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An SH2 Domain-dependent, Phosphotyrosine-independent Interaction between Vav1 and the Mer Receptor Tyrosine Kinase

2003; Elsevier BV; Volume: 278; Issue: 43 Linguagem: Inglês

10.1074/jbc.m305817200

1083-351X

Nupam P. Mahajan, H. Shelton Earp,

Phagocytosis and Immune Regulation

Mer belongs to the Mer/Axl/Tyro3 receptor tyrosine kinase family, which regulates immune homeostasis in part by triggering monocyte ingestion of apoptotic cells. Mutations in Mer can also cause retinitis pigmentosa, again due to defective phagocytosis of apoptotic material. Although, some functional aspects of Mer have been deciphered, how receptor activation lead to the physiological consequences is not understood. By using yeast two-hybrid assays, we identified the carboxyl-terminal region of the guanine nucleotide-exchange factor (GEF) Vav1 as a Mer-binding partner. Unlike similar (related) receptors, Mer interacted with Vav1 constitutively and independently of phosphotyrosine, yet the site of binding localized to the Vav1 SH2 domain. Mer activation resulted in tyrosine phosphorylation of Vav1 and release from Mer, whereas Vav1 was neither phosphorylated nor released from kinase-dead Mer. Mutation of the Vav1 SH2 domain phosphotyrosine coordinating Arg-696 did not alter Mer/Vav1 constitutive binding or Vav1 tyrosine phosphorylation but did retard Vav1 release from autophosphorylated Mer. Ligand-dependent activation of Mer in human monocytes led to Vav1 release and stimulated GDP replacement by GTP on RhoA family members. This unusual constitutive, SH2 domain-dependent, but phosphotyrosine-independent, interaction and its regulated local release and subsequent activation of Rac1, Cdc42, and RhoA may explain how Mer coordinates precise cytoskeletal changes governing the ingestion of apoptotic material by macrophages and pigmented retinal epithelial cells. Mer belongs to the Mer/Axl/Tyro3 receptor tyrosine kinase family, which regulates immune homeostasis in part by triggering monocyte ingestion of apoptotic cells. Mutations in Mer can also cause retinitis pigmentosa, again due to defective phagocytosis of apoptotic material. Although, some functional aspects of Mer have been deciphered, how receptor activation lead to the physiological consequences is not understood. By using yeast two-hybrid assays, we identified the carboxyl-terminal region of the guanine nucleotide-exchange factor (GEF) Vav1 as a Mer-binding partner. Unlike similar (related) receptors, Mer interacted with Vav1 constitutively and independently of phosphotyrosine, yet the site of binding localized to the Vav1 SH2 domain. Mer activation resulted in tyrosine phosphorylation of Vav1 and release from Mer, whereas Vav1 was neither phosphorylated nor released from kinase-dead Mer. Mutation of the Vav1 SH2 domain phosphotyrosine coordinating Arg-696 did not alter Mer/Vav1 constitutive binding or Vav1 tyrosine phosphorylation but did retard Vav1 release from autophosphorylated Mer. Ligand-dependent activation of Mer in human monocytes led to Vav1 release and stimulated GDP replacement by GTP on RhoA family members. This unusual constitutive, SH2 domain-dependent, but phosphotyrosine-independent, interaction and its regulated local release and subsequent activation of Rac1, Cdc42, and RhoA may explain how Mer coordinates precise cytoskeletal changes governing the ingestion of apoptotic material by macrophages and pigmented retinal epithelial cells. The Mer receptor tyrosine kinase was identified by molecular rather than functional assays (1Graham D.K. Dawson T.L. Mullaney D.L. Snodgrass H.R. Earp H.S. Cell Growth Differ. 1994; 5: 647-657PubMed Google Scholar), and hence its physiological function and that of its two family members, Axl and Tyro3, has been elucidated slowly. Mer is primarily expressed in monocytes and cells of epithelial and reproductive origin with the highest levels of Mer mRNA detected in testis, ovary, prostate, kidney, lung, and peripheral blood monocytes (1Graham D.K. Dawson T.L. Mullaney D.L. Snodgrass H.R. Earp H.S. Cell Growth Differ. 1994; 5: 647-657PubMed Google Scholar, 2Graham D.K. Bowman G.W. Dawson T.L. Stanford W.L. Earp H.S. Snodgrass H.R. Oncogene. 1995; 10: 2349-2359PubMed Google Scholar). Mer is not expressed in normal B- and T-cells but Mer mRNA expression was detected in variety of human tumor cells, including neoplastic T and B cell lines; it is also present in the majority of childhood acute lymphoid leukemia samples tested. 1D. K. Graham, T. Dawson, H. R. Snodgrass, and H. S. Earp, unpublished data. The Mer extracellular region, like Axl and Tyro3, comprises two immunoglobulin-like and two fibronectin type III repeats, a transmembrane domain, and an intracellular kinase domain with an unusual KWIAIES motif (1Graham D.K. Dawson T.L. Mullaney D.L. Snodgrass H.R. Earp H.S. Cell Growth Differ. 1994; 5: 647-657PubMed Google Scholar, 2Graham D.K. Bowman G.W. Dawson T.L. Stanford W.L. Earp H.S. Snodgrass H.R. Oncogene. 1995; 10: 2349-2359PubMed Google Scholar). Biochemical purification identified Gas-6 as a ligand for Axl and Tyro3; subsequent studies showed binding to Mer, but the affinity of Gas6 for Mer is considerably lower (29 nm) than its affinity for Axl and Tyro-3 (0.4 and 2.9 nm, respectively) (3Chen J. Carey K. Godowski P.J. Oncogene. 1997; 14: 2033-2039Crossref PubMed Scopus (155) Google Scholar, 4Godowski P.J. Mark M.R. Chen J. Sadick M.D. Raab H. Hammonds R.G. Cell. 1995; 82: 355-358Abstract Full Text PDF PubMed Scopus (195) Google Scholar, 5Nagata K. Ohashi K. Nakano T. Arita H. Zong C. Hanafusa H. Mizuno K. J. Biol. Chem. 1996; 271: 30022-30027Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar, 6Stitt T.N. Conn G. Gore M. Lai C. Bruno J. Radziejewski C. Mattsson K. Fisher J. Gies D.R. Jones P.F. Cell. 1995; 80: 661-670Abstract Full Text PDF PubMed Scopus (612) Google Scholar). To investigate the physiological function of Mer, our group generated a knockout mouse deleting the Mer tyrosine kinase domain (Mer kd) (7Camenisch T.D. Koller B.H. Earp H.S. Matsushima G.K. J. Immunol. 1999; 162: 3498-3503PubMed Google Scholar). Mer kd mice were extremely sensitive to endotoxin (lipopolysaccharide) treatment exhibiting excessive TNF-α production by monocytes resulting in lethal endotoxic shock. The spleens of Mer kd mice were enlarged in some animals demonstrating accumulation of apoptotic debris (7Camenisch T.D. Koller B.H. Earp H.S. Matsushima G.K. J. Immunol. 1999; 162: 3498-3503PubMed Google Scholar), and the spleens were markedly enlarged in Mer, Axl, and Tyro3 triple-knockout mice (8Lu Q. Gore M. Zhang Q. Camenisch T. Boast S. Casagranda F. Lai C. Skinner M.K. Klein R. Matsushima G.K. Earp H.S. Goff S.P. Lemke G. Nature. 1999; 398: 723-728Crossref PubMed Scopus (400) Google Scholar). Subsequently, a crucial role of Mer in phagocytosis of apoptotic cells was demonstrated in Mer kd mice. Monocytes bound but did not ingest apoptotic thymocytes, and the thymuses of dexamethasone-treated mice exhibited a marked diminution of apoptotic cell clearance (9Scott R.S. McMahon E.J. Pop S.M. Reap E.A. Caricchio R. Cohen P.L. Earp H.S. Matsushima G.K. Nature. 2001; 411: 207-211Crossref PubMed Scopus (931) Google Scholar). Phagocytosis of other particles was intact indicating a selective defect for apoptotic material in these animals (9Scott R.S. McMahon E.J. Pop S.M. Reap E.A. Caricchio R. Cohen P.L. Earp H.S. Matsushima G.K. Nature. 2001; 411: 207-211Crossref PubMed Scopus (931) Google Scholar). Indeed, the triple mutant mice lacking Mer, Axl, and Tyro3 receptors had very high levels of apoptotic cells in many organs, including liver, kidney, muscle, brain, spinal cord, and eye (10Lu Q. Lemke G. Science. 2001; 293: 306-311Crossref PubMed Scopus (546) Google Scholar). Failure to ingest apoptotic self material led to evidence of autoimmunity in Mer kd mice (7Camenisch T.D. Koller B.H. Earp H.S. Matsushima G.K. J. Immunol. 1999; 162: 3498-3503PubMed Google Scholar, 11Cohen P.L. Reap E.A. Caricchio R. Abraham V. Camenisch T.D. Earp H.S. Matsushima G. J. Exp. Med. 2002; 196: 135-140Crossref PubMed Scopus (513) Google Scholar) and evidence of profound autoactivation of the immune system in the triple-knockout mice (10Lu Q. Lemke G. Science. 2001; 293: 306-311Crossref PubMed Scopus (546) Google Scholar). The Royal College of Surgeons (RCS) 2The abbreviations used are: RCS, Royal College of Surgeons; RPE, retinal pigment epithelial; EGF, epidermal growth factor; GEF, guanine nucleotide-exchange factor; aa, amino acid(s); GST, glutathione S-transferase; DTT, dithiothreitol; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; EMC, EGFR-Mer-chimera; kdEMC, kinase-dead EMC; EGFR, epidermal growth factor receptor; PS, phosphatidylserine. rat exhibits a progressive and postnatal loss of vision because of a failure of retinal pigment epithelial (RPE) cells to ingest shed outer segments of photoreceptor cells (12D'Cruz P.M. Yasumura D. Weir J. Matthes M.T. Abderrahim H. LaVail M. Vollrath D. Hum. Mol. Genet. 2000; 9: 645-652Crossref PubMed Scopus (728) Google Scholar). This genetic defect was traced to a deletion of a Mer splice acceptor site next to the second exon resulting in the loss of functional Mer. Gas6-mediated activation of Mer can result in the ingestion of shed photoreceptor outer segments by the cultured rat RPE cells (13Hall M.O. Prieto A.L. Obin M.S. Abrams T.A. Burgess B.L. Heeb M.J. Agnew B.J. Exp. Eye Res. 2001; 73: 509-520Crossref PubMed Scopus (73) Google Scholar), and the retinal dystrophy phenotype can be corrected by delivery of replication-deficient adenovirus encoding rat Mer gene to the eyes of young RCS rats (14Vollrath D. Feng W. Duncan J.L. Yasumura D. D'Cruz P.M. Chappelow A. Matthes M.T. Kay M.A. LaVail M.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2584-2589Crossref Scopus (254) Google Scholar, 15Feng W. Yasumura D. Matthes M.T. LaVail M.M. Vollrath D. J. Biol. Chem. 2002; 277: 17016-17022Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Subsequently, separate mutations in Mer each predicted to abrogate Mer tyrosine kinase activity were identified in three families with retinitis pigmentosa (16Gal A. Li Y. Thompson D.A. Weir J. Orth U. Jacobson S.G. Apfelstedt-Sylla E. Vollrath D. Nat. Genet. 2000; 26: 270-271Crossref PubMed Scopus (551) Google Scholar). Individuals harboring these mutations suffer from progressive loss of vision, presumably due to defective phagocytosis of shed photoreceptor cells by RPE cells (16Gal A. Li Y. Thompson D.A. Weir J. Orth U. Jacobson S.G. Apfelstedt-Sylla E. Vollrath D. Nat. Genet. 2000; 26: 270-271Crossref PubMed Scopus (551) Google Scholar). To study Mer function we stably transfected the IL-3-dependent murine hematopoietic cell line 32Dc13 (32D) with an EGF receptor extracellular and transmembrane domain-Mer cytoplasmic domain chimera. In these cells, EGF-dependent Mer signaling prevented apoptosis upon IL-3 and serum withdrawal. In contrast to transfected full-length EGF receptor and other receptor tyrosine kinases, Mer prevented 32D cell apoptosis without stimulating cellular proliferation. When combined with IL-3, Mer signaling produced dramatic shape changes, suggesting involvement of Mer tyrosine kinase in cytoskeletal remodeling (17Guttridge K. Luft C.J. Dawson T.L. Kozlowska E. Mahajan N.P. Varnum B. Earp H.S. J. Biol. Chem. 2002; 277: 24057-24066Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). However, the mechanism by which Mer signaling brings about cytoskeletal changes in experimental (32D) or physiological (monocytes or macrophages) processes remains obscure. Here we demonstrate that Mer interacts constitutively with the SH2 domain of the guanine nucleotide exchange factor (GEF), Vav1. Surprisingly, this SH2 domain/Mer interaction is phosphotyrosine-independent. Mer activation leads to tyrosine phosphorylation and release of Vav1 and activation of Rho family members. This unusual constitutive, SH2 domain-dependent but phosphotyrosine-independent interaction and release may provide the circumscribed local cytoskeletal control necessary to trigger ingestion of apoptotic material bound to the surface of phagocytic monocytes or pigmented epithelial cells. Plasmids and Site-directed Mutagenesis—A chimeric receptor was constructed wherein the extracellular domain of Mer was replaced with a ligand binding and transmembrane domain of the rat EGF receptor (Fig. 1A), using a suitable SalI restriction site at juxtamembrane region of these two receptors. The entire coding region of chimeric receptor of 1142 amino acids was subcloned into pLXSN, a mammalian retroviral expression vector (Clontech). This E GFR-M er C himeric receptor was named EMC. For construction of the "bait" plasmid pNCMY, 290 amino acids of the carboxyl-terminal intracellular region of Mer (amino acids 546-836), which includes the entire kinase domain, was amplified by PCR and inserted in-frame to the Gal4 DNA-binding domain of the pAS2-1 vector (Fig. 1A). Full-length Vav1 and all the Vav1 truncation constructs were generated by PCR amplification using pJC11 plasmid (kindly provided by Prof. C. J. Der) as template and Pfx polymerase, which has proofreading ability (Invitrogen). The PCR products were digested with XhoI and HindIII and subcloned into the corresponding sites of pcDNA4.1/Myc-His vector (Invitrogen) in-frame with a myc epitope-encoding region (Fig. 1B). The Mer truncation constructs AMer (aa 529-999), BMer (aa 621-999), CMer (aa 690-999), DMer (aa 529-696), EMer (aa 755-999), and FMer (aa 777-999) were generated by PCR amplification using full-length Mer as template and Pfx polymerase. The PCR products were digested with XhoI and HindIII and subcloned into the corresponding sites of pcDNA4.1/Myc-His vector. The 3′ primers used in Mer truncation constructs had a stop codon thus no tag was present in AMer, BMer, CMer, DMer, EMer, and FMer (Fig. 5A). For expression of GST-Mer fusion proteins, intracellular region (carboxyl-terminal 415 amino acids) of Mer was PCR-amplified using Pfx polymerase. The PCR product was digested with BamHI and SalI and subcloned into pGEX-4T vector in appropriate reading frame (Fig. 1A). Mutagenesis of Mer and Vav1 proteins was performed using GeneEditor in vitro site-directed mutagenesis system (Promega). All the plasmid constructs were sequenced to confirm the authenticity.Fig. 5Vav1 recognize carboxyl-terminal region of Mer. A, schematic of Mer, Mer chimera, and various intracellular regions representing constructs used for co-immunoprecipitation assay. A-FMer constructs were co-transfected with Vav1 in 293T cells, and lysates were immunoprecipitated using anti-Mer (in the cases of A-CMer, EMer, and FMer) or anti-EGFR (in the case of DMer) antibodies. Immune complexes were analyzed using anti-myc, anti-Mer, and anti-Mer Ab2 antibodies. B, Vav1 co-immunoprecipitated with A, B, and CMer but failed to bind DMer, EMer, and FMer.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Two-hybrid Screening—Matchmaker two-hybrid system-2 was used for identification of interacting clones (Clontech). Y190 cells transformed with pNCMY produced expected fusion proteins of 69 kDa, detected by Western blotting with an antibody against the GAL4 DNA-binding domain (Upstate Biotechnology Inc.). A human bone marrow cDNA library was screened. 1 × 106 transformants were plated on SD/-Trp/-Leu/-His/-Ade plates. The colonies that grew on these plates were further screened and "true positive" were selected, based on the criterion that these colonies could not induce His or β-galactosidase production when plasmid DNA isolated from these "positive" colonies was transformed into yeast cells carrying pAS2-1 vector. To further confirm, the colony-lift β-galactosidase filter assay was performed according to manufacturer's protocol (Clontech). Antibodies, Cell Lines, Immunoprecipitations, and Immunoblot Analysis—A polypeptide containing the carboxyl-terminal 90 amino acids of Mer was expressed as GST fusion protein in DH5α Escherichia coli strain and plated onto ampicillin plates. Colonies were picked and grown overnight in 10 ml of LB containing ampicillin. Overnight grown culture was added to 300 ml of fresh LB containing ampicillin and grown for 2 h, which was followed by isopropyl-1-thio-β-d-galactopyranoside addition (0.1 mm final concentration). Culture was grown for 2 more h, and cells were harvested, lysed in Lysis Buffer, containing 25 mm Tris (pH 7.5), 150 mm NaCl, 0.4% Triton X-100, 1 mm DTT, 15% glycerol, phosphatase inhibitors (10 mm NaF, 1 mm Na2VO4), and protease inhibitor mix (25 μg/ml leupeptin, 25 μg/ml trypsin inhibitor, 25 μg ml pepstatin, 25 μg/ml aprotinin, 10 mm benzamidine, 1 mm phenylmethylsulfonyl fluoride). Lysates were incubated with glutathione beads for 2 h, followed by washing with lysis buffer and elution in PBS containing 10 mm glutathione. Eluted protein was dialyzed against 50 mm Tris, pH 7.5, 10% glycerol, 100 mm NaCl, and 1 mm DTT, to remove all glutathione. The purified polypeptide was then used to generate rabbit polyclonal antibody. Anti-Mer Ab2 (Clone 110) was obtained from FabGennix International. Anti-Vav1 antibody was purchased from UBI. Anti-phosphotyrosine (RC20) antibodies were purchased from BD Transduction Laboratories. Anti-Rac1, anti-RhoA, and anti-Cdc42 antibodies were purchased from Santa Cruz Biotechnology. Goat anti-rabbit immunoglobulin G (IgG)-HRP, and goat anti-mouse IgG-HRP antibodies were purchased from Amersham Biosciences. Anti-myc monoclonal antibody was obtained from Invitrogen. Human SV40-transformed embryonic kidney 293T cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. 32D cells were maintained in RPMI medium containing 15% fetal calf serum and 5% WEHI cell-spent media (IL-3). 293T cells were transfected using FuGENE (Invitrogen) as per the manufacturer's protocol. Thirty-two hours after transfection, cells were serum-starved for 16 h and treated for 20 min with EGF (100 ng/ml). Cells were harvested, immediately washed in ice cold phosphate-buffered saline (PBS), and lysed in receptor lysis buffer (RLB), containing 25 mm Tris (pH 7.5), 175 mm NaCl, 1% Triton X-100, 1 mm DTT, 15% glycerol, phosphatase inhibitors (10 mm NaF, 1 mm Na2VO4), and protease inhibitor mix (25 μg/ml leupeptin, 25 μg/ml trypsin inhibitor, 25 μg/ml pepstatin, 25 μg/ml aprotinin, 10 mm benzamidine, 1 mm phenylmethylsulfonyl fluoride). Equivalent amounts of protein were incubated with respective primary antibodies for 2 h or overnight at 4 °C, followed by incubation with protein A/G-Sepharose (Santa Cruz Biotechnology) for 30 min. The beads were washed three times in the RLB buffer, resuspended in the appropriate volume of Laemmli gel loading buffer, and subjected to SDS-polyacrylamide gel electrophoresis. The proteins were electrotransferred to nitrocellulose membranes and blocked in 3% bovine serum albumin (for pTyr blots) or 5% milk in TBST buffer (Tris-buffered saline, pH 7.5, 0.1% Tween 20) for 1 h. Blocked filters were probed with primary antibodies in the same buffer, followed by secondary antibody conjugated to HRP in blocking solution and developed using enhanced chemiluminescence (ECL) detection (Amersham Biosciences). The blots were re-probed with a second set of antibodies (anti-Mer or anti-myc) to confirm the presence of respective proteins. Purification of Recombinant Proteins LVav and KVav from Sf9 Insect Cells—Baculovirus expression constructs were generated using the pFast Bac method (Invitrogen-Brl). SH3-SH2-SH3 and SH3-SH3 domains of Vav1 were PCR-amplified using CVav and JVav as templates and were subcloned along with a 5′ six-histidine tag into the XbaI and SalI sites of pDR120 vector, named as LVav and KVav, respectively. Expression of LVav and KVav in recombinant baculovirus-infected Sf9 insect cell extracts was confirmed by Western analysis with anti-polyhistidine antibodies. 200 ml of baculovirus-infected Sf9 cells were used for the purification of the protein. Cells were harvested 3 days following infection with the virus. Cell pellets from (6 × 107 cells) were suspended in an extraction buffer containing 10 mm Tris-HCl, pH 8.0, 50 mm sodium phosphate, pH 8.0, 0.5 m NaCl, 10% glycerol, 20 mm β-mercaptoethanol, and protease inhibitor mixture (Roche Applied Science). The extract was sonicated for 1-min pulses and then clarified by centrifugation twice at 15,000 × g for 15 min. The supernatant was added to 2 ml (50% slurry) of pre-equilibrated Talon affinity beads (Clontech). Binding was done for 2 h in the cold room. The unbound proteins were removed by centrifugation at 2000 rpm for 2 min. The beads were washed with 10 ml of extraction buffer thrice. After final wash the beads were transferred to 15-ml chromatographic columns (Bio-Rad). The beads were subsequently washed with 10 bed volumes of wash buffer containing 50 mm sodium phosphate (pH 7.0), 500 mm NaCl, 20 mm imidazole, 10% glycerol, 20 mm β-mercaptoethanol. The bound proteins were eluted in buffer containing 20 mm Tris HCl, pH 8.0, 100 mm NaCl, 100 mm imidazole, 10% glycerol, and 20 mm β-mercaptoethanol and collected as 0.5-ml fractions. The fractions were analyzed by SDS-PAGE and Coomassie Blue staining. The fractions containing the purified protein were pooled and dialyzed against 50 mm Tris, pH 7.5, 10% glycerol, 100 mm NaCl, and 1 mm DTT. The protein concentration was determined by using the Bradford method. Small aliquots of the purified protein were stored at -80 °C. Purification of GST-Mer and in Vitro Binding Assay—GST-Mer construct was transformed into BL21(DE3) cells and plated onto ampicillin plates. Colonies were picked and grown overnight in 10 ml of LB containing ampicillin. Culture, grown overnight, was added to 300 ml of fresh LB containing ampicillin and grown for 2 h, which was followed by isopropyl-1-thio-β-d-galactopyranoside addition (0.1 mm final concentration). Culture was grown for 4 more hours, and cells were harvested, lysed in lysis buffer, containing 25 mm Tris (pH 7.5), 150 mm NaCl, 0.4% Triton X-100, 1 mm DTT, 15% glycerol, phosphatase inhibitors (10 mm NaF, 1 mm Na2VO4), and protease inhibitor mix. Lysates were incubated with glutathione beads for 2 h, followed by washing with lysis buffer and elution in PBS containing 10 mm glutathione. Eluted protein was dialyzed against 50 mm Tris, pH 7.5, 10% glycerol, 100 mm NaCl, and 1 mm DTT, to remove all glutathione. For the in vitro binding assay, 50 nm purified LVav or KVav were added to Talon beads resuspended in modified RLB containing 25 mm Tris (pH 7.5), 175 mm NaCl, 0.4% Triton X-100, 1 mm DTT, 15% glycerol, 1% protease-free bovine serum albumin, and protease inhibitor mix. After 1 h, Talon beads were washed to remove all unbound proteins. Purified GST-Mer (50 mm) was added to similarly treated Talon beads without Vav1 domain proteins or to Talon beads with bound LVav or KVav. Following incubation on ice for 30 min and then incubation with shaking at 4 °C for 1 h, beads were washed thrice with lysis buffer. Bound protein was dissociated from beads by boiling in SDS sample buffer and assessed by gel electrophoresis, transfer, and detection by immunoblotting with anti-Mer antibody. In a control experiment, the use of NaCl concentration above 175 mm prevented detection of GST-Mer/LVav interaction. Assay for Detection of Activated Rac1, RhoA, and Cdc42—The glutathione-Sepharose beads conjugated with GST-Pak (PBD) or GST-Rok (RBD) were used as specific probes for in vitro binding assays of activated Rac1, Cdc42, and RhoA, respectively. Stable cell lines expressing EMC and kdEMC were grown overnight in serum-free media with 5% WEHI cells spent media (IL-3). Next day, cells were stimulated with EGF ligand for 20 or 30 min. Cells were lysed in MLB buffer (25 mm HEPES, pH 7.5, 150 mm NaCl, 1% Igepal CA-630, 10 mm MgCl2, 1 mm EDTA, 10% glycerol, protease, and phosphatase inhibitor mixture). Samples of 500 μg of protein lysates were mixed with 20 μl of PBD beads and incubated at 4 °C for 45 min. The beads were washed three times in MLB buffer and analyzed by Western blotting to detect the bound Rac1 and Cdc42 GTPases. For RhoA, cells were lysed in RLB buffer (50 mm Tris, pH 7.2, 500 mm NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 10 mm MgCl2, 1 mm EDTA, 10% glycerol, protease, and phosphatase inhibitor mixture). Samples of 500 μg of protein lysates were mixed with 30 μl of RBD beads and incubated at 4 °C for 45 min. The beads were washed three times in wash buffer (50 mm Tris, pH 7.2, 150 mm NaCl, 1% Triton X-100, 0.1 mm phenylmethylsulfonyl fluoride, 10 mm MgCl2, 10% glycerol, protease, and phosphatase inhibitor mixture) and analyzed by Western blotting. Isolation of Human Macrophages—Human blood (buffy coat) was diluted to 50% with PBS and mixed gently. The mixture was gently layered over 50 ml of Ficoll (Histopaque-1077, Sigma). After centrifugation at 2000 rpm for 30 min the mononuclear cell layer was collected and washed four times with PBS and plated in RPMI media containing 10% fetal bovine serum. After 2 h of incubation, adherant monocytes and macrophages were retained and allowed to grow for 7-10 days in RPMI media, before Gas6 treatment. Mer Interacts with GEF and Vav1—Downstream effectors of Mer signaling were sought by yeast two-hybrid methods (18Fields S. Song O. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4860) Google Scholar). A human bone marrow cDNA library was screened with the Mer cytoplasmic domain (pNCMY vector, Fig. 1A) as bait. Of nine positive colonies, three encoded the carboxyl-terminal SH3-SH2-SH3 domains of Vav1 (CVavΔN depicted in Fig. 1B). This prototypic GEF is a 97-kDa protein with multiple structural motifs (Fig. 1B), including, from the carboxyl to the amino termini, the following domain/motifs: a calponin homology (CH), an acidic region (Ac), a Dbl-homology (DH), a pleckstrin-homology (PH), a zinc finger domain (ZF), a short proline-rich region (PR), and two SH3 domains flanking a single SH2 region (21Bustelo X.R. Mol. Cell. Biol. 2000; 20: 1461-1477Crossref PubMed Scopus (448) Google Scholar). Clones for two other Mer interacting proteins, Grb2 and Shc (19Ling L. Kung H. Mol. Cell. Biol. 1995; 15: 6582-6592Crossref PubMed Scopus (68) Google Scholar, 20Georgescu M. Kirsch K.H. Shishido T. Zong C. Hanafusa H. Mol. Cell. Biol. 1999; 19: 1171-1181Crossref PubMed Google Scholar), were also isolated. Because Gas6 had not yet been identified as a ligand for Axl, Tyro3, or Mer when our studies began, we created a ligand-activated EGFR-Mer-chimera (EMC), using the rat EGF receptor extracellular and transmembrane domains and the Mer intracellular domain (Fig. 1A). EMC was stably transfected into mouse 32D cells, and, when activated with ligand, it prevented apoptosis upon IL-3 withdrawal from this IL-3-requiring cell line. Unlike most receptor tyrosine kinases, Mer activation did not stimulate proliferation, but it did alter cell adherence and shape in these normally suspension-growing cells (17Guttridge K. Luft C.J. Dawson T.L. Kozlowska E. Mahajan N.P. Varnum B. Earp H.S. J. Biol. Chem. 2002; 277: 24057-24066Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). This positive effect on cytoskeletal components coupled with the fact that Mer knockout mice exhibited defects in local cytoskeletal regulation (phagocytosis), and the strong positive yeast two-hybrid signal, led us to pursue the interaction and potential physiological role of the Mer/Vav1 complex. For these studies full-length Vav1 and truncated constructs were generated using vector pcDNA4 (depicted in Fig. 1B) and tagged with an myc epitope. Each construct gave generally equivalent expression when transfected into 293T cells (Fig. 1C). The AVav construct, which was not myc-tagged and thus was detected using Vav1 antibodies, also expressed equivalently to Vav1 (Fig. 1D). Vav1 interaction with the Mer intracellular domain was studied by co-transfection with either EMC or an EMC with a K619M site-directed mutation that destroyed kinase activity (kinase-dead EMC or kdEMC). Activation of Mer Lead to Dissociation of Mer/Vav1 Complex and Vav1 Tyrosine Phosphorylation—To confirm Vav1/Mer interactions, EMC-expressing 32D cells were stimulated with EGF, and increased tyrosine phosphorylation of Vav1 was detected. Surprisingly, Vav1 co-immunoprecipitated with unactivated EMC. 3N. P. Mahajan and H. Shelton Earp, unpublished results. To study this interaction in more detail, myc-tagged Vav1 constructs were co-transfected in 293T cells with EMC or kdEMC. Vav1 was readily detected in Mer immunoprecipitates from unstimulated cells, but the level of Mer-associated Vav1 decreased considerably with ligand stimulation (Fig. 2A, first panel, lanes 1 and 2). When co-transfected with kdEMC, Vav1 was present in the kdEMC immunoprecipitates from both untreated and EGF-treated lysed cells (Fig. 2A, first panel, lanes 3 and 4). Activation of EMC increased Vav1 tyrosine phosphorylation (Fig. 2A, second panel, lanes 1 and 2). This was not observed in kdEMC-expressing cells (Fig. 2A, second panel, lanes 3 and 4). Thus, in unstimulated cells Vav1 is, at least in part, constitutively bound to Mer. Ligand activation and Mer autophosphorylation resulted in dissociation of tyrosine-phosphorylated Vav1 (compare Fig. 2A, first panel, lane 2 to second panel, lane 2). Upon co-transfection, the carboxyl-terminal SH3-SH2-SH3, CVav, was also readily detected in EMC immunoprecipitates from unstimulated cells and was tyrosine-phosphorylated and released from this association upon Mer stimulation (Fig. 2B, second panel). This indicates that at least one site for Mer-dependent Vav1 tyrosine phosphorylation differs from the N-terminal Tyr-174 known to be phosphorylated by LCK (21Bustelo X.R. Mol. Cell. Biol. 2000; 20: 1461-1477Crossref PubMed Scopus (448) Google Scholar). When co-transfected with kdEMC, CVav was bound to Mer regardless of ligand additi