Title: CD22 Is a Functional Ligand for SH2 Domain-containing Protein-tyrosine Phosphatase-1 in Primary T Cells
Abstract: The intracellular Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase (SHP-1) has been characterized as a negative regulator of T cell function, contributing to the definition of T cell receptor signaling thresholds in developing and peripheral mouse T lymphocytes. The activation of SHP-1 is achieved through the engagement of its tandem SH2 domains by tyrosine-phosphorylated proteins; however, the identity of the activating ligand(s) for SHP-1, within mouse primary T cells, is presently unresolved. The identification of SHP-1 ligand(s) in primary T cells would provide crucial insight into the molecular mechanisms by which SHP-1 contributes to in vivo thresholds for T cell activation. Here we present a combination of biochemical and yeast genetic analyses indicating CD22 to be a T cell ligand for the SHP-1 SH2 domains. Based on these observations we have confirmed that CD22 is indeed expressed on mouse primary T cells and capable of associating with SHP-1. Significantly, CD22-deficient T cells demonstrate enhanced proliferation in response to anti-CD3 or allogeneic stimulation. Furthermore, the co-engagement of CD3 and CD22 results in a raising of TCR signaling thresholds hence demonstrating a previously unsuspected functional role for CD22 in primary T cells. The intracellular Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase (SHP-1) has been characterized as a negative regulator of T cell function, contributing to the definition of T cell receptor signaling thresholds in developing and peripheral mouse T lymphocytes. The activation of SHP-1 is achieved through the engagement of its tandem SH2 domains by tyrosine-phosphorylated proteins; however, the identity of the activating ligand(s) for SHP-1, within mouse primary T cells, is presently unresolved. The identification of SHP-1 ligand(s) in primary T cells would provide crucial insight into the molecular mechanisms by which SHP-1 contributes to in vivo thresholds for T cell activation. Here we present a combination of biochemical and yeast genetic analyses indicating CD22 to be a T cell ligand for the SHP-1 SH2 domains. Based on these observations we have confirmed that CD22 is indeed expressed on mouse primary T cells and capable of associating with SHP-1. Significantly, CD22-deficient T cells demonstrate enhanced proliferation in response to anti-CD3 or allogeneic stimulation. Furthermore, the co-engagement of CD3 and CD22 results in a raising of TCR signaling thresholds hence demonstrating a previously unsuspected functional role for CD22 in primary T cells. SHP-1, 1The abbreviations used are: SHP-1, SH2 domain-containing protein-tyrosine phosphatase-1; SH2, Src homology 2; ITIM, immunoreceptor tyrosine-based inhibitory motif; PY, phosphotyrosine; PV, pervanadate; GST, glutathione S-transferase; Endo F, endo-β-N-acetylglucosaminidase F.1The abbreviations used are: SHP-1, SH2 domain-containing protein-tyrosine phosphatase-1; SH2, Src homology 2; ITIM, immunoreceptor tyrosine-based inhibitory motif; PY, phosphotyrosine; PV, pervanadate; GST, glutathione S-transferase; Endo F, endo-β-N-acetylglucosaminidase F. an intracellular protein-tyrosine phosphatase, has been demonstrated to be a negative regulator of TCR signaling thresholds (1Zhang J. Somani A.K. Siminovitch K.A. Semin. Immunol. 2000; 12: 361-378Crossref PubMed Scopus (284) Google Scholar). SHP-1 is normally maintained in a catalytically inactive state whereby activation minimally requires the engagement of the amino-terminal SH2 domain of SHP-1 by phosphotyrosine (PY)-containing ligand (2Pei D. Wang J. Walsh C.T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1141-1145Crossref PubMed Scopus (127) Google Scholar, 3Yang 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 (130) Google Scholar). It is predicted that SHP-1-activating ligand(s) exists on mouse naïve T cells based on substantial functional evidence indicating SHP-1 to be catalytically active in naïve T cells (1Zhang J. Somani A.K. Siminovitch K.A. Semin. Immunol. 2000; 12: 361-378Crossref PubMed Scopus (284) Google Scholar, 4Lorenz U. Ravichandran K.S. Burakoff S.J. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9624-9629Crossref PubMed Scopus (157) Google Scholar, 5Pani G. Fischer K.D. Mlinaric-Rascan I. Siminovitch K.A. J. Exp. Med. 1996; 184: 839-852Crossref PubMed Scopus (179) Google Scholar, 6Plas D.R. Williams C.B. Kersh G.J. White L.S. White J.M. Paust S. Ulyanova T. Allen P.M. Thomas M.L. J. Immunol. 1999; 162: 5680-5684PubMed Google Scholar, 7Carter J.D. Neel B.G. Lorenz U. Int. Immunol. 1999; 11: 1999-2014Crossref PubMed Scopus (71) Google Scholar, 8Johnson K.G. LeRoy F.G. Borysiewicz L.K. Matthews R.J. J. Immunol. 1999; 162: 3802-3813PubMed Google Scholar). It is currently assumed that SHP-1 is activated by one or more components of the TCR signaling pathway. Indeed, the intracellular protein-tyrosine kinase, ZAP-70, has been proposed to bind SHP-1 (9Plas D.R. Johnson R. Pingel J.T. Matthews R.J. Dalton M. Roy G. Chan A.C. Thomas M.L. Science. 1996; 272: 1173-1176Crossref PubMed Scopus (328) Google Scholar). However, the best evidence of SHP-1-associating molecules in other hemopoietic cells relates to the family of immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptors (10Blery M. Vivier E. Clin. Chem. Lab. Med. 1999; 37: 187-191Crossref PubMed Scopus (5) Google Scholar). In particular, ITIM receptors Ly49 and CD66a associate with SHP-1 in subpopulations of primary T cells, but to date there has been no definition of the ITIM receptors that activate SHP-1 in the majority of mouse primary T cells (11Long E.O. Annu. Rev. Immunol. 1999; 17: 875-904Crossref PubMed Scopus (830) Google Scholar). In the first instance, we have exploited SHP-1-deficient moth-eaten T cells to assist in the definition of genuine associations between SHP-1 and TCR signaling components in CD3/TCR-stimulated mouse primary T cells. Our results reveal no binding of the CD3 invariant chains or ZAP-70 to SHP-1 in mouse primary T cells following TCR/CD3 ligation. However, by employing pervanadate (PV) to induce a robust tyrosine phosphorylation of cellular proteins in primary T cells, we demonstrated that a glycosylated tyrosyl phosphoprotein of 150 kDa, (pp150) associates with SHP-1 in mouse peripheral T cells. We have identified pp150 as CD22 and confirmed CD22 to be expressed in primary T cells. The expression of CD22 has until now been thought to be restricted to the B cell lineage. However, consistent with CD22 expression in T cells, the co-engagement of CD22 and CD3 results in a raising of TCR signaling thresholds. Remarkably, the absence of CD22 in T cells results in increased responses to CD3 or allogeneic stimulation. These combined findings highlight a previously unrecognized inhibitory role for CD22 in T cells. Cells—C57BL/6J mice heterozygous at the moth-eaten locus were originally obtained from Dr. Leonard Shultz at The Jackson Laboratory (Bar Harbor, ME) and bred under pathogen-free conditions as a source of me/me mutants or me/+ controls. me/me mutants or littermate me/+ controls bearing the F5 TCR were as described in Ref. 8Johnson K.G. LeRoy F.G. Borysiewicz L.K. Matthews R.J. J. Immunol. 1999; 162: 3802-3813PubMed Google Scholar. me/me mutants or littermate me/+ controls were sacrificed between 9–13 days postpartum. CD22 deficient mice (CD22–/–) backcrossed for 10 generations onto the C57/BL6J genetic background were kindly provided by Professor Michael Neuberger, MRC Laboratory of Molecular Biology, Cambridge, UK. Lymph nodes from 8–12-week old CD22–/– and age-matched control mice were harvested as a source of T cells for proliferation assays. Spleens from 8–12-week old BALB/c mice provided a source of allogeneic feeder cells. For all other experiments, thymi and spleens from 4–12-week old mice kept under pathogen-free conditions were harvested, and cells were isolated as described in Ref. 8Johnson K.G. LeRoy F.G. Borysiewicz L.K. Matthews R.J. J. Immunol. 1999; 162: 3802-3813PubMed Google Scholar. All animal experimentation was in accordance with the UK Animal (Scientific Procedures) Act 1986 under Project Licenses PPL 40/2046 and 30/2125. Dr. Robert L. Geahlen in the Department of Medical Chemistry and Pharmacology at Purdue University kindly provided the mouse T cell lymphoma line, LSTRA, and Professor Elisabeth Simpson at Imperial College generously provided the Abelson virus-transformed B cells (12Scott D. McLaren A. Dyson J. Simpson E. Immunogenetics. 1991; 33: 54-61Crossref PubMed Scopus (24) Google Scholar). Generation of T Lymphoblasts—T lymphoblasts were generated as described in (13Sathish J.G. Johnson K.G. Fuller K.J. LeRoy F.G. Meyaard L. Sims M.J. Matthews R.J. J. Immunol. 2001; 166: 1763-1770Crossref PubMed Scopus (52) Google Scholar). Briefly, mouse splenocytes and thymocytes were stimulated by culturing with 2 μg/ml of concanavalin A for 72 h at 37 °C in complete RPMI 1640 medium supplemented with 360 IU/ml of rIL-2 (Chiron, Harefield, UK). The cells were washed thoroughly and cultured for a further 48 h in complete RPMI 1640 medium supplemented with IL-2. Fluorescence-activated cell sorter analysis performed on the cultured T lymphoblasts revealed no B cell contamination. B Cell Purification—B cells were purified from the spleens of C57Bl6/J and BALB/c mice by positive selection with CD45R Miltenyi microbeads according to the manufacturer's instructions (Miltenyi Biotec, Bisley, Surrey, UK). Purified B cells were lysed in Nonidet P-40 lysis buffer and normalized for protein concentration. Equal volumes of lysate were mixed with 6× Laemmli buffer and electrophoresed on a 10% acrylamide SDS-polyacrylamide gel. T Cell Stimulation and Lysis—A total of 5–10 × 107 T cells were stimulated for 2 min at 37 °C with 10 μg/ml of hamster anti-CD3ϵ mAb, 2C-11, and rabbit anti-hamster polyclonal serum (Sigma). 2C-11 was kindly provided by Dr. Doreen Cantrell, University of Dundee, Dundee, UK. YO1 cells (14Williams O. Tanaka Y. Bix M. Murdjeva M. Littman D.R. Kioussis D. Eur. J. Immunol. 1996; 26: 532-538Crossref PubMed Scopus (33) Google Scholar) previously pulsed with either 10 μm agonist (NP-68) or control (GAG) peptide were used as described in Ref. 8Johnson K.G. LeRoy F.G. Borysiewicz L.K. Matthews R.J. J. Immunol. 1999; 162: 3802-3813PubMed Google Scholar. Alternatively, cells were stimulated with 200 μm pervanadate for 10 min at 37 °C. Following stimulation, the cells were lysed as described in Ref. 13Sathish J.G. Johnson K.G. Fuller K.J. LeRoy F.G. Meyaard L. Sims M.J. Matthews R.J. J. Immunol. 2001; 166: 1763-1770Crossref PubMed Scopus (52) Google Scholar. Membrane fractions were prepared as described in Ref. 13Sathish J.G. Johnson K.G. Fuller K.J. LeRoy F.G. Meyaard L. Sims M.J. Matthews R.J. J. Immunol. 2001; 166: 1763-1770Crossref PubMed Scopus (52) Google Scholar. Immunoprecipitation, Deglycosylation, and Immunoblotting—SHP-1 was immunoprecipitated using a rabbit polyclonal anti-SHP-1 antibody as described in Ref. 15Matthews R.J. Bowne D.B. Flores E. Thomas M.L. Mol. Cell. Biol. 1992; 12: 2396-2405Crossref PubMed Scopus (325) Google Scholar or the C-19 antibody (Santa Cruz Biotechnology). CD22 was immunoprecipitated using polyclonal anti-CD22 antisera kindly provided by Dr. Paul Crocker, University of Dundee, Dundee, UK or with a purified rabbit antibody described in Ref. 16Jin L. McLean P.A. Neel B.G. Wortis H.H. J. Exp. Med. 2002; 195: 1199-1205Crossref PubMed Scopus (94) Google Scholar. Goat anti-serum to CD3ϵ was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Deglycosylation of SHP-1 immunoprecipitates was performed as described in Ref. 13Sathish J.G. Johnson K.G. Fuller K.J. LeRoy F.G. Meyaard L. Sims M.J. Matthews R.J. J. Immunol. 2001; 166: 1763-1770Crossref PubMed Scopus (52) Google Scholar. The immunoprecipitations were resolved by 12% SDS-PAGE, and membranes were probed with anti-PY antibody, 4G10 (Upstate Biotechnology, Lake Placid, NY), and detected by ECL (Amersham Biosciences). The mAb used for immunoblotting ZAP-70 was purchased from BD Transduction Laboratories (Franklin Lakes, NJ). CD22 was immunoblotted with a rabbit antibody raised against exon 12 of mouse CD22 (16Jin L. McLean P.A. Neel B.G. Wortis H.H. J. Exp. Med. 2002; 195: 1199-1205Crossref PubMed Scopus (94) Google Scholar) Yeast Trihybrid Screen—Yeast strain Ylck/BDSHP1SH2 was generated as described in Ref. 13Sathish J.G. Johnson K.G. Fuller K.J. LeRoy F.G. Meyaard L. Sims M.J. Matthews R.J. J. Immunol. 2001; 166: 1763-1770Crossref PubMed Scopus (52) Google Scholar, and all yeast manipulations were performed according to standard yeast protocols. Competent yeast cells were prepared and transformed using the lithium acetate/Tris-EDTA/polyethylene glycol protocol. The cDNA for the library was generated from poly(A)+ RNA extracted from LSTRA lymphoma cells and synthesized using an oligo(dT) primer. cDNA was unidirectionally cloned between the EcoRI and XhoI sites of the yeast cloning vector HybriZAP-2.1 (Stratagene). cDNA library screening was performed using Ylck/BDSHP1SH2 (13Sathish J.G. Johnson K.G. Fuller K.J. LeRoy F.G. Meyaard L. Sims M.J. Matthews R.J. J. Immunol. 2001; 166: 1763-1770Crossref PubMed Scopus (52) Google Scholar). Approximately 3 × 109 cells were transformed with 100 μg of LSTRA library DNA and plated on minimal agar containing yeast nitrogen base, 2% glucose, and 5 mm 3-amino-1,2,4-triazole, a competitive inhibitor of the HIS3 gene, one of the reporter genes in the Ylck/BDSHP1SH2 strain. Transformation plates were incubated at 30 °C for 10–14 days. Large colonies were selected from these plates and streaked onto smaller plates with or without methionine but supplemented with histidine. Colonies were left to grow for 3 days and then filter-lifted and screened for protein-protein interactions using the lacZ reporter. β-Galactosidase activity was monitored using a freeze-thaw fracture assay. Positive clones were deemed to be those yeast that only turned blue on media lacking methionine. Library plasmid DNA was recovered from yeast by enzymatic disruption of the cell wall by treatment with Zymolyase®-100T (ICN Biomedicals, Costa Mesa, CA), alkaline lysis extraction, and amplification in Max-Efficiency DH5α™ Escherichia Coli (Invitrogen). Three colonies from each positive hit from the yeast β-galactosidase filter assay were grown for 3 days at 30 °C before DNA was extracted using the QIAprep spin miniprep kit (Qiagen). Plasmids were re-introduced into the screening strain or Ylck with the Gal4 binding domain alone to confirm specificity. Sequence data were generated on a 3100 genetic analyzer using ABI Big Dye automated sequencing protocols (PE Applied Biosystems, Foster City, CA). Protein Purification and Identification—A GST fusion protein containing the two SH2 domains of SHP-1 prepared as described previously (17Lorenz U. Ravichandran K.S. Pei D. Walsh C.T. Burakoff S.J. Neel B.G. Mol. Cell. Biol. 1994; 14: 1824-1834Crossref PubMed Scopus (138) Google Scholar) was used to isolate the SHP-1-associated pp150 from LSTRA cell lysates. LSTRA lysates (20 mg) were precleared with glutathione-Sepharose beads. GST or the GST SH2 domain fusion protein (400 μg) was added and incubated overnight at 4 °C. The bound proteins were washed four times with lysis buffer supplemented with 500 mm NaCl, and deglycosylated as described above, resolved by 6% SDS-PAGE, and silver-stained. Protein bands were excised from SDS-polyacrylamide gels and digested with sequencing-grade trypsin (Promega) as described (18Peng J. Gygi S.P. J. Mass Spectrom. 2001; 36: 1083-1091Crossref PubMed Scopus (504) Google Scholar, 19Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7736) Google Scholar). Digested samples were loaded onto a fused silica microcapillary C18 column (Magic, Michrom BioResources, Auburn, CA) prepared in-house (75-μm inner diameter, 10-cm long). An Agilent 1100 high-pressure liquid chromatography system (Agilent Technologies, Palo Alto, CA) was used to deliver a gradient across a flow splitter to the column over 40 min. The column eluant was directed into an LCQ-Deca electrospray ion-trap mass spectrometer (ThermoFinnigan, San Jose, CA), and the eluting peptides were dynamically selected for fragmentation by the operating software. The acquired tandem mass spectrometer data were analyzed with the non-redundant mouse data base from NCBI using SEQUEST data base search tool for peptide identification (20Eng J.K. McCormack A.L. Yates III, J.R. J. Am. Soc. Mass Spectrom. 1994; 5: 976-989Crossref PubMed Scopus (5315) Google Scholar). Proliferation Assays—Splenic T cells from either BALB/c or C57BL/6J mice were stimulated in vitro for 72 h with a titration of plate-bound anti-CD3 in conjunction with either anti-CD22 or isotype control antibody (BD Biosciences Pharmingen). Alternatively, lymph node T cells from CD22-deficient and age-matched C57BL/6J control mice were stimulated in vitro for 72 h with a titration of plate-bound anti-CD3 in conjunction with either anti-CD22 or an isotype control antibody. Allogeneic T cell stimulation was performed by incubating lymph node T cells from CD22-deficient and age-matched C57BL/6J control mice with different ratios of irradiated BALB/c spleen stimulator cells. [3H]Thymidine was added at 1 μCi/well for the final 16 h of culture before harvesting, and the incorporated radioactivity was assessed. Flow Cytometry—Splenocytes from BALB/c, C57BL/6J, and CD22-deficient were stained simultaneously with anti-TCRPE and either anti-CD22Bio or isotypeBio antibody for 30 min on ice. The cells were washed twice with Cell Wash (BD Biosciences), and secondary staining with streptavidinRed670 (Invitrogen) was performed for 20 min on ice. The cells were washed and acquired on the flow cytometer (FACSCalibur, BD Biosciences) and analyzed by CellQuest software. ZAP-70 Is Not a Genuine SHP-1 Binding Ligand—The functional analyses of T cells from SHP-1-deficient moth-eaten mice and T cells expressing a catalytically inactive dominant negative isoform of SHP-1 have demonstrated a role for SHP-1 in contributing to the thresholds of TCR activation (1Zhang J. Somani A.K. Siminovitch K.A. Semin. Immunol. 2000; 12: 361-378Crossref PubMed Scopus (284) Google Scholar, 4Lorenz U. Ravichandran K.S. Burakoff S.J. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9624-9629Crossref PubMed Scopus (157) Google Scholar, 5Pani G. Fischer K.D. Mlinaric-Rascan I. Siminovitch K.A. J. Exp. Med. 1996; 184: 839-852Crossref PubMed Scopus (179) Google Scholar, 6Plas D.R. Williams C.B. Kersh G.J. White L.S. White J.M. Paust S. Ulyanova T. Allen P.M. Thomas M.L. J. Immunol. 1999; 162: 5680-5684PubMed Google Scholar, 7Carter J.D. Neel B.G. Lorenz U. Int. Immunol. 1999; 11: 1999-2014Crossref PubMed Scopus (71) Google Scholar, 8Johnson K.G. LeRoy F.G. Borysiewicz L.K. Matthews R.J. J. Immunol. 1999; 162: 3802-3813PubMed Google Scholar, 9Plas D.R. Johnson R. Pingel J.T. Matthews R.J. Dalton M. Roy G. Chan A.C. Thomas M.L. Science. 1996; 272: 1173-1176Crossref PubMed Scopus (328) Google Scholar). A corollary to these findings is that SHP-1 must be catalytically active in normal T cells as a consequence of a PY-dependent ligand engagement of its amino-terminal SH-2 domain. To reveal phosphotyrosine-containing proteins that bind to SHP-1 following TCR ligation we performed immunoprecipitations of SHP-1 from primary T cells isolated from SHP-1-deficient and littermate control T cells (Fig. 1A). In control T cells, a PY-containing protein of 72 kDa, established to be ZAP-70 by reprobing, was found co-immunoprecipitated with SHP-1 following CD3 triggering. However, ZAP-70 was also found co-immunoprecipitated with SHP-1 in lysates derived from SHP-1-deficient T cells perhaps because of the inadvertent immunoprecipitation of the activating anti-CD3 antibody, which itself co-immunoprecipitates ZAP-70 (21Straus D.B. Weiss A. J. Exp. Med. 1993; 178: 1523-1530Crossref PubMed Scopus (118) Google Scholar). To circumvent spurious co-precipitation, SHP-1-deficient and control thymocytes bearing a transgenic TCR, F5, were stimulated with a cognate peptide presented by YO1 cells (Fig. 1B). SHP-1 immunoprecipitations from peptide/antigen-presenting cell-stimulated thymocytes resulted in no co-immunoprecipitation of ZAP-70, although the tyrosine phosphorylation of ZAP-70 could be readily detected in a parallel anti-CD3 co-immunoprecipitation following peptide/antigen-presenting cell stimulation. We therefore concluded that the CD3/TCR triggering of mouse primary T cells does not induce the specific association of SHP-1 with CD3 or ZAP-70 proteins. pp150 Is the Major Tyrosine-phosphorylated Protein Associating with SHP-1 in Mouse Primary T Cells—A difficulty in identifying molecules associated with SHP-1 in primary T cells is that the physiological stimuli capable of inducing tyrosine phosphorylation of putative SHP-1 ligand(s) are unknown presently. However, the degree of tyrosine phosphorylation on a given protein is an outcome of the counteractive effects of protein-tyrosine kinases and protein-tyrosine phosphatases (22Chan A.C. Desai D.M. Weiss A. Annu. Rev. Immunol. 1994; 12: 555-592Crossref PubMed Scopus (497) Google Scholar). Consequently, inhibition of protein-tyrosine phosphatases by the potent inhibitor, pervanadate (PV), can lead to an accumulation of PY on those proteins, which are the normal substrates of protein-tyrosine kinases (23Secrist J.P. Burns L.A. Karnitz L. Koretzky G.A. Abraham R.T. J. Biol. Chem. 1993; 268: 5886-5893Abstract Full Text PDF PubMed Google Scholar). Hence, PV may be utilized to ascertain those molecules potentially capable of associating with SHP-1 under physiological conditions in primary T cells. SHP-1 immunoprecipitations were performed on PV-stimulated splenic T cell blasts derived from a number of mouse strains including C57BL/6J, CBA, NOD, BALB/c, A/J, C3H, SWR, NIH, DBA, FVP, and SJL. The T cell blasts were confirmed by fluorescence-activated cell sorter analysis to have no B cell contamination (data not shown). The results demonstrated that for the majority of strains the most prominent PY protein consistently associating with SHP-1 in mouse peripheral T cells is one of 150 kDa (pp150), although additional weaker phosphoproteins of 75 and 45 kDa also were occasionally co-immunoprecipitated (Fig. 2 and results not shown). In contrast to all other strains examined, strikingly, T cells from the strain C57BL/6J showed a much reduced level of tyrosine-phosphorylated pp150 associated with SHP-1 despite an equivalent immunoprecipitation of SHP-1 (Fig. 2 and results not shown). The differential association of pp150 and SHP-1 in T cells from C57BL/6J mice may be because of one or more genetic differences in the expression of the pp150 receptor, its ability to be tyrosine-phosphorylated and to associate with SHP-1, or a combination of these possibilities. Furthermore, anti-CD3 stimulation of T cells from the mouse strain BALB/c resulted in no pp150 associating with SHP-1, although pp150 was readily detected in association with SHP-1 following PV treatment of the same T cells (Fig. 3).Fig. 3SHP-1 associated pp150 is not tyrosine-phosphorylated upon anti-CD3 stimulation. T lymphoblasts from BALB/c mice were either left unstimulated or stimulated with anti-CD3 or PV, lysed, and subjected to immunoprecipitation with either pre-immune (PI) or anti-SHP-1 sera. Immune complexes were resolved on SDS-PAGE and immunoblotted for PY. The blot was subsequently stripped and reprobed for SHP-1.View Large Image Figure ViewerDownload (PPT) pp150 Is an N-Glycosylated, Membrane-associated Protein—We examined whether pp150 might represent a membrane-associated receptor. As the possession of N-linked carbohydrates is a common feature of plasma membrane receptors, SHP-1 was immunoprecipitated from a lysate of T cells of the CBA strain, and the immunoprecipitate was subjected to Endo F treatment. pp150 was indeed found to be N-glycosylated, as Endo F treatment accelerated its migration on SDS-PAGE to ∼130 kDa (Fig. 4A). We also performed SHP-1 immunoprecipitations on the P100 (membrane) fraction of PV-stimulated T cells and confirmed that pp150 is a membrane-associated protein (Fig. 4B). These results indicate that pp150 is a cell surface transmembrane receptor with an N-glycosylated extracellular domain. pp150 Is Identified as CD22—Previously, we reported a tyrosyl phosphoprotein around 150–160 kDa associated with SHP-1 in the mouse lymphoma, LSTRA, and it is possible that this protein corresponds to pp150 in primary T cells (17Lorenz U. Ravichandran K.S. Pei D. Walsh C.T. Burakoff S.J. Neel B.G. Mol. Cell. Biol. 1994; 14: 1824-1834Crossref PubMed Scopus (138) Google Scholar). To identify pp150, a cDNA library was generated in the yeast-cloning vector, HybriZAP-2.1 using mRNA from the LSTRA cells. The HybriZAP-2.1 vector directs the expression of hybrid proteins encompassing the yeast Gal4 activation domain. The cDNA library was used to transform a stable yeast strain, Ylck/BDSHP1SH2 (13Sathish J.G. Johnson K.G. Fuller K.J. LeRoy F.G. Meyaard L. Sims M.J. Matthews R.J. J. Immunol. 2001; 166: 1763-1770Crossref PubMed Scopus (52) Google Scholar), that expresses the active form of the protein-tyrosine kinase Lck under a regulatable promoter and a chimeric cDNA encoding the tandem SH2 domains (amino acids 1–222) of SHP-1 fused to the DNA binding domain of Gal4. In this yeast trihybrid vector system, the regulatable expression of Lck permits the identification of interactions between the SH2 domains of SHP-1 and tyrosine-phosphorylated ligands (13Sathish J.G. Johnson K.G. Fuller K.J. LeRoy F.G. Meyaard L. Sims M.J. Matthews R.J. J. Immunol. 2001; 166: 1763-1770Crossref PubMed Scopus (52) Google Scholar). The transformation of Ylck/BDSHP1SH2 with the LSTRA cDNA library resulted in the identification of partial cDNAs encoding for the ITIM-containing receptors gp49B1, CD72, and CD22 (Table I). All cDNAs encoded for the ITIM-containing cytoplasmic region of each receptor. Of the three proteins, only the known molecular mass of CD22 (150 kDa) (24Torres 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), as opposed to gp49B1 (49 kDa) (25Castells M.C. Wu X. Arm J.P. Austen K.F. Katz H.R. J. Biol. Chem. 1994; 269: 8393-8401Abstract Full Text PDF PubMed Google Scholar) and CD72 (45 kDa) (26Nakayama E. von Hoegen I. Parnes J.R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1352-1356Crossref PubMed Scopus (77) Google Scholar), is consistent with the size of the major tyrosyl phosphoprotein (pp150) found co-immunoprecipitating with SHP-1 in primary T cells. However, thus far, only CD72 has been reported to be expressed on mouse primary T cells (27Robinson W.H. Landolfi M.M. Parnes J.R. Immunogenetics. 1997; 45: 195-200Crossref PubMed Scopus (14) Google Scholar).Table IYeast trihybrid identification of SHP-1 SH2 domain-interacting proteinsCloneEncoded proteinResidue numbersNo. of isolatesA1-4gp49B1235-2804A5CD721-1991A6CD22767-8681 Open table in a new tab To confirm the identity of the LSTRA-derived pp150 protein, we purified this protein by affinity chromatography using a GST fusion protein column containing the SH2 domains of SHP-1 (17Lorenz U. Ravichandran K.S. Pei D. Walsh C.T. Burakoff S.J. Neel B.G. Mol. Cell. Biol. 1994; 14: 1824-1834Crossref PubMed Scopus (138) Google Scholar). LSTRA cell lysates were incubated with the purified GST fusion protein, and the bound protein was deglycosylated and resolved by SDS-PAGE. One major band corresponding to 150 kDa was visualized by silver staining before deglycosylation, and the band decreased to ∼110 kDa after deglycosylation (Fig. 5A). Mass spectrometric analysis identified both bands as CD22 (Fig. 5B), and immunoblot analysis using anti-CD22 antibodies confirmed this finding (Fig. 5C). Because CD22 is tyrosine-phosphorylated constitutively in LSTRA, one might expect SHP-1 to be bound constitutively to CD22 in LSTRA cells. Indeed, this can be readily demonstrated in reciprocal CD22 and SHP-1 immunoprecipitations from untreated LSTRA cells (Fig. 5D). To investigate whether the pp150 detected associating with SHP-1 on primary T cells corresponds to CD22, SHP-1 immunoprecipitations from T lymphoblasts generated from BALB/c and C57BL/6J strains were immunoblotted for CD22. Fig. 6A demonstrates that CD22 is indeed identical to SHP-1 associated pp150. Interestingly, the amount of CD22 associating with SHP-1 is significantly reduced in C57BL/6J-versus BALB/c-derived T cells. This result provides an explanation for the previous finding that the amount of tyrosine-phosphorylated pp150 associating with SHP-1 is reduced in C57BL/6J-derived T cells. The strain differences may reflect a reduction in the T cell expression of CD22 or an inability of CD22 to associate with SHP-1 in T cells of the C57BL/6J strain. To distinguish between these possibilities, CD22 was directly immunoprecipitated from T lymphoblasts generated from BALB/c and C57BL/6J strains and immunoblotted for CD22. Fig. 6B indicates that the level of expression of CD22 is reduced in T cells derived from the C57BL/6J strain. However, the strain differences in CD22 expression are restricted to T lymphocytes, because the level of expression of CD22 in B lymphocytes is equivalent between the two strains (Fig. 6C). Ligation of CD22 on Naïve T Cells Inhibits Anti-CD3-induced Proliferation—By employing a secondary amplification step during flow cytometry analysis, a low level expression of CD22 over backgro