Protein SH2D1A Phosphatase Is Regulated by the Adaptor SH2

CD150 Association with Either the SH2-Containing Inositol Phosphatase or the SH2-Containing Protein Tyrosine Phosphatase Is Regulated by the Adaptor This information is current as Protein SH2D1A of September 29, 2021. Larysa M. Shlapatska, Svitlana V. Mikhalap, Anna G. Berdova, Oleksander M. Zelensky, Theodore J. Yun, Kim E. Nichols, Edward A. Clark and Svetlana P. Sidorenko

J Immunol 2001; 166:5480-5487; ; Downloaded from doi: 10.4049/jimmunol.166.9.5480 http://www.jimmunol.org/content/166/9/5480 http://www.jimmunol.org/ References This article cites 55 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/166/9/5480.full#ref-list-1

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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. CD150 Association with Either the SH2-Containing Inositol Phosphatase or the SH2-Containing Protein Tyrosine Phosphatase Is Regulated by the Adaptor Protein SH2D1A1

Larysa M. Shlapatska,* Svitlana V. Mikhalap,* Anna G. Berdova,* Oleksander M. Zelensky,* Theodore J. Yun,† Kim E. Nichols,‡ Edward A. Clark,2† and Svetlana P. Sidorenko*

CD150 (SLAM/IPO-3) is a cell surface receptor that, like the B cell receptor, CD40, and CD95, can transmit positive or negative signals. CD150 can associate with the SH2-containing inositol phosphatase (SHIP), the SH2-containing protein tyrosine phosphatase (SHP-2), and the adaptor protein SH2 domain protein 1A (SH2D1A/DSHP/SAP, also called Duncan’s disease SH2-protein (DSHP) or SLAM-associated protein (SAP)). Mutations in SH2D1A are found in X-linked lymphoproliferative syndrome and

non-Hodgkin’s lymphomas. Here we report that SH2D1A is expressed in tonsillar B cells and in some B lymphoblastoid cell lines, Downloaded from where CD150 coprecipitates with SH2D1A and SHIP. However, in SH2D1A-negative B cell lines, including B cell lines from X-linked lymphoproliferative syndrome patients, CD150 associates only with SHP-2. SH2D1A protein levels are up-regulated by CD40 cross-linking and down-regulated by B cell receptor ligation. Using GST-fusion proteins with single replacements of tyrosine at Y269F, Y281F, Y307F, or Y327F in the CD150 cytoplasmic tail, we found that the same phosphorylated Y281 and Y327 are essential for both SHP-2 and SHIP binding. The presence of SH2D1A facilitates binding of SHIP to CD150. Apparently, SH2D1A may function as a regulator of alternative interactions of CD150 with SHP-2 or SHIP via a novel TxYxxV/I motif (immunoreceptor http://www.jimmunol.org/ tyrosine-based switch motif (ITSM)). Multiple sequence alignments revealed the presence of this TxYxxV/I motif not only in CD2 subfamily members but also in the cytoplasmic domains of the members of the SHP-2 substrate 1, sialic acid-binding Ig-like lectin, carcinoembryonic Ag, and leukocyte-inhibitory receptor families. The Journal of Immunology, 2001, 166: 5480–5487.

he B cell receptor (BCR),3 CD40 or CD95/Fas each can ments proliferation induced by CD40 mAb and IL-4 (3). On the play a dual role in the regulation of the B cell fate. The other hand, CD150-induced signals can synergize with and aug- T outcome after ligation of any of these receptors depends ment CD95-mediated apoptosis (5). Engaging CD150 with mAb on the stage of B cell differentiation, the combination and sequence promotes IL-2- and CD28-independent but cyclosporin A-sensi- by guest on September 29, 2021 of signals delivered via these receptors, and the involvement of tive proliferation of T cells (6). Furthermore, ligation of CD150 other molecules such as CD80, CD86, and IL-4R (1, 2). However, also induces IFN production by CD4ϩ T cell clones and Ig pro- little is known about cell surface receptors that may modulate B duction by activated B cells (4, 6, 7). Given that in Th1 cells cell fate at later stages of differentiation. CD150 is expressed at 7- to 25-fold higher levels than in Th2 cells A possible candidate for a regulator of mature B cells is CD150 (8), CD150 may be involved in expanding Th0/Th1 immune re- (signaling lymphocytic activation molecule/IPO-3) (3, 4). Ligation sponses (9). How CD150 mediates these effects in lymphocytes is of CD150 on resting B lymphocytes with CD150 mAb induces a not known. 2ϩ rapid elevation of intracellular free calcium ([Ca ]i) and aug- CD150 in T cells associates with the small SH2-containing adaptor protein 1A (SH2D1A), also called Duncan’s disease SH2- *Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology National protein (DSHP) or SLAM-associated protein (SAP) (10, 11). Mu- Academy of Sciences of the Ukraine, Kiev, Ukraine; †Department of Microbiology, University of Washington, Seattle, WA 98195; and ‡Pediatric Oncology, Children’s tations in the SH2D1A gene lead to X-linked lymphoproliferative Hospital of Philadelphia, Philadelphia, PA 19104 syndrome (XLP), an immunodeﬁciency associated with dysregu- Received for publication June 2, 2000. Accepted for publication February 23, 2001. lated proliferation of T and B lymphocytes in the setting of pri- The costs of publication of this article were defrayed in part by the payment of page mary EBV infection (10, 12–14). SH2D1A binds to a sequence charges. This article must therefore be hereby marked advertisement in accordance surrounding Y281 in the cytoplasmic tail of CD150 in a tyrosine- with 18 U.S.C. Section 1734 solely to indicate this fact. independent manner (15, 16). One possibility is that SH2D1A 1 This work was supported by Howard Hughes Medical Institute Grant 76195- functions as a signaling inhibitor by blocking and/or regulating 548101, and INTAS Grant 96-1493 (to S.P.S.), U.S. Civilian Research and Devel- opment foundation Grants UN2-437 (to S.P.S.) and USB-383 (to L.M.S.), and Na- binding of signal transducing molecules to SH2 docking sites (10, tional Institutes of Health Grant GM37905 (to E.A.C.). 13). Indeed SH2D1A may block recruitment of the protein tyrosine 2 Address correspondence and reprint requests to Dr. Edward A. Clark, Department of phosphatase SHP-2 to CD150 in T cells and the 2B4 receptor in Microbiology, Box 357242, University of Washington, Seattle, WA 98195. E-mail address: [email protected] NK cells (10, 11, 17). This block may lead to selective impairment 3 Abbreviations used in this paper: BCR, B cell antigen receptor; B-LCL, B lymphoblas- of 2B4-mediated NK cell activation and possibly T cell function in 2ϩ toid cell line; BL, Burkitt’s lymphoma cell line; [Ca ]i, intracellular free calcium; XLP patients (10, 18, 19). However, defects in T and NK signaling CD150ct, cytoplasmic tail of CD150; PY, phosphotyrosine; SH2D1A, SH2 domain pro- may not be completely responsible for such phenotypic manifesta- tein 1A; SHIP, SH2-containing inositol phosphatase; SHP-2, SH2-containing protein tyrosine phosphatase; XLP; X-linked lymphoproliferative syndrome; ITSM, immunorecep- tions of XLP as dysgammoglobulinemia and B cell non-Hodgkin tor tyrosine-based switch motif; SHPS, SHP-2 substrate 1; CEA, carcinoembryonic Ag; lymphomas (20, 21). NP-40, Nonidet P-40; HMM, hidden Markov model; Siglec, sialic acid-binding Ig-like lectin; ITIM, immunoreceptor tyrosine-based inhibitory motif; SIT, SHP-2-interacting Recently, we found that in B cells CD150 can bind not only transmembrane adaptor protein; LIR, leukocyte-inhibitory receptors. SHP-2 but also SH2-containing inositol phosphatase (SHIP) (5).

However, whether SH2D1A can compete with SHP-2 and/or SHIP CD150, or anti-CD95, followed by streptavidin-PerCP (Becton Dickinson, in B cells was unclear. Here we report that SH2D1A is expressed Mountain View, CA) and PE-labeled anti-CD38 (PharMingen, San Diego, in tonsillar B cells and in some B lymphoblastoid cell lines, where CA), anti-CD3, or anti-CD20 (Becton Dickinson). Cells were fixed in 1% paraformaldehyde for 20 min and permeabilized with 0.2% Tween 20 for it associates with CD150. In these cell lines, CD150 coprecipitates 15 min. Then rabbit anti-SH2D1A serum (affinity-purified using SH2D1A Ј with SHIP and SH2D1A, but in SH2D1A-negative cell lines peptide) was added followed by goat F(ab )2 anti-rabbit IgG. Cells were CD150 associated with SHP-2. Using mutational analysis, we washed twice with PBS containing 2.5% FCS and 0.2% Tween 20 and then found that both Y281 and Y327 in the CD150 cytoplasmic tail analyzed on a FACScan flow cytometer (Becton Dickinson). (CD150ct) are essential for binding of SHP-2 as well as SHIP. Biochemical methods Apparently, SH2D1A regulates SHIP vs SHP-2 binding to a Cell lysis, immunoprecipitation, SDS-PAGE, in vitro kinase assays, and TxYxxV/I motif (immunoreceptor tyrosine-based switch motif subcellular fractionations were performed as described (3, 27, 28). Western (ITSM)) in CD150. Multiple sequence alignments revealed that the blotting was performed with an ECL kit (Amersham, Arlington Heights, ITSM motif is not only in cytoplasmic tails of CD2 subfamily IL). For evaluation of kinase activities, immunoprecipitates were washed members (CD150, CD84, Ly-9/CD220, and 2B4/CD244), but also with Nonidet P-40 (NP-40) lysis buffer or with NP-40 lysis buffer containing 0.5 M NaCl, twice with high salt buffer (0.5 M LiCl), and once with in the SHPS (SHP-2 substrate 1), sialic acid-binding Ig-like lectin NP-40 lysis buffer and were subjected to in vitro kinase assays. (Siglec), carcinoembryonic Ag (CEA), and leukocyte-inhibitory receptor (LIR) families, underscoring the functional importance of Modeling of the CD150 cytoplasmic tail this motif. Preliminary CD150 homologue searches were performed with BLAST and PSI-BLAST over various protein databases including TrEMBL, Swiss-

Materials and Methods Prot, Kabad, and PDB. CD150 homologues were aligned with the ClustalX Downloaded from Abs and reagents program. Program alignment output was slightly modified manually with JalView and GeneDoc alignment viewers to align TxYxxV/I-containing Rabbit antisera against SHIP, SHP-2, and p38 mitogen-activated protein regions. We used HMMER package tools (29–31) to build hidden Markov kinase; were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). model (HMM) profiles for the multiple alignment of CD150, 2B4, CD84, Glutathione-agarose was purchased from Sigma (St. Louis, MO, protein A- and Ly9 and to search TrEMBL, Kabat, and Swiss-Prot databases using and protein G-Sepharose were from Pharmacia (Piscataway, NJ). For this profile as a query. As the number of identified homologues grew, new cross-linking of surface receptors, we used the following mAbs: IPO-3 sequences were added to the multiple alignments, and new HMM profiles anti-CD150 (IgG1); IPO-4 anti-CD95 (IgM) (22); and G28-5 anti-CD40 were built. Identification of CD150ct structural homologues was done by http://www.jimmunol.org/ Ј (IgG1) (23). F(ab )2 of goat anti-human IgM (Jackson ImmunoResearch, both sequence (BLAST, Fasta, CPHmodels, UCSC HMM) and structure West Grove, PA) were used for IgM cross-linking on human B cell lines. alignment H3P2 at UCLA-Department of Energy (32, 33). CD150 se- To generate a polyclonal Ab recognizing SH2D1A (DSHP), one of us quence fitting to the recognized fold was performed in a Swiss PDB viewer (K.E.N.) immunized rabbits with a 26-aa peptide conjugated to keyhole (34, 35) and submitted for modeling to the Swiss-Model server (http:// limpet hemocyanin (peptide sequence: EKKSSARSTQGTTGIREDPDV www.expasy.ch/swissmod/SWISS-MODEL.html). The quality of the mod- CLKAP). After three injections, serum was collected and the anti-SH2D1A eled structure was assessed by ERRAT (36) and Verify3D (37) programs. titer was determined using an ELISA with free peptide bound in the solid We used PovRay ray tracing software to prepare the presented picture. phase (Research Genetics, Huntsville, AL). Results and Discussion Plasmid constructs

SH2D1A is expressed in B cells where it associates with CD150 by guest on September 29, 2021 The GST-fusion protein construct of the cytoplasmic tail of CD150 (GST- CD150 is expressed on the surface of B cells and is up-regulated CD150ct) was prepared as described (5). Forward and reverse primers with the appropriate restriction sites for in-frame cloning into the pGEX-2T after activation (3). Using immunohistology and a microarray anal- plasmid were used to amplify the cDNA fragments using pfu polymerase ysis, CD150 was found in diffuse large B cell lymphoma (22, 38). (Stratagene, La Jolla, CA). Using PCR-based site-directed mutagenesis Because it was shown that in T cells CD150 associates with (24), we made constructs of GST-CD150ct fusion proteins with phenylal- SH2D1A, we tested whether SH2D1A is expressed in B cells and anine (F) replacements at tyrosines Y269, Y281, Y307, and Y327. Plas- mids with the correct nucleotide sequences were transformed into the bac- whether it associates with CD150 in B cells. We assess the ex- terial strain XLI-BlueMRFЈ (Stratagene) for fusion protein production. pression of SH2D1A protein in B lineage cells in a panel of B cell Plasmids containing GST-CD150ct were also transformed into the Esche- lines representing different stages of maturation. All studied B- richia coli strain TKX1 (Stratagene) for production of tyrosine-phospho- LCL, including cell lines from patients with XLP (IARC 739, rylated fusion proteins. Tyrosine phosphorylation of these fusion proteins XLP-D, XLP-8002, XLP-8005), expressed high levels of CD150 apparently was restricted only to the corresponding cytoplasmic tails, be- Ͼ cause GST was not tyrosine phosphorylated when expressed alone in the peak fluorescence intensity, 10.0). The BL lines Raji, Namalwa ϩ Ϫ same bacteria. Expression and purification of GST fusion proteins were (EBV ), and BJAB (EBV ) expressed CD150 at a moderate level performed as described (25). (peak fluorescence intentisy 7.0); all other B cell lines tested SH2D1A sequence analysis (REH, Ramos, B104, RPMI-8226) were CD150 negative. Western blot analysis of whole cell lysates revealed that SH2D1A was ex- DNA was extracted from EBV-immortalized cell lines according to stan- dard protocols. The SH2D1A coding sequence was PCR amplified using pressed in only two of the B lymphoblastoid cell lines studied, Expand Taq polymerase (Boehringer Mannheim, Indianapolis, IN) and MP-1 and CESS (Fig. 1A). Flow cytometry also showed intracel- primers flanking each of the four SH2D1A exons as described (13). lular expression of SH2D1A in MP-1, but not in the BJAB cell line Cell lines and stimulation (Fig. 1B). The surface phenotype of MP-1 cells resembles early germinal The pre-B cell lines REH and Namalwa; Burkitt’s lymphoma cell lines center cells: IgMϩIgDϪCD38ϩCD39lowCD95ϩCD150ϩ. The Ramos, BJAB, and Raji; the B lymphoma line B104; the B lymphoblastoid CESS cell line has a surface phenotype similar to memory B cells; cell lines (B-LCL) CESS, MP-1, T5-1, 6.16, and RPMI-1788; and the high Jurkat T cell line were maintained as described (26). B-LCL from XLP e.g., it expresses membrane IgG, but not IgM, and is CD39 .In patients included: IARC 739 (interstitial deletion of SH2D1A), XLP-D agreement with prior studies (13), immunohistochemical analysis (C3T mutation at the position 462), XLP-8005 (C3T mutation at the revealed SH2D1A expression in frozen sections from tonsils, position 471), XLP-8002 (no mutations in SH2D1A) (13). T cell-depleted lymph nodes, and diffuse large B cell lymphoma. SH2D1A was tonsillar cells were prepared as described (3, 25). present within the cytoplasm of scattered large cells localized to B Cell staining cell areas (data not shown). To evaluate the SH2D1A expression in For determination of cell surface phenotype and cytoplasmic expression of subsets of B cells in vivo, we performed three-color analysis of T ϩ SH2D1A, cells were surface stained with biotin-labeled anti-IgD, anti- cell-depleted tonsillar cells (2.8 Ϯ 0.3% of CD3 lymphocytes). 5482 SH2D1A REGULATES CD150 ASSOCIATION WITH SHIP AND SHP-2

lines tested, CD150 associated only with SHP-2 (Fig. 2A). Trace amounts of SHP-2 were detected together with CD150 in the CESS cell line that has a much lower level of SH2D1A expression than the MP-1 line. Apparently, this differential binding of SHIP with SH2D1A vs SHP-2 did not depend on CD150 tyrosine phosphorylation, since CD150 is constitutively phosphorylated on tyrosine in both the SH2D1Aϩ and SH2D1AϪ cell lines studied (Fig. 2B). To evaluate a possible role for differential phosphorylation of any of four tyrosine residues in CD150ct, we used tyrosine-phosphorylated GST-fusion proteins of CD150ct (5). In the absence of SH2D1A in cell lysates (cell line BJAB), this fusion protein precipitated SHP-2, but in the presence of SH2D1A (cell lysates from the MP-1 cell line), GST-CD150ctPY bound not only SHP-2 but also SHIP (Fig. 2C). Thus, the presence of SH2D1A- facilitated binding of SHIP to CD150. GST-CD150ctPY coprecipitation with both SHIP and SHP-2, apparently depends on the level of SH2D1A association with CD150. Preferential SHIP binding to the native CD150 molecules in SH2D1Aϩ cell lines may reflect associations in the context of intracellular localization. Downloaded from Short term ligation of IgM on SH2D1Aϩ MP-1 cells did not change the level of SH2D1A and SHP-2 coprecipitated with FIGURE 1. Expression of SH2D1A in B cells. A, The presence of CD150 but did reduce the level of SHIP associated with CD150 SH2D1A in B-LCL lines MP-1 and CESS was revealed by Western blot- (Fig. 3A). At the same time, the level of precipitable CD150 re- 5 ting with an anti-SH2D1A serum. NP-40 lysates of 5 ϫ 10 cells/well were mained constant (Fig. 3A). Under the same conditions, SHIP and resolved in 15% SDS-PAGE. Positive control was the T cell line Jurkat. SH2D1A levels in the cytosolic and particulate cell fractions re- http://www.jimmunol.org/ One of four experiments. B, Expression of SH2D1A in B cell lines BJAB mained constant (Fig. 3B). We did not detect BCR-induced relo- and MP-1. Rabbit IgG served as a negative control for purified anti- SH2D1A rabbit Ab. C, Expression of SH2D1A in tonsillar B cells. Cells calization of either SHIP or SH2D1A to detergent-insoluble gly- were stained for IgD, CD38, or CD150 surface expression and cytoplasmic colipid-enriched domains (Fig. 3B). In contrast, IgM cross-linking SH2D1A using three-color flow cytometry. Rabbit IgG was a negative had no effect on the level of either SHIP or SHP-2 association with Ϫ control for purified anti-SH2D1A Ab. Naive IgDϩCD38Ϫ B cells; mean, CD150 in the SH2D1A cell line BJAB (Fig. 3A, lower panels)or 85.4. Germinal center IgDϪCD38ϩ B cells, SH2D1Aϩ cells, 32.0%; mean, XLP B-LCLs (data not shown). This suggests that BCR ligation ϩ ϩ ϩ 94.4; peak channel (PKchan), 112.0. CD38 CD150 cells, SH2D1A can alter association of CD150 with SHIP, but the amount of SHIP Ϫ Ϫ cells, 46.14%; mean, 101.5; PKchan, 119.0. IgD CD38 cells, mean 70.3. associated with CD150 does not depend on SH2D1A levels. Thus, by guest on September 29, 2021 One of four experiments. SH2D1A does not simply compete with SHIP for binding to CD150. ϩ Because CD150 expression is up-regulated on naive B cells by These studies revealed SH2D1A in 11.5–18.0% of CD20 tonsil- either CD40 or BCR cross-linking (3), we tested whether SH2D1A lar B cells. Low levels of SH2D1A expression were found in naive IgDϩCD38ϪB cells (mean, 85.4). SH2D1A was detected in 32.0% expression in B cells could be induced and/or up-regulated. Up to of germinal center IgDϪCD38ϩ (9% of total B cells; mean, 94.4; 48 h after ligation of CD40, IgM, or CD150, SH2D1A expression was not induced in cell lines BJAB, Raji, or XLP-8002. In the peak, 112.0). The highest level of SH2D1A protein was detected in ϩ 46.1% of CD38ϩCD150ϩ cells (mean, 101.5; peak, 119.0) (Fig. SH2D1A cell line, CESS, ligation of CD40 or CD150 did not 1C). At the same time IgDϪCD38Ϫ cells, which include memory change the level of SH2D1A expression (data not shown). How- B cells, were SH2D1A negative (mean, 70.3) (Fig. 1C). These data ever, 48 h of stimulation via CD40 up-regulated SH2D1A expres- agree with immunohistochemical studies of frozen tonsillar sec- sion in MP-1 cells (Fig. 3C). Cross-linking of CD150 on these tions that did not reveal preferential localization of SH2D1A in cells did not affect SH2D1A levels, whereas IgM ligation down- any B cell zone, but did detect the highest level of SH2D1A ex- regulated SH2D1A expression (Fig. 3C). pression within large cells with cleaved nuclei localized in germinal centers (data not shown). Tyr281 and Tyr327 in the CD150ct are essential for both SHIP Association of CD150 with SHIP, SH2D1A, or SHP-2 in B cells and SHP-2 binding Because our data implied that CD150 and SH2D1A are coex- To clarify the molecular basis of SHIP vs SHP-2 binding to pressed in B cells, we tested whether these molecules associate in CD150, we constructed GST-fusion proteins of the CD150ct with B cells. In both SH2D1Aϩ cell lines (MP-1 and CESS), SH2D1A single replacements of tyrosine at Y269F, Y281F, Y307F, or coprecipitated with CD150. As expected, SH2D1A was not de- Y327F (Fig. 4A). Since SHIP and SHP-2 binding to CD150ct is tected with CD150 in SH2D1AϪ lines T5-1, IARC 739, XLP- phosphotyrosine dependent (5), all fusion proteins were expressed 8005, or BJAB (Fig. 2A). In some B cell lines, CD150 coprecipi- in both tyrosine-phosphorylated (PY) and nonphosphorylated tated with SHIP, and a tyrosine-phosphorylated fusion protein of forms. The major 145-kDa protein coprecipitated with GST- the CD150ct can bind SHP-2 (5). Also in COS-7 and mouse T CD150ct-PY and phosphorylated in the in vitro kinase assay pre- cells, CD150 binds SHP-2, and this association can be blocked by viously was identified as SHIP (Ref. 5; Fig. 2C). Mutations in any SH2D1A (10, 11). Immunoprecipitation experiments followed by one of the tyrosines did not affect binding of SH2D1A to CD150ct Western blot analysis clearly showed that in the SH2D1A-express- (Fig. 4B). However, SHIP bound to GST-CD150ct-PY, M1- ing cell lines MP-1 and CESS, CD150 coprecipitated with SHIP, PY(Y269F), and M3-PY(Y307F) in a phosphotyrosine-dependent and not SHP-2 (Fig. 2A). In contrast, in all SH2D1A-negative cell manner (Fig. 4B and data not shown). At the same time, we did not The Journal of Immunology 5483 Downloaded from

FIGURE 2. A, Coprecipitation of CD150 with SHP-2 vs SHIP and SH2D1A. CD150 was immunoprecipitated with mAb IPO-3 directly coupled to http://www.jimmunol.org/ Sepharose, and MOPC 21 myeloma protein directly coupled to Sepharose was used as a negative control; 50 ϫ 106 cells/immunoprecipitation. Western blot analysis with Abs against SHIP, SHP-2, and SH2D1A was performed using ECL. Whole cell lysates served as a positive control. All cell lines expressed CD150 at comparable levels. One of four experiments. B, Tyrosine phosphorylation of CD150 revealed in Western blot with mAb 4G10. CD150 was immunoprecipitated with mAb IPO-3 as described in A. C, Presence of SH2D1A-facilitated SHIP association with CD150. Nonphosphorylated and tyrosine-phosphorylated GST-fusion proteins of CD150ct were used for precipitations from MP-1 cells followed by Western blot with anti-SHIP, anti- SHP-2, and anti-SH2D1A serum. Tyrosine phosphorylation of fusion proteins was controlled with mAb 4G10 (Anti-PY). by guest on September 29, 2021

FIGURE 3. A, BCR ligation down-regulates association of SHIP with CD150 but does not affect CD150 association with SH2D1A and SHP-2. Western blot of CD150 immunoprecipitates. After activation of cells with anti-IgM sera, immunoprecipitations were performed as in Fig. 2. As a control for CD150 levels after BCR ligation, we included immunoprecipitation of surface-biotinylated CD150 followed by Western blot with streptavidin peroxidase. One of ﬁve experiments. B, At the same time BCR ligation did not change the level of SHIP and SH2D1A in particulate or cytosolic fractions or detergent-insoluble glycolipid-enriched domains (DIGs). The presence of Lyn served as a control for detergent-insoluble glycolipid-enriched domains preparation (lower panel). Western blot of subcellular fractions. One of three experiments. C, Regulation of SH2D1A expression via BCR and CD40. BCR ligation down- regulated and CD40 engagement up-regulated SH2D1A expression in MP-1 cells after 48 h of stimulation. Western blot analysis with anti-SH2D1A serum on NP-40 lysates. Anti-p38 mitogen-activated protein kinase (p38) blots controlled equal loading. One of three experiments. 5484 SH2D1A REGULATES CD150 ASSOCIATION WITH SHIP AND SHP-2 Downloaded from http://www.jimmunol.org/

FIGURE 4. A, Replacements of tyrosine by phenylalanine in GST-fusion protein constructs of the CD150ct. B, Y281 and Y327 in CD150ct are involved in SHIP recruitment. Nonphosphorylated and tyrosine-phosphorylated GST fusion proteins of CD150ct were used for precipitations from MP-1 cells followed by in vitro kinase assays on precipitates (SHIP) and Western blot with anti-SHIP (data not shown) and anti-SH2D1A serum. The tyrosine- phosphorylated fusion proteins GST-M2-PY (Y281F) and GST-M4-PY (Y327F) did not precipitate SHIP. One of ﬁve experiments. C, D, Both Y281 and Y327 in the CD150ct are involved in SHP-2 recruitment. Western blot analysis of fusion proteins precipitates with anti-SHP-2 and anti-SH2D1A Abs. Tyrosine-phosphorylated fusion proteins were used for precipitations from MP-1 (C and D, upper and middle panels) and BJAB (D, lower panel) cell lines. The tyrosine-phosphorylated fusion proteins GST-M2-PY (Y281F) and GST-M4-PY (Y327F) did not precipitate SHP-2 from the MP-1 cell line in the

presence of SH2D1A. At the same time, mutations in Y269 and Y307 (fusion proteins GST-M1-PY and GST-M3-PY did not affect binding of SHP-2 to by guest on September 29, 2021 CD150ct (upper panels of C and D). Recruitment of SH2D1A to CD150ct was revealed by Western blot with anti-SH2D1A serum. In the absence of SH2D1A (cell line BJAB), GST-M2-PY (Y281F) and GST-M4-PY (Y327F) were able to recruit SHP-2 (D). Tyrosine phosphorylation of fusion proteins were controlled with anti-PY mAb 4G10 (D, lower panel). One of four experiments. detect SHIP in the precipitates with M2-PY(Y281F) and M4- tonsillar B cells (Fig. 1). As in T cells, CD150 coprecipitates with PY(Y327F) (Fig. 4B). SHP-2 was also precipitated with GST- SH2D1A in B cells, and neither phosphorylation nor mutations of CD150ct-PY, M1-PY(Y269F), and M3-PY(Y307F) fusion pro- tyrosines in CD150 affect this association (Figs. 2 and 4). The SH2 teins, and again both M2-PY(Y281F) and M4-PY(Y327F) failed to domain of SH2D1A binds the sequence surrounding Y281 devoid bind SHP-2 in cell lysates from SH2D1Aϩ cells (Fig. 4, C and D). of tyrosine (15, 16) and also binds in a phosphotyrosine-dependent On the other hand, in the absence of SH2D1A (BJAB cell lysates) manner to the 2B4 receptor (17). Furthermore, SH2D1A can com- both M2-PY and M4-PY were able to bind SHP-2 (Fig. 4D), in- pete with SHP-2 for binding to the cytoplasmic tail of CD150 and dicating that SH2D1A and SHP-2 are competing not only for Y281 2B4 (10, 17). Here we found that in SH2D1A-expressing B cell but also for Y327. The fact that M2-PY and M4-PY bind more lines, CD150 coprecipitates with SHIP; however, in SH2D1AϪ SH2D1A than SHP-2-binding mutants also may reflect competi- cell lines, CD150 associates only with SHP-2, and the presence of tion for the same binding sites. These results suggest that the same SH2D1A facilitates SHIP binding to CD150ct (Fig. 2C). Probably, tyrosines within TxYxxV/I motif in CD150ct (Y281 and Y327) are the regulation of CD150 association with SHIP vs SHP-2 by required for SHIP and SHP-2 association with CD150. SH2D1A may be under control of BCR and CD40 signaling since: This study provides evidence of SH2D1A expression in human 1) short term signal via BCR decreases SHIP association with B cells. Using in vivo and in vitro approaches, we showed that CD150 (Fig. 3A); and 2) long term (48 h) BCR ligation reduces both SHIP and SHP-2 are able to bind CD150 in B cells. Point and CD40 cross-linking up-regulates the level of SH2D1A expres- mutations of tyrosines in GST-fusion proteins of the CD150 cy- sion (Fig. 3C). toplasmic tail revealed that tyrosine phosphorylation of the same residues Y281 and Y327 are essential for SHIP as well as SHP-2 binding. Replacements at residues Y269 and Y307 did not affect Modeling of the CD150ct either SHIP or SHP-2 association with CD150. How then is SHIP The cytoplasmic tail of CD150 has the paired tyrosine-based motif and SHP-2 binding to the same sites regulated? Our data indicate TxYxxV/I, which we propose to be designated as a “switch” motif that the adaptor protein SH2D1A is involved in this regulation. (ITSM). This motif with the help of the adaptor protein SH2D1A Despite the reported absence of SH2D1A expression in murine B may control binding of tyrosine vs inositol phosphatases to recep- cells (11), SH2D1A mRNA (12, 13) and protein are expressed in tors. This motif is different from the well-defined immunoreceptor human B cells, including the B cell lines MP-1 and CESS and tyrosine-based activation motifs D/ExxYxxL/I(x)6-8YxxL/I in BCR The Journal of Immunology 5485 and TCR complexes, which on phosphorylation recruit protein ty- The third most represented group of sequences belongs to the rosine kinases such as Syk and ZAP-70 (39, 40). However, the Siglec/CD33 family. The cytoplasmic domains of CD31/platelet en- CD150/2B4 motif has some similarities with immunoreceptor ty- dothelial cell adhesion molecule-1, CD33, Siglec-5 (OB/BP2), and rosine-based inhibitory motifs (ITIMs) I/VxYxxL/V(x)26-31I/ Siglec-9 all have a similar tyrosine-based motif distribution pattern. VxYxxL/V found within cytoplasmic domains of “inhibitory receptor A ITSM-like motif is situated 3–7 residues from the C terminus superfamily” members such as FcRIIb, CD22, CD72, killer Ig-related and is preceded by a conventional ITIM motif. Interestingly, simi- receptor, paired Ig-like receptors, p49B, Ig-like transcript (ILT), and lar to CD150ct, a GST platelet endothelial cell adhesion molecule/ leukocyte-associated Ig-like receptor (41). These ITIMs inhibit acti- CD31 cytoplasmic tail can bind both SHP-2 and SHIP, and SHIP vation receptors by recruiting SH2-containing tyrosine phosphatases interacts predominantly with the ITSM motif in CD31 (43). SHP-1 and SHP-2, and also SHIP (1, 41, 42). CEA superfamily members were also widely represented in the We performed a series of protein sequence databases searches to retrieved sequences with ITSM motifs. Bgp-1, Bgp-2, C-CAM 105 broaden the list of ITSM-containing molecules. Most of the pre- ecto-ATPase, and related molecules contain Y-based motifs the viously reported CD150 homologues, 2B4 (CD244), CD48, CD84, sequences and positions of which in the cytoplasmic domain are and Ly9 (CD220), belong to CD2 subfamily of the Ig superfamily. highly similar to the positions and sequences of ITSMs: tyrosines Multiple alignments of CD2 subfamily members with the highest at the C terminus followed by a group of positively charged residues. level of homology to CD150 (2B4, Ly9, CD84) were used to build Similar to CD150, which recently was shown to serve as alternative HMM profiles for position-specific matching searches against a measles virus receptor (44), both Bgp-1 and Bgp-2 are receptors for Swiss-Prot database with a HMMER program package. Because mouse hepatitis virus and also have truncated forms (45). we consider the ITSM motif the main functional unit within the Unlike sialoadhesin and CEA family members, other ITSM and Downloaded from CD150ct, the key criterion for sequence selection was the presence ITIM-containing molecules retrieved by the HMM search demon- of tyrosine-based motifs that fit the ITSM consensus (Fig. 5A). The strate experimentally confirmed inhibitory activity. CD150 common feature for members of the CD2 subfamily is a paired showed a weak sequence homology to several members of the ITSM and the existence of differentially spliced truncated forms recently established monocyte-inhibitory receptor/LIR/ILT family, leading to only a single ITSM for CD150, 2B4, and Ly-9 (Fig. 5, like paired Ig-like receptors B and programmed death-1 (PD-1)

A and B). receptor. However, ITIMs rather than ITSMs are presented more http://www.jimmunol.org/ Search results for CD150, 2B4, CD84, and Ly9 alignment revealed widely in the cytoplasmic domains of this group, and motif ho- homology to several members of SHPS-1 (SHP-2 substrate 1) family: mology to CD150 ITSMs is weaker than in the case of Siglecs or SHPS-1, BIT, and MYD. These sequences are highly homologous to CEAs (Fig. 5, A and B). Nevertheless, the structural similarity each other and contain two ITSM-like motifs alternated with ITIM between these sequences and CD150 creates a potentially impor- consensus motifs (Fig. 5, A and B). The cytoplasmic tail of SHPS-1, tant bridge between these two groups of molecules. ITSM-con- like CD150, is able to bind SHP-2 phosphatase. taining molecules also include the catalytically inactive tyrosine by guest on September 29, 2021

FIGURE 5. A, Multiple alignments of CD150 TxYxxV/I motifs (ITSM) and the tyrosine-containing motifs from the sequences found by HMM searches. B, Schematic chart representing the distribution of ITIM and ITSM-like motifs in the cytoplasmic domains of the molecules with homology to CD150. ᮀ, ITIM; ■, ITSM. PD-1, programmed death-1 receptor. C, Ribbon diagram illustrating the three-dimensional structure of CD150ct. The diagram was prepared using a Swiss PDB Viewer. 5486 SH2D1A REGULATES CD150 ASSOCIATION WITH SHIP AND SHP-2 kinase human receptor related to tyrosine kinase and the mem- dissociation constants for SH2 domains and their affinity to ITSMs brane adaptor protein called SHP-2-interacting transmembrane in CD150, we will be able to build a kinetic model of their com- adaptor protein (SIT) (46) (Fig. 5, A and B). The effector molecules petitive binding. that bind the ITIM motif in SIT have not been identified, but it is Why should SH2D1A binding to the sequence surrounding likely that SIT regulates TCR-mediated induction of IL-2 gene Y281 in CD150 prevent its association with SHP-2 but not com- transcription via this motif (47). pete with SHIP? Amino-terminal residues adjacent to Y281 in the The distribution of ITSM-like tyrosine-containing motifs in cytoplasmic tail of CD150 are critical for high affinity binding of CD150 homologues confirms that these motifs represent an im- SH2D1A to CD150 (16). At the same time, mutational analysis of portant and structurally unique but poorly studied group of ty- the Fc␥RIIB tail revealed that the residue Y279 within the ITIM rosine-containing regulatory motifs. Multiple alignment of ITSM- motif is required for SHP-2 but not SHIP binding to the cytoplas- like motifs from distant homologues of CD150 indicate the mic tail of Fc␥RIIB (50, 51). Therefore, high affinity binding of conservation pattern of this kind of motif: 1) in most of the motifs SH2D1A to residues amino-terminal to Y281 in CD150 may com- presented on Fig. 5A, at least one additional conserved position is pete with SHP-2 but not affect association of SHIP with CD150. evident: a threonine or serine in position Ϫ4 with respect to the Taken together, SH2D1A may play a role as a molecular “switch” ITSM tyrosine. This pattern is present in all ITSMs (Y281 and that regulates SHIP vs SHP-2 association with CD150. This model Y327 in CD150, 2B4, CD31) that have been shown to be func- is consistent with the reported involvement of SHIP in regulation tionally significant; 2) in members of CD2, Siglec, and CEA fam- of B cell development as well as immune responses to antigenic ilies, one ITSM is positioned 3–5 residues upstream of the C ter- challenge (52–54). In other words, CD150 may transmit SHIP- minus; 3) CD150, Ly9, 2B4, and murine Bgp-2 have alternatively dependent or SHP-2-mediated signals at distinct stages of B cell Downloaded from spliced forms, and shorter isoforms possess only a single ITSM; 4) maturation, and the adaptor protein SH2D1A may regulate this in several families (SHPS, Siglec, and LIR, both ITIM and ITSM switch. Using DT40 sublines, we are currently investigating what consensus motifs are present. CD150-mediated signal transduction pathways are regulated by We applied computational biology methods to explore possible SH2D1A. After this paper was submitted, Nagy et al. (55) reported mechanisms of CD150ct interactions with associated molecules the expression of SH2D1A in B cell lines derived from EBV-

and to explain the available experimental data. No sequences with positive Burkitt’s lymphomas. http://www.jimmunol.org/ significant homology to CD150 were found in Protein Data Bank (PDB) by either search algorithm we used, including the position- Acknowledgments specific scoring methods (30, 48); therefore, homology modeling We thank Dario Magaletti for expert technical assistance, Dr. O. V. Yurchenko was inapplicable. Threading of the CD150ct sequence by the H3P2 for immunohistochemical analysis, and Kate Elias for editorial assistance. method predicted an Ig-like ␤ sandwich fold for CD150ct. One structure (IgG1 H chain, PDB access code 1tet, residues 113–213) References with the best z score and alignment to CD150 was selected for 1. Healy, J. I., and C. C. Goodnow. 1998. Positive versus negative signaling by modeling. The model we built represents a Greek key fold, typical lymphocyte antigen receptors. Annu. Rev. Immunol. 16:645. for Ig-like domains, with two parallel ␤ sheets that contain two and 2. Craxton, A., K. L. Otipoby, A. Jiang, and E. A. Clark. 1999. Signal transduction by guest on September 29, 2021 pathways that regulate the fate of B lymphocytes. Adv. Immunol. 73:79. four strands (Fig. 5C). Checking the quality of the resulting struc- 3. Sidorenko, S. P., and E. A. Clark. 1993. Characterization of a cell surface gly- ture by ERRAT (36) and Verify3D (37) programs indicated that coprotein IPO-3, expressed on activated human B and T lymphocytes. J. Immu- the modeled structure might be natural. Realizing the limitations of nol. 151:4614. 4. Cocks, B. G., C. C. Chang, J. M. Carballido, H. Yssel, J. E. de Vries, and the used modeling method, nevertheless, we can use this model for G. Aversa. 1995. A novel receptor involved in T-cell activation. Nature 376:260. analysis of the experimental data from a structural perspective and 5. Mikhalap, S. V., L. M. Shlapatska, A. G. Berdova, C. L. Law, E. A. Clark, and make functional predictions. S. P. Sidorenko. 1999. CDw150 associates with Src-homology 2-containing inositol phosphatase and modulates CD95-mediated apoptosis. J. Immunol. Since Y281 and Y327 are essential for SHP-2 binding to 162:5719. CD150, it is possible that both SH2 domains of SHP-2 mediate this 6. Punnonen, J., B. G. Cocks, J. M. Carballido, B. Bennett, D. Peterson, G. Aversa, association. Using our model and SHP-2 structure revealed by crys- and J. E. de Vries. 1997. Soluble and membrane-bound forms of signaling lymphocytic activation molecule (SLAM) induce proliferation and Ig synthesis by tallography (49), we predicted that simultaneous binding of Y281 and activated human B lymphocytes. J. Exp. Med. 185:993. Y327 by two SHP-2 SH2 domains is unlikely, because the distance 7. Aversa, G., C. C. Chang, J. M. Carballido, B. G. Cocks, and J. E. de Vries. 1997. Engagement of the signaling lymphocytic activation molecule (SLAM) on acti- between tyrosine-binding pockets of N- and C-terminal domains of vated T cells results in IL-2-independent, cyclosporin A-sensitive T cell prolif- SHP-2 is 40 Å, compared with 8.18 Å, between Y281 and Y327 eration and IFN-␥ production. J. Immunol. 158:4036. residues in CD150ct model. Moreover, it was shown that only the 8. Hamalainen, H., S. Meissner, and R. Lahesmaa. 2000. Signaling lymphocytic activation molecule (SLAM) is differentially expressed in human Th1 and Th2 N-SH2, but not the C-SH2 domain of SHP-2 phosphopeptides binds cells. J. Immunol. Methods 242:9. to both ITIM- and ITSM-like motifs of CD31 (43). 9. Aversa, G., J. Carballido, J. Punnonen, C. C. Chang, T. Hauser, B. G. Cocks, and Apparently, displacement of SHP-2 by SHD1A (10) makes J. E. De Vries. 1997. SLAM and its role in T cell activation and Th cell responses. Immunol. Cell. Biol. 75:202. Y281 and Y327 available for binding by SHIP, which also requires 10. Sayos, J., C. Wu, M. Morra, N. Wang, X. Zhang, D. Allen, S. van Schaik, Y281 and Y327 (Fig. 4B). This possibility is supported by in vivo L. Notarangelo, R. Geha, M. G. Roncarolo, H. Oettgen, J. E. De Vries, G. Aversa, data demonstrating differential coprecipitation of CD150 with and C. Terhorst. 1998. The X-linked lymphoproliferative-disease gene product ϩ Ϫ SAP regulates signals induced through the co-receptor SLAM. Nature 395:462. SHIP in SH2D1A cell lines or with SHP-2 in SH2D1A cell 11. Castro, A. G., T. M. Hauser, B. G. Cocks, J. Abrams, S. Zurawski, T. Churakova, lines (Fig. 2). Although SHIP possesses only one SH2 domain, F. Zonin, D. Robinson, S. G. Tangye, G. Aversa, et al. 1999. Molecular and both Y281 and Y327 in CD150 are important for association with functional characterization of mouse signaling lymphocytic activation molecule (SLAM): differential expression and responsiveness in Th1 and Th2 cells. J. Im- SHIP (Fig. 4). Similarly, the association of SHIP with Fc␥RIIB munol. 163:5860. also requires two tyrosines; not only Y279 within the ITIM of the 12. Coffey, A. J., R. A. Brooksbank, O. Brandau, T. Oohashi, G. R. Howell, ␥ J. M. Bye, A. P. Cahn, J. Durham, P. Heath, P. Wray, et al. 1998. Host response Fc RIIB is required, but also Y296 is necessary for stable asso- to EBV infection in X-linked lymphoproliferative disease results from mutations ciation of SHIP with Fc␥RIIB (50). Interestingly, that SH2 domain in an SH2-domain encoding gene. Nat. Genet. 20:129. of SHIP has a much higher affinity to immobilized phosphopeptide 13. Nichols, K. E., D. P. Harkin, S. Levitz, M. Krainer, K. A. Kolquist, C. Genovese, A. Bernard, M. Ferguson, L. Zuo, E. Snyder, et al. 1998. Inactivating mutations of CD31 ITSM-like motif, than N-SH2 domain of SHP-2 (43). in an SH2 domain-encoding gene in X-linked lymphoproliferative syndrome. 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