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CD22 Regulates B Cell Receptor-mediated Signals via Two Domains That Independently Recruit Grb2 and SHP-1

2001; Elsevier BV; Volume: 276; Issue: 47 Linguagem: Inglês

10.1074/jbc.m105446200

ISSN

1083-351X

Autores

Kevin L. Otipoby, Kevin E. Draves, Edward A. Clark,

Tópico(s)

Immune Cell Function and Interaction

Resumo

Recognition of antigen by the B cell antigen receptor (BCR) determines the subsequent fate of a B cell and is regulated in part by the involvement of other surface molecules, termed coreceptors. CD22 is a B cell-restricted coreceptor that gets rapidly tyrosyl-phosphorylated and recruits various signaling molecules to the membrane following BCR ligation. Although CD22 contains three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), only the two carboxyl-terminal ITIM tyrosines are required for efficient recruitment of the SHP-1 phosphatase after BCR ligation. Furthermore, Grb2 is inducibly recruited to CD22 in human and murine B cells. Unlike SHP-1, Grb2 recruitment to CD22 is not inhibited by specific doses of the Src family kinase-specific inhibitor PP1. The tyrosine residue in CD22 required for Grb2 recruitment (Tyr-828) is distinct and independent from the two ITIM tyrosines required for efficient SHP-1 recruitment (Tyr-843 and Tyr-863). Individually both Lyn and Syk are required for maximal phosphorylation of CD22 following ligation of the BCR, and together Lyn and Syk are required for all of the constitutive and induced tyrosine phosphorylation of CD22. We propose that the cytoplasmic tail of CD22 contains two domains that regulate signal transduction pathways initiated by the BCR and B cell fate. Recognition of antigen by the B cell antigen receptor (BCR) determines the subsequent fate of a B cell and is regulated in part by the involvement of other surface molecules, termed coreceptors. CD22 is a B cell-restricted coreceptor that gets rapidly tyrosyl-phosphorylated and recruits various signaling molecules to the membrane following BCR ligation. Although CD22 contains three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), only the two carboxyl-terminal ITIM tyrosines are required for efficient recruitment of the SHP-1 phosphatase after BCR ligation. Furthermore, Grb2 is inducibly recruited to CD22 in human and murine B cells. Unlike SHP-1, Grb2 recruitment to CD22 is not inhibited by specific doses of the Src family kinase-specific inhibitor PP1. The tyrosine residue in CD22 required for Grb2 recruitment (Tyr-828) is distinct and independent from the two ITIM tyrosines required for efficient SHP-1 recruitment (Tyr-843 and Tyr-863). Individually both Lyn and Syk are required for maximal phosphorylation of CD22 following ligation of the BCR, and together Lyn and Syk are required for all of the constitutive and induced tyrosine phosphorylation of CD22. We propose that the cytoplasmic tail of CD22 contains two domains that regulate signal transduction pathways initiated by the BCR and B cell fate. B cell antigen receptor immunoreceptor tyrosine-based inhibitory motif protein tyrosine kinase surface IgM mitogen-activated protein kinase Lyn-deficient Syk-deficient Lyn and Syk double-deficient polyacrylamide gel electrophoresis Epstein-Barr virus polymerase chain reaction monoclonal antibody c-Jun NH2-terminal kinase extracellular signal-regulated kinase hemagglutinin fluorescence-activated cell sorter linker for activation of T cells 4-amino-5- (4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine The binding of antigenic determinants by the BCR1 on B cells is the initiating event in antigen-specific B cell responses. The generation of a productive humoral response is only one of many possible outcomes; engagement of the BCR complex by antigen can induce survival, proliferation, apoptosis, clonal nonresponsiveness (anergy), or differentiation into memory B cells or plasma cells (1Goodnow C.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2264-2271Crossref PubMed Scopus (369) Google Scholar). Not surprisingly, many factors determine which fate a B cell will choose, including the developmental stage of the B cell, the amount and form of the antigen, the microanatomical location of the B cell, and the availability of accessory cells. Somehow these factors in the extracellular milieu contribute to and alter the intracellular signal transduction pathways initiated by the BCR complex, resulting in differential gene expression and altered B cell fate. Recently the involvement of additional molecules expressed on the surface of B cells in delivering these extracellular cues and regulating the quality and/or quantity of BCR-initiated signals has become evident (2Craxton A. Otipoby K.L. Jiang A. Clark E.A. Adv. Immunol. 1999; 73: 79-152Crossref PubMed Google Scholar, 3Tedder T.F. Tuscano J. Sato S. Kehrl J.H. Annu. Rev. Immunol. 1997; 15: 481-504Crossref PubMed Scopus (271) Google Scholar, 4Carroll M.C. Adv. Immunol. 2000; 74: 61-88Crossref PubMed Google Scholar, 5Bolland S. Ravetch J.V. Adv. Immunol. 1999; 72: 149-177Crossref PubMed Google Scholar, 6Pan C. Baumgarth N. Parnes J.R. Immunity. 1999; 11: 495-506Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). These coreceptors, which include CD22, CD19, CD21, FcγRIIb, and CD72, regulate many signal transduction pathways in both “positive” and “negative” manners, thereby facilitating a range of B cell responses (2Craxton A. Otipoby K.L. Jiang A. Clark E.A. Adv. Immunol. 1999; 73: 79-152Crossref PubMed Google Scholar). Unlike receptor tyrosine kinases, the BCR has no intrinsic kinase. Engagement of the BCR rapidly recruits and activates protein tyrosine kinases (PTKs), which initiate perhaps all of the downstream biochemical events after antigen receptor stimulation (2Craxton A. Otipoby K.L. Jiang A. Clark E.A. Adv. Immunol. 1999; 73: 79-152Crossref PubMed Google Scholar, 7Kurosaki T. Annu. Rev. Immunol. 1999; 17: 555-592Crossref PubMed Scopus (369) Google Scholar). Three major families of PTKs are expressed in B cells: Lyn, Blk, Fyn, Lck, and Fgr of the Src family; Syk of the Syk/Zap70 family; and Btk of the Tec family (2Craxton A. Otipoby K.L. Jiang A. Clark E.A. Adv. Immunol. 1999; 73: 79-152Crossref PubMed Google Scholar, 7Kurosaki T. Annu. Rev. Immunol. 1999; 17: 555-592Crossref PubMed Scopus (369) Google Scholar). Activation of these kinases leads to tyrosine phosphorylation of various substrates, including coreceptors, adaptor molecules, and other enzymes (8DeFranco A.L. Curr. Opin. Immunol. 1997; 9: 296-308Crossref PubMed Scopus (282) Google Scholar). The phosphorylation of coreceptors produces potential binding sites for downstream signaling molecules and promotes the formation of signaling complexes at the lipid bilayer juxtaposed to potential substrates. CD22 is a B cell-restricted type I surface glycoprotein (3Tedder T.F. Tuscano J. Sato S. Kehrl J.H. Annu. Rev. Immunol. 1997; 15: 481-504Crossref PubMed Scopus (271) Google Scholar, 9Law C.L. Sidorenko S.P. Clark E.A. Immunol. Today. 1994; 15: 442-449Abstract Full Text PDF PubMed Scopus (6) Google Scholar, 10Cyster J.G. Goodnow C.C. Immunity. 1997; 6: 509-517Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). The extracellular portion of CD22 contains seven immunoglobulin (Ig)-like domains. The two amino-terminal Ig-like domains are capable of binding ligands with α2→6-linked sialic acid moieties expressed on epithelial, endothelial, B, and T cells (3Tedder T.F. Tuscano J. Sato S. Kehrl J.H. Annu. Rev. Immunol. 1997; 15: 481-504Crossref PubMed Scopus (271) Google Scholar). However, the specific ligands recognized by CD22 in vivo are not yet well defined (3Tedder T.F. Tuscano J. Sato S. Kehrl J.H. Annu. Rev. Immunol. 1997; 15: 481-504Crossref PubMed Scopus (271) Google Scholar). The cytoplasmic tail of CD22 contains six conserved tyrosine residues, some or all of which are rapidly phosphorylated upon engagement of the BCR (11Schulte R.J. Campbell M.A. Fischer W.H. Sefton B.M. Science. 1992; 258: 1001-1004Crossref PubMed Scopus (121) Google Scholar, 12Stamenkovic I. Seed B. Nature. 1990; 345: 74-77Crossref PubMed Scopus (165) Google Scholar, 13Wilson G.L. Fox C.H. Fauci A.S. Kehrl J.H. J. Exp. Med. 1991; 173: 137-146Crossref PubMed Scopus (127) Google Scholar, 14Torres R.M. Law C.L. Santos-Argumedo L. Kirkham P.A. Grabstein K. Parkhouse R.M. Clark E.A. J. Immunol. 1992; 149: 2641-2649PubMed Google Scholar). Phosphorylation of CD22 provides binding sites for many Src homology 2-containing molecules, including Lyn and Syk (15Tuscano J.M. Engel P. Tedder T.F. Agarwal A. Kehrl J.H. Eur. J. Immunol. 1996; 26: 1246-1252Crossref PubMed Scopus (73) Google Scholar, 16Law 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), the protein tyrosine phosphatase SHP-1 (16Law 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, 17Campbell M.A. Klinman N.R. Eur. J. Immunol. 1995; 25: 1573-1579Crossref PubMed Scopus (112) Google Scholar, 18Doody 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 (486) Google Scholar, 19Lankester A.C. van Schijndel G.M. van Lier R.A. J. Biol. Chem. 1995; 270: 20305-20308Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), phospholipase Cγ-1 (16Law 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), and phosphoinositide 3-kinase (15Tuscano J.M. Engel P. Tedder T.F. Agarwal A. Kehrl J.H. Eur. J. Immunol. 1996; 26: 1246-1252Crossref PubMed Scopus (73) Google Scholar). Furthermore, CD22 can physically associate with the BCR complex (20Leprince C. Draves K.E. Geahlen R.L. Ledbetter J.A. Clark E.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3236-3240Crossref PubMed Scopus (169) Google Scholar,21Peaker C.J. Neuberger M.S. Eur. J. Immunol. 1993; 23: 1358-1363Crossref PubMed Scopus (143) Google Scholar). By recruiting various signaling molecules to the BCR complex, CD22 may regulate BCR-mediated signal transduction pathways and B cell function. This hypothesis is supported by evidence from studies of CD22-deficient mice. Although cd22 −/− mice develop relatively normal numbers of peripheral B cells, cd22 −/−B cells have an abnormal surface phenotype: they have decreased expression of surface IgM (sIgM) and elevated expression of major histocompatibility complex class II molecules, indicative of B cells that have encountered antigen in vivo (22Otipoby 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, 23O'Keefe T.L. Williams G.T. Davies S.L. Neuberger M.S. Science. 1996; 274: 798-801Crossref PubMed Scopus (472) Google Scholar, 24Sato 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, 25Nitschke L. Carsetti R. Ocker B. Kohler G. Lamers M.C. Curr. Biol. 1997; 7: 133-143Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar). In agreement with a so-called “hyper-responsive” phenotype, engagement of the BCR on cd22 −/− B cells leads to greater increases in levels of intracellular free calcium ([Ca2+]i) compared with wild-type B cells (22Otipoby 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, 23O'Keefe T.L. Williams G.T. Davies S.L. Neuberger M.S. Science. 1996; 274: 798-801Crossref PubMed Scopus (472) Google Scholar, 24Sato 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, 25Nitschke L. Carsetti R. Ocker B. Kohler G. Lamers M.C. Curr. Biol. 1997; 7: 133-143Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar). Based on these data it has been proposed that CD22 functions primarily as a negative regulator of BCR signal transduction (18Doody 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 (486) Google Scholar, 26Tooze R.M. Doody G.M. Fearon D.T. Immunity. 1997; 7: 59-67Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). However, B cells from CD22-deficient mice proliferate less well compared with wild-type B cells when treated with the same reagent that leads to augmented release of [Ca2+]i (22Otipoby 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, 24Sato 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). Furthermore,cd22 −/− mice generate decreased antibody responses when immunized with a thymus-independent type II antigen (22Otipoby 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,25Nitschke L. Carsetti R. Ocker B. Kohler G. Lamers M.C. Curr. Biol. 1997; 7: 133-143Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar). These data suggest that CD22 can also function as a positive regulator of BCR-mediated signals. How CD22 might regulate B cell responses in both a positive and a negative manner has remained an enigma. We have looked at the role that single tyrosine residues play in the recruitment of signaling proteins and the role that CD22 plays in activating mitogen-activated protein kinase (MAPK) pathways. Here we present data suggesting that the cytoplasmic tail of CD22 contains two signaling domains that regulate signal transduction pathways initiated by the BCR. These findings may explain the paradoxical phenotype of CD22-deficient mice. cd22 +/− mice, which had been backcrossed to C57Bl/6 mice (Jackson Laboratories, Bar Harbor, ME) greater than seven generations, were crossed to generate cd22 +/+ and cd22 −/−mice (22Otipoby 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). Litter- and gender-matched, 7–12-week-old cd22 +/+ and cd22 −/−mice were used for all of the mouse studies. All mice were raised in the specific pathogen-free facility at the University of Washington. Mice were genotyped for the wild-type and mutant alleles of cd22 by subjecting tail DNA to three PCRs. Briefly, one forward primer that annealed in intron 2 (27Law C.L. Torres R.M. Sundberg H.A. Parkhouse R.M. Brannan C.I. Copeland N.G. Jenkins N.A. Clark E.A. J. Immunol. 1993; 151: 175-187PubMed Google Scholar) (5′-GCC ACG CTG GCC TCA GAC TCA TAG CAA TTC-3′) was used in three separate reactions with different reverse primers that annealed in intron 3 (5′-TCT CCA GAC CCA GCT ACC AGC CTG GGC CTC-3′), intron 4 (5′-GGT CCC TCT TTC GTG CCG CTA GAA CAG TTG-3′), or the neomycin cassette (5′-GGC CGG CTG GGT GTG GCG GAC CGC TAT CAG-3′). The PCRs generated a 207-base pair fragment from the wild-type allele with primers that annealed in intron 2/intron 3, a 342-base pair fragment from the mutant allele with primers that annealed in intron 2/neomycin, and 829- and 1309-base pair fragments from wild-type and mutant alleles, respectively, with primers that annealed in intron 2/intron 4. The EBV− Burkitt's lymphoma line BJAB was obtained from ATCC and cultured in RP10 (RPMI 1640 medium (Fisher Scientific) supplemented with 10% fetal calf serum (BioWhittaker, Walkersville, MD), 1 mm sodium pyruvate, and 1× nonessential amino acids (Irvine Scientific, Santa Ana, CA), 2 mml-glutamine (Gemini Bioproducts, Woodland, CA), and 100 units of penicillin-streptomycin (Life Technologies, Inc.)). Wild-type, Lyn−, Syk−, and Lyn-Syk− chicken DT40 cells (28Takata M. Sabe H. Hata A. Inazu T. Homma Y. Nukada T. Yamamura H. Kurosaki T. EMBO J. 1994; 13: 1341-1349Crossref PubMed Scopus (587) Google Scholar,29Qin S. Inazu T. Takata M. Kurosaki T. Homma Y. Yamamura H. Eur. J. Biochem. 1996; 236: 443-449Crossref PubMed Scopus (62) Google Scholar) were cultured in RP10 with 1% chicken serum (Life Technologies, Inc.) and 50 μm 2-mercaptoethanol. Purified mouse B cells were prepared as described previously (22Otipoby 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). mAbs to murine CD22 (2D6), human CD22 (HD39), c-Myc (9E10), and phosphotyrosine (4G10) and the myeloma protein mouse IgG1 (MOPC21) were prepared from mouse ascites (14Torres R.M. Law C.L. Santos-Argumedo L. Kirkham P.A. Grabstein K. Parkhouse R.M. Clark E.A. J. Immunol. 1992; 149: 2641-2649PubMed Google Scholar, 16Law 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). mAb to chicken IgM (M4) and control mouse IgM (FC-1) were purified over protein A-Sepharose columns from ammonium sulfate-precipitated culture supernatants (28Takata M. Sabe H. Hata A. Inazu T. Homma Y. Nukada T. Yamamura H. Kurosaki T. EMBO J. 1994; 13: 1341-1349Crossref PubMed Scopus (587) Google Scholar). In some experiments, mAbs were conjugated to biotin as described previously (30Hardy R.R. Weir D.M. Herzenberg L.A. Blackwell C. Herzenberg L.A. Handbook of Experimental Immunology. 1. Blackwell Scientific Publications, Oxford1986: 1-12Google Scholar). Rabbit antiserum to JNK1 (C-17) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Blotting antisera to phospho-ERK and phospho-JNK were purchased from Promega (Madison, WI), while the phospho-p38 MAPK antiserum was purchased from New England Biolabs (Beverly, MA). mAb to Grb2 was purchased from Pharmingen/Transduction Laboratories (San Diego, CA). Goat F(ab′)2 anti-mouse IgM serum, goat F(ab′)2anti-human IgM serum, goat F(ab′)2 IgG, and rat IgG were purchased from Jackson ImmunoResearch (West Grove, PA), and PP1 was purchased from BioMol Research Laboratories (Plymouth Meeting, PA). Six mutant constructs that contained single tyrosine to phenylalanine mutations were generated from pcDNAmCD22-HA (16Law 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), a full-length cDNA of murine cd22 in pcDNA-3 with a 3′EcoRI site, HA tag, stop codon, and XhoI site replacing the natural stop codon. Forward primers with the indicated mutation (Y773F, 5′-CAG GGA TGC TTC AAT CCG GCA-3′; Y783F, 5′-ACT GTT AGT TTT GCC ATC TTG-3′; Y817F, 5′-ACG GTC ACT TTC TCG GTG ATA-3′; Y828F, 5′-ATG GGG GAT TTT GAG AAT GTG-3′; Y843F, 5′-AGC ATC CAT TTC TCA GAG CTG-3′; and Y863F, 5′-GAC GTA GAC TTT GTG ACC CTC-3′) were used with the commercially available Sp6 reverse primer to generate six mutant fragments by PCR mutagenesis. After purifying the PCR products corresponding to the six mutated 3′-regions of cd22, another set of PCRs with pcDNAmCD22-HA as the template was set up using the mutated fragments, a forward primer that bound the 5′-end of pcDNAmCD22-HA (5′-GAT CGG ATC CGC GGC CTG GGA GAG AAA TAG CAG-3′), and the Sp6 reverse primer. Amplified full-length, HA-tagged wild-type or mutant murine cd22 cDNA products were then ligated into pcDNA3 after digesting both the PCR fragments and the vector with BamHI and XhoI. Since the HA tag encodes three tyrosine residues, it was removed and replaced with a c-myc tag and stop codon in each of the wild type and six mutants by setting up seven triple-ligation reactions, each containing the purified, untagged insert from BamHI- and EcoRI-digested wild-type or mutant HA-tagged cd22, EcoRI- and XhoI-digested c-myc tag/stop codon (generated by annealing 5′-TCG AGT TAC AAC AAG TCC TCT TCA GAA ATG AGC TTT TGC TCC TCT GCG-3′ and 5′-AAT TCG CAG AGG AGC AAA AGC TCA TTT CTG AAG AGG ACT TGT TGT AAC-3′), and BamHI- and XhoI-digested pcDNA3. Constructs were verified by restriction and sequence analysis. For cloning into the chicken expression vector pApuro-2 (28Takata M. Sabe H. Hata A. Inazu T. Homma Y. Nukada T. Yamamura H. Kurosaki T. EMBO J. 1994; 13: 1341-1349Crossref PubMed Scopus (587) Google Scholar), the full-length murine cd22 cDNA with SalI sites at 5′- and 3′-ends and an in-frame c-myc tag and stop codon just upstream of the 3′ SalI site was generated by PCR amplification from pmCD22–4 (14Torres R.M. Law C.L. Santos-Argumedo L. Kirkham P.A. Grabstein K. Parkhouse R.M. Clark E.A. J. Immunol. 1992; 149: 2641-2649PubMed Google Scholar) using appropriate primers (5′-GAT GAA TTC TCC CAC CCA GAC GAG ACA CCA TGC GCG TCC ATT ACC TGT GGC T-3′ and 5′-ACG CGT CGA CTC ACA GAT CCT CTT CTG AGA TGA GTT TTT GTT CCT CGA GGT GCT TGA GGG TCA CAT AGT-3′). After digesting the PCR fragment with SalI, it was cloned into SalI-digested pApuro-2. The pApuro-2-mCD22 construct was verified by restriction and sequence analysis. BJAB B cells in exponential phase were washed twice in electroporation medium (RPMI 1640 medium supplemented with 15% fetal calf serum, 1 mm sodium pyruvate, 1× nonessential amino acids, and 2 mml-glutamine) and resuspended at 3 × 107 ml−1. Next, 350 μl of the cell suspension was placed in a 0.4-cm cuvette with 50 μg of linearized plasmid DNA containing c-myc-tagged wild-type or mutant cd22 inserts in pcDNA-3 for 10 min on ice. Cells were then electroporated (Bio-Rad Gene Pulser with Capacitance Extender) at 230 V and 960 microfarads. After electroporation, cells were placed on ice for 10 min, resuspended in 20 ml of electroporation medium, and cultured at 37 °C. After 2 days, Geneticin (Life Technologies, Inc.) was added to final concentration of 3 mg ml−1. After ∼2 weeks, cells were cloned by limiting dilution. Clones were screened for similar expression levels of wild-type or mutant murine CD22 by surface staining with biotinylated anti-CD22 (2D6) and flow cytometry (FACScan, Becton Dickinson, Mountain View, CA). Stable transfectants were then maintained in 1 mg ml−1 Geneticin. Wild-type and mutant DT40 cells in exponential phase were washed once in RPMI 1640 medium. DT40 cells were electroporated with 10 μg of linearized plasmid DNA containing pApuro-2-mCD22 at 290 V and 960 microfarads and cultured as described above for transfection in BJAB cells. After 2 days, puromycin (Sigma) was added at 0.5 μg ml−1. Five days later, dead cells were removed by centrifugation over IsoPrep (Robbins Scientific, Sunnyvale, CA). Cells at the interface were washed twice and cultured at 37 °C. Cells were later stained for surface expression of murine CD22 with biotinylated CD22 mAb, sorted by a FACS (FACS Vantage Turbo S.E., Becton Dickinson) for high expression of CD22, and cloned by limiting dilution. Clones were screened and selected for similar expression levels of CD22. Cells in exponential phase were centrifuged and resuspended at 107 ml−1 in RP10. Cells were activated for the indicated number of minutes at 37 °C with 10 μg ml−1 of the indicated antiserum or mAb. Activation was stopped by the addition of at least 5 volumes of ice-cold phosphate-buffered saline. Cells were then washed once with phosphate-buffered saline and lysed at 30 × 106 ml−1 in Nonidet P-40 lysis buffer (50 mm Tris, pH 8.0, 5 mm EDTA, 150 mmNaCl, and 1.0% Nonidet P-40) with protease and phosphatase inhibitors (1 μg ml−1 aprotinin, 1 μg ml−1 pepstatin A, 1 μg ml−1 leupeptin, 2 mmphenylmethylsulfonyl fluoride, 10 mm NaF, 1 mmNa3VO4, 5 mmNa3PO4, and 100 μg ml−1 soybean trypsin inhibitor) for 1 h at 4 °C with constant rotation. After centrifugation at 14,000 rpm to remove nuclei, lysates were used for immunoprecipitations with the indicated mAb. Lysates with mAbs were incubated at 4 °C overnight with constant rotation. Samples were rotated for an additional 1 h at 4 °C with 50 μl of packed protein A- or protein G-Sepharose. Immunoprecipitates were washed three times with Nonidet P-40 lysis buffer, mixed with 100 μl of 1× reducing sample buffer (62.5 mm Tris, pH 6.8, 1.25% SDS, 12% glycerol, 0.05% bromphenol blue, and 5% 2-mercaptoethanol), boiled for 5 min, and frozen at −70 °C until used for SDS-PAGE. Immunoprecipitates were resolved on either a 12- or 20-cm 10% polyacrylamide gel and transferred to nylon membrane. Membranes were washed in TBST (50 mm Tris, pH 8.0, 150 mmNaCl, and 0.1% Tween 20), blocked in TBST-B (TBST with 5% bovine serum albumin) overnight, subjected to Western blotting with the indicated antiserum or mAb and the appropriate horseradish peroxidase-conjugated second antibody, and visualized by chemiluminescence (PerkinElmer Life Sciences). Densitometry was performed, and values were normalized against either Mst-1, for blots of whole cell lysates, or CD22, for blots of CD22 immunoprecipitations.In vitro kinase reactions were performed as described previously (31Jiang A. Craxton A. Kurosaki T. Clark E.A. J. Exp. Med. 1998; 188: 1297-1306Crossref PubMed Scopus (144) Google Scholar). The cytoplasmic tail of CD22 contains six conserved tyrosine residues and becomes rapidly tyrosine phosphorylated following BCR engagement (11Schulte R.J. Campbell M.A. Fischer W.H. Sefton B.M. Science. 1992; 258: 1001-1004Crossref PubMed Scopus (121) Google Scholar, 12Stamenkovic I. Seed B. Nature. 1990; 345: 74-77Crossref PubMed Scopus (165) Google Scholar, 13Wilson G.L. Fox C.H. Fauci A.S. Kehrl J.H. J. Exp. Med. 1991; 173: 137-146Crossref PubMed Scopus (127) Google Scholar, 14Torres R.M. Law C.L. Santos-Argumedo L. Kirkham P.A. Grabstein K. Parkhouse R.M. Clark E.A. J. Immunol. 1992; 149: 2641-2649PubMed Google Scholar). Three of these tyrosines, Tyr-783, Tyr-843, and Tyr-863 (numbering is based on the unprocessed murine CD22 protein), are located within ITIM motifs as defined by the consensus motif (V/L/I)XY XX(L/I/V) (where X is any amino acid) (32Thomas M.L. J. Exp. Med. 1995; 181: 1953-1956Crossref PubMed Scopus (114) Google Scholar). When the tyrosine residue within an ITIM motif is phosphorylated, the ITIM motif can bind molecules such as the SHP-1 protein tyrosine phosphatase and potentially play a role in activating SHP-1 (16Law 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, 18Doody 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 (486) Google Scholar). Using phosphopeptides equivalent to the six tyrosine-containing regions in CD22, it was previously reported that phosphopeptides corresponding to the three ITIM motifs of CD22 can compete with CD22 for the binding of SHP-1, whereas the analogous nonphosphorylated peptides do not (18Doody 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 (486) Google Scholar). These data suggested that Tyr-783, Tyr-843, and Tyr-863 were involved in the binding and activation of SHP-1 but did not clarify which of these tyrosines was required in vivo for recruitment of SHP-1 to CD22 following engagement of the BCR. To address this question, we used the sIgM-positive human B cell line BJAB to generate a stable transfectant expressing c-Myc-tagged wild-type murine CD22 and six stable transfectants expressing mutant forms of c-Myc-tagged murine CD22 with single tyrosine to phenylalanine mutations. All seven stable lines were selected for similar expression levels of the transfected wild-type or mutant murine CD22 molecules (Fig. 1 A). This system had several advantages relative to other transfection systems. Unlike non-B cell lines where CD22 has been studied, e.g. HeLa cells (33Blasioli J. Paust S. Thomas M.L. J. Biol. Chem. 1999; 274: 2303-2307Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), BJAB contains the same or very similar signal transduction machinery present in normal B cells. Also, the upstream and downstream components of the signal transduction machinery are present and at unmanipulated levels. Furthermore, BCR cross-linking, a relatively physiological stimulus, was used instead of pharmacological reagents to activate the B cells. Finally, the transfected BJAB lines express endogenous human CD22, which was used as an internal positive control for recruitment of SHP-1 and was easily distinguishable from the transfected wild-type or mutant murine CD22 by specific mAbs and antisera. Stimulation through the BCR on transfectants expressing wild-type murine CD22 led to efficient association of SHP-1 with both the transfected murine CD22 and endogenous human CD22 (Fig. 1 B). This was also observed for four of the six transfectants expressing different mutant forms of murine CD22. Transfectants with single tyrosine to phenylalanine mutations at position 773, 783, 817, or 828 (Y773F, Y783F, Y817F, or Y828F, respectively) efficiently recruited SHP-1 to both the mutant murine CD22 molecules and the endogenous human CD22 in response to BCR cross-linking (Fig. 1 B and data not shown). Thus, although a phosphopeptide of the “ITIM tyrosine” Tyr-783 blocked SHP-1 binding (18Doody 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 (486) Google Scholar), phosphorylation of Tyr-783 was not required for BCR-induced SHP-1 recruitment in vivo. However, after normalizing for the amount of immunoprecipitated CD22, we found that single tyrosine to phenylalanine mutations at position 843 (Y843F) or 863 (Y863F) decreased the amount of SHP-1 recruited to CD22 by 90 and 97%, respectively, compared with the amount bound by wild-type CD22 (Fig. 1 B). The endogenous human CD22 in these same cells maintained its ability to recruit SHP-1 (Fig.1 B). Thus, the upstream kinases required for phosphorylation of CD22 and recruitment of SHP-1 were not altered during

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