View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 408 (1997) 306-310 FEBS 18593 Roles of γ-carboxylation and a sex hormone-binding globulin-like domain in receptor-binding and in biological activities of Gas6 Kazuyo Tanabea, Kyoko Nagataa, Kazumasa Ohashia, Toru Nakanob, Hitoshi Aritab, Kensaku Mizunoa'* 8 Department of Biology, Faculty of Science, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-81, Japan bDiscovery Research Laboratory, Shionogi and Co., Ltd., Sagisu, Fukushima-ku, Osaka 553, Japan Received 17 March 1997; revised version received 11 April 1997 globulin (SHBG)-like domain [1]. The Gla domain, which is Abstract Gas6 is a ligand for an Axl/Sky receptor tyrosine kinase subfamily and has a structure composed of a Gla domain, present in various proteins involved in blood coagulation, is four EGF-like domains and a C-terminal sex hormone-binding generated by vitamin K-dependent γ-carboxylation of a clus- globulin (SHBG)-like domain. When examining the role of each ter of Glu residues and is thought to be involved in intra- domain in receptor-binding and biological activities of Gas6, we molecular and intermolecular protein-protein or protein- found that receptor-binding and mitogenic activities were membrane interactions through Ca2+-binding [16,17], The C- markedly reduced by inhibiting γ-carboxylation of the Gla terminal domains of protein S and Gas6 have a sequence domain, while a Gas6 mutant composed of only an SHBG-like similarity with human SHBG and rat androgen-binding pro- domain retained both of these activities. Thus, the SHBG-like tein, and these sequences are also distantly related to base- domain is apparently an entity indispensable for Gas6 activities, ment membrane proteins, such as laminin and merosin [18]. and γ-carboxylation of the Gla domain has a regulatory role in Based on the characteristic domain structure of Gas6 com- retaining the activity of native Gas6. pared with other known ligands for receptor tyrosine kinases, © 1997 Federation of European Biochemical Societies. it is interesting to know which domain of Gas6 is essential for its receptor-binding and biological activities and if γ-carbox- Key words: Gas6; Gla domain; γ-carboxylation; Sex hormone-binding globulin ylation in the Gla domain is involved in these activities. In this respect, Mark et al. recently reported data showing the importance of the C-terminal SHBG-like domain for receptor- binding and receptor-stimulating activities of Gas6 [19], but 1. Introduction the biological activity of this domain has not been shown. The role of γ-carboxylation in the Gla domain also remains un- The protein encoded by growth arrest-specific gene 6 (Gas6) known. We report here that the SHBG-like domain has full was initially identified as a gene product inducibly expressed mitogenic activity on fibroblasts and γ-carboxylation of the N- in fibroblasts in response to serum starvation [1]. Recent stud- terminal Gla domain significantly contributes to bring out the ies from our laboratory and others have demonstrated that activity of native Gas6. Gas6 is a ligand for Axl, Sky and Mer receptor tyrosine kin- ases [2-5], with the Gas6 binding affinities in the order of 2. Materials and methods Axl > Sky > Mer [5]. These receptors are typified by the cell adhesion molecule-related extracellular ligand-binding domain 2.1. Plasmid construction composed of two immunoglobulin-like motifs and two fibro- Gas6 mutants used in this study are schematically shown in Fig. nectin type III motifs [6-10]. Gas6 was recently found to act 2A. The 2.8-kb Gas6 cDNA [11] was isolated from a rat brain cDNA library, and subcloned into pBluescript SK1I~ (Stratagene) or as a growth-potentiating factor for thrombin-induced prolif- pGEM7 (Promega) at EcoRl site to generate pBS-Gas6 or pGEM- eration of vascular smooth muscle cells [11]. Gas6 also pre- Gas6. To construct pGEM-AG, a 280-bp EcoRl-Taql fragment of vented serum starvation-induced cell death of these cells [12]. pGEM-Gas6 coding for N-terminal 50 residues of Gas6 was isolated Mitogenic and survival activities of Gas6 for serum-starved and inserted into EcoRl-BamHl site of pBluescript with a synthetic EcoRV linker composed of 5'-CGGATATCG-3' and 5'-GATCCGA- fibroblasts and Schwann cells have also been described [13,14]. TATC-3'. The resulting plasmid (pBS-N) was cut with Clal and Gas6 has a structure similar to that of protein S, a vitamin EcoRV and the insert was ligated into the C/aI/£coRV-digested K-dependent plasma protein with anticoagulant activity, with pGEM-Gas6. To construct pGEM-ΔΕ and pGEM-AGE, pGEM- Gas6 was digested with Clal, blunt-ended, and ligated with a Xhol 43% amino acid identity [1,15]. Similar to protein S, Gas6 is linker. The resulting plasmid [pGEM(XhoI)-Gas6] was digested with composed of several defined structural domains; an N-termi- Xhol and Ball and ligated with a synthetic linker composed of nal γ-carboxyglutamic acid (Gla) domain, followed by a short 5'-TCGAGATATCGTGAGGACATCTTACCGTGTGTGCCCTTC- loop domain, four tandem repeats of epidermal growth factor AGCATGG-3' and 5'-CCATGCTGAAGGGCACACACGGTAA- (EGF)-like domains and a C-terminal sex hormone-binding GATGTCCTCACGATATC-3'. The plasmid was then digested with Xhol and EcoRV, and ligated with a 400-bp Xhol-EcoRN fragment of pGEM(XhoI)-Gas6 to generate pGEM-ΔΕ, or was ligated with a 250- ""Corresponding author. Fax: (81) 92-642-2645. bp XhoI-EcoRV fragment of pBS-N to generate pGEM-AGE. To E-mail: [email protected] construct pBS-AH-Myc, pBS-Gas6 was digested with Ball and Bam- HI, blunt-ended, and ligated with a Xbal linker composed of 5'- Abbreviations: BrdU, bromodeoxyuridine; DMEM, Dulbecco's mod- CCTCTAGA-3' and 5'-TCTAGAGG-3'. The resulting plasmid was ified Eagle's medium; EGF, epidermal growth factor; Gas6, the digested with Xbal and SacII, and ligated with a synthetic XballSacll protein encoded by growth arrest-specific gene 6; Gla, γ-carboxyglu- linker composed of 5'-CTAGTGAACAAAAGCTTATTTCTGAA- tamic acid; SHBG, sex hormone-binding globulin GAAGACTTGTGAGCGGCCGCGAATTCCGC-3' and 5'-GGAA- 0014-5793/97/S17.00 © 1997 Federation of European Biochemical Societies. All rights reserved. P//S0014-5793(97)00448-l K. Tanabe et al.lFEBS Letters 408 (1997) 306-310 307 TTCGCGGCCGCTCACAAGTCTTCTTCAGAAATAAGCTTTT- 2.3. Binding assays GTTCA-3' (coding for a C-terminal Myc epitope peptide, SSEQKLI- Axl-Fc fusion proteins were expressed in COS-7 cells and purified SEEDL) to generate pBS-AH-Myc. To construct plasmids for Myc- as described previously [3,5]. Gas6 mutants were incubated at 4°C tagged mutants (pGEM-Gas6-Myc, pGEM-AG-Myc, pGEM-AE-Myc overnight with 5 nM Axl-Fc fusion protein and Protein A-Sepharose and pGEM-AGE-Myc), a 820-bp Bfal fragment of pGEM-Gas6 was (30 μΐ of 50% slurry) in 200 μΐ of Hanks' balanced salt solution blunt-ended, and ligated with a Xbal linker. The resulting plasmid was containing 20 mM Hepes (pH 7.0), 0.02% NaN3, and 1% bovine digested with Sail and Xbal, and the 420-bp fragment was ligated into serum albumin. After centrifugation, the precipitates were washed pBluescript with a XballSacll linker described above to generate pBS- 4 times with cold phosphate-buffered saline (PBS), suspended in C-Myc. To generate pGEM-Gas6-Myc, pBS-C-Myc was digested with SDS sampling buffer, and subjected to SDS-PAGE. Bound proteins San and Notl and the insert was replaced with a SaWNotl fragment were detected by immunoblot analysis with anti-Gas6 antibody [3] or of pGEM-Gas6. pGEM-AG-Myc, pGEM-AE-Myc and pGEM-AGE- 9E10 anti-Myc epitope antibody. BIAcore binding analysis was car- Myc were generated, in a similar manner. The authenticity of ried out, as described previously [5]. Gas6 was passed over the immo- the expression plasmids was confirmed by nucleotide sequence analy- bilized Axl-Fc fusion protein at a flow rate of 2 μΐ/min for 25 min. The dissociation constants (ΚΛ) were calculated, using the manufac- turer's software (Pharmacia Biotech.). 2.2. Preparation of recombinant Gas6 and its mutants The cDNA constructs were subcloned into the PUC-SRa expres- 2.4. Tyrosine phosphorylation assay sion vector [3], and the plasmids were transfected into COS-7 cells, CHO cell lines (AM3) expressing Myc-tagged Axl (Axl-Myc) were using calcium phosphate methods. Cells were cultured for 3 days in constructed as described previously [5]. AM3 cells were cultured in serum-free Dulbecco's modified Eagle's medium (DMEM) with 2 μΜ Ham's F-12 medium supplemented with 10% fetal calf serum. The vitamin K2 (Menatetrenone, Eisai, Tokyo). Gas6(-Gla), which was cells were serum-starved, treated with Gas6 mutants for 10 min at deficient in γ-carboxylation of Glu residues, was expressed in serum- 37°C, then rinsed 3 times with cold PBS containing 1 mM orthova- free DMEM in the presence of 1 μΜ Warfarin (a competitive inhib- nadate, followed by lysing with cold lysis buffer (20 mM Hepes (pH itor of vitamin K, Sigma) in place of vitamin K2. Recombinant Gas6 7.2), 1% Nonidet P-40, 10% glycerol, 50 mM NaF, 1 mM phenyl- was purified from conditioned medium, as described elsewhere [11]. methylsulfonyl fluoride, 1 mM orthovanadate, and 10 μ^πύ leupep- Gas6(-Gla) was purified in a similar manner, except that its elution tin). The lysates were immunoprecipitated with an anti-Myc antibody was monitored by dot-blot or Western blot analysis using an anti- (9E10), run on SDS-PAGE, and immunoblotted with anti-phospho- Gas6 antibody. The absence of Gla residues in Gas6(-Gla) was con- tyrosine monoclonal antibody (PY20, ICN Biomedicals), as described firmed by amino acid analysis and amino acid sequencing [11].