Src Homology 2 Domain-Containing -Tyrosine Phosphatases, SHP-1 and SHP-2, Are Required for Endothelial Molecule-1/CD31-Mediated This information is current as Inhibitory Signaling of September 30, 2021. Tanya L. Henshall, Karen L. Jones, Ray Wilkinson and Denise E. Jackson J Immunol 2001; 166:3098-3106; ; doi: 10.4049/jimmunol.166.5.3098 Downloaded from http://www.jimmunol.org/content/166/5/3098

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Src Homology 2 Domain-Containing Protein-Tyrosine Phosphatases, SHP-1 and SHP-2, Are Required for Platelet Endothelial -1/CD31-Mediated Inhibitory Signaling1

Tanya L. Henshall, Karen L. Jones, Ray Wilkinson, and Denise E. Jackson2

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a newly assigned member of the Ig immunoreceptor tyrosine- based inhibitory motif superfamily, and its functional role is suggested to be an inhibitory that modulates immunore- ceptor tyrosine-based activation motif-dependent signaling cascades. To test whether PECAM-1 is capable of delivering inhibitory signals in B cells and the functional requirement of protein-tyrosine phosphatases (PTPs) for this inhibitory signaling, we gen- erated chimeric Fc␥RIIB1-PECAM-1 receptors containing the extracellular and transmembrane portions of murine Fc␥RIIB1 Downloaded from and the cytoplasmic domain of human PECAM-1. These chimeric receptors were stably expressed in chicken DT40 B cells either as wild-type or mutant cells deficient in SHP-1؊/؊, SHP-2؊/؊, SHIP؊/؊, or SHP-1/2؊/؊ and then assessed for their ability to inhibit Ag receptor (BCR) signaling. Coligation of wild-type Fc␥RIIB1-PECAM-1 with BCR resulted in inhibition of intracellular calcium release, suggesting that the cytoplasmic domain of PECAM-1 is capable of delivering an inhibitory signal that blocks BCR-mediated activation. This PECAM-1-mediated inhibitory signaling correlated with tyrosine phosphorylation of the http://www.jimmunol.org/ Fc␥RIIB1-PECAM-1 chimera, recruitment of SHP-1 and SHP-2 PTPs by the phosphorylated chimera, and attenuation of calcium mobilization responses. Mutational analysis of the two tyrosine residues, 663 and 686, constituting the immunoreceptor tyrosine- based inhibitory motifs in PECAM-1 revealed that both tyrosine residues play a crucial role in the inhibitory signal. Functional analysis of various PTP-deficient DT40 B cell lines stably expressing wild-type chimeric Fc␥RIIB1-PECAM-1 receptor indicated that cytoplasmic Src homology 2-domain-containing phosphatases, SHP-1 and SHP-2, were both necessary and sufficient to deliver inhibitory negative regulation upon coligation of BCR complex with inhibitory receptor. The Journal of Immunology, 2001, 166: 3098–3106. by guest on September 30, 2021 mmune cells recognize and respond to Ag-mediated activa- ExxYxxL/Ix6–8YxxL/I) are essential for initiating a cascade of tion by coaggregation of immunoreceptors such as B and T intracellular biochemical events, including activation of p60Src and I Ag receptors (BCR3 and TCR) and FcR. Liga- p72Syk protein-tyrosine kinases, activation of phosphatidylinositol tion of BCR Ag receptor initiates a cascade of intracellular sig- 3-kinase and phospholipase C-␥, generation of phosphoinositide naling pathways that directs biological responses, including B cell turnover, and calcium mobilization. Balancing the threshold of cel- proliferation, differentiation, Ab secretion, cell survival, or death lular activation and termination of BCR-mediated signals is prin- (for reviews, see Refs. 1–3). The BCR complex is composed of a cipally mediated by coligation of inhibitory receptors such as heterooligomeric complex of surface Ig-, IgM-, and immunorecep- Fc␥RIIB1, paired Ig-like receptor (PIR-B), Ig-like transcript ILT2, tor tyrosine-based activation motif (ITAM)-containing signaling biliary glycoprotein BGP-1 (CD66), and CD22 (4). These Ig im- components, Ig␣ and Ig␤ subunits. These ITAM motifs (D/ munoreceptor tyrosine-based inhibitory motif (ITIM) receptors be- long to the inhibitor receptor superfamily, which are characterized

by the presence of intracytoplasmic ITIM (I/VxxYxxL/V/IxϾ20I/ VxxYxxL/V/I) that recruit and activate protein-tyrosine phospha- Division of Haematology, Hanson Centre for Cancer Research, IMVS, Adelaide, South Australia tases (PTPs) such as SHP-1, SHP-2, and/or inositol polyphosphate Received for publication September 18, 2000. Accepted for publication December 5-phosphatases, SHIP, and SHIP2 (5). Coligation of BCR with 22, 2000. these inhibitory receptors leads to premature termination of inosi- 2ϩ The costs of publication of this article were defrayed in part by the payment of page tol trisphosphate production, inhibition of extracellular Ca in- charges. This article must therefore be hereby marked advertisement in accordance flux, and a blockage of blastogenesis (6). with 18 U.S.C. Section 1734 solely to indicate this fact. Our recent studies and others have defined that platelet endo- 1 This work was supported by Grant 981896 (to D.E.J.) from the National Health and Medical Research Council of Australia. D.E.J. is the recipient of a National Health thelial cell adhesion molecule-1 (PECAM-1/CD31) contains intra- and Medical Research Council R. D. Wright Fellowship. T.L.H. is a postgraduate cytoplasmic ITIM motifs, which recruit and activate the PTPs, student of School of Pharmacy and Medical Sciences, University of South Australia. SHP-1 and SHP-2, under physiologically relevant conditions (7– 2 Address correspondence and reprint requests to Dr. Denise Jackson, FAIMS, Divi- 11). Our biochemical analysis suggested that SHP-2 binds to PE- sion of Haematology, Hanson Centre for Cancer Research, IMVS, Frome Road, Ad- elaide, South Australia. 5000. E-mail address: [email protected] CAM-1 with high affinity compared with SHP-1, suggesting that when these PTPs are located in the same subcellular compartment, 3 Abbreviations used in this paper: BCR, B cell receptor; ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibitory mo- SHP-2 may be the preferred substrate for PECAM-1 (12, 13). tif; KIR, killer inhibitory receptor; mIg, membrane Ig; PECAM-1, platelet endothelial However, in vitro biochemical studies and PTP requirements for cell adhesion molecule-1; PIR-B, paired Ig-like receptor B; PTP, protein-tyrosine phosphatase; SHIP, Src homology 2 domain containing inositol polyphosphate 5’- inhibitory receptor signaling in the context of living cells are often phosphatase; SHP, Src homology 2 domain containing protein-tyrosine phosphatase. quite different. For example, previous studies have highlighted that

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 3099

while Fc␥RIIB1 ITIM motif was able to physically associate with (Sigma, St. Louis, MO) for chicken DT40 B cell lines. Cell surface ex- SHP-1 and SHIP, its functional analysis of chimeric receptors ex- pression of each transfected cell line was analyzed by flow cytometry stud- ies using 1 ␮g/ml 2.4G2 (anti-Fc␥RIIB1) Ab, followed by staining with 4 pressed in DT40 B cell lines devoid of SHP-1 or SHIP has dem- ␮ ␥ g/ml FITC-conjugated anti-rat IgG, and assessed on a FACScan (Becton onstrated that SHIP was the preferred substrate for Fc RIIB1 (14, Dickinson, San Jose, CA). 15). In addition, functional assessment of PIR-B revealed that SHP-1 and SHP-2 PTPs are required for PIR-B-inhibitory signal- Generation of SHP-1Ϫ/Ϫ-, SHP-2Ϫ/Ϫ-, SHP-1/2Ϫ/Ϫ-, and ing (16). Based upon these findings, we wanted to test the func- SHIPϪ/Ϫ-deficient DT40 cell lines tional importance of PECAM-1 cytoplasmic ITIM motifs and the requirement of PTPs for mediating PECAM-1-inhibitory Knockout PTP-deficient cell lines were created by homologous recombi- nation of targeting genomic constructs using DT40 chicken B cell line, as signaling. previously described (16). These PTP-deficient cell lines were purchased Previous studies have suggested that PECAM-1 may be expressed from Riken Cell Bank (Tuskuba Science City, Japan). Evidence of null on CD19-positive B cells derived from bone marrow (17), reactive mutant knockout cell lines was confirmed by Western blot analysis of plasma cells defined by CD38ϩϩϩ (18), and as a CD38 ligand found cellular lysates using polyclonal anti-SHP-1, anti-SHP-2, and anti-SHIP in the lymphoid compartment of follicular mantle B cells and plasma Abs that cross-react with chicken forms of the PTPs, as described above (Fig. 5A–C). cells (19). Our recent studies using primary human tonsillar tissue have demonstrated that PECAM-1 is expressed on discrete B lym- Peptido-precipitation, immunoprecipitation, and Western phocyte subpopulations with the phenotype of naive follicular mantle blotting analysis zone cells and not germinal center-specific cells (20). As memory 8 B display several intrinsic differences than naive B lym- A total of 4 ϫ 10 resting chicken DT40 B cells was solubilized in 1 ml of Downloaded from Triton lysis buffer (2% Triton X-100, 10 mM EGTA, 15 mM HEPES, 145 phocytes, including a lower threshold for cellular activation, an ability ␮ mM NaCl, 0.1 mM MgCl2, 1 mM PMSF, 20 g/ml leupeptin, and 2 mM to directly present Ag to Th cells, and a longer life span (21), it is sodium orthovanadate, pH 7.4) and constantly mixed on a nutator (Clay likely that the presence of inhibitory receptors such as PECAM-1 in Adams) at 4°C for 1 h. Triton-soluble and -insoluble (cytoskeletal) frac- naive B lymphocytes plays a negative regulatory role in their activa- tions were isolated by centrifugation at 15,000 ϫ g for 15 min at 4°C. tion and differentiation pathways. At present, there is no evidence to Following separation of the 15,000 ϫ g Triton-soluble fraction, the lysate was precleared with 50 ␮l of 50% slurry of streptavidin-agarose beads for

suggest that PECAM-1 may play a regulatory role in B cell activation, http://www.jimmunol.org/ 30 min at 4°C and then centrifuged at 4,000 rpm for 5 min. One milligram maturation, or differentiation. of precleared cell lysates was aliquoted into separate Eppendorf tubes and To test whether PECAM-1 is capable of delivering inhibitory incubated with 10 ␮g of each respective biotinylated PECAM-1 cytoplas- Ϯ signals in B cells and the functional requirement of PTPs for this mic domain peptide Tyr (PO4) overnight at 4°C. The design and prep- inhibitory signaling, we generated chimeric Fc␥RIIB1-PECAM-1 aration of PECAM-1 phosphopeptides have been previously described (11). Peptido-protein complexes were then isolated by addition of 50 ␮lof receptors containing the extracellular and transmembrane portions 50% slurry of streptavidin-agarose beads and incubated for1hat4°C, and of Fc␥RIIB1 and the cytoplasmic domain of PECAM-1. These then beads were washed five times with immunoprecipitation buffer. In chimeric receptors were stably expressed in DT40 B cells either as some experiments, DT40 cell lysates were precleared with 50 ␮lof50% wild-type or mutant cells deficient in SHP-1Ϫ/Ϫ, SHP-2Ϫ/Ϫ, protein G-Sepharose 4B beads and were then sequentially incubated with ␮ ␮ SHIPϪ/Ϫ, or SHP-1/2Ϫ/Ϫ and assessed for their ability to inhibit 4 g of 2.4G2 IgG and 50 l of 50% protein G-Sepharose 4 Fast Flow by guest on September 30, 2021 (Pharmacia LKB Biotechnology, Uppsala, Sweden). Bound were ITAM-dependent BCR signaling. Our studies demonstrate that the eluted from the beads by boiling for 10 min in 30 ␮l SDS reducing buffer PTPs, SHP-1 and SHP-2, are required for PECAM-1-mediated in- and resolved on a 10% SDS-PAGE gel, then transferred to polyvinylidene hibitory signaling. difluoride membrane and probed with each respective polyclonal anti- SHP-1, anti-SHP-2, or anti-SHIP Ab. The membrane was additionally in- cubated with a secondary HRP-conjugated goat anti-rabbit IgG (1:10,000) and visualized by ECL detection. Materials and Methods Cells, expression constructs, and Abs Calcium mobilization assays and receptor coligation Chicken DT40 B cell lines were maintained in RPMI 1640 medium sup- Cells (5 ϫ 106 cells/ml) were loaded with 3 ␮M fura 2-AM (Molecular plemented with 10% (v/v) heat-inactivated FCS, 1% (v/v) chicken serum, Probes, Eugene, OR) in medium at room temperature for 45 min. Cells 4 mM glutamine, 50 ␮M 2-ME, and antibiotics. Wild-type mouse were washed with PBS twice and resuspended in the same cell concentra- ␥ Fc RIIB1, pBabe, and pA PURO constructs were kindly provided by Jef- tion in PBS supplemented with 1 mM CaCl2 and 1 mM MgCl2. Two frey Ravetch (The Rockefeller University, New York, NY) and have been milliliters of cell suspension were added to a cuvette with a small stirrer. previously described (14). Human PECAM-1 Y663,686F construct has Calcium mobilization profiles were recorded at 510 nm emission wave- Ј been previously described (12). Intact and F(ab )2 rabbit anti-mouse IgM length excited by 340 nm and 380 nm using a Perkin-Elmer Luminescence (Zymed, San Francisco, CA), mouse anti-chicken IgM mAb M4 (mouse Spectrophotometer LS-50B (Department of Medical Biochemistry, IgM) (Southern Biotechnology Associates, Birmingham, AL), goat anti- Flinders University, Adelaide, Australia). The basis of coligation of BCR chicken IgM ␮-chain specific (Bethyl Laboratories, Montgomery, TX), with inhibitory receptor that contains the extracellular moiety of murine 2.4G2 (PharMingen, San Diego, CA), HRP anti-PY Plus Ab (Zymed), and Fc␥RIIB is based upon the fact that Fc␥RIIB can be effectively cross- polyclonal anti-human SHP-2 (Santa Cruz Biotechnology, Santa Cruz, CA) linked by Fc portions presented as multivalent ligands. In this case, the were purchased. Polyclonal anti-chicken SHP-1 Ab was kindly provided by anti-chicken IgM Ab is of mouse IgM isotype and requires cross-linking Ј Tomohiro Kurosaki (Kansai Medical University, Moriguchi, Japan). Poly- with intact rabbit anti-mouse IgM for coligation purposes, which F(ab )2 clonal anti-SHIP antiserum was kindly provided by Mark Coggeshall rabbit anti-mouse IgM is not capable of coligation. Stimulation of chicken Ј (Oklahoma Medical Research Foundation, Oklahoma City, OK). The DT40 B cells was conducted by cross-linking BCR with F(ab )2 rabbit cDNA of mouse Fc␥RIIB1 and human PECAM-1 receptor were fused at anti-mouse IgM (5 ␮g) and mouse anti-chicken IgM Ab (2 ␮g/ml). Coli- the carboxyl-terminal residue of the transmembrane domain of each protein gation of BCR to transfected chimeric receptor was obtained by preincu- by using overlap PCR with the following junction primers: Fc␥RIIB1- bation of intact rabbit anti-mouse IgM (5 ␮g) with chicken DT40 B cells PECAM-1, 5Ј-TGGTCTATCTCAGCCAAATGTTATTTTCTG-3Ј, and at room temperature for 10 min, followed by mouse anti-chicken IgM (2 ␥ Ј ␮ PECAM-1-Fc RIIB1primer,5 -ATAACATTTGGCTGAGATAGACCAA g/ml). Maximal (Rmax) calcium release from intracellular calcium store GGATA-3Ј. These chimeric cDNAs were then subcloned into pA PURO was measured in the presence of 20 ␮M ionomycin, and minimal calcium

expression vector. Sequence integrity was confirmed by automated ABI release (Rmin) was obtained by addition of 6.25 mM EGTA. Calibration nucleotide sequence analysis. A total of 20 ␮g of expression constructs was and calculation of calcium concentration were performed as described (22). transfected by electroporation at 250 V and 960 ␮F into the various wild- Cumulative calcium mobilization was evaluated by integration for 4 min of type and PTP-deficient chicken DT40 B cell lines. Expression of chimeric calcium mobilization over the baseline given before stimulation, as de- molecules was established by drug selection using 1 ␮g/ml puromycin scribed by the manufacturer. 3100 MECHANISM OF INHIBITION OF B CELL ACTIVATION BY PECAM-1

cell lineage, they are likely to express endogenous chicken PECAM-1 and Fc␥RIIB1. For this reason, we opted to introduce a chimeric receptor into the DT40 B cells expressing the cytoplas- mic domain of human PECAM-1 fused to the extracellular and transmembrane portions of murine Fc␥RIIB1, so that we could control ligand interactions through species-specific interactions. This chimeric molecule was transfected into chicken DT40 B cells, and selection commenced with puromycin to obtain stable trans- FIGURE 1. Schematic diagram of constructs for chimeric receptors used in this study. The extracellular and transmembrane portions of murine formants. Expression levels of stable transformants were assessed ␥ Fc␥RIIB1 were fused with the cytoplasmic domain of human PECAM-1. by flow cytometry analysis using rat anti-mouse Fc RIIB1 mAb, In addition, mutations of tyrosine residues in the putative cytoplasmic 2.4G2 (Fig. 2A). The expression levels for the various stable clones ITIM motifs (Y663, 686F) within human PECAM-1 were also fused with of chimeric receptors were similar and comparable with the levels murine Fc␥RIIB1. The amino acid sequences corresponding to the con- of PECAM-1 expression on normal naive primary B cells (19). sensus ITIM motifs are indicated by bold sequence. DT40 cells expressing wild-type Fc␥RIIB1-PECAM-1 were stim- ulated by BCR cross-linking alone (Fig. 2B, solid line) or coliga- tion of BCR and wild-type Fc␥RIIB1-PECAM-1 (Fig. 2B, dashed line), and calcium mobilization responses were recorded. To ex- NF-AT luciferase assays clude the possibility of spontaneous calcium mobilization in the absence of BCR cross-linking, we monitored unstimulated DT40 B Downloaded from A total of 1 ϫ 107 cells in 0.5 ml PBS was electroporated at 250 V and 975 ␮F in the presence of 20 ␮g of NF-AT luciferase construct, pxpGM55 cells over time and found no evidence of calcium release (data not IL140 ϫ 3 containing NF-AT-p binding sites (23) (supplied by Peter Cock- shown). In addition, ligation of Fc␥RIIB1 alone with 2.4G2 mAb erill, Hanson Center for Cancer Research, Adelaide, Australia) and then did not produce evidence of calcium release (data not shown). recultured in 30 ml of DT40 complete medium. After 24-h incubation at Coligation of wild-type Fc␥RIIB1-PECAM-1 with BCR resulted 39°C, cell suspensions were centrifuged and cells were resuspended in 1 ml of medium and aliquoted into six-well plates. For stimulation of DT40 B in inhibition of intracellular calcium release. To evaluate whether Ј ␮ ␮ http://www.jimmunol.org/ cells, F(ab )2 (5 g) anti-mouse IgM with 2 g/ml anti-chicken IgM (BCR PECAM-1 cytoplasmic domain inhibited BCR-induced calcium stimulation alone) or intact (5 ␮g) rabbit anti-mouse IgM with anti-chicken mobilization by release of calcium from intracellular stores and/or IgM (BCR ϩ inhibitory receptor) was incubated for6hat39°C. Cells were calcium influx, experiments were performed on DT40-Fc␥RIIB1- then lysed, normalized for protein content, and measured for luciferase activity using a luciferase reporter assay system (24). PECAM-1 in the absence of extracellular calcium. Incubation with EGTA before cross-linking of BCR with wild-type Fc␥RIIB1- PECAM-1 further decreased the calcium mobilization, indicating Results that wild-type Fc␥RIIB1-PECAM-1 acts on calcium release from The cytoplasmic domain of PECAM-1 can deliver an inhibitory intracellular stores (Fig. 2C). To test for specificity of this calcium signal in B cells

signal, a downstream effector of B cell calcium-dependent signal- by guest on September 30, 2021 To test whether the cytoplasmic domain of PECAM-1 is capable of ing pathway, NF-AT transcriptional factor activation was mea- inhibiting BCR activation, a chimeric molecule expressing the ex- sured, as it is dependent upon the calcium-sensitive translocation tracellular and transmembrane portions of murine Fc␥RIIB1 was of NF-AT cytosolic component to the nuclear compartment. In fused with the cytoplasmic domain of human PECAM-1 using an these experiments, transcriptional activation of the NF-AT lucif- overlap PCR strategy (Fig. 1). As DT40 B cells are of immature B erase reporter was inhibited by coligation of the BCR to this

FIGURE 2. The cytoplasmic domain of PECAM-1 is capable of inhibiting BCR activation. A, Cell surface expression of wild-type Fc␥RIIB1-PECAM-1 chi- meric receptor on parental DT40 B cell line using 1 ␮g/ml 2.4G2 and 4 ␮g/ml anti-rat FITC (filled profile) or with anti-rat FITC alone (open profile). B, Calcium mobilization profiles were measured after cross-linking of BCR (solid line) using predetermined concentra- Ј tions of F(ab )2 rabbit anti-mouse IgM and mouse anti- chicken IgM and coligation of inhibitory receptor (dashed line) using predetermined concentrations of intact rabbit anti-mouse IgM and mouse anti-chicken IgM to colocalize the chimeric receptor with BCR complex. C, Calcium release from intracellular stores was measured in the presence of 1 mM EGTA after cross-linking of BCR (solid line) and coligation of in- hibitory receptor (dashed line). D, NF-AT activation by BCR cross-linking and coligation of BCR with in- hibitory receptor, wild-type Fc␥RIIB1-PECAM-1. NF-AT activity was measured using a luciferase re- porter gene construct under the NF-AT promoter. NF-AT activity was normalized to that of BCR stim- ulation alone. Data represent the mean and SD of six replicate determinations. The Journal of Immunology 3101

wild-type Fc␥RIIB1-PECAM-1 chimeric molecule (Fig. 2D). Our results indicate that the cytoplasmic domain of PECAM-1 is capa- ble of delivering an inhibitory signal that blocks BCR-mediated ac- tivation in B cells. Recruitment of SHP-1 and SHP-2 PTPs to human PECAM-1 ITIM motifs in the context of chicken DT40 B cells The presence of ITIM-bearing sequences in the PECAM-1 cyto- plasmic domain suggests that upon tyrosine phosphorylation, SH2- containing PTPs, SHP-1 and SHP-2, or inositol polyphosphate 5Ј- phosphatase, SHIP, could be recruited. This hypothesis is ␣ ␤ supported by recent studies showing that upon IIb 3- mediated platelet aggregation, aggregation of the high affinity IgE receptor on mast cells, or aggregation of the TCR complex on T cells, PECAM-1 becomes tyrosine phosphorylated and recruits SHP-2 and/or SHP-1 in an ITIM-dependent manner (7, 8, 24). At present, there is no information on induction of PECAM-1 tyrosine phosphorylation in the context of B cells. As ITIM motifs and SH2

domain-containing molecules are highly conserved among various Downloaded from species, we would predict that when human PECAM-1 ITIM mo- tifs become tyrosine phosphorylated, they would be capable of recognizing and recruiting the chicken PTPs, SHP-1 and SHP-2. To test this hypothesis, resting chicken DT40 cell lysates were incubated with biotinylated human PECAM-1 cytoplasmic domain

Ϯ http://www.jimmunol.org/ peptides Tyr(PO4) residues encompassing the five tyrosine res- idues known to be present in the human PECAM-1 cytoplasmic domain (Y596, Y636, Y663, Y686, and Y701). These tyrosine- phosphorylated PECAM-1 peptides mimic activated forms of the PECAM-1 cytoplasmic domain. Bound SH2 domain-containing protein/peptide complexes were then recovered with streptavidin- agarose beads, and proteins resolved on an SDS-PAGE gel, and PTPs identified by respective Abs in immunoblot analysis. As shown in Fig. 3, only the tyrosine-phosphorylated Y663 and Y686 forms of human PECAM-1 peptides could bind the chicken PTPs, by guest on September 30, 2021 SHP-1 (A) and SHP-2 (B). However, the recruitment of chicken SHIP was not phosphotyrosine dependent and was recruited by residues surrounding the Y686 residue sequence (C). These results highlight that the SH2 domain-containing PTPs are highly con- served across species and recognize the human PECAM ITIM motifs. FIGURE 3. Recruitment of SHP-1 and SHP-2 PTPs to human PECAM-1 Using DT40 cells stably expressing Fc␥RIIB1-wild-type ITIM motifs in the context of chicken DT40 B cells. Solubilized chicken DT40 PECAM-1 chimeric receptor, the recruitment of PTPs was then cell lysates (1-mg aliquots) were absorbed with streptavidin-agarose beads confirmed in immunoprecipitation studies under conditions of coated with the following biotinylated peptides with and without phosphoty- BCR stimulation with and without coligation of the inhibitory re- rosine residues: (594–604, C595A, KApYFLRKAKAK); (631–641, EAN ceptor. As shown in Fig. 3D, SHP-1, PTP was recruited upon BCR SHpYGHNDD); (658–668, NSDVQpYTEVQV); (681–691, DTETVpYS stimulation and following coligation of the inhibitory receptor EVRK); (696–706, AVESRpYSRTEG), in which pY denotes a phos- (Fig. 3D, top panel). SHP-2, protein tyrosine phosphatase, was photyrosine residue. Protein peptido-complexes were separated by 10% SDS- recruited only upon conditions of coligation of inhibitory receptor PAGE, transferred to polyvinylidine difluoride membrane, and detected by polyclonal anti-SHP-1 (A), anti-SHP-2 (B), or anti-SHIP (C) Abs. The above with the BCR complex (Fig. 3D, middle panel). This apparent results are representative of three independent experiments. D, BCR stimula- difference in kinetics of assembly between SHP-1 and SHP-2 re- tion and coligation of inhibitory receptor induce recruitment of PTPs by the cruitment by the PECAM-1 cytoplasmic domain has been previously PECAM-1 cytoplasmic domain. DT40 cells expressing wild-type Fc␥RIIB1- described in other physiologically relevant conditions such as integrin ␣ ␤ PECAM-1 receptor were stimulated by BCR cross-linking and coligation of IIb 3-mediated platelet aggregation (11). In contrast, SHIP was con- BCR to wild-type Fc␥RIIB1-PECAM-1. These DT40 B cells expressing wild- stitutively associated with the chimeric receptor consistent with the type Fc␥RIIB1-PECAM-1 were preincubated with predetermined concentra- peptido-precipitation studies described above (Fig. 3D, lower panel). tion of rabbit anti-mouse IgM (5 ␮g) into respective tubes for 5 min at room temperature. BCR cross-linking was initiated by addition of 2 ␮g/ml of mouse Tyrosine residues 663 and 686 in the cytoplasmic domain of anti-chicken IgM and incubated for 3 min at room temperature. Reaction was PECAM-1 are essential in PECAM-1-mediated inhibitory stopped by the addition of equal volumes of Triton-lysis buffer. Wild-type signaling in B cells Fc␥RIIB1-PECAM-1 was then immunoprecipitated with 2.4G2, and pro- tein-Ab complexes were captured with protein G-Sepharose, separated by Our previous studies have defined that PECAM-1 contains two 663 686 SDS-PAGE, transferred to polyvinylidene difluoride membrane, and immu- ITIM consensus motifs (VQY TEV and TVY SEV) involving noblotted with polyclonal anti-chicken SHP-1, anti-SHP-2, and anti-SHIP tyrosine residues, 663 and 686, that upon phosphorylation are ca- Abs, followed by HRP-conjugated anti-rabbit conjugate and enhanced chemi- pable of recruiting and activating the PTPs, SHP-1 and SHP-2 (7, luminescence development. 11). In vitro surface plasmon resonance studies have suggested 3102 MECHANISM OF INHIBITION OF B CELL ACTIVATION BY PECAM-1

FIGURE 4. The tyrosine ITIM-containing resi- dues 663 and 686 are required for PECAM-1-inhib- itory signaling of BCR activation. A, Cell surface expression of Fc␥RIIB1-PECAM-1 chimeric wild- type and Y663,686F PECAM-1 mutant receptors on parental DT40 B cell line using 1 ␮g/ml 2.4G2 and 4 ␮g/ml anti-rat FITC (filled profile) or with anti-rat FITC alone (open profile). B, Calcium mobilization profiles were measured after cross-linking of BCR (solid line) and coligation of inhibitory receptor (dashed line) in DT40 B cells expressing various Fc␥RIIB1-PECAM-1 wild-type and Y663,686F PECAM-1 mutants. Downloaded from http://www.jimmunol.org/ differences in affinity between respective ITIM motifs in their as- quires clarification. Consistent with these observations, in vivo sociation with PTPs such as SHP-2 (12, 13). Mutation of either functional studies have revealed that ITIM motifs may deliver phosphotyrosine residue leads to loss of association of PTPs, sug- inhibitory signals to negatively regulate ITAM-dependent cell gesting that both tyrosine residues may be required (12). However, activation by selective PTP-substrate-dependent pathways. Sev- no functional evidence is available to assess the structural impor- eral examples include Fc␥RIIB1, which uses SHIP to mediate de- tance of each of the phosphotyrosine residues in PECAM-1-me- phosphorylation of phosphatidylinositol 3Ј-kinase and extracellu- diated inhibitory signaling in B cells. lar signal-regulated kinase activation pathways (14, 15), while sig- by guest on September 30, 2021 To test this hypothesis, a chimeric receptor was constructed con- nal regulatory protein-␣, killer inhibitory receptor (KIR), or PIR-B taining the extracellular and transmembrane domains of murine uses SHP-1 and SHP-2 to mediate dephosphorylation of protein- Fc␥RIIB1 and the cytoplasmic domain of human PECAM-1 con- tyrosine kinases, reduced activation of -activated protein taining Y663,686F mutant. This chimeric cDNA receptor was kinases, Erk1 and Erk2, and ITAM dephosphorylation (14–16, transfected into parental DT40 B cells, and selection commenced 25). Based upon our biochemical studies on PECAM-1 ITIM mo- with puromycin to obtain stable transformants. Parental DT40 B tifs, we would predict that SHP-2 and/or SHP-1 may be required cells expressing comparable levels of various Fc␥RIIB1- PECAM-1 wild-type and Y663,686F mutant (Fig. 4A) were stim- for delivery of inhibitory signals within the context of B cells. ulated by BCR alone (solid line) and following coligation of BCR To test our hypothesis, chimeric receptors containing the extra- ␥ with inhibitory receptor (dashed line). A significant 95% reversal cellular and transmembrane domains of murine Fc RIIB1 and the of inhibition of the calcium mobilization response was observed by cytoplasmic domain of human PECAM-1 were transfected into coligation of BCR with Fc␥RIIB1-PECAM-1 Y663,686F mutant wild-type chicken DT40 B cells and DT40 cells deficient in either Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ compared with Fc␥RIIB1-PECAM-1 wild type (Fig. 4B). These SHP-1 , SHP-2 , SHP-1/2 , or SHIP (Fig. 5, A–C). results suggest that both ITIM tyrosine residues, 663 and 686, are Following selection with puromycin, stable clones were obtained capable of mediating PECAM-1-inhibitory signaling in B cells. and refined by flow cytometry sorting using rat anti-mouse Fc␥RIIB1 Ab, 2.4G2 (2 ␮g/ml), so that all stable cell lines ex- SHP-1 and SHP-2 PTPs are required for PECAM-1-mediated pressed equivalent levels of chimeric receptor (Fig. 6A). In addi- inhibitory signaling tion, these cell lines were assessed for IgM expression by flow cytometry using goat anti-chicken IgM (10 ␮g/ml). Examination In vitro peptide-binding experiments have demonstrated that ITIM of flow cytometric profiles revealed comparable levels of cell sur- consensus sequences within Ig-ITIM-bearing receptors are capable face IgM expression and stability of BCR complex before func- of binding SHP-1 or SHP-2 PTPs and/or SHIP inositol 5Ј-phos- phatase with differing affinities. This is in contrast to coimmuno- tional analysis (Fig. 6B). Upon BCR stimulation (solid line) and precipitation studies from cells stimulated by coaggregation of coligation of inhibitory receptor with BCR complex (dashed line), ITAM-bearing with ITIM-bearing receptors such as BCR with the calcium mobilization responses were reduced in parental and Ϫ/Ϫ ␥ Fc␥RIIB1, in which the phosphorylated ITIM-bearing receptor ef- SHIP DT40 B cells expressing wild-type Fc RIIB1-PECAM-1 fector interactions may favor a predominant PTP-phosphotyrosine- (Fig. 6C). This inhibitory effect was partially reversed in SHP- Ϫ/Ϫ Ϫ/Ϫ dependent interaction or a combination of PTP-phosphotyrosine- 1 and SHP-2 DT40 B cells expressing wild-type dependent interactions depending upon kinetics of association and Fc␥RIIB1-PECAM-1, while it was completely abolished in SHP- Ϫ Ϫ dissociation. The potential role of non-SH2 determinants in PTP 1/2 / DT40 B cells expressing wild-type Fc␥RIIB1-PECAM-1 modulation of ITIM-bearing receptor inhibitory signaling still re- (Fig. 6C). Based upon these results, a quantitative summary of the The Journal of Immunology 3103

FIGURE 5. SHP-1, SHP-2, and SHIP protein expression in various DT40 B cell gene-targeted cell lines. A total of 100 ␮g of Triton-solubilized cell lysates from each DT40 cell line was elec- trophoresed by 10% SDS-PAGE, transferred to polyvinylidine difluoride membrane, and probed with specific polyclonal Abs against SHP-1 (A), SHP-2 (B), and SHIP (C), which cross-react with the chicken forms of the phosphatases. Downloaded from

inhibitory effect of wild-type Fc␥RIIB1-PECAM-1 chimeric re- (Fig. 4). In addition, analysis of various PTP-deficient DT40 B cell ceptor from various genetic background DT40 B cells was derived lines either singly or in combination indicated that cytoplasmic from three independent clones of each cell line, as shown in Fig. SH2 domain-containing phosphatases, SHP-1 and SHP-2, were

6D. These results suggest that both SHP-1 and SHP-2 PTPs are both necessary and sufficient to mediate inhibitory signaling by http://www.jimmunol.org/ required for mediating PECAM-1-inhibitory signaling in B cells. PECAM-1 ITIM motifs (Fig. 6). Based upon these findings, we can propose a model for PECAM-1-inhibitory signaling in the con- Discussion text of B cells. Upon BCR stimulation and coligation of inhibitory Accumulating evidence indicates that B cell coreceptors act as receptor with BCR complex, protein-tyrosine kinases are activated negative regulators of B cell activation and/or tolerance involving and induce progressive phosphorylation of tyrosine residues within distinct signaling pathways dependent upon the requirement of PECAM-1 ITIM motifs, which act as a molecular “scaffold” to SH2-tandem PTPs, SHP-1 and SHP-2, or SH2-domain-bearing coordinate the docking and activation of cytoplasmic SH2-tandem inositol polyphosphate 5-phosphatases, SHIP or SHIP2. At phosphatases, SHP-1 and SHP-2. The functional consequence of present, two distinct inhibitory signaling pathways have been iden- these PTP interactions involves dephosphorylation of target sub- by guest on September 30, 2021 tified. The Fc␥RIIB1-inhibitory signaling pathway is dependent on strates to prevent release of calcium from intracellular stores to SHIP, but not SHP-1 and SHP-2, while KIR-, PIR-B-, CD22-, and negatively regulate BCR-mediated cell activation. Our studies signal regulatory protein-␣-inhibitory signaling pathways are de- demonstrate that like the KIR and PIR-B class of molecules, the pendent upon either SHP-1 and/or SHP-2, but not SHIP. The fact PECAM-1 cytoplasmic domain appears to inhibit ITAM-mediated that PECAM-1 contains two intracytoplasmic ITIM motifs, and B cell signaling by preventing release of calcium from intracellular that upon tyrosine phosphorylation can recruit and activate cyto- stores only without affecting calcium influx from the extracellular plasmic SH2-domain-bearing PTPs suggests that these PTPs may compartment, as observed for Fc␥RIIB1 (14, 16, 27). be functionally important for in vivo delivery of inhibitory signals. Our in vitro studies have revealed that PECAM-1 has differing To date, little is known of the mechanism of PECAM-1-inhibitory kinetics of association and dissociation for SHP-1 and SHP-2 signaling pathways in the context of various cell types. As ITIM- PTPs. Typically, PECAM-1 precipitates higher amounts of SHP-2 bearing molecules have the capacity to negatively regulate cell and forms a more stable complex than with SHP-1. Under phys- activation induced by coaggregation with receptors bearing ITAM iological conditions, it would appear that the interaction of motifs, we would predict that PECAM-1 would be capable of reg- PECAM-1 with SHP-1 is of a transient nature. This observation is ulating ITAM-dependent cell activation and receptor tyrosine ki- further supported by the fact that a catalytically inactive C453S nase-mediated cell proliferation. In this study, we demonstrate that SHP-1 could be coimmunoprecipitated with PECAM-1 reconsti- the PECAM-1 cytoplasmic domain is capable of delivering an in- tuted in COS-7 cells (10). In this study, we have demonstrated that hibitory negative signal that can down-modulate BCR-mediated deficiency of SHP-2 is associated with a more significant reversal cell activation. Using the chicken DT40 B cell model, stable ex- in PECAM-1-inhibitory signaling than SHP-1, suggesting that it pression of chimeric Fc␥RIIB1-PECAM-1 receptors enabled us to may be the preferred PTP functionally used by PECAM-1. How- test whether the PECAM-1 cytoplasmic domain was capable of ever, our studies also demonstrate that SHP-1 contributes to the producing an inhibitory negative signal upon coligation of BCR attenuation of the calcium mobilization response (Fig. 6). In con- complex with inhibitory receptor. Our studies with the chimeric B trast, our functional data demonstrate that SHIP is dispensable for cell IgG coreceptor, Fc␥RIIB1, containing the cytoplasmic domain the PECAM-1-mediated inhibitory response. Therefore, under of human PECAM-1 have established a critical role for peptide conditions of ITIM-dependent phosphorylation of PECAM-1, sequences within the PECAM-1 cytoplasmic domain in mediating SHP-1 and SHP-2 appear to act in concert to augment the inhib- an inhibitory negative signal to suppress release of calcium from itory negative signaling. Our finding that SHIP is constitutively intracellular stores (Fig. 2). Our studies have also revealed that this associated with PECAM-1 via a nonphosphotyrosine-dependent inhibitory signaling was dependent upon the integrity of the interaction with 681–691 aa residues may implicate the possibility PECAM-1 intracytoplasmic ITIM motifs, as mutation of Tyr663 of a second regulatory mechanism under basal conditions (Fig. 3, and Tyr686 to Phe residues abrogated the inhibitory negative signal C and D). Interestingly, previous studies have demonstrated that 3104 MECHANISM OF INHIBITION OF B CELL ACTIVATION BY PECAM-1 Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 6. The PTPs, SHP-1 and SHP-2, are required for PECAM-1-inhibitory signaling of BCR activation. A, Cell surface expression of wild-type Fc␥RIIB1-PECAM-1 chimeras on various PTP-deficient DT40 B cells, as measured by flow cytometry after staining with 1 ␮g/ml 2.4G2 and 4 ␮g/ml anti-rat FITC (filled profile) or with anti-rat FITC alone (open profile). B, Cell surface expression of IgM on various parental or PTP-deficient DT40 B cells stably expressing wild-type Fc␥RIIB1-PECAM-1 chimeric receptors, as measured by flow cytometry after staining with 10 ␮g/ml goat anti-chicken IgM and 4 ␮g/ml anti-goat FITC (filled profile) or with anti-goat FITC alone (open profile). C, Calcium mobilization was measured after cross-linking BCR (solid line) and coligation of inhibitory receptor (dashed line). D, Comparison of inhibitory effects on calcium mobilization mediated by wild-type Fc␥RIIB1-PECAM-1 chimeric receptor expressed on parental and PTP-deficient DT40 B cells. The percentage of relative Ca2ϩ (%) was derived from the total calcium mobilization response obtained from coligation of BCR complex with inhibitory receptor divided by BCR stimulation alone and expressed as a percentage. The results are expressed as the mean of three independent experiments performed on three individual clones for each wild-type and PTP-deficient DT40 cell line expressing wild-type Fc␥RIIB1-PECAM-1 chimeric receptor. Error bars represent SD from the mean. loss of Tyr686 from exon 14 of PECAM-1 by mutation of B cell coreceptors have been demonstrated to suppress mem- Tyr6863Phe or phosphorylation of the tyrosine residue results in brane Ig (mIg) signaling via Fc␥RIIB1 coligation with mIg by a change in ligand specificity from heterophilic to homophilic IgG-Ag complexes or through cross-linking of mIg complex to binding (28). induce tyrosine phosphorylation of a constitutively associated B The Journal of Immunology 3105 cell coreceptor such as CD22 or CD72 (29, 30). In this study, 3. Reth, M., and J. Wienands. 1997. Initiation and processing of signals from the B suppression of mIg signaling was initiated by coligation of a chi- cell antigen receptor. Annu. Rev. Immunol. 15:453. meric Fc␥RIIB1-PECAM-1 receptor with mIg by soluble immune 4. Tsubata, T. 1999. Co-receptors on B lymphocytes. Curr. Opin. Immunol. 11:249. 5. Vivier, E., and M. Daeron. 1997. Immunoreceptor tyrosine-based inhibition mo- complexes. This approach enabled us to gain an insight into the tifs. Immunol. Today 18:286. mechanism of inhibition by human PECAM-1 cytoplasmic do- 6. Bolland, S., and J. V. Ravetch. 1999. Inhibitory pathways triggered by ITIM- main. However, the mechanism of mIg signaling via endogenous containing receptors. Adv. Immunol. 72:149. PECAM-1 coligation in primary B cells is not clear. Whether 7. Jackson, D. E., C. M. Ward, R. Wang, and P. J. Newman. 1997. The protein- tyrosine phosphatase SHP-2 binds platelet/endothelial cell adhesion molecule-1 PECAM-1 is constitutively associated with the BCR remains to be (PECAM-1) and forms a distinct signaling complex during platelet aggregation. determined and could not be addressed in this investigation. J. Biol. Chem. 272:6986. Examination of human tonsillar B cells has demonstrated a de- 8. Sagawa, K., T. Kimura, M. Swieter, and R. P. Siraganian. 1997. The protein- velopmental pattern of PECAM-1 expression found in naive rest- tyrosine phosphatase SHP-2 associates with tyrosine-phosphorylated adhesion molecule-1 PECAM-1 (CD31). J. Biol. Chem. 272:31086. ing B cells, which is lost upon differentiation from an immature B 9. Lu, T. T., M. Barreuther, S. Davis, and J. A. Madri. 1997. Platelet/endothelial cell cell into a germinal center memory B cell (20). These observations adhesion molecule-1 (PECAM-1) is phosphorylatable by c-Src, binds Src-Src taken together with results of this study would support the concept homology-2 domain, and exhibits immunoreceptor tyrosine-based activation mo- tif-like properties. J. Biol. Chem. 272:14442. that PECAM-1 acts as a B cell coreceptor in increasing the thresh- 10. Cao, M. Y., M. Huber, N. Beauchemin, J. Famiglietti, S. M. Albelda, and old of sensitivity to Ag that accompanies the differentiation of A. Veillette. 1998. Regulation of mouse PECAM-1 tyrosine phosphorylation immature B cells. As B cell coreceptors have been shown to mod- by the Src and Csk families of protein-tyrosine kinases. J. Biol. Chem. ulate the signaling threshold for B cell tolerance as well as B cell 273:15765. 11. Hua, C. T., J. R. Gamble, M. A. Vadas, and D. E. Jackson. 1998. Recruitment and Downloaded from activation, it is possible that PECAM-1 serves to control mainte- activation of SHP-1 protein-tyrosine phosphatase by human platelet endothelial nance of self-tolerance. The important regulatory role of B cell cell adhesion molecule-1 (PECAM-1/CD31). J. Biol. Chem. 273:28332. coreceptors has been demonstrated by targeted disruption of coin- 12. Jackson, D. E., K. R. Kupcho, and P. J. Newman. 1997. Characterization of hibitory receptor in mice, in which the absence of an inhib- phosphotyrosine binding motifs in the cytoplasmic domain of platelet/endothelial cell adhesion molecule-1 (PECAM-1) that are required for the cellular association itory receptor leads to uncoupling of signaling pathways respon- and activation of the protein-tyrosine phosphatase, SHP-2. J. Biol. Chem. 272: 24368.

sible for control of B cell tolerance and activation. Immunological http://www.jimmunol.org/ defects observed include enhanced humoral Ab and anaphylactic 13. Pumphrey, N. J., V. Taylor, S. Freeman, M. R. Douglas, P. F. Bradfield, ␥ Ϫ/Ϫ S. P. Young, J. M. Lord, M. J. O. Wakelam, I. N. Bird, M. Salmon, and responses in Fc RIIB1 mice, constitutively activated T cells C. D. Buckley. 1999. Differential association of cytoplasmic signalling molecules Ϫ/Ϫ and lymphoproliferation in CTLA-4 mice, and hyperrespon- SHP-1, SHP-2, SHIP and phospholipase C-␥1 with PECAM-1 (CD31). FEBS sive B cells and autoantibody production in CD22Ϫ/Ϫ mice (31– Lett. 450:77. 36). The phenotype of PECAM-1Ϫ/Ϫ B cells is currently under 14. Ono, M., H. Okada, S. Bolland, S. Yanagi, T. Kurosaki, and J. V. Ravetch. 1997. Deletion of SHIP or SHP-1 reveals two distinct pathways for inhibitory signaling. investigation. Cell 90:293. In conclusion, as PECAM-1 belongs to the newly defined 15. Gupta, N., A. M. Scharenberg, D. N. Burshtyn, N. Wagtmann, M. N. Lioubin, Ig-ITIM superfamily of receptors that possess intracytoplasmic L. R. Rohrschneider, J. P. Kinet, and E. O. Long. 1997. Negative signaling path-

ways of the killer cell inhibitory receptor and Fc␥RIIb1 require distinct phos- by guest on September 30, 2021 (I/V)xYxx(L/V) ITIM motif(s) and its cytoplasmic domain has the phatases. J. Exp. Med. 186:473. capacity to induce an ITIM- and PTP-dependent inhibitory nega- 16. Watt, S. M., J. Williamson, H. Genevier, J. Fawcett, D. L. Simmons, A. Hatzfeld, tive signal, we propose that it serves as a B cell coreceptor to S. A. Nesbitt, and D. R. Coombs. 1993. The heparin binding PECAM-1 adhesion ϩ negatively regulate the amplitude and duration of ITAM-mediated molecule is expressed by CD34 hematopoietic precursor cells with early my- eloid and B-lymphoid cell phenotypes. Blood 82:2649. BCR signaling responses upon coligation of BCR complex with 17. Govender, D., P. Harilal, M. Dada, and R. Chetty. 1997. CD31 (JC70) expression inhibitory receptor. Our studies indicate that PECAM-1 may op- in plasma cells: an immunohistochemical analysis of reactive and neoplastic erate as a negative regulator in hemopoietic cells that coexpress plasma cells. J. Clin. Pathol. 50:490. PECAM-1 and ITAM-bearing receptors providing they are posi- 18. Fernandez, J. E., S. Deaglio, D. Donati, I. S. Beusan, F. Corno, A. Aragega, M. Forni, B. Falini, and F. Malavasi. 1998. Analysis of the distribution of human tioned in close proximity. This concept is supported by recent ob- CD38 and of its ligand CD31 in normal tissues. J. Biol. Regul. Homeostatic servations of negative regulation of TCR-mediated calcium mobi- Agents 12:81. lization responses by PECAM-1 in Jurkat T cells (25). Additional 19. Jackson, D. E., L. M. Gully, T. L. Henshall, C. E. Mardell, and P. J. Macardle. 2000. Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is studies will be required to elucidate the physiological significance associated with a naive B cell phenotype in human tonsils. Tissue Antigens of PECAM-1 interactions in primary B cell biology. 56:105. 20. Liu, Y.-J., and C. Arpin. 1997. Germinal center development. Immunol. Rev. 156:111. 21. Maeda, A., M. Kurosaki, M. Ono, T. Takai, and T. Kurosaki. 1998. Requirement Acknowledgments of SH2-containing protein-tyrosine phosphatases SHP-1 and SHP-2 for paired We thank Dr. Tomohiro Kurosaki and Mari Kurosaki (Kansai Medical immunoglobulin-like receptor B (PIR-B)-mediated inhibitory signal. J. Exp. Med. University) for expert advice and supply of anti-chicken SHP-1 Ab; Dr. 187:1355. ϩ Silvia Bolland and Dr. Jeffrey Ravetch (The Rockefeller University, New 22. Grykiewicz, G., M. Poenie, and R. Y. Tsien. 1985. A new generation of Ca2 York, NY) for supply of pBabe, pA PURO, and murine Fc␥RIIB1-pA indicators with greatly improved fluorescence properties. J. Biol. Chem. 260: 3440. PURO cDNA constructs; and Dr. Mark Coggeshall (Oklahoma Medical 23. Bert, A. G., J. Burrows, C. S. Osborne, and P. N. Cockerill. 2000. Generation of Research Foundation) for supply of polyclonal anti-SHIP Ab. We thank an improved luciferase reporter gene plasmid that employs a novel mechanism Andrew Bert for technical advice on NF-AT luciferase assays, and Denise for high-copy replication. Plasmid 44:173. Barbulesca for technical assistance. 24. Duncliffe, K. N., A. G. Bert, M. A. Vadas, and P. N. Cockerill. 1997. A -specific enhancer in the interleukin 3 locus is activated cooperatively by Oct and NFAT elements within a DNase I-hypersensitive site. Immunity 6:175. 25. Newton-Nash, D. K., and P. J. Newman. 1999. A new role for platelet endothelial References cell adhesion molecule-1 (CD31): inhibition of TCR-mediated signal transduc- 1. Kurosaki, T. 1998. Molecular dissection of B cell antigen receptor signaling. Int. tion. J. Immunol. 163:682. J. Mol. Med. 1:515. 26. Lienard, H., P. Bruhn, O. Malbec, W. H. Fridman, and M. Daeron. 1999. Signal 2. Campbell, K. S. 1999. Signal transduction from the B cell antigen receptor. Curr. regulatory proteins negatively regulate immunoreceptor-dependent cell activa- Opin. Immunol. 11:256. tion. J. Biol. Chem. 273:32493. 3106 MECHANISM OF INHIBITION OF B CELL ACTIVATION BY PECAM-1

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