, which have been shown to be induced by the activation of AXL receptor by GAS6. These AXL signaling-mediated reactions were grouped into molecular association, catalysis/post translational modification (PTMs), protein-activation/inhibition, protein/gene regulation and protein translocation between cell organelles. Additional information, including cell lines used in the experiment, experiment type, the information about sites and residues of PTMs were also curated. Each signaling events described in GAS6-mediated AXL pathway were hyperlinked to PubMed entry of the corresponding articles. The curated reactions from each research article were further reviewed by an internal reviewer. An external reviewer called the Pathway Authority, an experienced scientist working in this specific pathway was involved to get the pathway reactions and pathway map to be reviewed. Manual curation of signaling events were enabled using a manual curation software called PathBuilder (Kandasamy et al. 2009). PathVisio was used to depict and visualize the signaling pathway map. Pathway map were submitted to WikiPathways Database.

Results and discussion

In the present study, the PubMed search using query terms showed 1170 articles, which were related to the AXL signalling pathway. These articles were reviewed based on our annotation criteria, and 83 articles, which had information relevant to GAS6-mediated AXL signaling shortlisted for detailed curation. The manual annotation provided a total of 12 molecular associations, 37 post-translational modifications (PTMs), 37 activation/inhibition reactions, 7 translocation events induced by GAS6-mediated AXL signaling. Information on altered expression of 81 genes at transcript level, 41 genes at the protein level were also gathered. The information pertaining to site and residue of PTMs were known only for 15 catalysis events. Of the 81 gene regulation events, 73 genes were found to be upregulated and 8 genes were downregulated. While in the case of 41 induced protein expression events, 9 proteins were downregulated and 33 proteins were upregulated. These events were incorporated into corresponding map of the GAS6-mediated AXL signaling pathway (Fig. 1). The comprehensive pathway map of GAS6-mediated AXL signaling can be obtained from the WikiPathways with the ID URL: https://www.wikipathways.org/index.php/Pathway:WP4847.

Fig. 1.

Schematic representation of AXL-mediated signaling pathway. Schematic representation of AXL induced signaling reactions. The signaling pathway map represents molecules involved in ligand-receptor interactions and AXL activated downstream molecular events including molecular association, enzyme catalysis, translocation, and gene regulation events. Information regarding the post-translational modification site and the residue is also shown in the pathway

Summary of AXL mediated signaling pathway

Binding of GAS6 to the extracellular domain of the AXL receptor results in autophosphorylation and activation, leading to the formation of AXL-mediated signaling complexes (Sasaki et al. 2006). While the majority of reported AXL activation by GAS6 involves homodimerization of the receptor, heterodimerization of AXL with TYRO3 in the presence of GAS6 has indeed been demonstrated (Brown et al. 2012). Intriguingly, this heterodimeric interaction may create a shift of AXL-stimulated downstream signaling from PI3K to MAPK, suggesting that binding partner of AXL may influence the outcome of GAS6-initiated signaling. Activation of MAPK1/3 (ERK1/2) downstream of GAS/AXL has been shown to involve in the proliferation of mouse mesangial cells, human umbilical vein endothelial cells (HUVECs), schwanncells, non-small cell lung cancer (NSCLC), cardiomyocytes, GnRH neurons and human neuroblastoma cells (Chen et al. 2018, Tian et al. 2016, Zhao et al. 2016, Li et al. 2015, Laurance et al. 2014, Yanagita et al. 1999, Allen et al. 1999). Activation of AXL receptor results in the reorganization of actin cytoskeleton and migration of GnRH neuronal cells to hypothalamus during development via Rac/p38 MAPK/MAPKAP kinase II/heat shock protein 25 pathway (Nielsen-Preiss et al. 2007, Allen et al. 2002, Allen et al. 1999). Studies have reported that GAS6-AXL activation implicated in chronic immune disorders and cardiac hypertrophy via the activation of MEK1/2–MAPK1/3 pathway (Zhao et al. 2016, Lemke and Rothlin 2008, Berk 2001). AXL activation induces the phosphorylation of AKT and MERTK, which leads to phosphorylation and nuclear translocation of RELA, and results in upregulation of ACTA2, COL1A1, TIMP1. These genes are involved in liver fibrosis in hepatic stellate cells (Barcena et al. 2015). In vascular smooth muscle cells, the AXL signaling inhibits the mineralization and thereby decreases the vascular calcification through PI3K/AKTsignaling pathway (Collett et al. 2007). The thrombin induced GAS6/AXL activation persuades the FoXo-1 phosphorylation and expression of VCAM-1 via PI3K/AKT pathway involved in venous thromboembolism (Bertin et al. 2015).

The altered expression of GAS6/AXL has been shown that it drives crucial pathways related to cancer cell survival, proliferation, migration, invasion, and angiogenesis in tumor cells (Linger et al. 2008). In breast cancer, AXL enhanced the expression of EMT transcriptional regulator Slug and MMP9, which induces tumor metastasis and chemosensitivity via activating PI3K/AKT/GSK3β and PI3K/AKT/NFKB1 signalling (Li et al. 2014, Tai et al. 2008). Similarly, the AXL activation stimulates PI3K/AKT pathway, results in the activation and nuclear translocation of NFKB1 by the phosphorylation of IKB-1alph (CHUK) and induces the upregulation of MMP9 protein, which is involved in cancer metastasis in NIH 3T3 mouse fibroblasts (Tai et al. 2008, Demarchi et al. 2001). AKT/MAPK1/3 signaling by the activation of AXL is reported in vascular smooth muscle cells and non-small cell lung cancer (Tian et al. 2016, Cavet et al. 2010). The altered AXL expression mediates cell resistance to both targeted and chemotherapy in stromal tumors and acute myeloid leukemia (Hong et al. 2008, Mahadevan et al. 2007). AXL also promotes invasion and survival of stage II pancreatic ductal adenocarcinoma cells via AKT activation (Zhou et al. 2016, Leconet et al. 2014). Epithelial to mesenchymal transition is driven by AXL activation by reducing the expression of epithelial markers and enhanced the expression of the mesenchymal markers in human colorectal cancer (CRC) (Nieto et al. 2016, Thiery et al. 2009, Martinelli et al. 2015).

Other than AKT and MAPKs, AXL also regulate the signaling via EGFR/PKC/mTOR pathway in head and neck and esophageal squamous cell carcinomas (Elkabets et al. 2015). S100A10 is a critical mediator of AXL signaling through SRC to promote plasmin production, endothelial cell invasion, and angiogenesis in renal cell carcinoma (Xiao et al. 2019). Activation of AXL receptor induces the phosphorylation of MDMX at Ser 314 and its association with MDM2, which suppresses p53 in melanoma (de Polo et al. 2017).

Conclusions

Axl is emerging as a promising therapeutic target. Multiple AXL inhibitors have been developed and are currently in clinical trials. The GAS6/AXL receptor activation regulates various processes including survival, growth, aggregation, migration in different cells through multiple downstream signaling pathways. In depth of literature mining from literature helped to develop an updated GAS6-mediated AXL signaling pathway map. We anticipated that the availability of GAS6-mediated AXL signaling pathway map in WikiPathways will aid as a reference guide to explore and expand the existing knowledge of the researchers. The updated AXL pathway map may accelerate the understaning of the complex mechanisms associated with roles of AXL signaling in both physiological and pathophysiological conditions.

Compliance with ethical standards

Conflict of interest

The authors report no conflicts of interest.