The Axl subfamily of mammalian RTKs is composed of
which participate in a signalling axis we shall refer to as the Gas6/Axl system. Axl RTK
subfamily members each possess a combination of two N-terminal immunoglobulin (Ig)-like domains and two ﬁbronectin type III
(FNIII) repeats in their extracellular regions. The Axl RTK
subfamily has long been in the shadow of ‘‘classical’’ growth factor receptors due to the rather enigmatic yet diverse array of biological effects their members have been shown to exert.
Most RTKs are single subunit receptors but some exist as multimeric complexes. Each monomer has a single hydrophobic transmembrane spanning domain composed of 25-38 amino acids, an extracellular N-terminal region and an intracellular C-terminal region. Axl possess a combination of two N-terminal immunoglobulin (Ig)-like domains and two ﬁbronectin type III (FNIII) repeats in their extracellular regions, that are characteristic for each subfamily of RTKs; these domains contain primarily a ligand-binding site, which binds extracellular ligands as a particular growth factor or hormone. The intracellular C-terminal region displays the highest level of conservation and comprises catalytic domains responsible for the kinase activity of these receptors, which catalyses receptor autophosphorylation and tyrosine phosphorylation of RTK substrates.
Gas6 and Protein S, GLA domain proteins, ligands for Axl RTKs
The biological ligands for Axl are two highly similar vitamin K-dependent proteins, Gas6 (‘product of growth arrest-speciﬁc gene 6’) and protein S, an abundant serum protein and a negative regulator of blood coagulation (43% amino acid identity). Both proteins are composed an N-terminal region containing multiple post-translationally modiﬁed g- carboxyglutamic acid residues (Gla). The Gla region possesses the ability to interact in a conformationally speciﬁc manner with negatively charged membrane phospholipids, which is thought to mediate the binding of both Gas6 and PS to apoptotic cells. In this way, they are thought to act as recognition bridges between apoptotic cells and the phagocyte cell that ingest them. In fact Vitamin K-dependent carboxylation/gamma-carboxyglutamic (GLA) domain is a protein domain that contains post-translational modifications of many glutamate residues by vitamin K-dependent carboxylation to form gamma-carboxyglutamate (Gla).
The Gla residues are responsible for the high-affinity binding of calcium ions. It starts at the N-terminal extremity of the mature form of proteins and ends with a conserved aromatic residue; a conserved Gla-x(3)-Gla-x-Cys motif is found in the middle of the domain which seems to be important for substrate recognition by the carboxylase. The 3D structures of several Gla domains have been solved. Calcium ions induce conformational changes in the Gla domain and are necessary for the Gla domain to fold properly. A common structural feature of functional Gla domains is the clustering of N-terminal hydrophobic residues into a hydrophobic patch that mediates interaction with the cell surface membrane.
Gla residues are found in some proteins that regulate blood coagulation, such as factors VII,
IX and X, prothrombin, protein C and protein S. It has been demonstrated that the importance of the Gla domain in these proteins lies in its ability to interact with Ca2+ and phospholipids. The interaction of Gas6 with its receptor requires Ca2+, suggesting the involvement of Gla residues in Gas6 receptor binding.
It has been demonstrated recently that glucocorticoids (like cortisol) induce their anti-inflammatory action by producing a protein that inhibits phospholipase A2,
the enzyme that produces arachidonic acid from phospholipids.
Thus, the availability of arachidonic acid, the substrate for prostaglandin and thromboxane biosynthesis, is reduced after exposure to glucocorticoids. The use of Cortisol inhibits phospholipase A2 and than the binding of the Gla domain with the membrane phospholipids. Glucocorticoids inibiths Gas6 and its related activity.
After the Gla region, there is a loop region followed by four Epidermal Growth Factor (EGF)-like repeats. This loop region is sensitive to thrombin cleavage only in PS, which is relevant to its role in coagulation, a role which Gas6 lacks. The C termini of Gas6 and PS house a globular sex hormone binding globulin (SHBG)-like region, comprising a pair of laminin G-like (LG) domains. The SHBG domain is believed to bind directly to and activate Axl, whilst a supporting role has been demonstrated for a properly g-carboxylated Gla region in the functional effects of Gas6.
Distinct roles of Axl in cell survival and uptake of apoptotic cells and immune regulation.
Gas6/proteinS – Axl interaction on the surface of several mesenchymal derived cell types leads to signalling for cell survival and possibly growth. In addition, soluble Axl ectodomain can be generated by extracellular protease action, leading to formation of a soluble Gas6 – Axl complex that blocks Gas6 ligand action.Gas6/proteinS acts as a bridging molecule between apoptotic cells and Axl RTK, causing cytoskeletal alterations that drive ingestion of the bound apoptotic cell. The apoptotic cell is decorated with negatively charged phospholipids on its outer surface, which interact with the Gla domain of Gas6/proteinS. In addition, a role for Axl subfamily RTKs has also been implicated in anti-inflammatory processes, whereby they inhibit induction of proinflammatory cytokines such as tumour necrosis factor-α.
In general, binding of ligand to an RTK monomer causes a conformational alteration that drives receptor dimerisation, and this has been shown for the Gas6–Axl pairing. Moreover, the potential for ligand-independent dimerisationand activation of Axl RTK also exists. There is also evidence for constitutive dimerisation of the intracellular regions of this molecule. Whether ligand-driven or not, dimerisation appears to stabilise interactions between cytoplasmic domain and leads to activation of the intrinsic kinase . Usually, the activated ICD (intracellular Axl domain) of one receptor monomer transphosphorylates multiple tyrosine residues on the neighbouring monomer. This autophosphorylation serves to increase the catalytic efﬁciency of the RTK by phosphorylating a conserved tyrosine residue inside the kinase domain as well as creating docking sites for signal transduction second messengers. Studies over the past 10 years have shed more light on the intracellular signalling cascades emanating from activation of the Axl RTK. Starting from the receptor itself, an early study revealed the p85a and p85b subunits of phosphatidylinositol 3-kinase (PI3K), and phospholipase C (PLC)-g as binding partners for Axl ICD .
Signalling interactions with the intracellular domain (ICD) of Axl RTK.
(A) Protein–protein interactions demonstrated for Axl and their intracellular consequences. Also shown are individual tyrosine phosphorylation sites that have been shown to recruit certain signalling proteins.
(B) Activating interactions between Axl ICD and : (i) another Axl ICD (ligand-independent) and (ii) IL-15 receptor alpha ICD (induced by IL-15 stimulation).
The interaction of Grb2 and PI3K with Axl, and uncovered p55g as an additional PI3K regulatory subunit that bound. Furthermore, there are a number of novel Axl binding partners in SOCS-1, Nck2, RanBPM, and C1-TEN. All of the above binding partners, with the exception of RanBPM, possessed phosphotyrosine-binding Src homology 2 (SH2) domains, indicating that these are the interfaces for Axl interaction. In addition, Axl ICD itself was one of the more common Axl-interacting proteins, suggesting a constitutive dimerisation process that may be biologically relevant and important for signalling. The activation of PI3K is an important event in Axl signalling, implicating cell survival, proliferation and migration. Moreover, the interactions with alternate isoforms of PI3K may serve to link Axl to discrete signalling pathways further downstream. Further support of previous ﬁndings comes from identiﬁcation of Grb2 adapter protein as an Axl interactor, which suggests a link with the Ras-extracellular regulated kinase (ERK) pathway. In addition, SOCS-1 was originally identiﬁed as a negative regulator of cytokine signalling, and therefore could serve a similar role in Axl signalling. Furthermore, Nck2 is an adapter protein composed of three tandem SH3 domains and an SH2 domain. These tandem domains may serve to tether Axl to other signalling complexes in the same way that Grb2 does. For example, the Axl–Nck2 interaction may connect Axl to a ternary complex consisting of the PINCH protein and integrin-linked kinase (ILK), which is a signalling platform at focal adhesions that regulates cytoskeletal dynamics and downstream signalling pathways. Recently was demonstrated a constitutive interaction between Axl and the IL-15 receptor a subunit. They showed that IL-15 could transactivate Axl and its associated signalling pathway, with concomitant phosphorylation of the IL-15 receptor. This example of a novel cross-talk mechanism broadens the repertoire of potential regulators of Gas6/Axl signalling, as well as implicating Axl RTKs in the signalling of other receptors. Gas6 stimulation of Axl actually inhibited activation of vascular endothelial growth factor receptor 2 and consequent endothelial cell morphogenesis. This inhibition appeared to be via Gas6/Axl-mediated activation of the SH2 domain-containing tyrosine phosphatase 2 (SHP-2). While Axl increases the survival of uveal melanoma cells, Gas6 stimulation of Axl caused a down-regulation of Cyr61, a member of the CCN protein family involved in tumour progression. In terms of signalling further downstream was observed that stimulation with EGF of an EGFR-Axl (extracellular–intracellular) chimera caused activation of the Ras/ERK pathway and cell proliferation; however in cells bearing full length Axl and stimulated with Gas6, PI3K was activated with no proliferation or ERK activation occurring. This showed that the type of signalling and its functional outcome depends on the nature of the stimulus and not solely on activation of the kinase per se. In cells that respond to Gas6 with increased survival, such as NIH 3T3 ﬁbroblasts, components of the PI3K pathway, incorporating the serine/threonine kinase Akt/PKB and the rapamycin-sensitive S6K, and Src appear to be required. Via Akt, Gas6 also causes an increase in the antiapoptotic protein Bcl-2 , as well as phosphorylation of the Bcl-2 family member, Bad. A transient ERK, JNK/SAPK and p38 MAPK activation also occurs, although blocking ERK did not inﬂuence Gas6- induced survival. PI3K also mediates Gas6-supported survival of human oligodendrocytes, endothelial cells, VSMC and lens epithelial cells. Although more unusual, ERK has in addition to PI3K been implicated in gonadotropin-releasing hormone neuronal survival. Additional signalling entities that have been implicated in Gas6/Axl survival signalling include the NF-kB transcription factor system. The NF-kB activation appeared to be a downstream consequence of PI3K and Akt signalling, with the Akt substrate glycogen synthase kinase 3 (GSK3) implicated as a link. In addition, a reduction in caspase 3 activity has been reported for Gas6 in endothelial cells. The growth effect of Gas6 was additive to that of EGF, suggesting that Gas6 can act as a growth factor through signalling pathways distinct from those utilised by EGF. ERK also mediates Gas6-induced human prostate cancer cell proliferation . Gas6 also activates the STAT3 (signal transducers and activators of transcription) transcription factor pathway. In response to Gas6, STAT3 is phosphorylated and translocates to the nucleus and induces STAT3-dependent transcriptional activation. In addition to survival and mitogenesis, gonadotropin-releasing hormone neuronal cells respond to Gas6/Axl by migration, and the signalling events associated with this phenotype included activation of the Rho family GTPase Rac and actin cytoskeletal reorganisation, downstream activation of p38 MAPK and MAPK-activated protein kinase 2 and phosphorylation of HSP25, a regulator of actin remodelling. Gas6 has also been shown to induce upregulation of the class A scavenger receptor in humans VSMC, an atherogenic process in which cells ingest lipids to become foam cells. This upregulation was attributed to the PI3K/Akt signalling pathway. Furthermore, Gas6 induces the phosphorylation and ubiquitination of Axl and its interaction with the ubiquitin ligase c-Cbl, which may be a mechanism for downregulation of Axl after activation .
Axl and cancer
As it’s shown in Axl singnalling, Axl appears to be the principal oncogene of its subfamily, being overexpressed in a variety of human cancers.
Detection and quantitation of plasma Axl may reflect altered regulation of Gas6–Axl system components under various clinical conditions, and may therefore be of diagnostic value. The Gas6–Axl system is now making its presence felt among the several growth factor–receptor pairings that are well established as role players in both development and disease. In particular, it appears that discrete functional outcomes can arise from a particular ligand–receptor combination on a particular cell type. Axl overexpression and activation appears to feature in many different types of cancer. Similarly, Axl activation, both with and without Gas6 stimulation, controls cell plastic processes typical of many growth factors. Gas6 itself is a growth, survival and chemotactic factor and also a possible recognition bridge between phagocytes and apoptotic cells. Gas6 could stimulate Axl RTK to actually suppress inflammation, through down-regulation of tumour necrosis factor α expression, achieved through induction of the Twist transcriptional repressor. Moreover, an additional novel role for Gas6–Axl was recently uncovered pertaining to natural killer (NK) cell differentiation. It was shown that expression of this receptor on NK precursor cells and their stimulation by bone marrow stromal cell-derived Gas6, were essential for NK cell functional maturation. Therefore, novel roles for the Gas6–Axl system in immune homeostasis on several levels is becoming increasingly apparent. Moreover, Axl overactivation can equally occur without ligand binding, which has implications for tumorigenesis. Further knowledge of this exquisite ligand–receptor system and the circumstances of its activation should provide the basis for development of novel therapies for the above diseases. Our current level of knowledge should stimulate future research efforts aimed at further elucidating this Axl RTK, which appear to be more important than every finding.
a possible role in the competition for fibrin availability ?