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Originally published In Press as doi:10.1074/jbc.M706584200 on November 15, 2007

J. Biol. Chem., Vol. 283, Issue 3, 1653-1659, January 18, 2008
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Novel Binding Site for Src Homology 2-containing Protein-tyrosine Phosphatase-1 in CD22 Activated by B Lymphocyte Stimulation with Antigen*Formula

Chenghua Zhu{ddagger}§, Motohiko Sato{ddagger}§, Teruhiko Yanagisawa{ddagger}§, Manabu Fujimoto||, Takahiro Adachi{ddagger}§, and Takeshi Tsubata{ddagger}§1

From the {ddagger}Laboratory of Immunology, School of Biomedical Science, and the §Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510, Japan, the ||Department of Dermatology, Kanazawa University Graduate School of Medical Science, Kanazawa, Ishikawa 920-8641, Japan, and Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Tokyo 113-8510, Japan

Received for publication, August 8, 2007 , and in revised form, October 1, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
CD22 (also known as siglec (sialic acid-binding immunoglobulin-like lectin) 2), a 140-kDa membrane glycoprotein expressed on B lymphocytes (B cells),2 is a negative regulator of B cell antigen receptor (BCR), and a crucial regulator of B cell activation (1, 2). Upon ligation of the BCR, tyrosine residues at the cytoplasmic region of CD22 are rapidly phosphorylated, thereby providing binding sites for various cytoplasmic signaling molecules, such as Src homology 2 (SH2)-containing protein-tyrosine phosphatase-1 (SHP-1), lipid phosphatase SH2-containing inositol polyphosphate 5-phosphatase, the tyrosine kinase Syk, and the adaptors Grb2 and Shc (3-6). Among these signaling molecules, SHP-1 is suggested to play a central role in negative regulation of BCR signaling, including Ca2+ signaling and mitogen-activated protein kinase cascades (7-12) through dephosphorylation of various signaling molecules and regulation of plasma membrane calcium-ATPase (13).

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 (Tyr783), 843 (Tyr843), and 863 (Tyr863) 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 Tyr843 and Tyr863 but not Tyr783 are required for recruitment of SHP-1 to CD22 (17). This result indicated that the region containing Tyr843 and Tyr863 constitutes the SHP-1-binding domain of CD22, whereas ITIM containing Tyr783 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-1-binding 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 Tyr843 and Tyr863 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 Tyr843 and Tyr863. Further, we demonstrate that Tyr783 at a previously defined ITIM (15) does not regulate phosphorylation of other tyrosines, such as Tyr843 and Tyr863, but plays an essential role, together with Tyr817, in SHP-1 binding and BCR regulation of the mutant CD22 carrying Tyr -> Phe mutations at both Tyr843 and Tyr863. These results clearly indicate that the region containing Tyr783 and Tyr817 constitutes a novel SHP-1-binding site that is activated upon BCR ligation by antigen but not anti-Ig Ab.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Retrovirus Vectors—The murine CD22 cDNA was obtained from pMX-CD22.2-REV (12) and was cloned into the bicistronic retrovirus vector pMXs-IG (23) (a gift of Dr. T. Kitamura, University of Tokyo), allowing expression of both green fluorescent protein and CD22 (pMXs-CD22-IG). A tyrosine to phenylalanine mutation (Tyr -> Phe mutation) at Tyr773, Tyr783, Tyr817, Tyr828, Tyr843, or Tyr863 was introduced to the CD22 cDNA by PCR using specific primers (supplemental Table S1). A pair of synthetic oligonucleotides (5'-GGCCGCCTATCCCTATGACGTGCCCGACTATGCCTGA-3' and 5'-GGCCTCAGGCATAGTCGGGCACGTCATAGGGATAGGC-3') encoding both hemagglutinin (HA) tag and a stop codon was annealed and inserted at the NotI site of the pMXs-IG vector containing wild type or mutant CD22 (Fig. 1).

Cell Culture—The mouse B cell lines K46µv and BAL17IgM were described previously (12). These cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 50 µM 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 mML-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. K46µv and BAL17IgM cells were incubated with the supernatant containing retrovirus for 4 h.

Flow Cytometry—Cells were stained with (4-hydroxy-3-nitrophenyl) acetyl (NP)-conjugated phycoerythrin or the combination of biotinylated anti-mouse CD22 monoclonal Ab (mAb) Cy34.1 (BD Biosciences) and Cy5-conjugated streptavidin (Jackson ImmunoResearch, West Grove, PA). Cells were analyzed by flow cytometry using a fluorescence-activated cell sorter LSR (BD Biosciences) or a CyAn (DAKO, Glostrup, Denmark).

Immunoprecipitation and Western Blotting—K46µv and BAL17IgM cells were stimulated with 0.2 µg/ml NP-conjugated bovine serum albumin (BSA), 25 µM pervanadate/H2O2 or 10 µg/ml anti-IgM Ab for 1, 3, and 10 min. Either unstimulated or stimulated cells were lysed in Triton X-100 lysis buffer (1% Triton X-100, 10% glycerol, 150 mM NaCl, 20 mM Tris-HCl, 2mM EDTA, 0.02% NaN3, 10 µg/ml phenylmethylsulfonyl fluoride, 1 mM Na3VO4) and were immunoprecipitated with rat anti-HA mAb 3F10 (Roche Applied Science) or rabbit anti-SHP-1 Ab (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) using protein G-Sepharose (Amersham Biosciences). Total cell lysates or immunoprecipitates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were incubated with mouse anti-phosphotyrosine mAb 4G10 (Upstate%20Biotechnology">Upstate Biotechnology, Inc., Lake Placid, NY), rat anti-HA mAb 3F10, rabbit anti-SHP-1 Ab, mouse anti-Grb2 Ab (BD Biosciences Pharmingen, San Diego, CA), Abs to phosphorylated ITIMs containing Tyr843 and Tyr863, respectively (26), anti-β-tubulin mAb TUB2.1 (Seikagaku Kogyo, Tokyo, Japan), anti-phospho-ERK, anti-phospho-AKT Ab, followed by a reaction with peroxidase-conjugated anti-rabbit IgG Ab (New England Biolabs, Beverly, MA), anti-mouse IgG Ab, or anti-rat IgG Ab (Southern Biotechnology Associates, Birmingham, AL). Proteins were visualized by an ECL system (Amersham Biosciences).

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 µg/ml NP-BSA or 10 µg/ml anti-IgM Ab and analyzed by flow cytometry using a fluorescence-activated cell sorter LSR (BD Biosciences).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
CD22 ITIMs Containing Tyr843 and Tyr863 Are Dispensable for CD22-mediated BCR Regulation—To investigate whether Tyr843 and Tyr863 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 -> Phe mutations at both Tyr843 and Tyr863 (CD22F5/6), and the other carries triple Tyr -> Phe mutations at Tyr783, Tyr843, and Tyr863 (CD22F2/5/6) (Fig. 1). Retrovirus expressing wild-type or mutant CD22 was transduced to the mouse B cell lines K46µv and BAL17IgM, both of which express membrane-bound IgM reactive to the hapten NP (12). Since K46µv but not BAL17IgM lacks expression of endogenous CD22 (12), we assessed expression of both transduced wild-type and mutated CD22 in K46µv 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 NP-reactive BCR were similar to those in the cells transduced with the empty vector alone (K46µv-vector and BAL17IgM-vector) (supplemental Figs. S1 and S2).


Figure 1
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  		FIGURE 1.
Schematic representation of Tyr -> Phe mutants of CD22.

 


Figure 2
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  		FIGURE 2.
Tyr843 and Tyr863 are dispensable for CD22 to negatively regulate antigen-induced phosphorylation of cellular substrates, including ERK and AKT. The indicated K46µv transfectants were treated with 0.2µg/ml NP-BSA for the indicated times. Total cell lysates were separated by SDS-PAGE and analyzed for tyrosine phosphorylation by Western blotting using an anti-phosphotyrosine mAb (A), anti-phospho-ERK Ab (B), and anti-phospho-AKT Ab (C). These blots were reprobed with anti-β-tubulin Ab to ensure equal loading. Representative data of more than three experiments are shown.

 
We stimulated K46µv-vector, K46µvCD22, K46µvCD22F5/6, and K46µvCD22F2/5/6 transfectants with an antigen NP-BSA and examined phosphorylation of cellular substrates. Surprisingly, antigen stimulation-induced phosphorylation of various cellular substrates was reduced in K46µvCD22F5/6 cells compared with K46µv-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 Tyr843 and Tyr863 of CD22 are dispensable for CD22-mediated signal inhibition in antigen-stimulated K46µv 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 K46µv transfectants (Fig. 2), suggesting that Tyr783 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 K46µv 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 Tyr843 and Tyr863 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/H2O2, 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 Tyr828 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 BCR-mediated substrate phosphorylation in these cells due to expression of endogenous CD22. Taken together, CD22F5/6 carrying Y/F mutations at both Tyr843 and Tyr863 but not CD22F2/5/6 was able to regulate BCR signaling and recruit SHP-1 upon antigen stimulation. Thus, Tyr843 and Tyr863 are dispensable for CD22 to bind to SHP-1 and to regulate BCR signaling in antigen-stimulated B cells, and Tyr783 is involved in CD22-mediated BCR regulation, probably by serving as a binding site for SHP-1.

Tyr783 Is Involved in CD22-mediated BCR Regulation without Influencing Phosphorylation of Other Tyrosine Residues in CD22—To address the role of Tyr783 in CD22-mediated signal regulation, we constructed the retrovirus vector encoding mutant CD22 containing a Tyr -> Phe mutation at Tyr783 alone (CD22F2) (Fig. 1) and transduced the retrovirus vector to K46µv cells. The expression levels of CD22 and NP-reactive BCR on K46µvCD22F2 transfectants were similar to those on K46µvCD22 cells (supplemental Fig. S1). When the K46µvCD22F2 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 K46µvCD22F2 cells showed weaker ERK phosphorylation and weaker calcium mobilization than K46µv cells transfected with the vector alone (Fig. 4, B and C). Thus, CD22F2 recruited SHP-1 and regulated BCR signaling, probably because ITIMs containing Tyr843 and Tyr863 in CD22F2 recruited SHP-1 and regulated BCR signaling. However, antigen-stimulated K46µvCD22F2 cells showed stronger calcium mobilization than K46µvCD22 cells (Fig. 4C). This result indicated that CD22F2 regulated calcium signaling less efficiently than wild-type CD22 and suggested that Tyr783 is involved in the regulation of BCR signaling even in the presence of Tyr843 and Tyr863. 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.


Figure 3
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  		FIGURE 3.
Tyr843 and Tyr863 are dispensable for CD22 to recruit SHP-1 in antigen-stimulated B cells. The indicated K46µv(A and B) and BAL17IgM (C and D) transfectants were treated with 0.2 µg/ml NP-BSA (A and C) or 25 µM pervanadate/H2O2 (B and 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.

 
To address whether Tyr783 regulates tyrosine phosphorylation of CD22, we examined phosphorylation of Tyr843 and Tyr863 using Abs that specifically recognize phosphorylated ITIMs containing Tyr843 and Tyr863, respectively (26). Phosphorylation of both Tyr843 and Tyr863 in CD22F2 in antigen-stimulated K46µv transfectants was comparable to that in wild-type CD22 (Fig. 4D), indicating that Tyr783 does not regulate phosphorylation of other tyrosines in the cytoplasmic region of CD22. Taken together, Tyr783 is involved in BCR regulation without influencing phosphorylation of other tyrosines in CD22.

Both Tyr783 and Tyr817 Are Required for CD22-mediated Signal Regulation in the Absence of Tyr843 and Tyr863—We next asked whether the ITIM containing Tyr783 regulates BCR signaling alone or in combination with yet uncharacterized ITIM. Besides tyrosine residues at the previously defined ITIMs, CD22 contains three tyrosine residues at positions 773, 817, and 828 in the cytoplasmic region. Among these tyrosine residues, the flanking sequence of Tyr773 does not fit the consensus ITIM sequence at all. We thus constructed retrovirus vector encoding CD22 Tyr -> Phe mutant containing only one tyrosine in the cytoplasmic region at Tyr783 (CD22F1/3/4/5/6), that containing two tyrosine residues at Tyr783 and Tyr817 (CD22F1/4/5/6), and that containing Tyr783 and Tyr828 (CD22F1/3/5/6) (Fig. 1), and K46µv cells were transduced with these vectors. The expression levels of both CD22 and NP-reactive BCR in these transfectants were similar to those of K46µvCD22 and K46µvCD22F5/6 cells (supplemental Fig. S1). When we stimulated the transfectants with an antigen NP-BSA, CD22F1/4/5/6 but not CD22F1/3/5/6 or CD22F1/3/4/5/6 negatively regulated both ERK phosphorylation (Fig. 5A) and calcium mobilization (Fig. 5B), although both CD22F5/6 and CD22F1/4/5/6 regulated calcium signaling less efficiently than wild type CD22. CD22F1/4/5/6 and CD22F1/3/5/6 but not CD22F1/3/4/5/6 were phosphorylated as strongly as wild-type CD22 (Fig. 5C). However, SHP-1 was recruited by CD22F1/4/5/6 containing Tyr783 and Tyr817 but not CD22F1/3/5/6 containing Tyr783 and Tyr828 (Fig. 5C). These results indicated that Tyr783 alone is not able to recruit SHP-1 or regulate BCR and requires co-presence of Tyr817 for recruiting SHP-1 and regulating BCR in the absence of Tyr843 and Tyr863. Since the flanking sequence of Tyr817 (VTYSVI) fits ITIM ((I/V/L/S)XYXX(L/V/I)) (27), the sequence surrounding Tyr817 appears to be a functional ITIM and to serve as a binding site for SHP-1 in combination with ITIM containing Tyr783 in antigen-stimulated B cells.

Both Tyr783 and Tyr817 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 K46µv transfectants were stimulated with anti-Ig Ab, wild-type CD22 but not CD22F5/6 carrying Tyr -> Phe mutations at both Tyr843 and Tyr863 co-precipitated SHP-1 (Fig. 6A) and regulated BCR signaling including ERK phosphorylation (Fig. 6B) and calcium mobilization (Fig. 6C). Thus, the ITIMs containing Tyr843 and Tyr863 but not those containing Tyr783 and Tyr817 are responsible for SHP-1 recruitment and BCR signal regulation in anti-Ig Ab-treated K46µv cells, in agreement with a previous finding by Otipoby et al. (17). Taken together, ITIMs containing Tyr783 and Tyr817 are involved in SHP-1 recruitment and BCR regulation upon BCR ligation by antigen but not anti-Ig Ab.


Figure 4
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  		FIGURE 4.
Tyr783 is involved in BCR regulation but does not regulate phosphorylation of Tyr843 or Tyr863. A, recruitment of SHP-1. The indicated K46µv transfectants were treated with 0.2 µg/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 K46µv transfectants were treated with 0.2 µg/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-β-tubulin Ab to ensure equal loading. Representative data of more than three experiments are shown. C, regulation of calcium mobilization. The indicated K46µv 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 µg/ml NP-BSA at 30 s (indicated by an arrow), and measurement of free calcium was continued for 300 s. D, phosphorylation of Tyr843 and Tyr863. The indicated K46µv transfectants were treated with 0.2 µg/ml NP-BSA for the indicated times. Total cell lysates were separated by SDS-PAGE and analyzed for phosphorylation of Tyr843 and Tyr863 by Western blotting using antibodies generated to the phosphopeptides containing Tyr843 and Tyr863, respectively. These blots were reprobed with anti-β-tubulin Ab to ensure equal loading. Representative data of more than three experiments are shown.

 


Figure 5
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  		FIGURE 5.
Both Tyr783 and Tyr817 are involved in BCR regulation and SHP-1 recruitment in antigen-stimulated B cells. A, regulation of ERK phosphorylation. The indicated K46µv transfectants were treated with 0.2 µg/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-β-tubulin Ab to ensure equal loading. Representative data of more than three experiments are shown. B, regulation of calcium mobilization. Indicated K46µv 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 µg/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 K46µv transfectants were treated with 0.2µg/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.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we demonstrate that the previously defined SHP-1-binding domain containing Tyr843 and Tyr863 is dispensable for CD22 to recruit SHP-1 and to regulate BCR signaling in the B cell lines K46µv and BAL17IgM and that the previously identified ITIM containing Tyr783 is involved in both SHP-1 binding and BCR regulation. Some of the tyrosine residues in 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, Tyr783 does not regulate phosphorylation of other tyrosine residues of CD22. In contrast, the peptide containing Tyr783 was previously shown to bind to SHP-1 (14). Thus, Tyr783 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 Tyr817 fits the consensus sequence of ITIM (27) and that both Tyr783 and Tyr817 are required for SHP-1 binding and BCR regulation of CD22F5/6 mutant carrying a Tyr -> Phe mutation at Tyr843 and Tyr863. 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 Tyr783 and Tyr817 for SHP-1 recruitment in CD22F5/6 therefore suggests that two ITIMs containing Tyr783 and Tyr817, respectively, bind to the two SH2 domains of SHP-1, thereby recruiting it and regulating BCR signaling. Thus, Tyr783 plays a role together with Tyr817 in CD22-mediated BCR regulation by constituting a novel SHP-1-binding site.


Figure 6
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  		FIGURE 6.
Both Tyr843 and Tyr863 are required for CD22 to recruit SHP-1 and regulate BCR signaling in anti-Ig Ab-treated B cells. A, recruitment of SHP-1. The indicated K46µv transfectants were treated with 10 µg/ml anti-IgM for the indicated times. Cells were lysed, and HA-tagged CD22 was immunoprecipitated 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. Representative data of more than three experiments are shown. B, regulation of ERK phosphorylation. The indicated K46µv transfectants were treated with 10 µg/ml anti-IgM for indicated times. Total cell lysates were separated by SDS-PAGE and analyzed by Western blotting using an anti-phospho-ERK Ab. The membranes were reprobed with anti-β-tubulin Ab to ensure equal loading. Representative data of more than three experiments are shown. C, regulation of calcium mobilization. The indicated K46µv transfectants were loaded with Fluo-4/AM, and intracellular free calcium was measured by flow cytometry using FACS LSR. Cells were treated with 10µg/ml anti-IgM at 30 s (indicated by an arrow), and measurement of free calcium was continued for 300 s.

 
Here we demonstrate that the ITIMs containing Tyr783 and Tyr817 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 Tyr843 and Tyr863 probably recruit SHP-1 with a lower phosphorylation level, whereas those containing Tyr783 and Tyr817 appear to require higher levels of phosphorylation for SHP-1 binding. Previously, Doody et al. (14) demonstrated that phosphorylated peptides containing Tyr783, Tyr843, and Tyr863, respectively, but not that containing Tyr817 block binding of CD22 to SHP-1. This suggests that ITIM containing Tyr817 binds to SHP-1 less efficiently than the other ITIMs and may require strong phosphorylation for SHP-1 recruitment. However, the flanking sequence of Tyr817 completely fits the consensus sequence of ITIM (27) and does not show a particular difference from other ITIMs; therefore, how the ITIM containing Tyr817 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 Tyr783 and Tyr817.


   FOOTNOTES
 
* This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology and from the Japan Society for the Promotion of Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1 and S2. Back

1 To whom correspondence should be addressed: Laboratory of Immunology, School of Biomedical Science, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. Tel.: 81-3-5803-5817; Fax: 81-3-5684-0717; E-mail: tsubata.imm{at}mri.tmd.ac.jp.

2 The abbreviations used are: B cell, B lymphocyte; ITIM, immunoreceptor tyrosine-based inhibition motif; BCR, B cell antigen receptor; SH2, Src homology 2; SHP-1, SH2 domain-containing protein-tyrosine phosphatase 1; HA, hemagglutinin; NP, 4-hydroxy-3-nitrophenyl acetyl; ERK, extracellular signal-regulated kinase; Ab, antibody; mAb, monoclonal Ab; BSA, bovine serum albumin. Back

3 J. Yu and T. Tsubata, unpublished observation. Back


   ACKNOWLEDGMENTS
 
We thank Drs. K. Ikuta (Kyoto University), G. P. Nolan (Stanford University), and T. Kitamura (University of Tokyo) for reagents; Drs. T. Adachi and C. Wakabayashi for technical advice; and A. Yoshino for technical help.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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