Soluble Axl Is Generated by ADAM10-Dependent Cleavage and Associates with Gas6 in Mouse Serum

Abstract

Axl receptor tyrosine kinase exists as a transmembrane protein and as a soluble molecule. We show that constitutive and phorbol 12-myristate 13-acetate-induced generation of soluble Axl (sAxl) involves the activity of disintegrin-like metalloproteinase 10 (ADAM10). Spontaneous and inducible Axl cleavage was inhibited by the broad-spectrum metalloproteinase inhibitor GM6001 and by hydroxamate GW280264X, which is capable of blocking ADAM10 and ADAM17. Furthermore, murine fibroblasts deficient in ADAM10 expression exhibited a significant reduction in constitutive and inducible Axl shedding, whereas reconstitution of ADAM10 restored sAxl production, suggesting that ADAM10-mediated proteolysis constitutes a major mechanism for sAxl generation in mice. Partially overlapping 14-amino-acid stretch deletions in the membrane-proximal region of Axl dramatically affected sAxl generation, indicating that these regions are involved in regulating the access of the protease to the cleavage site. Importantly, relatively high circulating levels of sAxl are present in mouse sera in a heterocomplex with Axl ligand Gas6. Conversely, two other family members, Tyro3 and Mer, were not detected in mouse sera and conditioned medium. sAxl is constitutively released by murine primary cells such as dendritic and transformed cell lines. Upon immobilization, sAxl promoted cell migration and induced the phosphorylation of Axl and phosphatidylinositol 3-kinase. Thus, ADAM10-mediated generation of sAxl might play an important role in diverse biological processes.Receptor tyrosine kinases (RTKs) play fundamental roles in diverse cell functions, including proliferation, differentiation, survival, migration, and metabolism (16). Axl RTK (also known as Ark, Ufo, and Tyro7) is the prototype of a family of transmembrane receptors, which also includes Tyro3 (also known as Sky, Brt, Etk, Tif, Dtk, and Rse) and Mer (c-Eyk, Nyk, and Tyro12) (34,44,64). They share a distinct molecular structure characterized by two immunoglobulin-like motifs and two fibronectin type III repeats in their extracellular domain and a cytoplasmic domain that contains a conserved catalytic kinase region (34, 44). Axl, Tyro3, and Mer are variably expressed in neural, lymphoid, vascular, and reproductive tissues and in different primary cells and tumor cell lines (11,41,42). Mutant mice that lack these three receptors have a defective phagocytic clearance of apoptotic cells and impaired spermatogenesis (41) and develop a severe lymphoproliferative disorder accompanied by broad-spectrum autoimmunity (42).A common heterophilic ligand for these RTK family members is Gas6, a vitamin K-dependent protein that is widely secreted by most tissues, including the lungs, intestine, and vascular endothelium (43). Gas6 is the product of growth arrest-specific gene 6, which was initially cloned from serumstarved fibroblasts and shares about 44% sequence identity and similar domain organization with protein S, a negative regulator of blood coagulation (48). Recent studies indicate that the Gas6/A...

Regulation of Axl receptor tyrosine kinase expression by miR-34a and miR-199a/b in solid cancer

“…Hypothetically, the presence of Axl in the cytoplasm (more frequent in tumors with high miR-34a) could be due to proteosomal degradation or other proteolytic degradation (for example by ADAM10 or maybe other functional changes caused by specific miRs in tumors). We did not extend specific data on this aspect at this point of time in our present study (O'Bryan et al, 1995;Budagian et al, 2005), but consider it as an important aspect that those patients with high miR-34a expression appear to have less functional (membranebound) Axl protein as suggested by IHC, the molecular mechanism leading to this still being in need to be investigated.…”

TAM Receptor Tyrosine Kinases: Biologic Functions, Signaling, and Potential Therapeutic Targeting in Human Cancer

“…Another candidate is soluble Axl (sAxl), which is present at high levels in vivo. Incubation of Axl-expressing cells with immobilized Axl-Fc induces phosphorylation of Axl and PI3K suggesting that Axl-Fc/Axl interaction activates the kinase domain of the full-length receptor (Budagian et al, 2005a). Thus, sAxl may mediate interactions between full-length Axl and the extracellular matrix.…”

“…Soluble forms of Axl and Mer, produced by proteolytic cleavage and release of the ectodomain, can be detected in murine and human plasma (Budagian et al, 2005a;Costa et al, 1996;O'Bryan et al, 1995;Sather et al, 2007). Although a truncated form of Tyro-3 was found in the cytoplasm when expressed in 293 cells (Taylor et al, 1995a), extracellular soluble Tyro-3 was not detected in human plasma (Sather et al, 2007).…”

“…Although alternative splicing of Axl (O'Bryan et al, 1991;Schulz et al, 1993) and Tyro-3 (Biesecker et al, 1995;Lewis et al, 1996b) have been reported, the transcripts generated encode transmembrane proteins. Soluble TAM receptors bind to Gas6 and can act as a ligand sink and inhibit normal cellular functions of the full-length RTK (Budagian et al, 2005a;Sather et al, 2007). In the same regard, soluble TAM receptors may have therapeutic potential in pathological conditions, such as cancer, where TAM receptor activity is upregulated.…”

Plasma concentrations of growth arrest specific protein 6 and the soluble form of its tyrosine kinase receptor Axl as markers of large abdominal aortic aneurysms

“…In mice, this process is dependent on the ADAM10 enzyme, but the shedding mechanism for the human sAxl has not been elucidated 23 .…”