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Identification and Characterization of Leukocyte-associated Ig-like Receptor-1 as a Major Anchor Protein of Tyrosine Phosphatase SHP-1 in Hematopoietic Cells

2000; Elsevier BV; Volume: 275; Issue: 23 Linguagem: Inglês

10.1074/jbc.m001313200

1083-351X

Ming-jiang Xu, Runxiang Zhao, Zhizhuang Joe Zhao,

Immune Cell Function and Interaction

SHP-1, an SH2 domain-containing tyrosine phosphatase, has a crucial role in hematopoiesis. Here we report that SHP-1 is associated with two major tyrosine-phosphorylated proteins in hematopoietic cells treated with the tyrosine phosphatase inhibitor, pervanadate. One of the proteins corresponds to leukocyte-associated Ig-like receptor-1 (LAIR-1), a recently cloned transmembrane protein. Molecular cloning revealed four isoforms of the protein. LAIR-1 is hyper-phosphorylated on tyrosyl residues in cells overexpressing a catalytically inactive mutant form of SHP-1 as well as in pervanadate-treated cells. An antibody against the extracellular domain of the protein also induced its tyrosine phosphorylation. Tyrosine-phosphorylated LAIR-1 specifically interacts with SHP-1 but not with SHP-2, a structurally related tyrosine phosphatase. Using site-specific mutagenesis, we demonstrated that Tyr233 and Tyr263, each embedded in an immunoreceptor tyrosine-based inhibitory motif, are responsible for tyrosine phosphorylation of LAIR-1 and recruitment of SHP-1. Both tyrosyl residues are required for SHP-1 binding. Protein kinases responsible for tyrosine phosphorylation of LAIR-1 may belong to the Src family since PP1, a Src family kinase inhibitor, significantly inhibited its phosphorylation. As a major binding protein of SHP-1 on the plasma membrane, LAIR-1 may play an important role in hematopoietic cell signaling. SHP-1, an SH2 domain-containing tyrosine phosphatase, has a crucial role in hematopoiesis. Here we report that SHP-1 is associated with two major tyrosine-phosphorylated proteins in hematopoietic cells treated with the tyrosine phosphatase inhibitor, pervanadate. One of the proteins corresponds to leukocyte-associated Ig-like receptor-1 (LAIR-1), a recently cloned transmembrane protein. Molecular cloning revealed four isoforms of the protein. LAIR-1 is hyper-phosphorylated on tyrosyl residues in cells overexpressing a catalytically inactive mutant form of SHP-1 as well as in pervanadate-treated cells. An antibody against the extracellular domain of the protein also induced its tyrosine phosphorylation. Tyrosine-phosphorylated LAIR-1 specifically interacts with SHP-1 but not with SHP-2, a structurally related tyrosine phosphatase. Using site-specific mutagenesis, we demonstrated that Tyr233 and Tyr263, each embedded in an immunoreceptor tyrosine-based inhibitory motif, are responsible for tyrosine phosphorylation of LAIR-1 and recruitment of SHP-1. Both tyrosyl residues are required for SHP-1 binding. Protein kinases responsible for tyrosine phosphorylation of LAIR-1 may belong to the Src family since PP1, a Src family kinase inhibitor, significantly inhibited its phosphorylation. As a major binding protein of SHP-1 on the plasma membrane, LAIR-1 may play an important role in hematopoietic cell signaling. protein-tyrosine phosphatase leukocyte-associated Ig-like receptor-1 immunoreceptor tyrosine-based inhibitory motif SHP-1 is an SH2 domain-containing protein tyrosine phosphatase that is highly expressed in hematopoietic cells and, at a much lower level, in non-hematopoietic cells (1.Adachi M. Fischer E.H. Ihle J. Imai K. Jirik F. Neel B. Pawson T. Shen S.-H. Thomas M. Ullrich A. Zhao Z. Cell. 1996; 85: 15Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 2.Michel S. Curr. Opin. Cell Biol. 1996; 183: 182-188Google Scholar, 3.Frearson J.A. Alexander D.R. Bioessays. 1997; 19: 417-427Crossref PubMed Scopus (62) Google Scholar, 4.Zhao Z. Shen S.-H. Fischer E.H. Adv. Protein Phosphatases. 1995; 9: 297-317Google Scholar, 5.Neel B.G. Semin. Cell Biol. 1993; 4: 419-432Crossref PubMed Scopus (106) Google Scholar, 6.Feng G.S. Pawson T. Trends Genet. 1994; 10: 54-58Abstract Full Text PDF PubMed Scopus (169) Google Scholar). Studies indicate that SHP-1 is a key negative regulator of cell signaling. Motheaten and viable motheaten mice, which have mutations of the SHP-1 gene, develop a severe autoimmune and immunodeficiency syndrome with an extremely high proliferation rate of all hematopoietic cells (7.Tsui H.W. Siminovitch K.A. Souza L. Tsui F.W.L. Nat. Genet. 1993; 4: 124-129Crossref PubMed Scopus (514) Google Scholar, 8.Shultz L.D. Schweitzer P.A. Rajan T.V. Yi T. Ihle J.N. Matthews R.J. Thomas M.L. Beier D.R. Cell. 1993; 73: 1445-1454Abstract Full Text PDF PubMed Scopus (686) Google Scholar). This is consistent with the negative role of SHP-1 in cell signaling mediated by interleukin-3 receptor (9.Yi T. Mui A.L. Krystal G. Ihle J.N. Mol. Cell. Biol. 1993; 13: 7577-7586Crossref PubMed Google Scholar), c-Kit (10.Lorenz U. Bergemann A.D. Steinberg H.N. Flanagan J.G. Li X.T. Galli S.J. Neel B.G. J. Exp. Med. 1996; 184: 1111-1126Crossref PubMed Scopus (93) Google Scholar), erythropoietin receptor (11.Klingmuller U Lorenz U. Cantley L.C. Neel B.G. Lodish H.F. Cell. 1995; 80: 729-738Abstract Full Text PDF PubMed Scopus (838) Google Scholar), interferon-α/β receptor (12.David M. Chen H.E. Goelz S. Larner A.C. Neel B.G. Mol. Cell. Biol. 1995; 15: 7050-7058Crossref PubMed Scopus (317) Google Scholar), colony-stimulating factor-1 (CSF-1) receptor (13.Chen H.E. Chang S. Trub T. Neel B.G. Mol. Cell. Biol. 1996; 7: 3685-3697Crossref Scopus (181) Google Scholar), B-cell antigen receptor (14.Cyster J.G. Goodnow C.C. Immunity. 1995; 2: 13-24Abstract Full Text PDF PubMed Scopus (349) Google Scholar), T-cell antigen receptor (15.Pani G. Fischer K.D. Mlinaricrascan I. Siminovitch K.A. J. Exp. Med. 1996; 184: 839-852Crossref PubMed Scopus (179) Google Scholar, 16.Plas D.R. Johnson R. Pingel J.T. Matthews R.J. Dalton M. Roy G. Chan A.C. Thomas M.L. Science. 1996; 272: 1173-1176Crossref PubMed Scopus (330) Google Scholar), NK cell inhibitory receptor, and CD22 (17.Burshtyn D.N. Scharenberg A.M. Wagtmann N. Rajagopalan S. Berrada K. Yi T. Kinet J.P. Long E.O. Immunity. 1996; 4: 77-85Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar, 18.Doody G.M. Justement L.B. Delibrias C.C. Matthews R.J. Lin J. Thomas M.L. Fearon D.T. Science. 1995; 269: 242-244Crossref PubMed Scopus (485) Google Scholar). Our previous studies demonstrated that SHP-1 displayed very low activity in vitro due to internal suppression, and we proposed that SHP-1 remains in an inactive form in the resting state and can be activated upon simulation (19.Zhao Z. Bouchard P. Diltz C.D. Shen S.H. Fischer E.H. J. Biol. Chem. 1993; 268: 2816-2820Abstract Full Text PDF PubMed Google Scholar). Studies by others and us demonstrated that SHP-1 can be activated by anionic phospholipidsin vitro or by binding to tyrosine-phosphorylated receptors and other proteins concomitant with translocation of SHP-1 to the plasma membrane in cells (20.Zhao Z. Shen S.H. Fischer E.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4251-4255Crossref PubMed Scopus (108) Google Scholar, 21.Tomic S. Greiser U. Lammers R. Kharitonenkov A. Imyanitov E. Ullrich A. Bohmer F.D. J. Biol. Chem. 1995; 270: 21277-21284Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 22.Pei D. Lorenz U. Klingmuller U. Neel B.G. Walsh C.T. Biochemistry. 1994; 33: 15483-15493Crossref PubMed Scopus (185) Google Scholar, 23.Zhao Z. Shen S.H. Fischer E.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5007-5011Crossref PubMed Scopus (53) Google Scholar). Therefore, binding of SHP-1 to tyrosine-phosphorylated receptors is a major mechanism by which SHP-1 is regulated. Indeed, SHP-1 has been shown to bind to a number of tyrosine-phosphorylated growth factor receptors including c-Kit, EPO-R, IL-3-R, c-Fym, and gp130 (9.Yi T. Mui A.L. Krystal G. Ihle J.N. Mol. Cell. Biol. 1993; 13: 7577-7586Crossref PubMed Google Scholar, 10.Lorenz U. Bergemann A.D. Steinberg H.N. Flanagan J.G. Li X.T. Galli S.J. Neel B.G. J. Exp. Med. 1996; 184: 1111-1126Crossref PubMed Scopus (93) Google Scholar, 11.Klingmuller U Lorenz U. Cantley L.C. Neel B.G. Lodish H.F. Cell. 1995; 80: 729-738Abstract Full Text PDF PubMed Scopus (838) Google Scholar, 12.David M. Chen H.E. Goelz S. Larner A.C. Neel B.G. Mol. Cell. Biol. 1995; 15: 7050-7058Crossref PubMed Scopus (317) Google Scholar, 13.Chen H.E. Chang S. Trub T. Neel B.G. Mol. Cell. Biol. 1996; 7: 3685-3697Crossref Scopus (181) Google Scholar, 14.Cyster J.G. Goodnow C.C. Immunity. 1995; 2: 13-24Abstract Full Text PDF PubMed Scopus (349) Google Scholar, 15.Pani G. Fischer K.D. Mlinaricrascan I. Siminovitch K.A. J. Exp. Med. 1996; 184: 839-852Crossref PubMed Scopus (179) Google Scholar, 16.Plas D.R. Johnson R. Pingel J.T. Matthews R.J. Dalton M. Roy G. Chan A.C. Thomas M.L. Science. 1996; 272: 1173-1176Crossref PubMed Scopus (330) Google Scholar, 17.Burshtyn D.N. Scharenberg A.M. Wagtmann N. Rajagopalan S. Berrada K. Yi T. Kinet J.P. Long E.O. Immunity. 1996; 4: 77-85Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar, 18.Doody G.M. Justement L.B. Delibrias C.C. Matthews R.J. Lin J. Thomas M.L. Fearon D.T. Science. 1995; 269: 242-244Crossref PubMed Scopus (485) Google Scholar). It is intriguing that the ligands of these growth factor receptors promote cell signaling and activate SHP-1 as a very early event, which presumably turns off the signal. Furthermore, in most cases, only a very small fraction of SHP-1 is bound to these receptors. Therefore, the complete mechanism of action following the association of SHP-1 with these receptors is still not fully understood (9.Yi T. Mui A.L. Krystal G. Ihle J.N. Mol. Cell. Biol. 1993; 13: 7577-7586Crossref PubMed Google Scholar, 10.Lorenz U. Bergemann A.D. Steinberg H.N. Flanagan J.G. Li X.T. Galli S.J. Neel B.G. J. Exp. Med. 1996; 184: 1111-1126Crossref PubMed Scopus (93) Google Scholar, 11.Klingmuller U Lorenz U. Cantley L.C. Neel B.G. Lodish H.F. Cell. 1995; 80: 729-738Abstract Full Text PDF PubMed Scopus (838) Google Scholar, 12.David M. Chen H.E. Goelz S. Larner A.C. Neel B.G. Mol. Cell. Biol. 1995; 15: 7050-7058Crossref PubMed Scopus (317) Google Scholar, 13.Chen H.E. Chang S. Trub T. Neel B.G. Mol. Cell. Biol. 1996; 7: 3685-3697Crossref Scopus (181) Google Scholar, 14.Cyster J.G. Goodnow C.C. Immunity. 1995; 2: 13-24Abstract Full Text PDF PubMed Scopus (349) Google Scholar, 15.Pani G. Fischer K.D. Mlinaricrascan I. Siminovitch K.A. J. Exp. Med. 1996; 184: 839-852Crossref PubMed Scopus (179) Google Scholar, 16.Plas D.R. Johnson R. Pingel J.T. Matthews R.J. Dalton M. Roy G. Chan A.C. Thomas M.L. Science. 1996; 272: 1173-1176Crossref PubMed Scopus (330) Google Scholar, 17.Burshtyn D.N. Scharenberg A.M. Wagtmann N. Rajagopalan S. Berrada K. Yi T. Kinet J.P. Long E.O. Immunity. 1996; 4: 77-85Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar, 18.Doody G.M. Justement L.B. Delibrias C.C. Matthews R.J. Lin J. Thomas M.L. Fearon D.T. Science. 1995; 269: 242-244Crossref PubMed Scopus (485) Google Scholar). To better understand the regulation and function of SHP-1, we have investigated the association of SHP-1 with tyrosine-phosphorylated proteins in cells treated with pervanadate, a powerful protein-tyrosine phosphatase (PTP)1 inhibitor that induces robust tyrosine phosphorylation of cellular proteins. Our results demonstrate that SHP-1 is associated with two major tyrosine-phosphorylated proteins in hematopoietic cells, including Jurkat T-cells, HL-60 cells, and human peripheral T-cells. We further identified that one of the proteins corresponds to leukocyte-associated Ig-like receptor-1 (LAIR-1), a recently cloned immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing transmembrane protein that is implicated in NK cell and T-cell functions (24.Meyaard L. Adema G.J. Chang C. Woollatt E. Sutherland G.R. Lanier L.L. Phillips J.H. Immunity. 1997; 7: 283-290Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 25.Meyaard L. Hurenkamp J. Clevers H. Lanier L.L. Phillips J.H. J. Immunol. 1999; 162: 5800-5804PubMed Google Scholar). Molecular cloning revealed four isoforms of this protein, presumably resulting from alternative splicing, and these isoforms of LAIR-1 have distinct expression in hematopoietic cells. More importantly, we have generated an antibody that induces tyrosine phosphorylation of LAIR-1 and consequent recruitment and activation of the tyrosine phosphatase SHP-1. We also have characterized the mechanism by which LAIR-1 interacts with SHP-1. As a specific and a major anchor protein of SHP-1 in plasma membrane, LAIR-1 may have an important role in hematopoietic cell signaling. Human 293 embryonic kidney cells, Jurkat T cells, and HL-60 cells were obtained from American Type Culture Collection. Jurkat and HL-60 cells were maintained in RPMI, and 293 cells were maintained in Dulbecco's modified Eagle's medium. All the media were supplemented with 10% fetal bovine serum and 100 unit/ml each of penicillin and streptomycin antibiotics. Human T lymphocytes were purified from peripheral blood of healthy volunteers as previously reported (26.Sui X. Krantz S.B. Zhao Z. Blood. 1997; 90: 651-657Crossref PubMed Google Scholar). Briefly, heparinized blood was layered onto Ficoll-Hypaque, and light-density mononuclear cells were separated by centrifugation. The collected mononuclear cells were then centrifuged through 10% bovine serum albumin to delete platelets. T lymphocytes were obtained by sheep erythrocyte rosetting followed by red cell hemolysis. Monoclonal anti-phosphotyrosine 4G10 was obtained from Upstate Biotechnology Inc. (Lake Placid, NY), and rabbit polyclonal anti-SHP-1, anti-SHP-2, and anti-SHIP antibodies were purchased from Santa Cruz Biotechnology Inc. Different maltose-binding protein fusion proteins containing the full-length Cys-455-to-Ser mutant, tandem SH2 domains, and N-terminal SH2 domain of SHP-1 were purified by using amylose resin as described previously (27.Law C.L. Sidorenko S.P. Chandran K.A. Zhao Z. Shen S.H. Fischer E.H. Clark E.A. J. Exp. Med. 1996; 183: 547-560Crossref PubMed Scopus (176) Google Scholar). The catalytically inactive Cys-455-to-Ser SHP-1 mutant used for transfection of 293 cells was constructed with the pRC/CMV vector (Invitrogen). Buffer A consisted of 25 mm β-glycerolphosphate (pH 7.3), 10 mmEDTA, 2 mm EGTA, 1 mm benzamidine, 0.1 mm phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, 1 μm pepstatin A, and 1 μg/ml aprotinin. Human LAIR-1 cDNAs were amplified from human bone marrow, Jurkat cell, and HL-60 cell Marathon-ready cDNA libraries (CLONTECH Laboratories, Inc., Palo Alto, CA) by polymerase chain reaction with primers 5′-GCCATGTCTCCCACCCCAC-3′ and 5′-GTCAGTGTCTGGCAACGGCTGC-3′ derived from the 5′- and 3′-coding regions of LAIR-1, respectively (24.Meyaard L. Adema G.J. Chang C. Woollatt E. Sutherland G.R. Lanier L.L. Phillips J.H. Immunity. 1997; 7: 283-290Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). The polymerase chain reaction was performed with thermo-DNA polymerase Pfu (Stratagene), and the products were cloned into the pBluescript KS vector (Stratagene), which was digested withEcoRV. The cDNA clones were sequenced using the automated DNA sequencing facilities of the Vanderbilt-Ingram Cancer Center. The intracellular and extracellular portions of LAIR-1c (see below), corresponding to amino acid residues 170–269 and 1–141, respectively, were expressed inEscherichia coli as glutathione S-transferase (GST) fusion proteins by using the pGex-2T vector (Amersham Pharmacia Biotech) and purified with glutathione-Sepharose. For antibody production, rabbits were injected with the fusion proteins mixed with Freund's adjuvant (Pierce). Anti-sera raised against the intracellular part of LAIR-1 was designated 145, whereas that against the extracellular fusion protein was named 148. Unless otherwise noted, immunoprecipitation was carried out with 145, whereas immunoblotting and cell treatment were performed with 148. LAIR-1c was subcloned into the pCDNA3 vector (Invitrogen). Tyr233 → Phe and Tyr263 → Phe mutations of LAIR-1c were performed by polymerase chain reaction with primers containing the desired mutations. All these clones were verified by DNA sequencing. Cell transfection was carried out according to the standard calcium phosphate co-precipitation technique as described (28.Zhao R. Zhao Z.J. Biochem. J. 1999; 338: 35-39Crossref PubMed Google Scholar). Briefly, 293 cells were grown to ∼30% confluence, and 25 μg plasmid DNAs were used for transfecting cells in each 10-cm plate. The cells were harvested 48 h after transfection. To induce tyrosine phosphorylation of LAIR-1, cells were treated with 0.1 mm pervanadate for 30 min. A 50 mm stock solution of pervanadate was made by mixing equal volumes of 0.1m sodium vanadate and 0.2 mH2O2 and incubating at room temperature for 20 min before the addition to the cells (29.Zhao Z. Tan Z. Diltz C.D. You M. Fischer E.H. J. Biol. Chem. 1996; 271: 22251-22255Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). When cells were stimulated with anti-LAIR-1 antibody 148 or its pre-serum, they were serum-starved for 5 h at 37 °C before treatment. Stimulated cells were washed with ice-cold phosphate-buffered saline immediately and then were subjected to extraction as described below. Cells were washed with cold phosphate-buffered saline and then lysed in Buffer A supplemented with 0.1 m NaCl and 1% Triton X-100. Extracts were cleared by centrifugation and then were immunoprecipitated with the specific antibodies pre-bound to protein A-Sepharose. Following overnight incubation, the beads were washed three times with the extraction buffer supplemented with 0.15m NaCl. For Western blot analyses, samples were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were probed with various primary antibodies and were detected by using the ECL system with horseradish peroxidase-conjugated secondary antibodies (Amersham Pharmacia Biotech). Cells were lysed in Buffer A by using a Dounce homogenizer (23.Zhao Z. Shen S.H. Fischer E.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5007-5011Crossref PubMed Scopus (53) Google Scholar). To avoid nonspecific protein binding, 0.1 m NaCl was added to the homogenate. This was followed by centrifugation at 800 × g for 20 min to remove the nuclear pellets. The postnuclear extracts were further centrifuged at 100,000 ×g for 60 min to give rise to a clear cytosolic supernatant and a pelleted membrane fraction. The pellets, washed once with buffer A and then dissolved in Buffer A supplemented with 1% Triton X-100, were referred to as membrane extracts. Equal proportions of membrane and cytosol fractions were subjected to immunoprecipitation with anti-LAIR-1 or anti-SHP-1 and to Western blotting analyses with anti-SHP-1. For PTP activity assays, sodium vanadate was omitted from the buffers. Assays were performed with anti-SHP-1 and anti-LAIR-1 immunoprecipitates by using the ENDpYINASL (where pY is phosphotyrosine) peptide as the substrate as described previously (30.Zhao Z. Larocque R. Ho W.-T. Fischer E.H. Shen S.-H. J. Biol. Chem. 1994; 269: 8780-8785Abstract Full Text PDF PubMed Google Scholar). Although SHP-1 has been shown to bind a number of tyrosine-phosphorylated proteins on the cell surface, the stoichiometry appeared very low. We recently isolated a major SHP-2-binding protein designated PZR, and we demonstrated a near stoichiometric association of PZR with SHP-2 in pervanadate-stimulated cells (31.Zhao Z.J. Zhao R. J. Biol. Chem. 1998; 273: 29367-29372Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). We used the same strategy to identify SHP-1-binding proteins. We chose Jurkat cells, HL-60 cells, and primary peripheral T cells together with several non-hematopoietic cells for our study. As a powerful inhibitor of PTPs, pervanadate, induced robust tyrosine phosphorylation of cellular proteins. SHP-1-binding proteins were co-immunoprecipitated by anti-SHP-1 antibody, and tyrosine phosphorylation was analyzed by immunoblotting with anti-phosphotyrosine. As shown in Fig. 1, SHP-1 is co-precipitated with two tyrosine-phosphorylated proteins of 40 and 95 kDa in Jurkat cells, of 45 and 95 kDa in HL-60 cells, and of 40, 45, and 95 kDa in peripheral T cells. However, in non-hematopoietic cell lines including 293, HepG2, A431, HT-1080, and HeLa cells, only the 95-kDa protein was co-immunoprecipitated with SHP-1 (data not shown, see Ref. 31.Zhao Z.J. Zhao R. J. Biol. Chem. 1998; 273: 29367-29372Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). This suggests that the 95-kDa SHP-1-binding protein may be widely expressed, whereas the 40- and 45-kDa proteins seem to be restricted to hematopoietic cells. The 68-kDa phosphoprotein shown in Fig. 1 was SHP-1 as revealed by immunoblotting with anti-SHP-1. As a powerful, non-selective inhibitor of PTPs, pervanadate induces robust tyrosine phosphorylation of numerous cellular proteins. In this regard, the 40-kDa, 45-kDa, and 95-kDa proteins may be major SHP-1-binding proteins in the three types of hematopoietic cells examined. Next, we tried to identify the 40-kDa and 45-kDa proteins associated with SHP-1. We used antibodies to search for candidate phosphotyrosine-containing signaling molecules in this molecular weight range. We ruled out PZR, conexin 43, FcγRIIB (CD32), and CD7 by doing Western blotting analyses with their specific antibodies. Our last candidate was LAIR-1, a recently cloned leukocyte-specific ITIM-containing protein of ∼40 kDa (24.Meyaard L. Adema G.J. Chang C. Woollatt E. Sutherland G.R. Lanier L.L. Phillips J.H. Immunity. 1997; 7: 283-290Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 25.Meyaard L. Hurenkamp J. Clevers H. Lanier L.L. Phillips J.H. J. Immunol. 1999; 162: 5800-5804PubMed Google Scholar). Since antibody for Western blotting analyses of LAIR-1 was not available, we first generated an antibody in rabbits by using glutathioneS-transferase fusion proteins containing the intracellular or extracellular domain of LAIR-1. Re-blotting of membranes with anti-LAIR-1 antibody indeed indicated that the 40-kDa protein in Jurkat and primary T-cells and the 45-kDa protein in HL-60 cells and primary T cells both corresponded to LAIR-1 (Fig. 1). Anti-LAIR-1 immunoprecipitation further verified the results by showing not only tyrosine phosphorylation of LAIR-1 but also co-immunoprecipitation with SHP-1 that was also phosphorylated on tyrosine (Fig.2). When probed for the presence of other phosphatases including SHP-2 and SHIP, using their specific antibodies in anti-LAIR-1 immunoprecipitates from these three kinds of cells, neither SHP-2 nor SHIP was associated with LAIR-1 following pervanadate stimulation (data not shown). We thus identified that one of the major binding proteins of SHP-1 corresponded to LAIR-1. SHP-1, but neither SHP-2 nor SHIP, was specifically associated with LAIR-1 upon pervanadate stimulation. The fact that LAIR-1 from Jurkat and HL-60 cells ran at different molecular sizes may suggest different degrees of glycosylation at the protein level and/or alternate splicing at the mRNA level. We then tried to clone LAIR-1 by using polymerase chain reaction with primers designed according to the published LAIR-1 sequence (24.Meyaard L. Adema G.J. Chang C. Woollatt E. Sutherland G.R. Lanier L.L. Phillips J.H. Immunity. 1997; 7: 283-290Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). From Jurkat T cells and human bone marrow libraries, we pulled out predominantly a cDNA clone with a sequence that was different from the published sequences of LAIR-1a and 1b. We designated our sequence as LAIR-1c. We also cloned LAIR-1b as a minor form (three out of 50 clones) from Jurkat cell cDNAs. From HL-60 cells, we cloned LAIR-1a as the major form and LAIR-1b as a minor form. We also isolated a minor form that we designated LAIR-1d from HL-60 cell cDNAs. The DNA sequences of LAIR-1c and LAIR-1d were deposited in the GenBank™ data base under accession numbers AF251509 and AF251510, respectively. Sequence alignment showed that these four different forms of LAIR-1 are identical except for certain gaps and inserts characteristic of alternative RNA splicing (data not shown). These different cDNA sequences give different protein products as shown in Fig.3. LAIR-1c differs from LAIR-1b by a single amino acid residue, but LAIR-1d lacks essentially the entire intracellular segment and, thus, has no ITIMs. We focused on LAIR-1c in our continued studies since it is a major form in bone marrow and in T-cells. Embedded in the ITIM sequence, Tyr 233 and Tyr 263 are the two unique tyrosyl residues in the cytoplasmic domain of LAIR-1c. They most likely represent the tyrosine phosphorylation sites. To verify this, we mutated one or both of these residues to phenylalanine by site-directed mutagenesis. All the cDNA constructs were made with the pcDNA 3 vector. The mutant constructs (LAIR-1cF233, F263, and F233/F263) together with the control vector and the native LAIR-1c construct were used to transfect 293 cells, and the transfected cells were treated with pervanadate. The cell extracts were subjected to immunoprecipitation with anti-LAIR-1 145 and Western blot analyses with anti-phosphotyrosine, anti-LAIR-1 148, and anti-SHP-1. As shown in Fig. 4, although anti-LAIR-1 immunoblotting revealed expression of equivalent amounts of LAIR-1c or its mutants in the cells, mutation of either Tyr233 or Tyr263 significantly reduced the tyrosine phosphorylation, whereas mutation of both residues totally abolished the tyrosine phosphorylation of LAIR-1c. More importantly, any of the mutations caused a total loss of SHP-1 association with LAIR-1c. These results indicate that Try233 and Tyr263 are responsible for tyrosine phosphorylation of LAIR-1c. The fact that binding of SHP-1 with LAIR-1c was abolished by mutation of a single site indicates that simultaneous phosphorylation of both sites is required for recruitment of SHP-1 to LAIR-1c. The last lane on the right of Fig. 4 shows inhibition of pervanadate-induced tyrosine phosphorylation of LAIR-1 by Src family kinase inhibitor PP1. This indicates that protein kinases responsible for tyrosine phosphorylation of LAIR-1 may belong to the Src family. The binding of SHP-1 with LAIR-1c is presumably mediated by the interaction between SH2 domains of SHP-1 and the ITIMs of LAIR-1c. To verify this, we determined the binding of LAIR-1 to different maltose-binding protein fusion proteins containing the full-length catalytically inactive mutant (SHP-1M), tandem SH2 domains (2SH2), and N-terminal SH2 domain (N-SH2) of SHP-1. These fusion proteins and control maltose-binding protein immobilized on amylose resin beads were incubated for 1 h with extracts obtained from pervanadate-treated or non-treated Jurkat cells. Western blot analyses with anti-LAIR-1 and anti-phosphotyrosine revealed that only the full-length SHP-1M and the 2SH2 domain fusion proteins could efficiently bind LAIR-1 (Fig.5), whereas the fusion proteins with a single SH2 domain could not. This study indicates that SH2 domains of SHP-1 mediate the interaction with LAIR-1 and that the tandem SH2 domains of SHP-1 are both required for efficient binding. Our previous studies showed that the Cys-to-Ser mutant of SHP-1 displays dominant negative effects and causes hyperphosphorylation of specific cellular proteins on tyrosine (31.Zhao Z.J. Zhao R. J. Biol. Chem. 1998; 273: 29367-29372Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). To further reveal the specific interaction of LAIR-1 with SHP-1 inside the cells, LAIR-1c cDNA constructs were co-transfected into 293 cells with the plain vector or with dominant negative mutant SHP-1M(C-S). Cell lysates were prepared from the transfected cells, and Western blot analyses were performed with the cell extracts or anti-SHP-1 and anti-LAIR-1 immunoprecipitates. As shown in Fig. 6, strong tyrosine phosphorylation of a 40-kDa protein that corresponded to LAIR-1c was seen by direct Western blotting analysis of cell extracts obtained from cells co-transfected with LAIR-1c and SHP-1(C-S). Phosphorylation of this protein was not detected in cells co-transfected with LAIR-1c and the plain vector. This indicates that dominant negative SHP-1(C-S) induces tyrosine phosphorylation of LAIR-1c in intact cells, presumably by binding through the SH2 domain to the tyrosine phosphorylation site of LAIR-1, thereby preventing the latter from being dephosphorylated. Immunoprecipitation with LAIR-1 and SHP-1 antibodies further confirmed the results. The fact that SHP-1(C-S), but not native SHP-1, causes hyperphosphorylation of LAIR-1 also suggests that LAIR-1 is a physiological substrate of SHP-1in vivo. In the studies we have described so far, tyrosine phosphorylation of LAIR-1 was induced either by treating cells with pervanadate or by expressing the dominant negative mutant of SHP-1. As a cell surface molecule, tyrosine phosphorylation of LAIR-1 might be induced by its dimerization. To confirm this possibility, we investigated whether LAIR-1 is phosphorylated by anti-LAIR-1 antibody 148, which was raised against the extracellular portion of LAIR-1. Jurkat cells were pre-starved and then treated with 148 sera or 148 pre-immune sera. Cell lysates were precipitated by anti-SHP-1 or anti-LAIR-1 antibodies, and the precipitates were then subjected to Western blot analyses sequentially with anti-phosphotyrosine, anti-LAIR-1, and anti-SHP-1. As shown in Fig.7 A, upon stimulation with 148, strong tyrosine phosphorylation of LAIR-1 was detected that was accompanied by association of SHP-1 with LAIR-1. In contrast, cells treated with pre-immune serum displayed no tyrosine phosphorylation of LAIR-1, indicating that the antibody-induced phosphorylation is specific. Tyrosine phosphorylation of LAIR-1 resulted in the association of SHP-1, and the amount of SHP-1 co-precipitated with LAIR-1 correlated with the degree of LAIR-1 phosphorylation. We did not detect the association of LAIR-1 with other phosphatases, including SHP-2 and SHIP by Western blotting using specific antibodies. These results indicate that cross-linking of LAIR-1, with its antibodies against its extracellular segment, induced tyrosine phosphorylation of LAIR-1 and recruitment of SHP-1. When the time course of antibody-induced tyrosine phosphorylation of LAIR-1 was studied (Fig. 7 B), tyrosine phosphorylation of LAIR-1 was clearly observed at 5 min of stimulation, peaked at 10 min, and gradually decreased after 1 h. This transient tyrosine phosphorylation of LAIR-1 induced by its antibody is similar to that of growth factor and cytokine receptors induced by their ligands. SHP-1 stays inactive during the resting states of cells and is activated upon cell stimulation by binding to tyrosine-phosphorylated receptors on the plasma membrane. Since our data showed that anti-LAIR-1 148 induced tyrosine phosphorylation of LAIR-1 and resulted in recruitment of SHP-1, these changes should alter SHP-1 activity as well as its localization. To verify this, Jurkat cells were starved for 5 h, then stimulated with 148 or 148 pre-serum. Cell lysates were fractionated into cytosolic and membrane fractions by ultracentrifugation. Equal proportions of extracts were subjected to SDS-polyacrylamide gel electrophoresis and analyzed by Western blotting with anti-SHP-1. As shown in Fig. 8 A, most of the SHP-1 in pre-serum-treated Jurkat cells was distributed in the cytosolic fraction, with a marginal amount in the membrane fraction. However, about 30% of SHP-1 was found in the membrane fraction in anti-serum 148-treated cells. We further measured the activity of SHP-1 in the anti-LAIR-1 and anti-SHP-1 immunoprecipitates from the membrane fraction by using a 32P-labeled peptide substrate (Fig.8 B). Stimulation with LAIR-1 antibody caused a 5-fold increase in total SHP-1 activity in the membrane fraction. The majority of this activity is associated with tyrosine-phosphorylated LAIR-1. These results indicate that phosphorylation of LAIR-1 may produce a major SHP-1 activator in hematopoietic cells. In the present study, we have demonstrated that one of the major binding proteins of SHP-1 in hematopoietic cells corresponds to LAIR-1, and we have cloned two new isoforms of the protein. As an Ig-superfamily protein with two ITIMs, LAIR-1 specifically recruits SHP-1 but not SHP-2 or SHIP. This interaction is mediated by Tyr233 and Tyr263 embedded in the ITIMs that account for the entire tyrosine phosphorylation of LAIR-1. Furthermore, tyrosine phosphorylation of both sites is required to interact with SHP-1 through its SH2 domains. Although SHP-1 has been shown to bind a number of tyrosine-phosphorylated receptor proteins, in many cases the binding is mediated through a single SH2 domain, and the stoichiometry of binding is relatively low. In fact, the same binding sites may also mediate interaction with SHP-2. By binding through both SH2 domains, LAIR-1 confers great specificity to SHP-1. This is reminiscent of the specific interaction of PZR to SHP-2 (31.Zhao Z.J. Zhao R. J. Biol. Chem. 1998; 273: 29367-29372Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 32.Zhao R. Zhao Z.J. J. Biol. Chem. 2000; 275: 5453-5459Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Three-dimensional structures suggest mechanisms by which tandem SH2 domains may display higher specificities than individual SH2 domains (33.Hof P. Pluskey S. Dhe-Paganon S. Eck M.J. Shoelson S.E. Cell. 1998; 92: 441-450Abstract Full Text Full Text PDF PubMed Scopus (733) Google Scholar). In vitro studies with phosphopeptides reveal that tandem SH2 domains bind bisphosphotyrosyl peptides 20–50-fold stronger than individual SH2 domains (34.Ottinger E.A. Botfield M.C. Shoelson S.E. J. Biol. Chem. 1998; 273: 729-735Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Interestingly, one of the isoforms of LAIR-1, namely LAIR-1d, has no intracellular ITIMs and, thus, lacks the ability to bind SHP-1. We think that LAIR-1d may have a dominant negative role in the signaling mediated by ITIM-bearing isoforms of LAIR-1. The ITIM was initially defined by Burshtyn et al. (17.Burshtyn D.N. Scharenberg A.M. Wagtmann N. Rajagopalan S. Berrada K. Yi T. Kinet J.P. Long E.O. Immunity. 1996; 4: 77-85Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar, 36.Burshtyn D.N. Long E.O. Trends Cell Biol. 1997; 7: 473-479Abstract Full Text PDF PubMed Scopus (61) Google Scholar) as a V/IXYXXL consensus sequence. This motif is generally believed to play a negative role in signal transduction by recruiting terminating enzymes such as protein-tyrosine phosphatase SHP-1 and SHP-2 and inositol phosphatase SHIP (37.Unkeless J.C. Jin J. Curr. Opin. Immunol. 1997; 9: 338-343Crossref PubMed Scopus (126) Google Scholar, 38.Vely F. Vivier E. J. Immunol. 1997; 159: 2075-2077PubMed Google Scholar, 39.Isakov N. Immunol. Res. 1997; 16: 85-100Crossref PubMed Scopus (82) Google Scholar, 40.Long E.O. Annu. Rev. Immunol. 1999; 17: 875-904Crossref PubMed Scopus (840) Google Scholar). Recently, emergence of an increasing number of ITIM-bearing receptors has further emphasized the important regulatory role of this motif in various signal transduction pathways and subsequent cellular responses (41.Izzi L. Turbide C. Houde C. Kunath T. Beauchemin N. Oncogene. 1999; 18: 5563-5572Crossref PubMed Scopus (70) Google Scholar, 42.Taylor V.C. Buckley C.D. Douglas M. Cody A.J. Simmons D.L. Freeman S.D. J. Biol. Chem. 1999; 274: 11505-11512Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 43.Ulyanova T. Blasioli J. Woodford-Thomas T.A. Thomas M.L. Eur. J. Immunol. 1999; 29: 3440-3449Crossref PubMed Scopus (115) Google Scholar, 44.Newton-Nash D.K. Newman P.J. J. Immunol. 1999; 163: 682-688PubMed Google Scholar, 45.Adachi T. Flaswinkel H. Yakura H. Reth M. Tsubata T. J. Immunol. 1998; 160: 4662-4665PubMed Google Scholar). Prior studies have shown that LAIR-1 acts as an inhibitory receptor in hematopoietic cells (24.Meyaard L. Adema G.J. Chang C. Woollatt E. Sutherland G.R. Lanier L.L. Phillips J.H. Immunity. 1997; 7: 283-290Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 25.Meyaard L. Hurenkamp J. Clevers H. Lanier L.L. Phillips J.H. J. Immunol. 1999; 162: 5800-5804PubMed Google Scholar, 35.van der Vuurst de Vries A.R. Clevers H. Logtenberg T. Meyaard L. Eur. J. Immunol. 1999; 29: 3160-3167Crossref PubMed Scopus (78) Google Scholar), and we believe that its inhibitory function is mediated by recruiting SHP-1. As a major negative regulator of hematopoietic cell signaling, SHP-1 remains in an inactive state in resting cells and is activated by binding to tyrosine-phosphorylated proteins on the plasma membrane (19.Zhao Z. Bouchard P. Diltz C.D. Shen S.H. Fischer E.H. J. Biol. Chem. 1993; 268: 2816-2820Abstract Full Text PDF PubMed Google Scholar). Activation of SHP-1 is important because it presumably turns off signal transduction, which may lead to cellular growth arrest. In fact, motheaten and viable motheaten mice develop a severe autoimmune and immunodeficiency syndrome characterized by an extremely high proliferation rate of all hematopoietic cells due to disruption of SHP-1 activity (7.Tsui H.W. Siminovitch K.A. Souza L. Tsui F.W.L. Nat. Genet. 1993; 4: 124-129Crossref PubMed Scopus (514) Google Scholar, 8.Shultz L.D. Schweitzer P.A. Rajan T.V. Yi T. Ihle J.N. Matthews R.J. Thomas M.L. Beier D.R. Cell. 1993; 73: 1445-1454Abstract Full Text PDF PubMed Scopus (686) Google Scholar). Widely expressed in hematopoietic cells, including T, B, NK, mast, and myeloid cells (24.Meyaard L. Adema G.J. Chang C. Woollatt E. Sutherland G.R. Lanier L.L. Phillips J.H. Immunity. 1997; 7: 283-290Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar), LAIR-1 may be a major upstream activator of SHP-1 and, thereby, modulate hematopoiesis. Although SHP-1 is believed to be a potential target for therapeutic drug development, how to specifically induce its activation has remained a major challenge. By treating cells with anti-LAIR-1 antibody, we have provided a way to specifically activate SHP-1. The physiological meaning of the antibody-induced LAIR-1 phosphorylation, with concurrent activation of SHP-1, are under study and may have therapeutic implications. We are grateful to Dr. Sanford B. Krantz for his critical review of the manuscript.