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• W2009162022 abstract "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. 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. CD22 (also known as siglec (sialic acid-binding immunoglobulin-like lectin) 2), a 140-kDa membrane glycoprotein expressed on B lymphocytes (B cells), 2The abbreviations used are: B cellB lymphocyteITIMimmunoreceptor tyrosine-based inhibition motifBCRB cell antigen receptorSH2Src homology 2SHP-1SH2 domain-containing protein-tyrosine phosphatase 1HAhemagglutininNP4-hydroxy-3-nitrophenyl acetylERKextracellular signal-regulated kinaseAbantibodymAbmonoclonal AbBSAbovine serum albumin. is a negative regulator of B cell antigen receptor (BCR), and a crucial regulator of B cell activation (1Nitschke L. Curr. Opin. Immunol. 2005; 17: 290-297Crossref PubMed Scopus (163) Google Scholar, 2Nitschke L. Tsubata T. Trends Immunol. 2004; 25: 543-550Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). 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 (3Blasioli J. Paust S. Thomas M.L. J. Biol. Chem. 1999; 274: 2303-2307Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 4Law C.L. Sidorenko S.P. Chandran K.A. Zhao Z. Shen S.H. Fischer E.H. Clark E.A. J. Exp. Med. 1996; 183: 547-560Crossref PubMed Scopus (176) Google Scholar, 5Poe J.C. Fujimoto M. Jansen P.J. Miller A.S. Tedder T.F. J. Biol. Chem. 2000; 275: 17420-17427Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 6Yohannan J. Wienands J. Coggeshall K.M. Justement L.B. J. Biol. Chem. 1999; 274: 18769-18776Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). 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 (7Nitschke L. Carsetti R. Ocker B. Kohler G. Lamers M.C. Curr. Biol. 1997; 7: 133-143Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 8O'Keefe T.L. Williams G.T. Davies S.L. Neuberger M.S. Science. 1996; 274: 798-801Crossref PubMed Scopus (475) Google Scholar, 9Otipoby K.L. Andersson K.B. Draves K.E. Klaus S.J. Farr A.G. Kerner J.D. Perlmutter R.M. Law C.L. Clark E.A. Nature. 1996; 384: 634-637Crossref PubMed Scopus (363) Google Scholar, 10Sato S. Miller A.S. Inaoki M. Bock C.B. Jansen P.J. Tang M.L. Tedder T.F. Immunity. 1996; 5: 551-562Abstract Full Text PDF PubMed Scopus (391) Google Scholar, 11Tooze R.M. Doody G.M. Fearon D.T. Immunity. 1997; 7: 59-67Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 12Wakabayashi C. Adachi T. Wienands J. Tsubata T. Science. 2002; 298: 2392-2395Crossref PubMed Scopus (149) Google Scholar) through dephosphorylation of various signaling molecules and regulation of plasma membrane calcium-ATPase (13Chen J. McLean P.A. Neel B.G. Okunade G. Shull G.E. Wortis H.H. Nat. Immunol. 2004; 5: 651-657Crossref PubMed Scopus (86) Google Scholar). B lymphocyte immunoreceptor tyrosine-based inhibition motif B cell antigen receptor Src homology 2 SH2 domain-containing protein-tyrosine phosphatase 1 hemagglutinin 4-hydroxy-3-nitrophenyl acetyl extracellular signal-regulated kinase antibody monoclonal Ab bovine serum albumin. 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 (14Doody G.M. Justement L.B. Delibrias C.C. Matthews R.J. Lin J. Thomas M.L. Fearon D.T. Science. 1995; 269: 242-244Crossref PubMed Scopus (489) Google Scholar). Indeed, the flanking sequences of these tyrosine residues fit the consensus sequence of the immunoreceptor tyrosine-based inhibition motif (ITIM) (15Thomas M.L. J. Exp. Med. 1995; 181: 1953-1956Crossref PubMed Scopus (114) Google Scholar), the motif often found at the binding sites for SH2-containing phosphatases, including SHP-1 (16Sweeney M.C. Wavreille A.S. Park J. Butchar J.P. Tridandapani S. Pei D. Biochemistry. 2005; 44: 14932-14947Crossref PubMed Scopus (114) Google Scholar), and more importantly phosphotyrosyl peptides containing these tyrosine residues are capable of binding to SHP-1 in vitro (14Doody G.M. Justement L.B. Delibrias C.C. Matthews R.J. Lin J. Thomas M.L. Fearon D.T. Science. 1995; 269: 242-244Crossref PubMed Scopus (489) Google Scholar). 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 (17Otipoby K.L. Draves K.E. Clark E.A. J. Biol. Chem. 2001; 276: 44315-44322Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). 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 (18Hokazono Y. Adachi T. Wabl M. Tada N. Amagasa T. Tsubata T. J. Immunol. 2003; 171: 1835-1843Crossref PubMed Scopus (44) Google Scholar). 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 (19Smith K.G. Tarlinton D.M. Doody G.M. Hibbs M.L. Fearon D.T. J. Exp. Med. 1998; 187: 807-811Crossref PubMed Scopus (226) Google Scholar, 20Nishizumi H. Horikawa K. Mlinaric-Rascan I. Yamamoto T. J. Exp. Med. 1998; 187: 1343-1348Crossref PubMed Scopus (174) Google Scholar, 21Cornall R.J. Cyster J.G. Hibbs M.L. Dunn A.R. Otipoby K.L. Clark E.A. Goodnow C.C. Immunity. 1998; 8: 497-508Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, 22Chan V.W. Lowell C.A. DeFranco A.L. Curr. Biol. 1998; 8: 545-553Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). 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 (15Thomas M.L. J. Exp. Med. 1995; 181: 1953-1956Crossref PubMed Scopus (114) Google Scholar) 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. Retrovirus Vectors—The murine CD22 cDNA was obtained from pMX-CD22.2-REV (12Wakabayashi C. Adachi T. Wienands J. Tsubata T. Science. 2002; 298: 2392-2395Crossref PubMed Scopus (149) Google Scholar) and was cloned into the bicistronic retrovirus vector pMXs-IG (23Onishi M. Kinoshita S. Morikawa Y. Shibuya A. Phillips J. Lanier L.L. Gorman D.M. Nolan G.P. Miyajima A. Kitamura T. Exp. Hematol. 1996; 24: 324-329PubMed Google Scholar) (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 (12Wakabayashi C. Adachi T. Wienands J. Tsubata T. Science. 2002; 298: 2392-2395Crossref PubMed Scopus (149) Google Scholar). 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) (24Morita S. Kojima T. Kitamura T. Gene Ther. 2000; 7: 1063-1066Crossref PubMed Scopus (1376) Google Scholar) and Phoenix (a gift of Dr. G. P. Nolan, Stanford University School of Medicine) (25Grignani F. Kinsella T. Mencarelli A. Valtieri M. Riganelli D. Lanfrancone L. Peschle C. Nolan G.P. Pelicci P.G. Cancer Res. 1998; 58: 14-19PubMed Google Scholar) 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 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 (26Fujimoto M. Kuwano Y. Watanabe R. Asashima N. Nakashima H. Yoshitake S. Okochi H. Tamaki K. Poe J.C. Tedder T.F. Sato S. J. Immunol. 2006; 176: 873-879Crossref PubMed Scopus (29) Google Scholar), 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). 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 (12Wakabayashi C. Adachi T. Wienands J. Tsubata T. Science. 2002; 298: 2392-2395Crossref PubMed Scopus (149) Google Scholar). Since K46μv but not BAL17IgM lacks expression of endogenous CD22 (12Wakabayashi C. Adachi T. Wienands J. Tsubata T. Science. 2002; 298: 2392-2395Crossref PubMed Scopus (149) Google Scholar), 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). 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 (5Poe J.C. Fujimoto M. Jansen P.J. Miller A.S. Tedder T.F. J. Biol. Chem. 2000; 275: 17420-17427Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) (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. 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 (26Fujimoto M. Kuwano Y. Watanabe R. Asashima N. Nakashima H. Yoshitake S. Okochi H. Tamaki K. Poe J.C. Tedder T.F. Sato S. J. Immunol. 2006; 176: 873-879Crossref PubMed Scopus (29) Google Scholar). 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)) (27Neel B.G. Gu H. Pao L. Trends Biochem. Sci. 2003; 28: 284-293Abstract Full Text Full Text PDF PubMed Scopus (971) Google Scholar), 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. (17Otipoby K.L. Draves K.E. Clark E.A. J. Biol. Chem. 2001; 276: 44315-44322Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). 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. 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 (28Yamanashi Y. Fukuda T. Nishizumi H. Inazu T. Higashi K. Kitamura D. Ishida T. Yamamura H. Watanabe T. Yamamoto T. J. Exp. Med. 1997; 185: 1387-1392Crossref PubMed Scopus (90) Google Scholar), in which a phosphorylated tyrosine recruits the kinase that phosphorylates other tyrosine residues in the same molecule (29Mayer B.J. Hirai H. Sakai R. Curr. Biol. 1995; 5: 296-305Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 30Songyang Z. Carraway K.L. Eck 3rd, M.J. Harrison S.C. Feldman R.A. Mohammadi M. Schlessinger J. Hubbard S.R. Smith D.P. Eng C. Lorenzo M.J. Ponder B.A.J. Mayer B.J. Cantiley L.C. Nature. 1995; 373: 536-539Crossref PubMed Scopus (848) Google Scholar, 31Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2391) Google Scholar). 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 (14Doody G.M. Justement L.B. Delibrias C.C. Matthews R.J. Lin J. Thomas M.L. Fearon D.T. Science. 1995; 269: 242-244Crossref PubMed Scopus (489) Google Scholar). 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 (27Neel B.G. Gu H. Pao L. Trends Biochem. Sci. 2003; 28: 284-293Abstract Full Text Full Text PDF PubMed Scopus (971) Google Scholar) 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 (27Neel B.G. Gu H. Pao L. Trends Biochem. Sci. 2003; 28: 284-293Abstract Full Text Full Text PDF PubMed Scopus (971) Google Scholar, 32Yang J. Liu L. He D. Song X. Liang X. Zhao Z.J. Zhou G.W. J. Biol. Chem. 2003; 278: 6516-6520Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). 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. 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 (18Hokazono Y. Adachi T. Wabl M. Tada N. Amagasa T. Tsubata T. J. Immunol. 2003; 171: 1835-1843Crossref PubMed Scopus (44) Google Scholar). 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 (33Yu J. Sawada T. Adachi T. Gao X. Takematsu H. Kozutsumi Y. Ishida H. Kiso M. Tsubata T. Biochem. Biophys. Res. Commun. 2007; 360: 759-764Crossref PubMed Scopus (17) Google Scholar). In contrast, BCR ligation with anti-Ig Ab induces weak phosphorylation of CD22 (18Hokazono Y. Adachi T. Wabl M. Tada N. Amagasa T. Tsubata T. J. Immunol. 2003; 171: 1835-1843Crossref PubMed Scopus (44) Google Scholar) probably because of exclusion of CD22 from BCR-containing lipid rafts. 3J. Yu and T. Tsubata, unpublished observation. 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. (14Doody G.M. Justement L.B. Delibrias C.C. Matthews R.J. Lin J. Thomas M.L. Fearon D.T. Science. 1995; 269: 242-244Crossref PubMed Scopus (489) Google Scholar) 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 (27Neel B.G. Gu H. Pao L. Trends Biochem. Sci. 2003; 28: 284-293Abstract Full Text Full Text PDF PubMed Scopus (971) Google Scholar) 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. 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. Download .pdf (1.57 MB) Help with pdf files" @default.
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• W2009162022 title "Novel Binding Site for Src Homology 2-containing Protein-tyrosine Phosphatase-1 in CD22 Activated by B Lymphocyte Stimulation with Antigen" @default.
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