Cutting Edge: The B Cell Surface Protein CD72 Recruits the Tyrosine Phosphatase SHP-1 upon Tyrosine Phosphorylation This information is current as Takahiro Adachi, Heinrich Flaswinkel, Hidetaka Yakura, Michael of September 28, 2021. Reth and Takeshi Tsubata J Immunol 1998; 160:4662-4665; ; http://www.jimmunol.org/content/160/10/4662 Downloaded from References This article cites 30 articles, 12 of which you can access for free at: http://www.jimmunol.org/content/160/10/4662.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Print ISSN: 0022-1767 Online ISSN: 1550-6606. c Cutting Edge: The B Cell Surface Protein CD72 Recruits the Tyrosine Phosphatase SHP-1 upon Tyrosine Phosphorylation1 Takahiro Adachi,* Heinrich Flaswinkel,2† Hidetaka Yakura,‡ Michael Reth,† and Takeshi Tsubata3* Downloaded from (FcgR)IIB and CD22 in B cells, ILT2 (LIR-1) in B cells and Activation signals of lymphocytes are negatively regulated by monocytes, ILT3 in myeloid cells, gp49 in NK and mast cells, and the membrane molecules carrying the immunoreceptor ty- LAIR-1 expressed in the majority of leukocytes (1–3). Those mol- rosine-based inhibition motif (ITIM). Upon tyrosine phosphor- ecules contain the conserved immunoreceptor tyrosine-based inhi- ylation, ITIMs recruit SH2-containing phosphatases such as bition motif (ITIM) in the cytoplasmic domain (4–6). When the SHP-1, resulting in down-modulation of cell activation. We tyrosine residue in the ITIM is phosphorylated, the ITIM recruits http://www.jimmunol.org/ showed that the cytoplasmic domain of the CD72 molecule car- Src homology 2 (SH2)-containing phosphatases, such as protein ries an ITIM and is associated in vitro with SHP-1 upon ty- tyrosine phosphatase SHP-1, and SH2-containing inositol 5-phos- rosine phosphorylation. Moreover, cross-linking of B cell Ag phatase (SHIP) (7–12). Those phosphatases have been shown to receptor (BCR) enhances both tyrosine phosphorylation of down-modulate the cell activation presumably by dephosphorylat- CD72 and association of CD72 with SHP-1 in B cell line ing the signaling molecules. NK cells express ITIM-containing WEHI-231. These results indicate that CD72 recruits SHP-1 molecules of two distinct families: KIR, gp49, and LAIR-1 are upon tyrosine phosphorylation induced by BCR signaling, sug- members of the Ig superfamily, while Ly-49 and CD94/NKG2 are gesting that CD72 is a negative regulator of BCR signaling. type II membrane molecules carrying a C-type lectin domain (2, The Journal of Immunology, 1998, 160: 4662–4665. 3). Although B cells express members of the Ig superfamily car- by guest on September 28, 2021 rying ITIMs such as FcgRIIB, CD22, and ILT-2 (3), an inhibitory receptor carrying C-type lectin domain has not yet been identified. ost lymphocyte activation signals such as those deliv- CD72 (Lyb-2) is a 45-kDa type II membrane protein carrying a ered via the Ag receptor involve phosphorylation of C-type lectin domain and is expressed on B lineage cells as a M signaling molecules by specific kinases. The activa- homodimer (13–16). Treatment with anti-CD72 has been shown to tion signals are negatively regulated by various membrane mole- augment the activation of B cells induced by B cell Ag receptor cules such as the killer cell inhibitory receptor (KIR),4 CD94/ (BCR) signaling (17–19). Recently, we demonstrated that an anti- NKG2, and Ly-49 in NK cells, the low-affinity Fc receptor for IgG CD72 Ab blocks BCR-mediated cell death of mature B cells (20). These findings suggest that CD72 ligation modulates BCR signal- ing. Here, we demonstrate that the cytoplasmic domain of CD72 *Department of Immunology, Medical Research Institute, Tokyo Medical and Dental carries an ITIM and recruits SHP-1 upon tyrosine phosphorylation. University, Bunkyo-ku, Tokyo, Japan; †Department of Molecular Immunology, Bi- Moreover, BCR signaling enhances phosphorylation of CD72 and ology III, Freiburg University, Freiburg, Germany; and ‡Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo, Japan its recruitment of SHP-1. Since it has been suggested that SHP-1 negatively regulates activation signals, including the one via BCR Received for publication December 18, 1997. Accepted for publication March 17, 1998. (1–3, 7, 9, 21, 22), BCR signaling may be negatively regulated The costs of publication of this article were defrayed in part by the payment of page by CD72. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Materials and Methods 1 This work was supported in part by grants from the Ministry of Education, Science, Cells Sports, and Culture of Japan, the Science and Technology Agency of Japan, and the Uehara Memorial Foundation. The mouse B lymphoma cell line WEHI-231 was cultured as described 2 Present address: Institute for Medical Microbiology, Immunology, and Hygiene, previously (23). In some experiments, WEHI-231 was cultured with 10 m 9 Technical University of Munich, D-81675 Munich, Germany. g/ml of F(ab )2 fragments of goat anti-mouse IgM Ab (ICN Pharmaceu- a a 3 Address correspondence and reprint requests to Dr. Takeshi Tsubata, Department of ticals, Aurora, OH) or anti-mouse CD72 mAb 9-6.1 (18). The CD72 Immunology, Medical Research Institute, Tokyo Medical and Dental University, cDNA was obtained from total RNA prepared from a DBA/2 spleen by 1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan. E-mail address: tsubata.imm@ mri.t- RT-PCR using a set of primers (59-CCGAATTCATGGCTGACGCTAT md.ac.jp CACG-39 and 59-AAGCGGCCGCTATATCCGGTTCAGTTCAG-39 a 4 Abbreviations used in this paper: KIR, killer cell inhibitory receptor; BCR, B (15). The CD72 cDNA was subcloned into an expression vector, pMik- cell Ag receptor; ITIM, immunoreceptor tyrosine-based inhibition motif; GST, neo (a gift of Dr. K. Maruyama, Tokyo Medical and Dental University) glutathione S-transferase; SH2, Src homology 2; SHIP, SH2-containing inositol (pMikCD72). Transfection of WEHI-231 was done as described 5-phosphatase. previously (23). Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 c The Journal of Immunology 4663 In vitro phosphorylation and association of glutathione S- transferase (GST) fusion proteins The cDNA encoding the cytoplasmic domain of CD72 was obtained by PCR with a set of primers (59-CCGAATTCATGGCTGACGCTAT CACG-39 and 59-CCGTCGACGTTTTGCAAGCAGACCGT-39) and sub- cloned into the EcoRI-SalI sites of the pGEX vector (Pharmacia, Uppsala, Sweden), resulting in pGEX-CD72 coding for a GST-CD72 fusion protein. For constructing the pGEX-Y7, pGEX-Y32, and pGEX-Y39 expression plasmids encoding GST fusion proteins containing peptides surrounding Y7 (AITYADLRFV), Y32 (CEAYEDGELT), and Y39 (ELTYENVQVS), respectively, we synthesized oligonucleotide pairs encoding the peptides (59-AATTCGCTATCACGTATGCAGACCTGCGCTTTGTGG-39 and 59- TCGACCACAAAGCGCAGGTCTGCATACGTGATAGCG-39 for Y7; 59-AATTCTGTGAGGCCTATGAAGATGGGGAACTCACCG-39 and 59- TCGACGGTGAGTTCCCCATCTTCATAGGCCTCACAG-39 for Y32; and 59-AATTCGAACTCACCTACGAGAACGTGCAAGTGTCTG-39 and 59- TCGACAGACACTTGCACGTTCTCGTAGGTGACTTCG-39 for Y39). Oligonucleotide pairs were annealed and inserted into the EcoRI-XhoI sites of the pGEX vector. The GST fusion protein (10 mg) were phosphorylated by incubation with Sf9 cell lysates containing Lyn or Syk in in vitro kinase buffer FIGURE 1. Phosphorylation of CD72 fusion proteins by Lyn or Syk. (24). The cDNA encoding murine SHP-1 (25) was generated from total RNA Ten micrograms of GST (lanes 1, 3, and 5) or GST-CD72 fusion protein Downloaded from of WEHI-231 by RT-PCR using a set of primers (59-CCGAATTCGAAC (lanes 2, 4, and 6) was incubated with the lysates of Sf9 cells infected with 9 9 CCCAGGATGGTGAGG-3 and 5 -AAGATCGACTCACTTCCTCTT a baculovirus carrying lyn (lanes 3 and 4)orsyk (lanes 5 and 6) in the in 9 GAGAGA-3 ) and subcloned into the EcoRI-SalI sites of the pBluescript vec- vitro kinase buffer (30) for 15 min at 37°C. Proteins were subjected to tor. The 35S-labeled SHP-1 proteins were synthesized in the presence of 35 SDS-PAGE, followed by Western blotting. As a negative control, the same L-[ S]methionine using TNT-coupled in vitro translation kit (Promega, Mad- ison, WI) and incubated with phosphorylated or unphosphorylated GST fusion amount of untreated GST and GST-CD72 was analyzed in parallel (lane 1 proteins together with glutathione-coupled beads (Pharmacia) for 30 min at and 2). Upper panel, The membrane was probed with anti-phosphotyrosine 4°C. Proteins precipitated with glutathione-coupled beads were subjected to mAb 4G10. Lower panel, The SDS-polyacrylamide gel was stained with http://www.jimmunol.org/ SDS-PAGE, and the 35S-labeled proteins were visualized by autoradiography. Coomassie brilliant blue. Immunoprecipitation and Western blotting Cell lysates were incubated with 5 mg of anti-CD72 mAb 9-6.1 or anti- CD22 mAb Lyb-8.2 (PharMingen, San Diego, CA) and 30 ml of protein Among three tyrosine residues in the cytoplasmic domain of G-Sepharose (Pharmacia), and immunoprecipitation was done as described CD72 (amino acid residues number 7, 32, and 39), surrounding previously (26). Immune complexes were subjected to SDS-PAGE. West- sequences of Y7 fit to the consensus sequence of ITIM.