(Received for publication, July 11, 1995)
From the
Sky (also called Rse, Brt, and Tyro3) is a member of a subfamily of related receptor tyrosine kinases, including Axl/Ufo/Ark and c-Eyk/Mer. We obtained evidence that Gas6 (the product of growth arrest-specific gene 6) is a ligand of the Sky receptor tyrosine kinase. Gas6, but not protein S (an anticoagulant protein structurally similar to Gas6), specifically bound to the soluble form of Sky (Sky-Fc), composed of the extracellular domain of Sky fused to the Fc domain of human immunoglobulin G1. The native and recombinant Gas6, but not protein S, stimulated tyrosine phosphorylation of Sky ectopically expressed in Chinese hamster ovary cells. Stimulation of Sky in response to Gas6 was inhibited by Sky-Fc. The half-maximal concentration of Gas6 that stimulated Sky was about 1 nM. Thus, Gas6 as a ligand for Sky specifically binds to and stimulates Sky receptor tyrosine kinase.
Receptor tyrosine kinases play a central role in transducing the external signals across cell membranes into intracellular signaling systems and these signals lead to cell proliferation, differentiation, and other responses(1) . Based on similarities of the sequences of kinase catalytic domains and the structural motifs in the extracellular domains, these receptors can be classified into subfamilies(1) . The members of an Axl/Sky receptor subfamily, which include Axl (also called Ufo and Ark)(2, 3, 4) , Sky (also called Rse, Brt, and Tyro3) (5, 6, 7, 8, 9) , and c-Eyk (also called Mer)(10, 11) , contain the characteristic extracellular ligand-binding domain composed of two immunoglobulin-like motifs and two fibronectin type III motifs. Axl was originally identified as the oncogene for human myelogenic leukemia(2, 3) , and the gene for c-Eyk was isolated as a proto-oncogene for the avian viral oncogene v-eyk(10) . Overexpression of Axl and Sky led to cell transformation(2, 9, 12) . As the Axl/Sky family receptors have an oncogenic potential, they may be involved in tumor progression and in normal cell proliferation. Northern blot analysis revealed that the Sky transcripts are predominantly expressed in the brain(5, 6, 7, 8, 9) , while those for Axl and c-Eyk are more widely distributed in various tissues(2, 3, 4, 10, 11) .
The functional roles of the Axl/Sky subfamily of receptor tyrosine
kinases have been given much attention, especially the identification
of ligands. The ligands for Axl and Sky were recently reported to be
the product of growth arrest-specific gene 6 (Gas6) ()and protein S, respectively(13, 14) .
Protein S is a vitamin K-dependent plasma glycoprotein that has
anticoagulant activity by acting as a cofactor of activated protein C
(APC)-catalyzed inactivation of coagulation factors Va and
VIIIa(15) . Gas6, originally identified as a gene product
expressed in response to growth arrest, has structural similarity to
protein S with 42-43% identity (16) and was seen to
function as a potentiating factor for thrombin-induced proliferation of
vascular smooth muscle cells (VSMC)(17) .
Prior to investigating the physiological functions of Sky and its reported ligand protein S, we examined the potential of protein S to bind to Sky and to stimulate tyrosine phosphorylation of Sky. In contrast to an earlier report(14) , we found no specific binding of protein S to Sky or stimulation of Sky tyrosine phosphorylation by protein S. Gas6, not protein S, did show potent activity to bind to Sky and to induce the phosphorylation of Sky. We describe here our evidence that Gas6 is the ligand for Sky.
To determine the binding potential of protein S and Gas6 to Sky, we prepared a chimeric protein (Sky-Fc) that contains the extracellular ligand-binding domain of Sky fused to the Fc region of human immunoglobulin IgG1 heavy chain. Affinity adsorption and precipitation analysis using Fc fusion proteins in the presence of Protein A-Sepharose showed that Sky-Fc, but not Met-Fc (a control chimeric protein composed of the extracellular domain of c-Met and the Fc region of human IgG1), specifically co-precipitated Gas6 protein (Fig. 1A). On the other hand, protein S was not co-precipitated by either Sky-Fc or Met-Fc (Fig. 1B). These observations suggest that Gas6 but not protein S can specifically bind to the extracellular domain of Sky.
Figure 1:
Binding of Gas6 to Sky-Fc fusion
protein. A, rat Gas6 (2 nM) purified from conditioned
media of VSMC was incubated with Protein A-Sepharose in the absence (lane4) or presence of 5 nM Sky-Fc (lane2) or Met-Fc (lane3) fusion
protein. After centrifugation, the precipitates were run on SDS-PAGE
and the bound Gas6 was visualized by immunoblotting with anti-Gas6
antibody (-Gas6). Lane1 shows the
immunoblot of the purified Gas6 used in this assay. B, protein
S (2 nM) purified from human plasma was treated as in A, and the precipitates were immunoblotted with anti-protein S
antibodies (
-PS). Elution positions of rat Gas6 (rGas6) and human protein S (hPS) are indicated by arrows. The upper and lower bands of protein S correspond to
the intact and thrombin-cleaved form, respectively. Molecular sizes
(kDa) of marker proteins are indicated on the left.
Ligands for receptor tyrosine kinases bind to their cognate receptors, then rapidly stimulate tyrosine phosphorylation of these receptors. To determine if Gas6 and/or protein S could stimulate tyrosine phosphorylation of Sky, we constructed CHO cells stably expressing the full-length Sky receptor tyrosine kinase. Immunoblot analysis using the anti-Sky antibody raised against the C-terminal peptide of Sky revealed that the Sky protein with an apparent molecular mass of 140 kDa was detected in Sky-transfected (B31) cells, while the parental CHO cells showed no detectable immunoreactive band (Fig. 2A). Cell surface biotinylation experiments revealed that a 140-kDa protein immunoprecipitated with anti-Sky antibody was exposed at the cell surface (data not shown). The tyrosine phosphorylation of Sky expressed on B31 cells was assessed by immunoblotting with anti-phosphotyrosine antibody after immunoprecipitation of the cell lysates with anti-Sky antibody. As shown in Fig. 2B, phosphorylation of Sky was induced when the B31 cells were treated with Gas6 (compare lanes1 and 2), but not when treated with protein S (lane4), while the amount of Sky in the immunoprecipitates remained unchanged. No immunoreactive band with anti-phosphotyrosine antibody was detected in the anti-Sky immunoprecipitates of the parental CHO cells treated with Gas6 (Fig. 2B, lane5). These results clearly show that Gas6 but not protein S can stimulate tyrosine phosphorylation of the Sky receptor tyrosine kinase expressed in B31 cells. Additionally, phosphorylation of Sky in response to Gas6 was almost completely blocked in the presence of excess amounts of Sky-Fc (Fig. 2B, lane3). This means that Sky phosphorylation was induced by direct interaction between Gas6 and Sky and not by indirect cross-phosphorylation of Sky by other receptor tyrosine kinases.
Figure 2:
Tyrosine phosphorylation of Sky on B31
cells in response to purified Gas6. A, immunoblot analysis of
Sky expression on CHO cells and B31 cells. Cell lysates of parental CHO
cells (lane1) and sky cDNA-transfected CHO
cells (B31 cells) (lane2) were run on SDS-PAGE and
immunoblotted with anti-Sky antibody (-Sky). B,
B31 cells (lanes 1-4) and CHO cells (lanes5 and 6) were treated for 10 min at 37 °C
with 2 nM Gas6 purified from conditioned media of VSMC or
protein S (PS) purified from human plasma, as indicated. In lane3, Gas6 (2 nM) was preincubated with
excess amounts of Sky-Fc (20 nM). Cell lysates were
immunoprecipitated with anti-Sky antibody, run on SDS-PAGE, and
immunoblotted with anti-phosphotyrosine antibody (
-pY, upperpanel) or anti-Sky antibody (
-Sky, lowerpanel). Elution position
of Sky is indicated by an arrow. Molecular sizes (kDa) of
marker proteins are indicated on the left.
To exclude the possibility that the Gas6 preparation purified from VSMC used in this study might be contaminated by the protein activating Sky, recombinant Gas6 was expressed in COS cells and examined to observe if it would stimulate Sky phosphorylation. Recombinant protein S was also expressed and examined, in parallel experiments. Expression of Gas6 and protein S in culture supernatants of COS cells transfected with each expression plasmid was confirmed by immunoblot analysis (Fig. 3A). As shown in Fig. 3B, the culture supernatants of COS cells expressing recombinant Gas6 induced tyrosine phosphorylation of Sky, whereas the supernatants of mock-transfected COS cells and the supernatants of COS cells expressing recombinant protein S had no detectable activity. These observations also show that Gas6 but not protein S stimulates Sky tyrosine phosphorylation.
Figure 3:
Tyrosine phosphorylation of Sky on B31
cells in response to recombinant Gas6. A, immunoblot analysis
of recombinant rat Gas6 and human protein S transiently expressed in
COS-7 cells. The culture supernatants of COS cells transfected with rat
Gas6 or human protein S cDNA expression plasmid were concentrated, run
on SDS-PAGE, and immunoblotted with anti-Gas6 or anti-protein S
antibody. B, B31 cells were treated for 10 min at 37 °C
with the culture supernatants of mock-transfected COS cells (lane2) or the cells transfected with human protein S (lane3) or rat Gas6 expression plasmid (lane4). C, effects of EGTA on tyrosine
phosphorylation of Sky in response to Gas6. B31 cells were treated for
10 min at 37 °C with 2 nM recombinant rat Gas6 in the
presence or absence of 10 mM EGTA. In B and C, cell lysates were immunoprecipitated with -Sky, run on
SDS-PAGE, and immunoblotted with
-pY (upperpanel) or
-Sky antibody (lowerpanel), as described in Fig. 2. Elution position
of Sky is indicated by an arrow. Molecular sizes (kDa) of
marker proteins are indicated on the left.
Protein S
contains 11 -carboxyglutamic acid (Gla) residues in the N-terminal
Gla domain, one
-hydroxyaspartic acid residue in the first
epidermal growth factor (EGF)-like domain, and three
-hydroxyasparagine residues in three other EGF-like
domains(15) . These modified residues appear to be involved in
Ca
binding(15) . As these residues are
conserved in the sequence of Gas6, Gas6 may also bind Ca
ions through these residues(16, 17) . To examine
the effects of Ca
ion on Sky-stimulating activity of
Gas6, recombinant Gas6 was treated with EGTA and lost all activity (Fig. 3C). Therefore, Ca
ion binding
is essential for the conformation and activity of Gas6 to bind to and
stimulate Sky.
Recombinant Gas6 purified to apparent homogeneity
from the culture medium of COS cells transfected with Gas6 expression
plasmid stimulated Sky phosphorylation in a dose-dependent manner (Fig. 4). Tyrosine phosphorylation of Sky was detectable at 0.5
nM of recombinant Gas6 and the half-maximal stimulation was
obtained at approximately 1 nM. The concentration of Gas6
required for stimulation of Sky is comparable to the K value (0.3 nM) of the binding of Gas6 to membranes of
VSMC, for which Gas6 has growth potentiating activity(17) .
Figure 4:
Dose-dependent tyrosine phosphorylation of
Sky in response to purified recombinant rat Gas6. Recombinant Gas6 was
purified from the culture supernatants of COS-7 cells transfected with
rat Gas6 expression plasmid. Serum-starved B31 cells were treated for
10 min at 37 °C with the indicated concentrations of purified
recombinant Gas6. Cell lysates were immunoprecipitated and
immunoblotted with -pY (upperpanel) or
-Sky antibody (lowerpanel), as described in Fig. 2. Elution position of Sky is indicated by an arrow. Molecular sizes (kDa) of marker proteins are indicated
on the left. B, relative levels of tyrosine
phosphorylation of Sky are plotted as a function of the concentration
of Gas6 added. The levels of tyrosine phosphorylation of Sky were
calculated by dividing the density of anti-phosphotyrosine
immunoreactive band by the density of anti-Sky immunoreactive band. The
density was evaluated from data in A by NIH image software.
The level of tyrosine phosphorylation of Sky when treated with 10
nM recombinant rat Gas6 was taken as
100%.
In light of all these findings, we propose that Gas6, but not protein S, is a ligand for Sky, specifically binding to Sky and stimulating tyrosine phosphorylation. Our observations differ from those of Stitt et al.(14) . They reported that protein S but not Gas6 efficiently binds to and stimulates mouse Sky (Tyro3). Although the Sky receptor they used was of a different species (mouse Sky used by Stitt et al.(14) versus human Sky used in our study), the species difference would not likely explain the different results in ligand specificity, because sequences of human and mouse Sky are highly homologous (85% identity within their extracellular domains). If all results are compiled, human protein S does bind to mouse Sky (as described by Stitt et al.), but does not bind to human Sky (as described here). As the purified human protein S used in our study retained both APC cofactor activity and inhibitory activity of the prothrombinase complex activity(20) , it is unlikely that the purified protein S we used was structurally damaged. Thus, at present we have no valid explanation for the discrepancy between the results obtained in this study and those by Stitt et al.(14) .
Two different research groups reported that Gas6 is the ligand for Axl, a receptor closely related to Sky(13, 14) . The effective dose of Gas6 to Sky shown in this study is comparable to the reported value for Gas6-Axl interaction(13) . Thus, Gas6 may be a common ligand for the two related receptors, Sky and Axl. The binding of a ligand to two distinct members of a receptor subfamily is also seen for fibroblast growth factor (FGF) family ligands (acidic FGF and basic FGF), both of which bind to two members of an FGF receptor subfamily, Flg and Bek, with similar affinity constants(28) , and the ligands for Eph family receptors (B61 and EHK1-L), which bind to two members of an Eph receptor subfamily, Eck and EHK-1, with similar affinity constants (29) . Although neither the functions of Gas6 nor the physiological significance of receptor redundancy are well understood, Gas6 may exhibit diverse functions in a cell and tissue-dependent manner through two distinct receptors, Sky and Axl, whose expression patterns differ significantly.
Identification of
the ligand for Sky should pave the way to initiate research on the
functional roles of Sky and its ligand Gas6. As Sky is expressed
predominantly in neurons in restricted regions of the brain (6) , future study will focus on the biological function of
Gas6 on neurons. As Sky is also expressed in some extents in other
tissues, such as testis, ovary and kidney, and in certain types of
cells(5, 6, 7, 8, 9, 12) ,
the functional roles of Gas6 to these tissues and cells may be
identified. Gas6 potentiates cell proliferation of VSMC stimulated by
Ca-mobilizing growth factors, such as thrombin, and
may be involved in intimal thickening of the vascular wall accompanying
atherosclerosis or restenosis(17) . To develop antagonists for
Gas6 that will aid in overcoming these vascular diseases, it is also
important to clearly define which receptor, Sky or Axl, mediates the
action of Gas6 on VSMC. In addition, it will be interesting to search
for other members of the protein S-related protein family, which may
function as ligands for Sky-related receptors such as c-Eyk.