CD22 Forms a Quaternary Complex with SHIP, Grb2, and Shc
2000; Elsevier BV; Volume: 275; Issue: 23 Linguagem: Inglês
10.1074/jbc.m001892200
ISSN1083-351X
AutoresJonathan C. Poe, Manabu Fujimoto, Paul J. Jansen, Ann S. Miller, Thomas F. Tedder,
Tópico(s)Protein Tyrosine Phosphatases
ResumoCD22 is a cell surface molecule that regulates signal transduction in B lymphocytes. Tyrosine-phosphorylated CD22 recruits numerous cytoplasmic effector molecules including SHP-1, a potent phosphotyrosine phosphatase that down-regulates B cell antigen receptor (BCR)- and CD19-generated signals. Paradoxically, B cells from CD22-deficient mice generate augmented intracellular calcium responses following BCR ligation, yet proliferation is decreased. To understand further the mechanisms through which CD22 regulates BCR-dependent calcium flux and proliferation, interactions between CD22 and effector molecules involved in these processes were assessed. The adapter proteins Grb2 and Shc were found to interact with distinct and specific regions of the CD22 cytoplasmic domain. Src homology-2 domain-containing inositol polyphosphate-5′-phosphatase (SHIP) also bound phosphorylated CD22, but binding required an intact CD22 cytoplasmic domain. All three molecules were bound to CD22 when isolated from BCR-stimulated splenic B cells, indicating the formation of a CD22·Grb2·Shc·SHIP quaternary complex. Therefore, SHIP associating with CD22 may be important for SHIP recruitment to the cell surface where it negatively regulates calcium influx. Although augmented calcium responses in CD22-deficient mice should facilitate enhanced c-Jun N-terminal kinase (JNK) activation, BCR ligation did not induce JNK activation in CD22-deficient B cells. These data demonstrate that CD22 functions as a molecular "scaffold" that specifically coordinates the docking of multiple effector molecules, in addition to SHP-1, in a context necessary for BCR-dependent SHIP activity and JNK stimulation. CD22 is a cell surface molecule that regulates signal transduction in B lymphocytes. Tyrosine-phosphorylated CD22 recruits numerous cytoplasmic effector molecules including SHP-1, a potent phosphotyrosine phosphatase that down-regulates B cell antigen receptor (BCR)- and CD19-generated signals. Paradoxically, B cells from CD22-deficient mice generate augmented intracellular calcium responses following BCR ligation, yet proliferation is decreased. To understand further the mechanisms through which CD22 regulates BCR-dependent calcium flux and proliferation, interactions between CD22 and effector molecules involved in these processes were assessed. The adapter proteins Grb2 and Shc were found to interact with distinct and specific regions of the CD22 cytoplasmic domain. Src homology-2 domain-containing inositol polyphosphate-5′-phosphatase (SHIP) also bound phosphorylated CD22, but binding required an intact CD22 cytoplasmic domain. All three molecules were bound to CD22 when isolated from BCR-stimulated splenic B cells, indicating the formation of a CD22·Grb2·Shc·SHIP quaternary complex. Therefore, SHIP associating with CD22 may be important for SHIP recruitment to the cell surface where it negatively regulates calcium influx. Although augmented calcium responses in CD22-deficient mice should facilitate enhanced c-Jun N-terminal kinase (JNK) activation, BCR ligation did not induce JNK activation in CD22-deficient B cells. These data demonstrate that CD22 functions as a molecular "scaffold" that specifically coordinates the docking of multiple effector molecules, in addition to SHP-1, in a context necessary for BCR-dependent SHIP activity and JNK stimulation. B cell antigen receptor mitogen-activated protein kinase extracellular signal-regulated kinase c-Jun N-terminal kinase immunoreceptor tyrosine-based inhibition motif phospholipase C-γ2 Src homology 2 Src homology-2 domain-containing inositol polyphosphate-5′-phosphatase glutathioneS-transferase polyacrylamide gel electrophoresis Src homology 3 B cell antigen receptor (BCR)1-generated signals activate Syk, Btk, and Src family protein tyrosine kinases during B lymphocyte activation (1.Bolland S. Pearse R.N. Kurosaki T. Ravetch J.V. Immunity. 1998; 8: 509-516Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 2.DeFranco A.L. Curr. Opin. Immunol. 1997; 9: 296-308Crossref PubMed Scopus (281) Google Scholar, 3.Reth M. Wienands J. Annu. Rev. Immunol. 1997; 15: 453-479Crossref PubMed Scopus (366) Google Scholar, 4.Kurosaki T. Annu. Rev. Immunol. 1999; 17: 555-592Crossref PubMed Scopus (365) Google Scholar, 5.Tamir I. Cambier J.C. Oncogene. 1998; 17: 1353-1364Crossref PubMed Scopus (103) Google Scholar). Kinase activation in turn propagates multiple downstream signaling events including tyrosine phosphorylation of important transmembrane receptors, phosphoinositide generation, increases in calcium mobilization into the cytoplasm, and the activation of mitogen-activated protein kinases (MAPKs). Upon cell activation, MAPKs translocate to the nucleus and activate specific transcription factors (6.Kim K.-M. Alber P. Weiser P. Reth M. Eur. J. Immunol. 1993; 23: 911-916Crossref PubMed Scopus (114) Google Scholar, 7.Saxton T.M. van Oostveen I. Bowtell D. Aebersold R. Gold M.R. J. Immunol. 1994; 153: 623-636PubMed Google Scholar, 8.Su B. Karin M. Curr. Opin. Immunol. 1996; 8: 402-411Crossref PubMed Scopus (712) Google Scholar, 9.Tordai A. Franklin R.A. Patel H. Gardner A.M. Johnson G.L. Gelfand E.W. J. Biol. Chem. 1994; 269: 7538-7543Abstract Full Text PDF PubMed Google Scholar). Subfamilies of MAPKs include the extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38, which become fully activated as a result of dual phosphorylation on single threonine and tyrosine residues (reviewed in Ref. 10.Dolmetsch R.E. Lewis R.S. Goodnow C.C. Healy J.I. Nature. 1997; 386: 855-858Crossref PubMed Scopus (1542) Google Scholar). BCR-induced calcium mobilization is essential for JNK, but not ERK or p38, activation (10.Dolmetsch R.E. Lewis R.S. Goodnow C.C. Healy J.I. Nature. 1997; 386: 855-858Crossref PubMed Scopus (1542) Google Scholar, 11.Hashimoto A. Okada H. Jiang A. Kurosaki M. Greenberg S. Clark E.A. Kurosaki T. J. Exp. Med. 1998; 188: 1287-1295Crossref PubMed Scopus (183) Google Scholar, 12.Jiang A. Craxton A. Kurosaki T. Clark E.A. J. Exp. Med. 1998; 188: 1297-1306Crossref PubMed Scopus (144) Google Scholar). Also critical to each of these events in B cells are cell surface receptors such as CD22 that modify and provide a context for BCR signal transduction (13.Tedder T.F. Semin. Immunol. 1998; 10: 259-265Crossref PubMed Scopus (44) Google Scholar). CD22 is a transmembrane glycoprotein that is expressed on the surface of mature B cells (14.Tedder T.F. Tuscano J. Sato S. Kehrl J.H. Annu. Rev. Immunol. 1997; 15: 481-504Crossref PubMed Scopus (266) Google Scholar). The ∼140-amino acid cytoplasmic domain of CD22 contains six tyrosines, some of which are rapidly phosphorylated following BCR or CD22 ligation (15.Schulte R.J. Campbell M.A. Fischer W.H. Sefton B.M. Science. 1992; 258: 1001-1004Crossref PubMed Scopus (120) Google Scholar, 16.Wilson G.L. Fox C.H. Fauci A.S. Kehrl J.H. J. Exp. Med. 1991; 173: 137-146Crossref PubMed Scopus (126) Google Scholar). These tyrosines are localized within immunoreceptor tyrosine-based inhibition motifs (ITIM) and immunoreceptor tyrosine-based activation motif-like sequences (17.Leprince 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 (168) Google Scholar, 18.Peaker C.J.G. Neuberger M.S. Eur. J. Immunol. 1993; 23: 1358-1363Crossref PubMed Scopus (142) Google Scholar), suggesting negative and positive signaling functions. Consistent with a negative regulatory role for CD22, phosphorylated CD22 recruits SHP-1, a potent phosphotyrosine phosphatase that is proposed to limit BCR signaling (19.Doody G.M. Justement L.B. Delibrias C.C. Mathews R.J. Lin J. Thomas M.L. Fearon D.T. Science. 1995; 269: 242-244Crossref PubMed Scopus (480) Google Scholar, 20.Lankester A.C. van Schijndel G.M.W. van Lier R.A.W. J. Biol. Chem. 1995; 270: 20305-20308Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 21.Campbell M.A. Klinman N.R. Eur. J. Immunol. 1995; 25: 1573-1579Crossref PubMed Scopus (112) Google Scholar, 22.Law 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, 23.Blasioli J. Paust S. Thomas M.L. J. Biol. Chem. 1999; 274: 2303-2307Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). In addition, eliminating B cell expression of CD22 results in augmented calcium responses after BCR cross-linking (24.Sato S. Miller A.S. Inaoki M. Bock C.B. Jansen P.J. Tang M.L.K. Tedder T.F. Immunity. 1996; 5: 551-562Abstract Full Text PDF PubMed Scopus (386) Google Scholar, 25.Sato S. Tuscano J.M. Inaoki M. Tedder T.F. Semin. Immunol. 1998; 10: 287-297Crossref PubMed Scopus (90) Google Scholar, 26.Otipoby 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 (358) Google Scholar, 27.O'Keefe T.L. Williams G.T. Davies S.L. Neuberger M.S. Science. 1996; 274: 798-801Crossref PubMed Scopus (465) Google Scholar, 28.Nitschke L. Carsetti R. Ocker B. Kohler G. Lamers M.C. Curr. Biol. 1997; 7: 133-143Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). Consistent with a positive role for CD22, phosphorylated CD22 physically interacts with effector molecules including Lyn, Syk, phosphatidylinositol 3-kinase, phospholipase C-γ2 (PLC-γ2), and Grb2 (17.Leprince 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 (168) Google Scholar, 18.Peaker C.J.G. Neuberger M.S. Eur. J. Immunol. 1993; 23: 1358-1363Crossref PubMed Scopus (142) Google Scholar, 22.Law 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, 23.Blasioli J. Paust S. Thomas M.L. J. Biol. Chem. 1999; 274: 2303-2307Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 29.Yohannan 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, 30.Tuscano J. Engel P. Tedder T.F. Kehrl J.H. Blood. 1996; 87: 4723-4730Crossref PubMed Google Scholar, 31.Tuscano J.M. Engel P. Tedder T.F. Agarwal A. Kehrl J.H. Eur. J. Immunol. 1996; 26: 1246-1252Crossref PubMed Scopus (73) Google Scholar). In addition, proliferative responses and overall protein tyrosine phosphorylation are reduced in B cells from CD22-deficient mice after BCR cross-linking (24.Sato S. Miller A.S. Inaoki M. Bock C.B. Jansen P.J. Tang M.L.K. Tedder T.F. Immunity. 1996; 5: 551-562Abstract Full Text PDF PubMed Scopus (386) Google Scholar, 25.Sato S. Tuscano J.M. Inaoki M. Tedder T.F. Semin. Immunol. 1998; 10: 287-297Crossref PubMed Scopus (90) Google Scholar, 26.Otipoby 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 (358) Google Scholar, 27.O'Keefe T.L. Williams G.T. Davies S.L. Neuberger M.S. Science. 1996; 274: 798-801Crossref PubMed Scopus (465) Google Scholar, 28.Nitschke L. Carsetti R. Ocker B. Kohler G. Lamers M.C. Curr. Biol. 1997; 7: 133-143Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). Tyrosine phosphorylation of Src homology-2 (SH2) domain-containing inositol polyphosphate-5′-phosphatase (SHIP), PLC-γ2, CD79a, and CD79b is decreased in CD22-deficient B cells following BCR cross-linking, whereas Syk, Lyn, Fyn, Blk, and SHP-1 are phosphorylated at wild type levels (32.Sato S. Jansen P.J. Tedder T.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13158-13162Crossref PubMed Scopus (99) Google Scholar). In addition, CD22 interacts with Grb2 following BCR stimulation of the K46 B lymphoma cell line (29.Yohannan 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). Thus, interactions between CD22 and multiple effector molecules may contribute to both positive and negative signaling pathways. Critical interactions between CD22 and other effector molecules may provide a molecular explanation for augmented calcium responses yet decreased proliferation by CD22-deficient B cells in response to BCR ligation. Although decreased recruitment of SHP-1 to the plasma membrane due to the absence of CD22 could explain augmented calcium responses, evidence suggests that other molecules are also involved. SHP-1 and SHIP differentially down-regulate calcium responses by preventing release from intracellular stores and from the influx of extracellular free calcium, respectively (33.Ono M. Okada H. Bolland S. Yanagi S. Kurosaki T. Ravetch J.V. Cell. 1997; 90: 293-301Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). Like the killer cell inhibitory receptor class of molecules, which block the release of calcium from intracellular stores (34.Blery M. Delon J. Trautmann A. Cambiaggi A. Olcese L. Biassoni R. Moretta L. Chavrier A. Moretta A. Daeron M. Vivier E. J. Biol. Chem. 1997; 272: 8989-8996Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), it has been suggested that CD22 inhibits calcium release solely through SHP-1 activity (33.Ono M. Okada H. Bolland S. Yanagi S. Kurosaki T. Ravetch J.V. Cell. 1997; 90: 293-301Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). However, CD22 modulates the mobilization of both intracellular and extracellular calcium (27.O'Keefe T.L. Williams G.T. Davies S.L. Neuberger M.S. Science. 1996; 274: 798-801Crossref PubMed Scopus (465) Google Scholar), suggesting that CD22 up-regulates both SHP-1 and SHIP activity. A SHIP·Grb2·Shc ternary complex forms following BCR ligation in primary B cells, which may be required for recruitment of SHIP to the plasma membrane where it down-regulates calcium mobilization (35.Harmer S.L. DeFranco A.L. J. Biol. Chem. 1998; 274: 12183-12191Abstract Full Text Full Text PDF Scopus (49) Google Scholar). To evaluate further how CD22 regulates calcium mobilization, we have assessed the ability of SHIP to associate with CD22 in the current study. Since calcium mobilization is dramatically elevated in CD22-deficient B cells and calcium mobilization is essential for JNK activation (10.Dolmetsch R.E. Lewis R.S. Goodnow C.C. Healy J.I. Nature. 1997; 386: 855-858Crossref PubMed Scopus (1542) Google Scholar, 11.Hashimoto A. Okada H. Jiang A. Kurosaki M. Greenberg S. Clark E.A. Kurosaki T. J. Exp. Med. 1998; 188: 1287-1295Crossref PubMed Scopus (183) Google Scholar, 12.Jiang A. Craxton A. Kurosaki T. Clark E.A. J. Exp. Med. 1998; 188: 1297-1306Crossref PubMed Scopus (144) Google Scholar), we have also assessed whether CD22 loss enhances JNK activation following BCR ligation. These studies reveal that phosphorylated CD22 specifically associates with SHIP, Grb2, and Shc to form a quaternary complex. Intriguingly, despite augmented calcium mobilization in CD22-deficient B cells, JNK activation was selectively impaired, which may explain their decreased proliferative responses. Thus, adapter protein-mediated interactions between CD22 and other effector molecules are likely to contribute to the regulatory activities of CD22. CD22-deficient (B6 × 129) and wild type control mice (B6 × 129) were generated by breeding mice heterozygous for the CD22 deficiency as described (24.Sato S. Miller A.S. Inaoki M. Bock C.B. Jansen P.J. Tang M.L.K. Tedder T.F. Immunity. 1996; 5: 551-562Abstract Full Text PDF PubMed Scopus (386) Google Scholar, 36.Engel P. Zhou L.-J. Ord D.C. Sato S. Koller B. Tedder T.F. Immunity. 1995; 3: 39-50Abstract Full Text PDF PubMed Scopus (482) Google Scholar). All mice were 2 months of age when used and were housed in a specific pathogen-free barrier facility. All studies and procedures were approved by the Animal Care and Use Committee of Duke University. A cDNA that encoded a GST-CD22 fusion protein (GST-CD22cyt) containing the complete mouse CD22 cytoplasmic domain starting at amino acid 708 (14.Tedder T.F. Tuscano J. Sato S. Kehrl J.H. Annu. Rev. Immunol. 1997; 15: 481-504Crossref PubMed Scopus (266) Google Scholar) in plasmid pGEX-3X was expressed in TKX1 tyrosine kinase-competent Escherichia coli (Stratagene, La Jolla, CA). Fusion protein expression (GST-CD22cyt) was induced by culturing the bacteria in LB medium containing 0.1 mmisopropyl-1-thio-β-d-galactopyranoside, 50 μg/ml ampicillin, and 12.5 μg/ml tetracycline for 3 h at 37 °C before fusion protein purification. Tyrosine phosphorylation of GST-CD22cyt (GST-CD22cyt(Tyr(P))) was induced by culturing the bacteria with 10 μg/ml indoleacrylic acid in Trp starvation medium (M9 minimal salts medium containing 1 mm MgSO4, 0.2% glucose, 0.1% casamino acids, 1.5 μm thiamine HCl, 50 μg/ml ampicillin, and 12.5 μg/ml tetracycline) for 2 h at 37 °C. The cells were pelleted by centrifugation and resuspended in ice-cold 1% Triton X-100 lysis buffer containing 50 mmHEPES (pH 7.5), 150 mm NaCl, 10% glycerol, 1 mm EDTA, 1 mm EGTA, 1.5 mmMgCl2, 0.5 mm sodium orthovanadate, 10 mm NaF, 0.5 mm phenylmethylsulfonyl fluoride, 10 mm pyrophosphate, and 10 μg/ml each of leupeptin, aprotinin, and antipain. The cells were disrupted five times by sonication for 20-s bursts with the cells placed on ice for 1.5 min between sonication cycles. The fusion proteins were purified by column chromatography with glutathione-Sepharose beads (Amersham Pharmacia Biotech) and eluted with buffer containing 50 mm Tris-HCl (pH 8.0), 5 mm glutathione, and 0.5 mm each of sodium orthovanadate and phenylmethylsulfonyl fluoride. Biotin-conjugated peptides corresponding to regions of the mouse CD22 cytoplasmic domain were synthesized by the University of North Carolina Peptide Synthesis Facility (Chapel Hill, NC) in both tyrosine-phosphorylated and non-phosphorylated forms. Hybidomas producing CD22-specific monoclonal antibodies were generated by the fusion of NS-1 myeloma cells with spleen cells from a CD22-deficient mouse that was immunized three times with the GST-CD22cyt fusion protein in adjuvant. Supernatant fluid from four hybridomas reacted with GST-CD22cyt in enzyme-linked immunosorbent assays but not GST alone. Each anti-CD22 antibody-producing hybridoma was subcloned twice by limiting dilution and used to generate tissue culture supernatant fluid for these studies. Antibody isotypes were determined using a mouse monoclonal antibody isotyping kit (Amersham Pharmacia Biotech). Horseradish peroxidase-conjugated anti-phosphotyrosine (4G10), anti-SHP1, anti-Grb2, and anti-Shc antibodies were from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-SHIP, anti-Lyn, anti-SOS, anti-BLNK, anti-ERK2, anti-JNK2, and anti-glutathioneS-transferase (GST) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated goat anti-rabbit IgG and anti-mouse IgG antibodies were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Phospho-specific antibodies reactive with the fully activated forms of MAPK subfamilies were from Promega (Madison, WI). Antisera used also included F(ab′)2 fragments of goat anti-mouse IgM antibodies (Cappel, Durham, NC). B cells were purified from single cell splenocyte suspensions by removing T cells with anti-Thy1.2 antibody-coated magnetic beads (Dynal Inc., Lake Success, NY). B cell suspensions were analyzed by immunofluorescence staining with flow cytometric analysis following isolation and were always >94% B220+. The B cells were resuspended (2 × 107/ml) in RPMI 1640 medium containing 5% fetal calf serum. Following incubation for 5 min at 37 °C, B cells were cultured with media alone or containing F(ab′)2 fragments of goat anti-mouse IgM antibodies (40 μg/ml) at 37 °C and then lysed in buffer containing 1% Nonidet P-40, 150 mm NaCl, 50 mm Tris-HCl (pH 8.0), 1 mm sodium orthovanadate, 2 mm EDTA, 50 mm NaF, and protease inhibitors as described (37.Bradbury L.E. Kansas G.S. Levy S. Evans R.L. Tedder T.F. J. Immunol. 1992; 149: 2841-2850PubMed Google Scholar). For the detection of MAPK subfamily kinase activation, B cell lysates were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) followed by electrophoretic transfer to nitrocellulose with indirect immunoblotting using antibodies against active forms of MAPK subfamily members. For immunoprecipitation studies using antibodies reactive with proteins of interest, B cell lysates were pre-cleared using protein G-Sepharose beads (50 μl; Amersham Pharmacia Biotech) and then incubated with 5 μg/ml antibody for 2 h at 4 °C, followed by the addition of protein G-Sepharose beads (50 μl) for an additional 2 h. The beads were washed five times with 1 ml of lysis buffer and resuspended in 100 μl of SDS-PAGE sample buffer followed by boiling for 5 min. For coprecipitation studies using CD22 fusion proteins, B cell lysates were pre-cleared using glutathione-Sepharose beads (50 μl; Amersham Pharmacia Biotech) and then incubated with 25 μg/ml GST-CD22cyt or GST-CD22cyt(Tyr(P)) for 2 h at 4 °C, followed by the addition of glutathione-Sepharose beads (50 μl) for an additional 2 h. The beads were washed five times with 1 ml of lysis buffer and resuspended in 100 μl of SDS-PAGE sample buffer followed by boiling for 5 min. For coprecipitation studies using CD22 peptides, B cell lysates were pre-cleared with avidin-agarose beads (50 μl; Pierce) and then incubated with each peptide (20 μm) for 2 h at 4 °C, followed by the addition of avidin-agarose beads (50 μl) for an additional 2 h. The beads were washed five times with 1 ml of lysis buffer and resuspended in 100 μl of SDS-PAGE sample buffer followed by boiling for 5 min. Precipitated proteins were subjected to SDS-PAGE with subsequent electrophoretic transfer to nitrocellulose membranes. After blocking the membranes with buffer containing 2% (w/v) bovine serum albumin, protein presence was detected either by direct immunoblotting with horseradish peroxidase-conjugated anti-phosphotyrosine antibody or by indirect immunoblotting using antibodies reactive with the proteins of interest. Incubation periods for primary and secondary antibodies were 1 h at 25 °C with extensive washing of the membranes following each step. Immunoblots were developed using enhanced chemiluminescence kits from Pierce. To verify equivalent amounts of protein in each lane, the same blots were stripped and reprobed with antibodies against proteins of interest. Relative band intensities of MAPK immunoblots were determined using NIH Image software (version 1.60). Lyn phosphorylation of CD22 peptides was assessed using in vitro kinase reactions. Each reaction mixture (30 μl) contained CD22 non-phosphorylated peptides or control (phosphorylated) peptides at the indicated concentrations, Src kinase reaction buffer (10 μl), a 1:10 dilution of Src manganese/ATP mixture, 2 units of purified Lyn kinase (all from Upstate Biotechnology, Inc.), and 10 μCi of [γ-32P]ATP (ICN Biomedicals, Inc., Costa Mesa, CA). The reactions were carried out for 20 min at 25 °C before termination using 30 μl of 4.75m guanidine HCl. 20 μl of each reaction mixture was then spotted onto SAMTM Biotin Capture Membranes (Promega). The membranes were washed three times with 2 m NaCl, once with 1 m NaCl + 0.75% phosphoric acid, and once with 2m NaCl containing 1% Tween 20. Radioactivity was quantified by scintillation counting. To evaluate effector molecule interactions with the CD22 cytoplasmic domain, a fusion protein consisting of GST and the entire 140-amino acid cytoplasmic domain of mouse CD22 was generated (Fig.1). The fusion protein was produced in bacteria in non-phosphorylated (GST-CD22cyt) and tyrosine-phosphorylated (GST-CD22cyt(Tyr(P))) forms as described under "Experimental Procedures." Migration of the GST-CD22cyt(Tyr(P)) protein in SDS-PAGE gels was significantly slower than for GST-CD22cyt protein due to tyrosine phosphorylation (Fig.2 C). In addition, peptides containing either single tyrosine motif or tandem tyrosine motif sequences corresponding to specific regions of the CD22 cytoplasmic domain were used in these studies in non-phosphorylated and tyrosine-phosphorylated forms (Fig. 1).Figure 2Characterization of the CD22-specific MB22-1 monoclonal antibody. A, purified splenic B cells (1 × 107 cells/sample) from wild type or CD22-deficient mice were incubated in media alone or with anti-IgM antibodies before detergent lysis. Whole cell lysates (1 × 106 cell equivalents) were subjected to SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and immunoblotted with a 1:50 dilution of MB22-1 hybridoma culture supernatant fluid. Antibody reactivity was revealed using a horseradish peroxidase-conjugated secondary antibody with detection by enhanced chemiluminescence. B, splenic B cell lysates were prepared as in A with immunoprecipitation (IP) using a 1:10 dilution of MB22-1 hybridoma supernatant fluid followed by capture with protein G-Sepharose beads. Precipitated proteins were subjected to SDS-PAGE, transferred to nitrocellulose, and subsequently immunoblotted with an anti-phosphotyrosine monoclonal antibody. C,GST-CD22 cytoplasmic domain fusion proteins or a control GST-CD19 cytoplasmic domain fusion protein were subjected to SDS-PAGE (5 μg per lane) and transferred to nitrocellulose membranes. The membrane was first immunoblotted using MB22-1 hybridoma supernatant fluid. The membrane was subsequently stripped and reprobed with anti-GST antibody as indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Since the two currently reported anti-mouse CD22 monoclonal antibodies react with the extracellular domain of mouse CD22 and do not react with CD22 in Western blots, new antibodies were generated that react with CD22 under reducing conditions. Monoclonal antibody secreting hybridomas were generated by immunizing CD22-deficient mice with the GST-CD22cyt fusion protein. Of 546 hybridomas screened, four secreted IgG1 antibodies that reacted with the GST-CD22cyt fusion protein in enzyme-linked immunosorbent assays but not GST alone (31 antibodies): MB22-1, MB22-2, MB22-3, and MB22-4. One of the four antibodies, MB22-1 reacted predominantly with a band of M r 145,000 and to a lesser extent with a band of M r 130,000 in immunoblots of mouse splenocytes from wild type but not CD22-deficient mice (Fig. 2 A). These bands appear to represent CD22 with differences in post-translational processing, and the M r 145,000 protein was further resolved into two similar-sized bands in some experiments. The MB22-1 antibody effectively immunoprecipitated the M r 145,000 isoform of CD22 from B cell lysates as detected by immunoblotting with an anti-phosphotyrosine antibody (Fig. 2 B). The MB22-1 antibody did not immunoprecipitate phosphoproteins from CD22-deficient B cells (Fig. 2 B). Also, MB22-1 did not react with CD22 in lysates of human B cell lines (data not shown). The MB22-1 antibody reacted equally with the GST-CD22cyt and GST-CD22cyt(Tyr(P)) fusion proteins bound to nitrocellulose but did not react with a CD19-GST fusion protein (Fig. 2 C). Since CD22 interacts with Grb2 following BCR stimulation in K46 cells (29.Yohannan 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), and primary B cells generate a SHIP·Grb2·Shc ternary complex following BCR ligation (35.Harmer S.L. DeFranco A.L. J. Biol. Chem. 1998; 274: 12183-12191Abstract Full Text Full Text PDF Scopus (49) Google Scholar), we assessed whether CD22 could serve as a membrane-proximal docking site for the SHIP·Grb2·Shc complex. The ability of the SHIP·Grb2·Shc complex to associate with CD22 in primary cells was evaluated by immunoprecipitation of SHIP, Grb2, and Shc from cell lysates of resting and anti-IgM activated splenic B cells, followed by detection of CD22 in the complexes by immunoblotting. SHIP, Grb2, and Shc each coprecipitated CD22 at low levels from resting B cells but at significantly higher levels following BCR stimulation for 5 min (Fig.3, A–C). Equivalent levels of SHIP and Grb2 were precipitated from resting and activated B cells as revealed by stripping the immunoblots and reprobing with anti-SHIP or anti-Grb2 antibodies (Fig. 3, A and B). The anti-Shc antibody used in these studies was inadequate for reprobing Western blots, so the cell lysates were reprecipitated with an anti-Vav antibody and immunoblotted with the anti-Vav antibody to verify equal protein concentrations in the cell lysates (Fig. 3 C). The GTP exchange factor SOS, an activator of the ERK pathway, has also been reported to interact with CD22 following BCR stimulation in K46 cells (29.Yohannan 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). However, SOS did not coprecipitate CD22 at detectable levels in primary B cells, so the anti-SOS antibody was used as a negative control in these experiments (Fig. 3). Although it is possible that SOS may interact with CD22 in primary splenic B cells at later time points of activation, these results suggest that a fundamental difference in signaling events exists between primary splenic B cells and the K46 cell line. The ability of CD22 to coprecipitate SHIP, Grb2, and Shc from lysates of resting and anti-IgM activated splenic B cells was also assessed by immunoblotting. CD22 immunoprecipitated from activated B cell lysates coprecipitated SHIP, but SHIP was not coprecipitated from lysates of unstimulated B cells (data not shown). Shc and Grb2 interactions with CD22 could not be effectively evaluated i
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