Novel Binding Site for Src Homology 2-containing Protein-tyrosine Phosphatase-1 in CD22 Activated by B Lymphocyte Stimulation with Antigen*

CD22, a B lymphocyte membrane glycoprotein, contains immunoreceptor tyrosine-based inhibition motifs (ITIMs) in the cytoplasmic region and recruits Src homology 2-containing protein-tyrosine phosphatase-1 (SHP-1) to the phosphorylated ITIMs upon ligation of B lymphocyte antigen receptor (BCR), thereby negatively regulating BCR signaling. Among the three previously identified ITIMs, both ITIMs containing tyrosine residues at position 843 (Tyr843) and 863 (Tyr863), respectively, are shown to be required for CD22 to recruit SHP-1 and regulate BCR signaling upon BCR ligation by anti-Ig antibody (Ab), indicating that CD22 has the SHP-1-binding domain at the region containing Tyr843 and Tyr863. Here we address the requirement of CD22 for SHP-1 recruitment and BCR regulation upon BCR ligation by antigen, which induces much stronger CD22 phosphorylation than anti-Ig Ab does. We demonstrate that the CD22 mutant in which both Tyr843 and Tyr863 are replaced by phenylalanine (CD22F5/6) recruits SHP-1 and regulates BCR signaling upon stimulation with antigen but not anti-Ig Ab. This result strongly suggests that CD22 contains another SHP-1 binding domain that is specifically activated upon stimulation with antigen. Both of the flanking sequences of Tyr783 and Tyr817 fit the consensus sequence of ITIM, and the CD22F5/6 mutant requires these tyrosine residues for SHP-1 binding and BCR regulation. Thus, these ITIMs constitute a novel conditional SHP-1-binding site of CD22 that is activated upon BCR ligation by antigen but not by anti-Ig Ab.

Among six tyrosine residues in the cytoplasmic region of CD22, the tyrosine residues involved in SHP-1 binding have been defined. First, Doody et al. demonstrated that the tyrosine residues at the positions 783 (Tyr 783 ), 843 (Tyr 843 ), and 863 (Tyr 863 ) are the potential binding sites for SHP-1 (14). Indeed, the flanking sequences of these tyrosine residues fit the consensus sequence of the immunoreceptor tyrosine-based inhibition motif (ITIM) (15), the motif often found at the binding sites for SH2-containing phosphatases, including SHP-1 (16), and more importantly phosphotyrosyl peptides containing these tyrosine residues are capable of binding to SHP-1 in vitro (14). Later, Otipoby et al. examined SHP-1 recruitment to CD22 in B cells when CD22 is phosphorylated by BCR ligation, and demonstrated that both Tyr 843 and Tyr 863 but not Tyr 783 are required for recruitment of SHP-1 to CD22 (17). This result indicated that the region containing Tyr 843 and Tyr 863 constitutes the SHP-1-binding domain of CD22, whereas ITIM containing Tyr 783 is dispensable for SHP-1 recruitment. However, this study was done using B cells in which BCR is ligated by anti-Ig antibody (Ab) but not antigen.
Recently, we demonstrated that BCR ligation by antigen induces stronger CD22 phosphorylation than anti-Ig Ab-induced BCR ligation through generation of a distinct BCR signaling (18). Weak CD22 phosphorylation upon stimulation with anti-Ig Ab was not due to quantitatively weak BCR signaling but rather due to qualitatively distinct signaling from that induced by antigen, because stimulation with anti-Ig Ab strongly phosphorylated various signaling molecules, including extracellular signal regulatory kinase (ERK), but only weakly phosphorylated CD22. Many anti-Ig Abs bind to the membrane-proximal part of BCR, whereas antigens bind to the antigen-binding site at the membrane-distal part of BCR. Therefore, we proposed that these anti-Ig Abs disrupt interaction between CD22 and BCR, thereby preventing phosphorylation of CD22 by BCR-associated kinase Lyn (19 -22). Thus, SHP-1binding sites in CD22 need to be examined in B cells after BCR ligation by antigens, because tyrosine phosphorylation of CD22 is crucial for its binding to SHP-1. We here demonstrate that, in B cells stimulated with antigen but not anti-Ig Ab, the CD22 mutant in which both Tyr 843 and Tyr 863 are replaced by phenylalanine is capable of recruiting SHP-1 and negatively regulating BCR signaling, suggesting that CD22 contains another SHP-1 binding domain specifically activated by antigen stimulation other than that containing Tyr 843 and Tyr 863 . Further, we demonstrate that Tyr 783 at a previously defined ITIM (15) does not regulate phosphorylation of other tyrosines, such as Tyr 843 and Tyr 863 , but plays an essential role, together with Tyr 817 , in SHP-1 binding and BCR regulation of the mutant CD22 carrying Tyr 3 Phe mutations at both Tyr 843 and Tyr 863 . These results clearly indicate that the region containing Tyr 783 and Tyr 817 constitutes a novel SHP-1-binding site that is activated upon BCR ligation by antigen but not anti-Ig Ab.
Cell Culture-The mouse B cell lines K46v and BAL17IgM were described previously (12). These cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 50 M 2-mercaptoethanol, and 1 mM glutamine. The retrovirus packaging cell lines PLAT-E (a gift of Dr. T. Kitamura) (24) and Phoenix (a gift of Dr. G. P. Nolan, Stanford University School of Medicine) (25) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, and 100 units of penicillin/streptomycin. For retrovirus transduction, the packaging cells were transfected with retrovirus vectors using Fugene 6 (Roche Applied Science). Cells were cultured for 48 h, and the culture supernatant was collected. K46v and BAL17IgM cells were incubated with the supernatant containing retrovirus for 4 h.
Measurement of Intracellular Calcium Mobilization-Cells were incubated in culture medium containing 5 mM Fluo-4/AM (Molecular Probes, Inc., Eugene, OR) for 30 min. Cells were stimulated with 0.2 g/ml NP-BSA or 10 g/ml anti-IgM Ab and analyzed by flow cytometry using a fluorescence-activated cell sorter LSR (BD Biosciences).

CD22 ITIMs Containing Tyr 843 and Tyr 863 Are Dispensable for CD22-mediated BCR Regulation-To investigate whether
Tyr 843 and Tyr 863 are required for CD22-mediated signal regulation in antigen-stimulated B cells, we generated two retrovirus vectors expressing HA-tagged mutant CD22. One carries double Tyr 3 Phe mutations at both Tyr 843 and Tyr 863 (CD22F5/6), and the other carries triple Tyr 3 Phe mutations at Tyr 783 , Tyr 843 , and Tyr 863 (CD22F2/5/6) (Fig. 1). Retrovirus expressing wild-type or mutant CD22 was transduced to the mouse B cell lines K46v and BAL17IgM, both of which express membrane-bound IgM reactive to the hapten NP (12). Since K46v but not BAL17IgM lacks expression of endogenous CD22 (12), we assessed expression of both transduced wild-type and mutated CD22 in K46v and BAL17 transfectants by flow cytometry using anti-CD22 Ab and Western blotting using anti-HA Ab, respectively. In these transfectants, the expression levels of mutant CD22 molecules were comparable with that of wild-type CD22, and the expression levels of NPreactive BCR were similar to those in the cells transduced with the empty vector alone (K46v-vector and BAL17IgM-vector) (supplemental Figs. S1 and S2).
We stimulated K46v-vector, K46vCD22, K46vCD22F5/6, and K46vCD22F2/5/6 transfectants with an antigen NP-BSA and examined phosphorylation of cellular substrates. Surprisingly, antigen stimulation-induced phosphorylation of various cellular substrates was reduced in K46vCD22F5/6 cells compared with K46v-vector, and the reduction of phosphorylation by CD22F5/6 was as strong as that by wild-type CD22 ( Fig.  2A). Further, CD22F5/6 reduced antigen-induced phosphorylation of both ERK and AKT as efficiently as wild-type CD22 (Fig. 2, B and C). These results strongly suggested that both Tyr 843 and Tyr 863 of CD22 are dispensable for CD22-mediated signal inhibition in antigen-stimulated K46v cells. In contrast, expression of CD22F2/5/6 failed to reduce the phosphorylation levels of various cellular substrates, including ERK and AKT, in antigen-stimulated K46v transfectants (Fig. 2), suggesting that Tyr 783 is required for BCR regulation by CD22F5/6. We next examined association of mutant CD22 molecules with SHP-1 and, as a control, their association with Grb2. When K46v transfectants were stimulated with NP-BSA, CD22F5/6 was phosphorylated and co-precipitated with both Grb2 and SHP-1 as efficiently as wild-type CD22 (5) (Fig. 3A). This result indicated that both Tyr 843 and Tyr 863 are dispensable for recruiting SHP-1 to CD22 upon antigen stimulation. In contrast, CD22F2/5/6 was phosphorylated weakly and was not co-precipitated with SHP-1. Treatment with pervanadate/ H 2 O 2 , a phosphatase inhibitor, phosphorylated CD22F2/5/6 as strongly as wild type CD22 and CD22F5/6 and induced efficient co-precipitation of Grb2 with CD22F2/5/6, probably due to the presence of Tyr 828 responsible for Grb2 binding in this mutant (Fig. 3B). However, SHP-1 was co-precipitated with wild type CD22 and CD22F5/6 but not CD22F2/5/6, indicating that CD22F2/5/6 does not recruit SHP-1 even if strongly phosphorylated. Essentially the same result on SHP-1 recruitment was obtained in BAL17IgM transfectants (Fig. 3, C and D), although we could not determine the impact of mutant CD22 on BCRmediated substrate phosphorylation in these cells due to expression of endogenous CD22. Taken together, CD22F5/6 carrying Y/F mutations at both Tyr 843 and Tyr 863 but not CD22F2/5/6 was able to regulate BCR signaling and recruit SHP-1 upon antigen stimulation. Thus, Tyr 843 and Tyr 863 are dispensable for CD22 to bind to SHP-1 and to regulate BCR signaling in antigen-stimulated B cells, and Tyr 783 is involved in CD22-mediated BCR regulation, probably by serving as a binding site for SHP-1.
Tyr 783 Is Involved in CD22-mediated BCR Regulation without Influencing Phosphorylation of Other Tyrosine Residues in CD22-To address the role of Tyr 783 in CD22-mediated signal regulation, we constructed the retrovirus vector encoding mutant CD22 containing a Tyr 3 Phe mutation at Tyr 783 alone (CD22F2) (Fig. 1) and transduced the retrovirus vector to K46v cells. The expression levels of CD22 and NP-reactive BCR on K46vCD22F2 transfectants were similar to those on K46vCD22 cells (supplemental Fig. S1). When the K46vCD22F2 cells were stimulated with NP-BSA, CD22F2 was phosphorylated and associated with SHP-1 almost as efficiently as wild-type CD22 (Fig. 4A). Antigen-stimulated K46vCD22F2 cells showed weaker ERK phosphorylation and weaker calcium mobilization than K46v cells transfected with the vector alone (Fig. 4, B and C). Thus, CD22F2 recruited SHP-1 and regulated BCR signaling, probably because ITIMs containing Tyr 843 and Tyr 863 in CD22F2 recruited SHP-1 and regulated BCR signaling. However, antigen-stimulated K46vCD22F2 cells showed stronger calcium mobilization than K46vCD22 cells (Fig. 4C). This result indicated that CD22F2 regulated calcium signaling less efficiently than wildtype CD22 and suggested that Tyr 783 is involved in the regulation of BCR signaling even in the presence of Tyr 843 and Tyr 863 .  In contrast, CD22F2 down-modulated ERK phosphorylation almost as strongly as wild-type CD22 (Fig. 4B), suggesting that activation of ERK is more sensitive to SHP-1-mediated signal regulation than calcium signaling.
To address whether Tyr 783 regulates tyrosine phosphorylation of CD22, we examined phosphorylation of Tyr 843 and Tyr 863 using Abs that specifically recognize phosphorylated ITIMs containing Tyr 843 and Tyr 863 , respectively (26). Phosphorylation of both Tyr 843 and Tyr 863 in CD22F2 in antigenstimulated K46v transfectants was comparable to that in wildtype CD22 (Fig. 4D), indicating that Tyr 783 does not regulate phosphorylation of other tyrosines in the cytoplasmic region of CD22. Taken together, Tyr 783 is involved in BCR regulation without influencing phosphorylation of other tyrosines in CD22.
Both Tyr 783 and Tyr 817 Are Not Involved in BCR Regulation upon BCR Ligation by Anti-Ig Ab-We asked whether SHP-1 binding sites of CD22 in antigen-stimulated B cells is different from those in B cells in which BCR is ligated by anti-Ig Ab. When K46v transfectants were stimulated with anti-Ig Ab, wild-type CD22 but not CD22F5/6 carrying Tyr 3 Phe mutations at both Tyr 843 and Tyr 863 co-precipitated SHP-1 (Fig. 6A) and regulated BCR signaling including ERK phosphorylation (Fig. 6B) and calcium mobilization (Fig. 6C). Thus, the ITIMs containing Tyr 843 and Tyr 863 but not those containing Tyr 783 and Tyr 817 are responsible for SHP-1 recruitment and BCR signal regulation in anti-Ig Ab-treated K46v cells, in agreement with a previous finding by Otipoby et al. (17). Taken together, ITIMs containing Tyr 783 and Tyr 817 are involved in SHP-1 recruitment and BCR regulation upon BCR ligation by antigen but not anti-Ig Ab.

DISCUSSION
In this study, we demonstrate that the previously defined SHP-1-binding domain containing Tyr 843 and Tyr 863 is dispensable for CD22 to recruit SHP-1 and to regulate BCR signaling in the B cell lines K46v and BAL17IgM and that the previously identified ITIM containing Tyr 783 is involved in both SHP-1 binding and BCR regulation. Some of the tyrosine residues in  D) for the indicated times. Cells were lysed, and HA-tagged mutant and wild-type CD22 molecules were immunoprecipitated (IP) with anti-HA (3F10) Ab. Immunoprecipitates were analyzed by immunoblotting using anti-phosphotyrosine mAb, anti-SHP-1 Ab, or anti-Grb2 Ab. The same membranes were reprobed with anti-HA Ab to ensure equal loading. Alternatively, cell lysates were immunoprecipitated with anti-SHP-1 Ab. Immunoprecipitates were analyzed for HA-tagged CD22 by immunoblotting using anti-HA Ab. The same membranes were reprobed with anti-SHP-1 Ab to ensure equal loading. The data are representative of more than three experiments.
the molecules, such as HS1, augment their tyrosine phosphorylation by a mechanism known as processive phosphorylation (28), in which a phosphorylated tyrosine recruits the kinase that phosphorylates other tyrosine residues in the same molecule (29 -31). However, Tyr 783 does not regulate phosphorylation of other tyrosine residues of CD22. In contrast, the peptide containing Tyr 783 was previously shown to bind to SHP-1 (14). Thus, Tyr 783 does not augment binding of SHP-1 to other tyrosine residues of CD22 but rather serves as a SHP-1-binding site, thereby contributing to CD22-mediated signal regulation. We further demonstrated here that the flanking sequence of Tyr 817 fits the consensus sequence of ITIM (27) and that both Tyr 783 and Tyr 817 are required for SHP-1 binding and BCR regulation of CD22F5/6 mutant carrying a Tyr 3 Phe mutation at Tyr 843 and Tyr 863 . Both biochemical and structural studies previously demonstrated that efficient activation of SHP-1 requires engagement of both of the SH2 domains by peptides containing two phosphotyrosine residues (27,32). Requirement of both Tyr 783 and Tyr 817 for SHP-1 recruitment in CD22F5/6 there- K46v transfectants were treated with 0.2 g/ml NP-BSA for the indicated times. Cells were lysed, and HA-tagged CD22 was immunoprecipitated (IP) with anti-HA (3F10) Ab. Immunoprecipitates were analyzed by immunoblotting using anti-phosphotyrosine mAb or anti-SHP-1 Ab. The same membranes were reprobed with anti-HA Ab to ensure equal loading. The data are representative of more than three experiments. B, regulation of ERK phosphorylation. The indicated K46v transfectants were treated with 0.2 g/ml NP-BSA for the indicated times. Total cell lysates were separated by SDS-PAGE and analyzed for ERK phosphorylation by Western blotting using an antiphospho-ERK Ab. These blots were reprobed with anti-␤-tubulin Ab to ensure equal loading. Representative data of more than three experiments are shown. C, regulation of calcium mobilization. The indicated K46v transfectants were loaded with Fluo-4/AM, and intracellular free calcium was measured by flow cytometry using FACS LSR. Cells were treated with 0.2 g/ml NP-BSA at 30 s (indicated by an arrow), and measurement of free calcium was continued for 300 s. D, phosphorylation of Tyr 843 and Tyr 863 . The indicated K46v transfectants were treated with 0.2 g/ml NP-BSA for the indicated times. Total cell lysates were separated by SDS-PAGE and analyzed for phosphorylation of Tyr 843 and Tyr 863 by Western blotting using antibodies generated to the phosphopeptides containing Tyr 843 and Tyr 863 , respectively. These blots were reprobed with anti-␤-tubulin Ab to ensure equal loading. Representative data of more than three experiments are shown. A, regulation of ERK phosphorylation. The indicated K46v transfectants were treated with 0.2 g/ml NP-BSA for the indicated times. Total cell lysates were separated by SDS-PAGE and analyzed for ERK phosphorylation by Western blotting using an anti-phospho-ERK Ab. These blots were reprobed with anti-␤-tubulin Ab to ensure equal loading. Representative data of more than three experiments are shown. B, regulation of calcium mobilization. Indicated K46v transfectants were loaded with Fluo-4/AM, and intracellular free calcium was measured by flow cytometry using FACS LSR. Cells were treated with 0.2 g/ml NP-BSA at 30 s (indicated by an arrow), and measurement of free calcium was continued for 300 s. C, recruitment of SHP-1. The indicated K46v transfectants were treated with 0.2 g/ml NP-BSA for the indicated times. Cells were lysed, and HA-tagged CD22 was immunoprecipitated (IP) with anti-HA (3F10) Ab. Immunoprecipitates were analyzed by immunoblotting using anti-phosphotyrosine mAb or anti-SHP-1 Ab. The same membranes were reprobed with anti-HA Ab to ensure equal loading. The data are representative of more than three experiments.

Novel SHP-1-binding Site in CD22
fore suggests that two ITIMs containing Tyr 783 and Tyr 817 , respectively, bind to the two SH2 domains of SHP-1, thereby recruiting it and regulating BCR signaling. Thus, Tyr 783 plays a role together with Tyr 817 in CD22-mediated BCR regulation by constituting a novel SHP-1-binding site.
Here we demonstrate that the ITIMs containing Tyr 783 and Tyr 817 recruit SHP-1 and regulate BCR signaling upon BCR ligation by antigens but not anti-Ig Ab. We previously demonstrated that antigen stimulation induces stronger CD22 phosphorylation than stimulation with anti-Ig Ab by generating a qualitatively distinct signaling (18). When B cells are stimulated with antigens, CD22 is translocated to the BCR-containing lipid rafts and is strongly phosphorylated by the tyrosine kinase Lyn, which is concentrated in lipid rafts (33). In contrast, BCR ligation with anti-Ig Ab induces weak phosphorylation of CD22 (18) probably because of exclusion of CD22 from BCR-containing lipid rafts. 3 ITIMs containing Tyr 843 and Tyr 863 probably recruit SHP-1 with a lower phosphorylation level, whereas those containing Tyr 783 and Tyr 817 appear to require higher levels of phosphorylation for SHP-1 binding. Previously, Doody et al. (14) demonstrated that phosphorylated peptides containing Tyr 783 , Tyr 843 , and Tyr 863 , respectively, but not that containing Tyr 817 block binding of CD22 to SHP-1. This suggests that ITIM containing Tyr 817 binds to SHP-1 less efficiently than the other ITIMs and may require strong phosphorylation for SHP-1 recruitment. However, the flanking sequence of Tyr 817 completely fits the consensus sequence of ITIM (27) and does not show a particular difference from other ITIMs; therefore, how the ITIM containing Tyr 817 binds to SHP-1 less efficiently is not yet known. Further studies are necessary to elucidate the mechanism and functional significance of stimulation-dependent activation of the novel inhibitory domain of CD22 containing Tyr 783 and Tyr 817 .