HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 275, Issue 23, 17440-17446, June 9, 2000

This Article Abstract Full Text (PDF) All Versions of this Article: 275/23/17440    most recent M001313200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Xu, M.-j. Articles by Zhao, Z. J. Search for Related Content PubMed PubMed Citation Articles by Xu, M.-j. Articles by Zhao, Z. J. Social Bookmarking                      What's this?

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

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

From the Hematology/Oncology Division, Department of Medicine, Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, Tennessee 37232-6305

Received for publication, February 15, 2000, and in revised form, April 6, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 (1-6). 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, 8). This is consistent with the negative role of SHP-1 in cell signaling mediated by interleukin-3 receptor (9), c-Kit (10), erythropoietin receptor (11), interferon-/ receptor (12), colony-stimulating factor-1 (CSF-1) receptor (13), B-cell antigen receptor (14), T-cell antigen receptor (15, 16), NK cell inhibitory receptor, and CD22 (17, 18).

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). Studies by others and us demonstrated that SHP-1 can be activated by anionic phospholipids in vitro or by binding to tyrosine-phosphorylated receptors and other proteins concomitant with translocation of SHP-1 to the plasma membrane in cells (20-23). 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 (9-18). 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 (9-18).

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, 25). 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- 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). 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). 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 -glycerolphosphate (pH 7.3), 10 mM EDTA, 2 mM EGTA, 1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 1 µM pepstatin A, and 1 µg/ml aprotinin.

Molecular Cloning of LAIR-1-- 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). 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 with EcoRV. The cDNA clones were sequenced using the automated DNA sequencing facilities of the Vanderbilt-Ingram Cancer Center.

Production of Anti-LAIR-1 Antibodies-- The intracellular and extracellular portions of LAIR-1c (see below), corresponding to amino acid residues 170-269 and 1-141, respectively, were expressed in Escherichia 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.

Expression of LAIR-1 in 293 Cells and Stimulation of Cells-- 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). 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 0.1 M sodium vanadate and 0.2 M H₂O₂ and incubating at room temperature for 20 min before the addition to the cells (29). 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.

Cell Extraction, Immunoprecipitation, and Western Blotting-- 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 0.15 M 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).

Fractionation of Cell Extracts and PTP Activity Assays-- Cells were lysed in Buffer A by using a Dounce homogenizer (23). 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).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

SHP-1 Is Associated with Two Major Tyrosine-phosphorylated Proteins in Hematopoietic Cells-- 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). 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). 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.

View larger version (93K): [in this window] [in a new window]   Fig. 1.   SHP-1 is associated with two major tyrosine-phosphorylated proteins in hematopoietic cells, and one of the proteins corresponds to LAIR-1. Human primary T cells, Jurkat cells, and HL-60 cells were treated with 0.1 mM pervanadate for 30 min. Cell extracts were immunoprecipitated with anti-SHP-1 antibody, and the immunoprecipitates were analyzed by immunoblotting with antibodies against phosphotyrosine (Anti-PY), SHP-1, and LAIR-1 as indicated. IgG denotes the heavy chain of immunoglobulin G.

Identification of the SHP-1 Binding 40-kDa and 45-kDa Proteins as LAIR-1-- 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, FcRIIB (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, 25). Since antibody for Western blotting analyses of LAIR-1 was not available, we first generated an antibody in rabbits by using glutathione S-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.

View larger version (79K): [in this window] [in a new window]   Fig. 2.   Specific association of LAIR-1 with SHP-1. Human primary T cells, Jurkat cells, and HL-60 cells were treated with pervanadate as described in Fig. 1. Cell extracts were immunoprecipitated with anti-LAIR-1 antibody, and the immunoprecipitates were analyzed by immunoblotting with antibodies against phosphotyrosine (Anti-PY), SHP-1, and LAIR-1 as indicated. The positions of SHP-1 and LAIR-1 are shown.

Molecular Cloning of Four Isoforms of LAIR-1-- We then tried to clone LAIR-1 by using polymerase chain reaction with primers designed according to the published LAIR-1 sequence (24). 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^TM 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.

View larger version (47K): [in this window] [in a new window]   Fig. 3.   Protein sequence alignment of LAIR-1 a, b, c, and d. Colons and minus signs denote identical amino acid residues and gaps, respectively.

Tyr²³³ and Tyr²⁶³ Are the Tyrosine Phosphorylation Sites of LAIR-1, and Both Are Required for Binding of SHP-1-- 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.

View larger version (56K): [in this window] [in a new window]   Fig. 4.   Tyr233 and Tyr263 of LAIR-1 are the tyrosine phosphorylation sites, and both are required for binding of SHP-1. Human 293 cells were transfected with the pCDNA3 vector or constructs containing LAIR-1c, LAIR-1cF233, LAIR-1cF263, and LAIR-1cF233/F263. Cells were treated with 0.1 mM pervanadate for 30 min. Cells extracts were immunoprecipitated with anti-LAIR-1, and the immunoprecipitates (IP) were subjected to Western blot analyses with anti-phosphotyrosine (Anti-PY), anti-LAIR-1, and anti-SHP-1 as indicated. The last lane on the right shows inhibition of pervanadate-induced tyrosine phosphorylation of LAIR-1 by Src family kinase inhibitor PP1 (20 µM).

SH2 Domains of SHP-1 Are Required for Binding to LAIR-1-- 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.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   SH2 domains of SHP-1 mediate its association with LAIR-1. Maltose-binding protein (MBP) and maltose-binding protein fusion proteins containing catalytically inactive SHP-1 mutant (SHP-1M), N-terminal SH2 domain (N-SH2), and tandem SH2 domain (2SH2) of SHP-1 were immobilized onto amylose resin beads and incubated with cell extracts obtained from Jurkat cells treated with 0.1 mM pervanadate. The beads were washed with Buffer A supplemented with 0.1% Triton X-100 and 0.15 M NaCl. This was followed by Western blot analyses with anti-LAIR-1.

LAIR-1 Is Hyper-phosphorylated on Tyrosyl Residues in 293 Cells Overexpressing a Catalytically Inactive Mutant Form of SHP-1-- 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). 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-1 in vivo.

View larger version (50K): [in this window] [in a new window]   Fig. 6.   Expression of a catalytically inactive Cys-to-Ser mutant of SHP-1 (SHP-1M) causes hyperphosphorylation of LAIR-1. Human 293 cells were transfected with pCDNA3-LAIR-1c plus pCDNA3 vector or pCDNA3-SHP-1M. The extracts were immunoprecipitated with anti-LAIR-1 or anti-SHP-1. The extracts and the immunoprecipitates (IP) were subjected to Western blot analyses with anti-phosphotyrosine (Anti-PY), anti-LAIR-1, and anti-SHP-1 as indicated.

LAIR-1 Is Tyrosine-phosphorylated upon Cross-linking with an Anti-LAIR-1 Antibody-- 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. 7A, 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.

View larger version (45K): [in this window] [in a new window]   Fig. 7.   Anti-LAIR-1 antibody 148 induces tyrosine phosphorylation of LAIR-1. Jurkat cells were serum-starved and then treated with a 1:1000 dilution of anti-serum 148 or pre-immune serum for 10 min (A) or with anti-serum 148 for the indicated periods of time (B). Cell extracts were immunoprecipitated with anti-LAIR-1 or anti-SHP-1. The immunoprecipitates (IP) were analyzed by Western blotting with anti-phosphotyrosine (Anti-PY), anti-SHP-1, or anti-LAIR-1 as indicated.

When the time course of antibody-induced tyrosine phosphorylation of LAIR-1 was studied (Fig. 7B), 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.

Stimulation of Cells with Anti-LAIR-1 Antibody Induces Translocation and Activation of SHP-1-- 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. 8A, 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. 8B). 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.

View larger version (28K): [in this window] [in a new window]   Fig. 8.   Treatment of Jurkat cells with anti-LAIR-1 antibody 148 induces translocation and activation of SHP-1. Jurkat cells were treated as described in Fig. 7. Cells were homogenized and fractionated into cytosolic and membrane fractions. Equal proportions of extracts were analyzed by immunoblotting with anti-SHP-1 (A). Equal amounts of membrane extracts were immunoprecipitated with anti-LAIR-1 and anti-SHP-1, and activities of SHP-1 in the immunoprecipitates (IP) were measured using the 32P-labeled ENDpYINASL (where pY is phosphotyrosine) peptide as a substrate (B).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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, 32). Three-dimensional structures suggest mechanisms by which tandem SH2 domains may display higher specificities than individual SH2 domains (33). In vitro studies with phosphopeptides reveal that tandem SH2 domains bind bisphosphotyrosyl peptides 20-50-fold stronger than individual SH2 domains (34). 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, 36) 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 (37-40). 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 (41-45). Prior studies have shown that LAIR-1 acts as an inhibitory receptor in hematopoietic cells (24, 25, 35), 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). 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, 8). Widely expressed in hematopoietic cells, including T, B, NK, mast, and myeloid cells (24), 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.

	ACKNOWLEDGEMENT

We are grateful to Dr. Sanford B. Krantz for his critical review of the manuscript.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants HL-57393, CA75218 (to Z. J. Z), and CA-68485 (to Vanderbilt-Ingram Cancer Center).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF251509 and AF251510.

To whom correspondence should be addressed: Hematology/Oncology Division, Dept. of Medicine, 547, MRB II, 2220 Pierce Ave., Nashville, TN 37232-6305. Tel.: 615-936-1797; Fax: 615-936-3853; E-mail: joe.zhao@mcmail.vanderbilt.edu.

Published, JBC Papers in Press, April 10, 2000, DOI 10.1074/jbc.M001313200

	ABBREVIATIONS

The abbreviations used are: PTP, protein-tyrosine phosphatase; LAIR-1, leukocyte-associated Ig-like receptor-1; ITIM, immunoreceptor tyrosine-based inhibitory motif.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				L. Meyaard The inhibitory collagen receptor LAIR-1 (CD305) J. Leukoc. Biol., April 1, 2008; 83(4): 799 - 803. [Abstract] [Full Text] [PDF]

				N. Tedla, C.-W. Lee, L. Borges, C. L. Geczy, and J. P. Arm Differential expression of leukocyte immunoglobulin-like receptors on cord blood-derived human mast cell progenitors and mature mast cells J. Leukoc. Biol., February 1, 2008; 83(2): 334 - 343. [Abstract] [Full Text] [PDF]

				D. Imhof, A.-S. Wavreille, A. May, M. Zacharias, S. Tridandapani, and D. Pei Sequence Specificity of SHP-1 and SHP-2 Src Homology 2 Domains: CRITICAL ROLES OF RESIDUES BEYOND THE pY+3 POSITION J. Biol. Chem., July 21, 2006; 281(29): 20271 - 20282. [Abstract] [Full Text] [PDF]

				V. C. J. Fawcett and U. Lorenz Localization of Src Homology 2 Domain-Containing Phosphatase 1 (SHP-1) to Lipid Rafts in T Lymphocytes: Functional Implications and a Role for the SHP-1 Carboxyl Terminus J. Immunol., March 1, 2005; 174(5): 2849 - 2859. [Abstract] [Full Text] [PDF]

				R. J. Lebbink, T. de Ruiter, A. Verbrugge, W. S. Bril, and L. Meyaard The Mouse Homologue of the Leukocyte-Associated Ig-Like Receptor-1 Is an Inhibitory Receptor That Recruits Src Homology Region 2-Containing Protein Tyrosine Phosphatase (SHP)-2, but Not SHP-1 J. Immunol., May 1, 2004; 172(9): 5535 - 5543. [Abstract] [Full Text] [PDF]

				A. Verbrugge, T. d. Ruiter, H. Clevers, and L. Meyaard Differential contribution of the immunoreceptor tyrosine-based inhibitory motifs of human leukocyte-associated Ig-like receptor-1 to inhibitory function and phosphatase recruitment Int. Immunol., November 1, 2003; 15(11): 1349 - 1358. [Abstract] [Full Text] [PDF]

				R. Zhao, X. Fu, L. Teng, Q. Li, and Z. J. Zhao Blocking the Function of Tyrosine Phosphatase SHP-2 by Targeting Its Src Homology 2 Domains J. Biol. Chem., October 31, 2003; 278(44): 42893 - 42898. [Abstract] [Full Text] [PDF]

				G. Ferjoux, F. Lopez, J.-P. Esteve, A. Ferrand, E. Vivier, F. Vely, N. Saint-Laurent, L. Pradayrol, L. Buscail, and C. Susini Critical Role of Src and SHP-2 in sst2 Somatostatin Receptor-mediated Activation of SHP-1 and Inhibition of Cell Proliferation Mol. Biol. Cell, September 1, 2003; 14(9): 3911 - 3928. [Abstract] [Full Text] [PDF]

				R. Zhao, A. Guerrah, H. Tang, and Z. J. Zhao Cell Surface Glycoprotein PZR Is a Major Mediator of Concanavalin A-induced Cell Signaling J. Biol. Chem., March 1, 2002; 277(10): 7882 - 7888. [Abstract] [Full Text] [PDF]

				E. C. J. M. de Vet, B. Aguado, and R. D. Campbell G6b, a Novel Immunoglobulin Superfamily Member Encoded in the Human Major Histocompatibility Complex, Interacts with SHP-1 and SHP-2 J. Biol. Chem., November 2, 2001; 276(45): 42070 - 42076. [Abstract] [Full Text] [PDF]

				M. W.-C. Su, C.-L. Yu, S. J. Burakoff, and Y.-J. Jin Targeting Src Homology 2 Domain-Containing Tyrosine Phosphatase (SHP-1) into Lipid Rafts Inhibits CD3-Induced T Cell Activation J. Immunol., March 15, 2001; 166(6): 3975 - 3982. [Abstract] [Full Text] [PDF]

				J. G. Sathish, K. G. Johnson, K. J. Fuller, F. G. LeRoy, L. Meyaard, M. J. Sims, and R. J. Matthews Constitutive Association of SHP-1 with Leukocyte-Associated Ig-Like Receptor-1 in Human T Cells J. Immunol., February 1, 2001; 166(3): 1763 - 1770. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 275/23/17440    most recent M001313200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Xu, M.-j. Articles by Zhao, Z. J. Search for Related Content PubMed PubMed Citation