Leukocyte-associated Ig-like receptor-1 has SH2 domain-containing phosphatase-independent function and recruits C-terminal Src kinase

LAIR-1 has inhibitory function independent of SHP-1 and SHP-2

LAIR-1 recruits both SHP-1 and SHP-2 4, 13–15. To determine the contribution of each phosphatase in LAIR-1 signaling, we investigated the function of LAIR-1 in the chicken B cell line DT40. DT40 mutants have been generated that lack SHP-1, SHP-2 or both phosphatases (19 and Fig. 1A). We stably transfected the DT40 cells with an mFcγRIIB/hLAIR-1 chimera that contained the extracellular and transmembrane domains of mFcγRIIB and the intracellular domain of LAIR-1. The mFcγRIIB/hLAIR-1 chimera can be co-ligated to the BCR by cross-linking the BCR with mouse IgM anti-chicken IgM in combination with intact rabbit Ig anti-mouse IgM, which will also bind to the FcγRIIB moiety of the chimera through the Fc part. No co-ligation of the chimera occurs when rabbit F(ab’)₂ anti-mouse IgM is used as a secondary antibody 20, 21. The expression of the mFcγRIIB/hLAIR-1 chimera was similar on all clones (Fig. 1B).

Using this system, we found that signaling by LAIR-1 inhibited BCR-induced calcium mobilization (Fig. 2A). The inhibitory function depended on the LAIR-1 ITIM, as mutation of the tyrosines within these ITIM abrogated the inhibition of calcium mobilization (Fig. 2B). We next investigated the contribution of each phosphatase to LAIR-1-mediated inhibition. In DT40 cells that were deficient for either SHP-1 or SHP-2, LAIR-1 inhibited the calcium mobilization to a similar extent as in wild-type cells (Fig. 2C, D), suggesting that SHP-1 and SHP-2 have redundant functions in LAIR-1 signaling. Surprisingly, LAIR-1 still had an inhibitory effect on BCR-induced calcium mobilization in cells lacking both phosphatases (Fig. 2E), indicating that LAIR-1 has a phosphatase-independent inhibitory function.

SHP-1 and SHP-2 exert their inhibitory function by dephosphorylation of key molecules involved in cellular activation 22. We therefore investigated the effect of LAIR-1-mediated signaling on BCR-induced tyrosine phosphorylation. Cross-linking of the chicken BCR resulted in the phosphorylation of a number of proteins, which was inhibited upon co-ligation of the mFcγRIIB/hLAIR-1 chimera (Fig. 3, left panel). Again, the inhibitory function depended on phosphorylation of the ITIM, as the LAIR-1 mutant lacking functional ITIM had no effect on tyrosine phosphorylation (Fig. 3, middle panel). However, in the absence of SHP-1 and SHP-2, co-ligation of the wild-type mFcγRIIB/hLAIR-1 chimera still reduced BCR-induced tyrosine phosphorylation. Thus, the ITIM of LAIR-1 are able to recruit at least one other molecule that mediates the inhibition of protein tyrosine phosphorylation and cellular activation.

LAIR-1 does not recruit SHIP

An alternative molecule involved in the signaling of several ITIM-bearing receptors is the 5′-inositol phosphatase SHIP. To investigate whether SHIP binds to phosphorylated LAIR-1, we transfected 293T cells with FLAG-tagged LAIR-1 (Fig. 4A). FLAG-tagged human FcγRIIB was used as a positive control 23, 24. The cells were treated with pervanadate to induce extensive phosphorylation of the receptors 15, and the FLAG-tagged proteins were immunoprecipitated. While a significant amount of SHIP co-immunoprecipitated with FcγRIIB, no SHIP was found in association with LAIR-1 (Fig. 4B). In agreement with this observation, the mFcγRIIB/hLAIR-1 chimera inhibited BCR-induced calcium mobilization in SHIP-deficient DT40 cells (Fig. 4C, D), while co-ligation of wild-type FcγRIIB has no inhibitory effect in these cells 20. Thus, we conclude that SHIP does not play a role in LAIR-1-mediated signaling.

Human and mouse LAIR-1 bind Csk

To identify proteins that selectively bind to phosphorylated LAIR-1, we performed a yeast-tri-hybrid screen with a human fetal brain library using the intracellular tail of human LAIR-1 as bait. We used the Ylck4.1 strain, in which expression of Lck can be induced, enabling the identification of phosphotyrosine-dependent interactions. Protein interactions were detected by the expression of the β-galactosidase reporter gene. We identified two clones that were positive in the reporter assay. Both clones contained a segment of Csk carrying the SH2 domain and the kinase domain (amino acids 84–450). This Csk fragment bound to LAIR-1 in a phosphorylation-dependent manner, as the yeast cells did not show β-galactosidase activity when grown on media containing methionine, which suppresses Lck expression. The intracellular tail of mouse LAIR-1 (mLAIR-1) also bound Csk in yeast (Fig. 5A).

To confirm the interaction of Csk with LAIR-1 in eukaryotic cells, we performed co-immunoprecipitation studies in rat basophilic leukemia (RBL) clones stably expressing human LAIR-1 or a human LAIR-1/mouse LAIR-1 chimera. Both molecules were selectively co-immunoprecipitated using anti-Csk antibodies (Fig. 5B). As the interaction between LAIR-1 and Csk is phosphorylation dependent in yeast, our results suggest that there must be constitutive LAIR-1 phosphorylation in RBL cells. Indeed, we have found that in unstimulated RBL cells, although barely detectable, there is phosphorylation of LAIR-1. This is sufficient for a low level of SHP-1 recruitment, which requires the phosphorylation of the ITIM (15 and data not shown). As shown below, Csk did not interact with the LAIR-1 mutant in which both tyrosines were mutated, indicating that the interaction with Csk also requires phosphorylation of LAIR-1 in RBL cells.

The interaction of Csk with LAIR-1 requires the SH2 domain of Csk and the tyrosine residue in the N-terminal ITIM of LAIR-1

Csk consists of an SH3 domain, an SH2 domain and a kinase domain. The Csk fragment that was obtained in the yeast-tri-hybrid screen contained the SH2 domain and the kinase domain. As the interaction of Csk with LAIR-1 is phosphotyrosine dependent, we investigated whether Csk binds to LAIR-1 through its SH2 domain. We therefore transformed yeast cells, stably expressing the Gal4 binding domain-LAIR-1 fusion protein, with Gal4 activation domain fusion proteins of either full-length Csk, Csk lacking the 83 N-terminal amino acids containing the SH3 domain (CskΔSH3) or Csk lacking the SH2 domain (CskΔSH2). As shown in Fig. 6A, full-length Csk and CskΔSH3 bound to LAIR-1, while CskΔSH2 did not. This indicates that indeed the SH2 domain of Csk binds to phosphorylated LAIR-1.

We have previously shown that the ITIM of LAIR-1 contribute differentially to the recruitment of SHP-1 and SHP-2 15. To investigate which tyrosine is required for the interaction with Csk, we performed co-immunoprecipitations in RBL cells stably expressing wild-type LAIR-1 or mutants of LAIR-1 with tyrosine-to-phenylalanine mutations in the N-terminal ITIM (FY), the C-terminal ITIM (YF) or both (FF). The expression level of each LAIR-1 mutant was similar 15. The mutant in which the C-terminal ITIM was mutated still bound Csk, while mutation of the N-terminal ITIM abrogated Csk binding (Fig. 6B). Thus, the SH2 domain of Csk binds to the phosphorylated tyrosine residue in the N-terminal ITIM of LAIR-1.

cDNA constructs

Human LAIR-1 and LAIR-1 containing tyrosine-to-phenylalanine mutations in the ITIM have been described before 4, 15. The mFcγRIIB/hLAIR-1 chimera containing the extracellular and transmembrane domain of mFcγRIIB and the intracellular domain of LAIR-1 (amino acids 187–289) was generated by PCR as previously described for the mFcγRIIB/PECAM-1 chimera 21 and cloned into the pMx expression vector. Human FcγRIIB cDNA has been described before 39. N-terminal FLAG-tagged FcγRIIB and LAIR-1 were generated by cloning the cDNA to the 3′ site of the FLAG sequence in the pMx vector (generated at the DNAX Research Institute, Palo Alto, CA). cDNA encoding human Csk was generated by Neet et al. 40. cDNA encoding Csk lacking the SH2 domain (amino acids 80–163, CskΔSH2) or the SH2 domain of Csk alone (amino acids 77–172) were generated by PCR.

Cell lines and culture

The DT40 chicken B cell line and SHP1^–/–, SHP2^–/–, SHP1,2^–/– and SHIP^–/– DT40 cells have been described before 19, 20 and were purchased from RIKEN Cell Bank (Tsukuba Science City, Japan). The DT40 cells were grown on RPMI 1640 (Gibco, Paisley, UK) supplemented with 10% fetal calf serum (Integro, Dieren, The Netherlands), 1% chicken serum (Sigma), 4 mM glutamine (Gibco), 50 μM β-mercaptoethanol and antibiotics. To generate DT40 cells expressing the mFcγRIIB/hLAIR-1 chimera, the cells were transfected by electroporation. The cells were grown on selective media and cloned by the limiting dilution method, 24 h after transfection. To confirm expression of the chimera, the cells were stained with FITC-conjugated anti-mFcγRIIB 2.4G2 antibodies or FITC-conjugated isotype control (Pharmingen BD) and analyzed by flow cytometry.

RBL-2H3 is a rat basophilic leukemia cell line 41. RBL transfectants stably expressing either wild-type LAIR-1, LAIR-1 mutants or a hLAIR-1/mLAIR-1 chimera have been described previously 15, 16. Human embryonic kidney 293T cells were obtained from American Type Culture Collection (Manassas, VA). 293T cells, and RBL cells were grown in RPMI 1640 (Gibco) supplemented with 10% fetal calf serum (Integro) and antibiotics.

Antibodies

Polyclonal anti-chicken SHP-1 antibodies were a generous gift of Dr. T. Kurosaki (Kansai Medical University, Moriguchi, Japan). Polyclonal anti-SHP-2 and PLCγ2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The PY20 anti-phosphotyrosine antibody was purchased from BD Transduction Laboratories (Franklin Lakes, NJ). Mouse anti-chicken IgM mAb M4 was purchased from Southern Biotechnology Associates (Birmingham, AL). Intact and F(ab’)₂ rabbit anti-mouse IgM were obtained from Zymed (San Francisco, CA). Polyclonal anti-SHIP antibodies were kindly provided by Dr. J. Cambier (University of Colorado Health Sciences Center, Denver, CO). Monoclonal mouse IgG1 antibody directed against human LAIR-1, 8A8, has been described before 16. For immunoprecipitation polyclonal rabbit anti-Csk antibodies were used (purchased from Santa Cruz Biotechnology). For Western blot monoclonal anti-Csk antibody (BD Transduction Laboratories) was used. Horseradish peroxidase (HRP)-conjugated rabbit anti-mouse antibody (DAKO) or HRP-conjugated goat anti-rabbit antibody (Pierce) were used as secondary antibodies in Western blotting.

Calcium mobilization assay

DT40 cells (10⁷/mL) were loaded with 3 μM Fluo 4-AM (Molecular Probes, Eugene, OR) in PBS for 20 min at 37°C. The cells were washed and resuspended in PBS supplemented with 1 mM MgCl₂ and 1 mM CaCl₂ . Triggering of the B cell receptor (BCR) with or without co-ligation of the mFcγRIIB/hLAIR-1 chimera was performed as described previously 20, 21. Co-ligation of the mFcγRIIB/hLAIR-1 chimera is achieved by cross-linking the FcγRIIB moiety of the chimera by the Fc part of the intact antibody used to cross-link the BCR, while F(ab’)₂ fragments do not cross-link the chimera. Briefly, the cells were incubated with intact or F(ab’)₂ rabbit anti-mouse IgM (final concentrations 9.5 μg/mL and 6.3 μg/mL respectively) for 5 min at room temperature. Mouse IgM anti-chicken IgM was added (clone M4, final concentration 25 μg/mL) and the fluorescence intensity was measured with 10-s intervals in the Fluoroskan Ascent (Thermo Labsystems, Franklin, MA) with excitation by 485 nm and emission at 527 nm.

Yeast-tri-hybrid assay

The yeast strain Ylck4.1 42 and the pYTH9B vector were generously provided by Dr K. Fuller (GlaxoSmithKline, UK). cDNA encoding the intracellular parts of human and mouse LAIR-1 (amino acids 187–287 and 165–263, respectively) were cloned in frame to the DNA binding domain of Gal4 in the pYTH9B vector. Stable transformants were generated by integration of the constructs into the Trp locus.

For screening, yeast cells stably expressing the BD-hLAIR-1 fusion protein were transformed with 50 μg human fetal brain library in the pACT2 vector (BD Biosciences) using a lithium acetate/Tris EDTA/polyethylene glycol protocol 42. Transformants were grown on selective media containing 50 mM 3-amino-1,2,4-triazole (Sigma) for 10–14 days. Large colonies were re-streaked on selective plates, grown for 48 h and filter-lifted on Hybond-N membranes (Amersham). β-Galactosidase activity was determined by a freeze-thaw fracture assay. Yeast clones that turned blue in this assay were considered as possible positives. Library DNA was recovered by mechanically disrupting the yeast cells with acid-washed glass beads. To exclude false positives, the recovered library DNA was transformed into yeast cells that expressed the DNA binding domain of Gal4 alone. The proteins that interacted selectively with LAIR-1 were then identified by automated DNA sequencing of the library DNA.

For Csk interaction studies in yeast, full-length Csk, CskΔSH2 and the SH2 domain were cloned in frame to the activation domain of Gal4 in the pACT2 vector (BD Biosciences). Of these constructs or the Csk construct obtained in the screen (CskΔSH3), 1 μg was transformed in yeast cells expressing the DNA binding domain/hLAIR-1 or DNA binding domain/mLAIR-1 fusion protein. Three colonies were spotted on media and grown in the presence or absence of methionine for 48 h. Interactions were identified by the β-galactosidase assay as described above.

Immunoprecipitation and Western blot analysis

To assess the expression of SHP-1 in the DT40 cell lines, the cells were treated with 50 μM pervanadate and lysed in TNE lysis buffer (50 mM Tris-HCl, pH 8, 1% Nonidet P-40, 2 mM EDTA, 0.02% sodium azide) supplemented with protease inhibitors (complete mini EDTA-free protease inhibitor cocktail tablets, Roche, Mannheim, Germany) and 1 mM phenylmethylsulfonyl fluoride. Protein A/G PLUS-agarose beads (Santa Cruz Biotechnologies) were coated with anti-chicken SHP-1 antibody and immunoprecipitation was performed for 90 min in the presence of 0.5% BSA. Immune complexes were washed three times with lysis buffer supplemented with 1 mM phenylmethylsulfonyl fluoride and boiled in non-reducing Laemmli sample buffer. Western blotting was performed and phosphorylated SHP-1 was detected by anti-phosphotyrosine antibodies. To assess the expression of SHP-2, the cells were directly lysed in Laemmli sample buffer and equal amounts of cell lysate were analyzed by Western blotting.

To investigate the LAIR-1-mediated effects on BCR-induced tyrosine phosphorylation, DT40 cells were incubated with intact or F(ab’)₂ rabbit anti-mouse IgM, followed by mouse IgM anti-chicken IgM as described for the calcium mobilization assay and left at 37°C for 1 min. The cells were immediately washed in ice-cold PBS containing 250 μM orthovanadate and lysed in non-reducing Laemmli sample buffer.

For the identification of LAIR-1/Csk interactions, RBL cells stably transfected with LAIR-1 or LAIR-1 mutants were washed in PBS and 25 × 10⁶ cells were lysed in TNE lysis buffer. Immunoprecipitation was performed as described above using polyclonal anti-Csk antibodies.

To investigate binding of SHIP to LAIR-1 293T cells were transfected with FLAG-tagged LAIR-1 or FcγRIIB using FuGENE 6 (Roche) as transfection agent. To confirm expression of the FLAG-tagged receptors the cells were stained with M2 anti-FLAG antibody (Sigma) followed by phycoerythrin (PE)-conjugated goat anti-mouse IgG (Southern Biotechnology Associates) and analyzed by flow cytometry. After 48 h, the cells were treated with 50 μM pervanadate and lysed in Triton lysis buffer (containing 1% Triton X-100, 10 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.02% sodium azide) supplemented with protease inhibitors as described above. Immunoprecipitation was performed as described before 15 using precoated beads with anti-FLAG antibodies (Sigma). The samples were boiled in reducing Laemmli sample buffer.

Proteins were resolved by SDS-polyacrylamide gel electrophoresis and transferred to Immobilon-P membranes (Millipore, Bedford, MA). Western blot analyses were performed with anti-phosphotyrosine, anti-SHP-2, anti-PLCγ2, anti-SHIP, 8A8 anti-LAIR-1 or monoclonal anti-Csk antibodies, followed by HRP-linked secondary antibodies. Proteins were detected by enhanced chemiluminescence (Amersham, Little Chalfort, UK).