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

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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 © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. ●

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 (Fc␥R)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 Fc␥RIIB, 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 ␮ Ј 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 (5Ј-CCGAATTCATGGCTGACGCTAT md.ac.jp CACG-3Ј and 5Ј-AAGCGGCCGCTATATCCGGTTCAGTTCAG-3Ј 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).

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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 (5Ј-CCGAATTCATGGCTGACGCTAT CACG-3Ј and 5Ј-CCGTCGACGTTTTGCAAGCAGACCGT-3Ј) 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 (5Ј-AATTCGCTATCACGTATGCAGACCTGCGCTTTGTGG-3Ј and 5Ј- TCGACCACAAAGCGCAGGTCTGCATACGTGATAGCG-3Ј for Y7; 5Ј-AATTCTGTGAGGCCTATGAAGATGGGGAACTCACCG-3Ј and 5Ј- TCGACGGTGAGTTCCCCATCTTCATAGGCCTCACAG-3Ј for Y32; and 5Ј-AATTCGAACTCACCTACGAGAACGTGCAAGTGTCTG-3Ј and 5Ј- TCGACAGACACTTGCACGTTCTCGTAGGTGACTTCG-3Ј for Y39). Oligonucleotide pairs were annealed and inserted into the EcoRI-XhoI sites of the pGEX vector. The GST fusion protein (10 ␮g) 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 (5Ј-CCGAATTCGAAC (lanes 2, 4, and 6) was incubated with the lysates of Sf9 cells infected with Ј Ј CCCAGGATGGTGAGG-3 and 5 -AAGATCGACTCACTTCCTCTT a baculovirus carrying lyn (lanes 3 and 4)orsyk (lanes 5 and 6) in the in Ј 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 ␮g of anti-CD72 mAb 9-6.1 or anti- CD22 mAb Lyb-8.2 (PharMingen, San Diego, CA) and 30 ␮l 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. Moreover, ern blotting was done using peroxidase-conjugated anti-phosphotyrosine mAb 4G10, rabbit anti-SHP-1 (Upstate Biotechnology, Lake Placid, NY), the sequences surrounding Y39 (LTYENV) are similar to the con-

and anti-SHIP (a kind gift of Dr. J. V. Ravetch, Rockefeller University, sensus sequence of ITIM. To determine the tyrosines responsible by guest on September 28, 2021 New York, NY) or rabbit anti-CD72 Abs generated against GST-CD72. for association with SHP-1, we prepared GST fusion proteins con- taining peptides surrounding either Y7, Y32, or Y39. When the Results and Discussion fusion proteins were incubated with Lyn, both Y7 and Y39, but not The surrounding sequences of Y7 (ITYADL) in the cytoplasmic Y32, were phosphorylated (Fig. 2D). However, SHP-1 was copre- domain of CD72 completely match the consensus sequence of cipitated with the GST fusion protein containing Y7, but not Y32 ITIM (V/IxYxxL/V). Since KIR ITIM has been shown to be phos- or Y39 (lanes 1–3, Fig. 2C). SHP-1 may thus associate with the phorylated by the Src family tyrosine kinase Lck (27), we exam- ITIM surrounding Y7 but not the ITIM-like sequence containing ined whether the cytoplasmic domain of CD72 is phosphorylated Y39 upon phosphorylation. in vitro by the Src family tyrosine kinase Lyn and the cytoplasmic To test whether the cytoplasmic domain of CD72 recruits SH2- tyrosine kinase Syk expressed in B cells. GST fusion proteins con- containing phosphatases upon phosphorylation in vivo, we trans- taining the cytoplasmic domain of CD72 (GST-CD72) were incu- fected the CD72a cDNA into the B lymphoma line WEHI-231 bated with Sf9 cell lysates expressing either Lyn or Syk, and the expressing CD72b but not CD72a. Cell lysates from positive trans- phosphorylation of GST-CD72 was examined by Western blotting fectants (WEHI-CD72) were precipitated with anti-CD72 mAb (9- using anti-phosphotyrosine Ab. Whereas GST alone was not phos- 6.1) recognizing CD72a but not CD72b (28). Western blotting of phorylated by Lyn or Syk (Fig. 1, lanes 3 and 5), GST-CD72 was the immunoprecipitates revealed that CD72 is weakly phosphory- phosphorylated by both tyrosine kinases (lanes 4 and 6), indicat- lated in untreated WEHI-CD72 (4G10 blot in Fig. 3). However, ing that CD72 is a potential substrate of Lyn and Syk expressed in tyrosine phosphorylation of CD72 was not increased by treatment B cells. with anti-CD72 mAb (data not shown). Because 1) CD72 is co- Tyrosine-phosphorylated ITIMs have been shown to recruit modulated with BCR (29), and 2) CD72 is phosphorylated in vitro SH2-containing phosphatases such as SHP-1 (1, 2, 7–9, 11, 12). To by BCR-associated kinases Lyn and Syk (Fig. 1), we hypothesized ask whether SHP-1 is able to associate with the cytoplasmic do- that BCR cross-linking enhances phosphorylation of CD72. In- 35 Ј main of CD72 upon tyrosine phosphorylation, we synthesized S- deed, treatment of WEHI-CD72 cells with F(ab )2 fragments of labeled SHP-1 by in vitro translation and incubated the phospha- anti-IgM Abs enhanced tyrosine phosphorylation of CD72 to- tase with GST-CD72 either unphosphorylated or phosphorylated gether with other substrates coprecipitated with CD72 (4G10 blot by Lyn. Although equivalent amounts of phosphorylated or unphos- in Fig. 3). We thus assessed whether SH2-containing phosphatases phorylated GST-CD72 were incubated with SHP-1 (Fig. 2A), are coprecipitated with CD72 in BCR-cross-linked WEHI-CD72 SHP-1 was coprecipitated with tyrosine-phosphorylated GST- cells. Because CD22 has been shown to be phosphorylated and CD72 but not by unphosphorylated GST-CD72 (Fig. 2B). This recruit SHP-1 upon BCR cross-linking, we precipitated the lysates result indicates that the cytoplasmic tail of CD72 can bind to with anti-CD22 Ab in parallel as a positive control. In cell lysates SHP-1 upon tyrosine phosphorylation as is the case for other mem- prepared from untreated WEHI-CD72 cells, a small amount of brane molecules carrying ITIMs (1, 2, 7–9, 11, 12). SHP-1 was coprecipitated with CD72, whereas anti-CD22 failed to 4664 CUTTING EDGE

FIGURE 3. BCR signaling enhances tyrosine phosphorylation of CD72 and its association with SHP-1. WEHI-CD72 cells were incubated with or ␮ Ј ␣␮ without 10 g/ml of F(ab )2 fragments of anti-IgM Abs ( ) for 3 min in Downloaded from RPMI 1640 medium, washed with PBS, and lysed in Triton X-100 lysis buffer. The lysates were incubated with 5 ␮g of either anti-CD72 mAb or anti-CD22 mAb together with 30 ␮l of protein G-Sepharose. Immune com- plexes were collected by centrifugation, washed three times with PBS, separated by SDS-PAGE, and subjected to Western blotting with indicated Abs. The bands representing CD72, determined by probing the membrane

with rabbit anti-mouse CD72, are indicated. Note that two bands represent http://www.jimmunol.org/ CD72, presumably due to differences in the m.w. between CD72a and CD72b (28). CD72b, which forms a dimer with CD72a, may also be pre- cipitated with anti-CD72a Ab.

vivo as is the case for CD22. In contrast, we failed to detect SHIP in the anti-CD72 precipitates from WEHI-CD72 either untreated Ј by guest on September 28, 2021 FIGURE 2. Tyrosine-phosphorylated CD72 associates with SHP-1 in or treated with F(ab )2 fragments of anti-IgM, although a consid- vitro. GST-CD72 was phosphorylated as described in the legend to Figure erable amount of SHIP is expressed in WEHI-231 (data not 1 except that the reaction was prolonged to 30 min. A, GST-CD72 fusion shown). Thus, it is probable that CD72 does not recruit SHIP re- proteins treated with Lyn or untreated were subjected to SDS-PAGE, fol- gardless of the phosphorylation of CD72. Taken together, tyrosine- 35 lowed by Western blotting using rabbit anti-mouse CD72 Abs. B, S- phosphorylated CD72 may recruit SHP-1 but not SHIP. labeled SHP-1 proteins were incubated with either phosphorylated (lane 2) We demonstrated that CD72 recruits SHP-1 upon tyrosine phos- or unphosphorylated GST-CD72 (lane 1) together with glutathione-cou- pled beads for 30 min at 4°C. The beads were washed with PBS three times phorylation both in vitro and in vivo. The ITIM sequence contain- and incubated with SDS-PAGE sample buffer. Proteins were separated by ing Y7 is responsible for association with SHP-1, in agreement SDS-PAGE. 35S-labeled proteins were visualized by autoradiography. C, with the previous findings that phosphorylated ITIMs in other GST-Y7 (lane 1), GST-Y32 (lane 2), GST-Y39 (lane 3), and GST-CD72 membrane molecules associate with SH2-containing phosphatases (lane 4) fusion proteins were incubated with Lyn and subsequently with such as SHP-1 (1–3, 7, 8, 11, 12). We also demonstrated that BCR 35S-labeled SHP-1. Proteins were precipitated with glutathione-coupled signaling enhances phosphorylation of CD72 and its association 35 beads and subjected to SDS-PAGE. S-labeled proteins were visualized with SHP-1 in WEHI-231 cells. Because 1) CD72 is comodulated by autoradiography. D, GST-Y7 (lane 1), GST-Y32 (lane 2), and GST- with BCR (29) and 2) BCR-associated kinases Lyn and Syk are Y39 (lane 3) fusion proteins were incubated with Lyn and subjected to able to phosphorylate CD72 (Fig. 1), BCR-associated kinases ac- SDS-PAGE, followed by Western blotting using 4G10 mAb. tivated by BCR cross-linking may phosphorylate CD72. Tyrosine- phosphorylated CD72 may subsequently recruit SHP-1, resulting precipitate SHP-1 (anti-SHP-1 blot). When we treated WEHI- in down-modulation of cell activation induced by BCR signaling. CD72 with anti-Ig, a markedly increased amount of SHP-1 was CD72 may, therefore, negatively regulate the cell activation in- coprecipitated with CD72 or CD22 from the cell lysates, although duced by BCR ligation. The negative regulation of CD72 on BCR equal amounts of cell lysates were tested. Indeed, almost the same signaling may be enhanced or suppressed by treatment with anti- amount of CD72 was precipitated from the lysates of anti- CD72 Ab. Indeed, anti-CD72 Ab have been shown to modulate the Ig-treated WEHI-CD72 as from the lysates of untreated WEHI- BCR signaling, resulting in augmentation of activation and abro- CD72 (anti-CD72 blot). Upon BCR cross-linking, CD72 appears gation of cell death in BCR-cross-linked B cells (17–20). CD22, an to be phosphorylated less efficiently than CD22 (4G10 blot), pre- ITIM-containing member of the Ig superfamily, is also associated sumably due to the fact that CD22 carries six tyrosines in the with BCR and recruits SHP-1 upon BCR cross-linking, resulting in cytoplasmic tail whereas CD72 contains only three tyrosines. down-modulation of B cell activation induced by BCR signaling However, the amount of SHP-1 coprecipitated with CD72 in un- (30). Moreover, treatment with anti-CD22 Ab enhances B cell ac- treated and BCR-cross-linked WEHI-CD72 correlates with the ex- tivation by BCR signaling by perturbing the negative regulation by tent of phosphorylation of CD72 (4G10 blot). It is therefore most CD22. CD72 may thus play a similar role to CD22 in the regula- likely that SHP-1 is recruited to CD72 upon phosphorylation in tion of B cell function. 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