Hematopoietic Cell Phosphatase Is Recruited to CD22 following B Cell Antigen Receptor Ligation
1995; Elsevier BV; Volume: 270; Issue: 35 Linguagem: Inglês
10.1074/jbc.270.35.20305
ISSN1083-351X
AutoresArjan C. Lankester, Gijs M.W. van Schijndel, René A. W. van Lier,
Tópico(s)Cell death mechanisms and regulation
ResumoHematopoietic cell phosphatase is a nonreceptor protein tyrosine phosphatase that is preferentially expressed in hematopoietic cell lineages. Motheaten mice, which are devoid of (functional) hematopoietic cell phosphatase, have severe disturbances in the regulation of B cell activation and differentiation. Because signals transduced via the B cell antigen receptor are known to guide these processes, we decided to analyze molecular interactions between the hematopoietic cell phosphatase and the B cell antigen receptor. Ligation of the B cell antigen receptor induces moderate tyrosine phosphorylation of hematopoietic cell phosphatase and the formation of a multimolecular complex containing additional 68-70- and 135-kDa phosphoproteins. In resting B cells most of the hematopoietic cell phosphatase proteins reside in the cytosolic compartment, whereas after B cell antigen receptor cross-linking, a small fraction translocates toward the membrane where it specifically binds to the 135-kDa phosphoprotein. This 135-kDa glycoprotein was identified as CD22, a transmembrane associate of the B cell antigen receptor complex. Together these findings provide the first direct evidence that this cytoplasmic tyrosine phosphatase is involved in antigen receptor-mediated B cell activation, suggesting that in vivo B cell antigen receptor constituents or associated molecules may serve as substrate for its catalytic activity. Hematopoietic cell phosphatase is a nonreceptor protein tyrosine phosphatase that is preferentially expressed in hematopoietic cell lineages. Motheaten mice, which are devoid of (functional) hematopoietic cell phosphatase, have severe disturbances in the regulation of B cell activation and differentiation. Because signals transduced via the B cell antigen receptor are known to guide these processes, we decided to analyze molecular interactions between the hematopoietic cell phosphatase and the B cell antigen receptor. Ligation of the B cell antigen receptor induces moderate tyrosine phosphorylation of hematopoietic cell phosphatase and the formation of a multimolecular complex containing additional 68-70- and 135-kDa phosphoproteins. In resting B cells most of the hematopoietic cell phosphatase proteins reside in the cytosolic compartment, whereas after B cell antigen receptor cross-linking, a small fraction translocates toward the membrane where it specifically binds to the 135-kDa phosphoprotein. This 135-kDa glycoprotein was identified as CD22, a transmembrane associate of the B cell antigen receptor complex. Together these findings provide the first direct evidence that this cytoplasmic tyrosine phosphatase is involved in antigen receptor-mediated B cell activation, suggesting that in vivo B cell antigen receptor constituents or associated molecules may serve as substrate for its catalytic activity. INTRODUCTIONAntigen receptor-mediated B cell activation critically depends on the regulated activities of both protein tyrosine kinases and protein tyrosine phosphatases. Early after BCR 1The abbreviations used are: BCRB cell antigen receptorHCPhematopoietic cell phosphatasemAbmonoclonal antibodyPAGEpolyacrylamide gel electrophoresisILinterleukinCLBCentral Laboratory of the Netherlands Red Cross Blood Transfusion Service. cross-linking a large number of cellular proteins become phosphorylated on tyrosine residues(1Pleiman C.M. D'Ambrosio D. Cambier J.C. Immunol. Today. 1994; 15: 393-399Abstract Full Text PDF PubMed Scopus (29) Google Scholar). This change in phosphorylation status of cellular proteins has two potential consequences. First, it may alter the enzymatic activity of certain proteins (e.g. PLCγ (2Padeh S. Levitsky A. Gazit A. Mills G.B. Roifman C.M. J. Clin. Invest. 1991; 87: 1114-1118Crossref PubMed Scopus (61) Google Scholar)). Second, the induction of tyrosine phosphorylation provides a mechanism to accomplish specific interactions with SH2 domain-containing proteins and can result in an altered subcellular distribution of proteins or protein complexes(3Koch C.A. Anderson D. Moran M.F. Ellis C. Pawson T. Science. 1991; 252: 668-674Crossref PubMed Scopus (1429) Google Scholar). It has been shown previously that two types of PTK are physically and functionally associated with the BCR. These include the src family members lyn, fyn, blk, and lck(4Yamanashi Y. Kakiuchi T. Mizuguchi J. Yamamoto T. Toyoshima K. Science. 1991; 251: 192-194Crossref PubMed Scopus (335) Google Scholar, 5Burkhardt A.L. Brunswick M. Bolen J.B. Mond J.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7410-7414Crossref PubMed Scopus (354) Google Scholar) and the ZAP70-related PTK syk(6Hutchcroft J.E. Harrison M.L. Geahlen R.L. J. Biol. Chem. 1991; 266: 14846-14849Abstract Full Text PDF PubMed Google Scholar, 7Hutchcroft J.E. Harrison M.L. Geahlen R.L. J. Biol. Chem. 1992; 267: 8613-8619Abstract Full Text PDF PubMed Google Scholar, 8Kolanus W. Romeo C. Seed B. Cell. 1993; 74: 171-183Abstract Full Text PDF PubMed Scopus (301) Google Scholar, 9Law C.L. Sidorenko S.P. Chandran K.A. Draves K.E Chan A.C. Weiss A. Edelhoff S. Disteche C.M. Clark E.A. J. Biol. Chem. 1994; 269: 12310-12319Abstract Full Text PDF PubMed Google Scholar).In contrast to the considerable number of protein tyrosine kinase that are known to be involved in BCR signaling, studies on the contribution of protein tyrosine phosphatase have so far been restricted to the CD45 protein. Expression of CD45 is required for BCR signaling, because BCR-induced tyrosine phosphorylation is severely affected in B cells lacking CD45(10Justement L.B. Campbell K.S. Chien N.C. Cambier J.C. Science. 1991; 252: 1839-1842Crossref PubMed Scopus (244) Google Scholar). The recent observation that CD45 may be physically associated with the BCR supports this notion(11Brown V.K. Ogle E.W. Burkhardt A.L. Rowley R.B. Bolen J.B. Justement L.B. J. Biol. Chem. 1994; 269: 17238-17244Abstract Full Text PDF PubMed Google Scholar). A potential role for a second class of protein tyrosine phosphatase was suggested by the recent identification of the intracellular protein tyrosine phosphatase 1C-hematopoietic cell phosphatase (HCP) (12Yi T. Cleveland J.L. Ihle J.N. Mol. Cell. Biol. 1992; 12: 836-846Crossref PubMed Scopus (306) Google Scholar) and Syp (protein tyrosine phosphatase 1D)(13Freeman R.M. Plutzky Jr., J. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11239-11243Crossref PubMed Scopus (326) Google Scholar). HCP is mainly expressed in cells of hematopoietic origin, whereas Syp is ubiquitously expressed. Both protein tyrosine phosphatases are characterized by the presence of two SH2 domains, which provide them with the capacity to become recruited toward tyrosine-phosphorylated substrates(14Yi T. Ihle J.N. Mol. Cell. Biol. 1993; 13: 3350-3358Crossref PubMed Scopus (234) Google Scholar, 15Yi T. Mui A.L.F. Krystal G. Ihle J.N. Mol. Cell. Biol. 1993; 13: 7577-7586Crossref PubMed Google Scholar). Interestingly, Motheaten mice and viable Motheaten mice, which do not express or express aberrant forms of HCP protein, respectively(16Tsui H.W. Siminovitch K.A. De Souza L. Tsui F.W.L. Nature Genet. 1993; 4: 124-129Crossref PubMed Scopus (514) Google Scholar, 17Schultz L.D. Schweitzer P.A. Rajan T.V. Yi T. Ihle J.N. Matthews R.J. Thomas M.L. Beier D.R. Cell. 1993; 73: 1445-1454Abstract Full Text PDF PubMed Scopus (686) Google Scholar), are characterized by defects in lymphocyte development, including premature thymic involution, impaired mitogen and alloantigen-induced T cell responses, and diminished numbers of B cell precursors(18Sidman C.L. Schultz L.D. Unanue E.R. J. Immunol. 1978; 121: 2392-2404PubMed Google Scholar, 19Greiner D.L. Goldschneider I. Komschlies K.L. Medlock E.S. Bollum F.J. Schultz L. J. Exp. Med. 1986; 164: 1129-1144Crossref PubMed Scopus (46) Google Scholar). Clinically, Motheaten mice suffer from severe autoimmune diseases and severe combined immunodeficiency syndromes(20Schultz L.D. Green M.C. J. Immunol. 1976; 116: 936-943PubMed Google Scholar). At present, the molecular role of HCP in B cell signaling and differentiation is unknown. Because signals transmitted via the BCR are known to guide B cell development and differentiation, we decided to analyze the possible involvement of HCP in BCR signaling.MATERIALS AND METHODSCellsThe Burkitt lymphoma cell line Daudi was routinely cultured in Iscove's modified Dulbecco's medium supplemented with 10% fetal calf serum and antibiotics. Tonsillar B cells were isolated from tonsils of healthy donors and purified as described previously (21van Noesel C.J.M. Brouns G.S. van Schijndel G.M.W. Bende R.J. Mason D.Y. Borst J. van Lier R.A.W. J. Exp. Med. 1992; 175: 1511-1519Crossref PubMed Scopus (44) Google Scholar, 22Lankester A.C. van Schijndel G.M.W. Rood P.M.L. Verhoeven A.J. van Lier R.A.W. Eur. J. Immunol. 1994; 24: 2818-2825Crossref PubMed Scopus (50) Google Scholar).AntibodiesThe mAb specific for μH chain (CLB-MH15), CD3 (CLB-T3.4/2a), CD14 (CLB-mon/1), CD16 (CLB-FcRgran/1), CD19 (CLB-CD19), CD22 (CLB-CD22), and HLA-Dr (CLB-HLA-DR) were generated at the CLB (Amsterdam, The Netherlands). The δH chain mAb (δTA-4) was obtained from the ATCC. Antibodies directed against phosphotyrosine (RC20) and Shc were from Signal Transduction Laboratories (Lexington, KY), and phosphatidylinositol 3-kinase antibodies were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Antibodies specific for HCP were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).Immunoprecipitation and Western BlottingIntact cells and subcellular fractions were lysed with IMMUNOPRECIPITATION BUFFER (final concentration, 1% Nonidet P-40, 0.01 M triethanolamine-HCl, pH 7.8, 0.15 M NaCl, 5 mM EDTA, 1 mM 1-chloro-3-tosylamido-7-amino-2-heptanone, 0.02 mg/ml ovomucoid trypsin inhibitor, 1 mM phenylmethylsulfonyl fluoride, 0.02 mg/ml leupeptin, 0.4 mM vanadate, 10 mM NaF, 10 mM pyrophosphate, 25 μM phenylarsine oxide) as described previously(22Lankester A.C. van Schijndel G.M.W. Rood P.M.L. Verhoeven A.J. van Lier R.A.W. Eur. J. Immunol. 1994; 24: 2818-2825Crossref PubMed Scopus (50) Google Scholar). Postnuclear debris and subcellular fractions were precleared by three incubations with 50 μl of a 10% (v/v) suspension of protein A-CL4B Sepharose beads (Pharmacia Biotech Inc.) coated with nonimmune mouse Ig, and once with protein A-Sepharose. Next, cell lysates were sequentially incubated with specific antibodies (15 min) and protein A-Sepharose (1.5 h). After washing in immunoprecipitation buffer, the immunoprecipitates were resuspended in sample buffer and separated on SDS-PAGE. Western blotting was performed as described previously(23Lankester A.C van Schijndel G.M.W. van Noesel C.J.M. van Lier R.A.W. Eur. J. Immunol. 1994; 24: 812-816Crossref PubMed Scopus (101) Google Scholar). In short, after transfer to Hybond C nitrocellulose blots (Amersham Corp.), employing a semidry electroblotting chamber (Multiphore II, Pharmacia) and blocking with 1% bovine serum albumin (Organon, Oss, The Netherlands), the proteins were detected with specific antibodies. Immunoreactive proteins were visualized by enhanced chemiluminescence (ECL, Amersham; POD, Boehringer Mannheim). For sequential analysis of the same blot with distinct antibodies, deprobing was performed according to the manufacturer's instructions.B Cell ActivationThe cells were washed twice in Hepes solution (132 mM NaCl, 6 mM KCl, 1 mM MgSO4, 1 mM CaCl2, 1.2 mM K2HPO4, 20 mM Hepes, pH 7.4, supplemented with 0.5% human serum albumin and 0.1% glucose) and kept at 4°C. Subsequently, the cells were incubated with purified biotinylated mAb for 3 min, pelleted by rapid centrifugation, and resuspended in Hepes solution containing 25 μg/ml streptavidin at 37°C for the indicated period of time. Following activation, the cells were either pelleted and lysed or resuspended in ice-cold sonication buffer.Subcellular FractionationFollowing stimulation, B cells (2-3 × 107) were resuspended in ice-cold sonication buffer (5% w/v sucrose, 10 mM Hepes, 1 mM EGTA in phosphate-buffered saline, supplemented with protease and phosphatase inhibitors). After sonication of the suspension (3 × 15 s at 21 kHz frequency and 9 μm peak-to-peak amplitude) and removal of unbroken cells and nuclei, 1 ml of postnuclear supernatant was layered on a discontinuous sucrose gradient consisting of 1.5 ml of 40% (w/v) sucrose and 1.5 ml of 15% (w/v) sucrose. After centrifugation (35,000 × g, 50 min), 80% of the supernatant (as source of cytosol) and the interface of the sucrose layers (as source of membranes) were collected and analyzed as indicated elsewhere(24Bolscher B.G.J.M. Denis S.W. Verhoeven A.J. Roos D. J. Biol. Chem. 1990; 265: 15782-15787Abstract Full Text PDF PubMed Google Scholar).RESULTS AND DISCUSSIONBCR Cross-linking Induces Tyrosine Phosphorylation of HCP and the Formation of a Multimolecular HCP ComplexPrevious reports have indicated that HCP may serve as substrate for src family protein tyrosine kinase(25Lorenz 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, 26Matozaki T. Uchida T. Fujioka Y. Kasuga M. Biochem. Biophys. Res. Commun. 1994; 204: 874-881Crossref PubMed Scopus (31) Google Scholar). Since the BCR is functionally and physically coupled to several of these src family protein tyrosine kinase we have analyzed whether BCR ligation results in tyrosine phosphorylation of HCP. HCP was isolated from activated and nonactivated Daudi cells and was subsequently analyzed in anti-phosphotyrosine blots. Following activation, a moderately tyrosine phosphorylated HCP protein was detected that migrated with an apparent molecular mass of 65 kDa (Fig. 1, upper panel (arrow)). This protein reacted with anti-HCP antibodies (Fig. 1, lower panel). An additional tyrosine phosphorylated protein (arrow∗) was visualized that migrated only slightly slower than HCP but was nonreactive with anti-HCP antibodies (Fig. 1, lower panel). Next to HCP and the 68-70-kDa protein a very prominent tyrosine-phosphorylated protein with an apparent molecular mass of 130-135 kDa (<rif;) was detected in anti-HCP immunoprecipitates following BCR cross-linking. Similar results were obtained when HCP was isolated from tonsillar B cells (data not shown).Subcellular Distribution of Activation-induced HCP ComplexesSeveral studies have demonstrated that HCP interacts with tyrosine-phosphorylated transmembrane receptors in an activation-dependent manner(14Yi T. Ihle J.N. Mol. Cell. Biol. 1993; 13: 3350-3358Crossref PubMed Scopus (234) Google Scholar, 15Yi T. Mui A.L.F. Krystal G. Ihle J.N. Mol. Cell. Biol. 1993; 13: 7577-7586Crossref PubMed Google Scholar, 27Yi T. Zhang J. Miura O. Ihle J.N. Blood. 1995; 85: 87-95Crossref PubMed Google Scholar). Similarly, the phosphoproteins detected in anti-HCP immunoprecipitates might represent constituents of the BCR complex that serve to recruit HCP toward potential substrates associated with the BCR complex. When the anti-HCP immunoprecipitates were analyzed in anti-phosphotyrosine blots, HCP complexes with distinct features were observed in membrane and cytosolic fractions, respectively (Fig. 2, upper panel). Most of the HCP proteins resided in the cytosolic fraction, although after activation a slight decrease was observed (Fig. 2, lower panel). In activated B cells, cytosolic HCP proteins were moderately phosphorylated on tyrosine residues (arrow) and were associated with the 68-70-kDa phosphoprotein (arrow∗). However, a small amount of the HCP proteins (5-10%) was detected in the membrane fraction. In contrast to what was observed in the cytosolic fraction, neither tyrosine phosphorylation of the HCP protein nor of the 68-70-kDa protein was detected in the membrane fraction. Although it can not be excluded that the tyrosine phosphorylation of HCP proteins residing in the membrane fraction is below detection level, these findings argue against a preferential membrane translocation of tyrosine phosphorylated HCP proteins, which appears to be the case for Shc proteins(22Lankester A.C. van Schijndel G.M.W. Rood P.M.L. Verhoeven A.J. van Lier R.A.W. Eur. J. Immunol. 1994; 24: 2818-2825Crossref PubMed Scopus (50) Google Scholar, 28Saxton T.M. van Oostveen I. Bowtell D. Aebersold R. Gold M.R. J. Immunol. 1994; 153: 623-636PubMed Google Scholar).Figure 2:Distinct HCP complexes are localized in the membrane and cytosolic fraction. Daudi cells were stimulated for the indicated periods of time as described in Fig. 1and, prior to lysis, subcellular fractions were prepared by sonication. Next, HCP proteins were specifically recovered from the precleared membrane (m) and cytosolic (c) fractions and analyzed in anti-phosphotyrosine (upper panel) and anti-HCP Western blots (lower panel). Symbols are used as described in Fig. 1. Three additional experiments gave similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Tyrosine-phosphorylated CD22 Acts as Membrane Target for HCPIn marked contrast to the 68-70-kDa phosphoprotein, the tyrosine-phosphorylated 130-135-kDa phosphoprotein was exclusively detected in association with the membrane-translocated HCP protein (Fig. 2, <rif;), indicating that this phosphoprotein possibly represents the membrane target of HCP. The fact that this 130-135-kDa phosphoprotein was not detected in HCP complexes from activated Jurkat cells (data not shown) suggested that this protein could be a B cell-specific transmembrane molecule. Among the B cell-specific transmembrane molecules that serve as a substrate for BCR-induced protein tyrosine kinase activity and are known to be involved in BCR signaling, the BCR complex-associated CD22 appeared to be a possible candidate(29Leprince 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, 30Peaker C.J.G. Neuberger M.S. Eur. J. Immunol. 1993; 23: 1358-1363Crossref PubMed Scopus (142) Google Scholar). To investigate this hypothesis, anti-HCP and CD22 immunoprecipitates were isolated from activated Daudi cells, and half of each immunoprecipitate was directly analyzed in anti-phosphotyrosine blots. In accordance with previous reports, CD22 was detected as a 135-kDa tyrosine-phosporylated protein (Fig. 3, left panel)(31Schulte R.J. Campbell M.A. Fischer W.H. Sefton B.M. Science. 1992; 258: 1001-1004Crossref PubMed Scopus (120) Google Scholar). Comparison with the HCP-associated 135-kDa phosphoprotein demonstrated that both proteins migrated at the same position in the SDS-PAGE, both under nonreducing (Fig. 3, left panel) and reducing conditions (data not shown). Treatment of the remaining half of the immunoprecipitates with N-glycanase prior to analysis in anti-phosphotyrosine blots revealed that both CD22 and the HCP-associated 135-kDa protein were deglycosylated and then still migrated at the same position following SDS-PAGE (Fig. 3, right panel). The apparent molecular mass of 100-105 kDa corresponds with the reported protein backbone of CD22(32Schwartz-Albiez R. Dorken B. Moldenhauer G. Knapp W. Dorken B. Gilks W.R. Rieber E.P. Schmidt R.E. Stein M. Kr. von dem Borne A.E.G. Leucocyte Typing IV. Oxford University Press, Oxford1989: 65-67Google Scholar). Definite evidence for the interaction of CD22 with HCP was obtained when CD22 immunoprecipitates, isolated from activated B cells, were analyzed in anti-HCP blots. In agreement with the subcellular fractionation experiments (see Fig. 2) only a small part of the total amount of HCP protein was found to interact with CD22 (Fig. 4). Densitometric analysis indicated that 5-10% of the total cellular HCP pool may associate with CD22 upon activation. The observation that in these parallel immunoprecipitations the phosphotyrosine content of HCP-associated CD22 is comparable with that of directly isolated CD22 suggests that most of the tyrosine-phosphorylated CD22 is bound by HCP.Figure 3:The HCP-associated 135-kDa glycoprotein comigrates with CD22. HCP and CD22 were specifically isolated from lysates of Daudi cells following activation as described in Fig. 1. Subsequently, the immunoprecipitates were separated by SDS-PAGE either directly (left panel) or after treatment with N-glycanase (right panel), and analyzed in anti-phosphotyrosine Western blots. Two additional experiments gave similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4:HCP is associated with tyrosine-phosphorylated CD22. HCP and CD22 were specifically isolated from lysates of Daudi cells following activation as described in Fig. 1. The immunoprecipitates were separated by SDS-PAGE and analyzed in anti-phosphotyrosine Western blots (upper panel). Subsequently, the membrane was reprobed with HCP antibodies (lower panel). Densitometric analysis indicated that CD22-bound HCP represented 5-10% of the directly isolated HCP proteins. Two additional experiments gave similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The present finding that HCP serves as a substrate for BCR-induced protein tyrosine kinase activity, together with the identification of tyrosine-phosphorylated CD22 as the specific docking site for HCP within the BCR complex, provides the first direct evidence for a role of this cytoplasmic tyrosine phosphatase in BCR signaling. Likely, one or more phosphotyrosine-incorporating motifs within the CD22 cytoplasmic tail directly mediate the interaction with one or both SH2 domains of HCP. Indeed, some of these CD22 motifs share homology with the recently described erythropoietin receptor-derived phosphopeptides that display binding specificity for the amino-terminal SH2 domain of HCP(27Yi T. Zhang J. Miura O. Ihle J.N. Blood. 1995; 85: 87-95Crossref PubMed Google Scholar, 29Leprince 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). The recruitment of HCP via CD22 into the BCR complex suggests that one or more BCR constituent(s) and/or associated tyrosine- phosphorylated proteins may serve as substrate for its tyrosine phosphatase activity. So far, we failed to detect substantial protein tyrosine phosphatase activity of HCP directed against BCR constituents in vitro (data not shown). However, the recent observation that recombinant HCP has the capacity to dephosphorylate the IL-3 receptor β-chain, c-fms and c-kit in vitro provides a precedent for this possibility(14Yi T. Ihle J.N. Mol. Cell. Biol. 1993; 13: 3350-3358Crossref PubMed Scopus (234) Google Scholar, 15Yi T. Mui A.L.F. Krystal G. Ihle J.N. Mol. Cell. Biol. 1993; 13: 7577-7586Crossref PubMed Google Scholar). The involvement of the phosphorylation status of HCP in its protein tyrosine phosphatase activity is still unresolved. The IL-3-induced association between the IL-3 receptor β-chain and HCP occurs without any significant alteration in HCP tyrosine phosphorylation and activity, while a marginal induction of HCP tyrosine phosphorylation was detected following c-fms and c-kit ligation, again without effect on its activation status. Our experiments indicate that tyrosine-phosphorylated HCP is preferentially localized in the cytosolic compartment (Fig. 2-4). Therefore tyrosine phosphorylation of HCP might facilitate the potential interactions with other cytosolic proteins incorporating SH2 domains.Previous studies in Motheaten mice, which lack HCP protein, have demonstrated the importance of HCP in B cell differentiation(18Sidman C.L. Schultz L.D. Unanue E.R. J. Immunol. 1978; 121: 2392-2404PubMed Google Scholar, 20Schultz L.D. Green M.C. J. Immunol. 1976; 116: 936-943PubMed Google Scholar, 33Sidman C.I. Schultz L.D. Unanue E.R. J. Immunol. 1978; 121: 2399-2404PubMed Google Scholar). Interestingly, most of the B cells in these mice belong to the CD5+ subset, which is thought to be responsible for the production of autoreactive antibodies(34Sidman C.L. Schultz L.D. Hardy R.R. Hayakawa K. Herzenberg L.A. Science. 1986; 232: 1423-1425Crossref PubMed Scopus (260) Google Scholar). Several studies have reported structural and functional differences between the BCR in CD5+ and conventional B cells, respectively(23Lankester A.C van Schijndel G.M.W. van Noesel C.J.M. van Lier R.A.W. Eur. J. Immunol. 1994; 24: 812-816Crossref PubMed Scopus (101) Google Scholar, 35Zupo S. Dono M. Azzoni L. Chiorazzi N. Ferrarini M. Eur. J. Immunol. 1991; 21: 351-359Crossref PubMed Scopus (36) Google Scholar, 36Bhat N.M. Kantor A.B. Bieber M.M. Stall A.M. Herzenberg L.A. Teng N.N.H. Int. Immunol. 1992; 4: 243-252Crossref PubMed Scopus (129) Google Scholar, 37Defrance T. Vanbervliet B. Durand I. Briolay J. Banchereau J. Eur. J. Immunol. 1992; 22: 2831-2839Crossref PubMed Scopus (52) Google Scholar, 38Antin J.H. Emerson S.G. Martin P. Gadol N. Ault K.A. J. Immunol. 1986; 136: 505-510PubMed Google Scholar). This may indicate that BCR signals required for differentiation of the former subset operate relatively independent of HCP or that the presence of CD5 within the BCR complex somehow compensates for this defect. However, another explanation could be that HCP is involved in the BCR-mediated deletion of autoreactive B cells. Lack of HCP expression might thus deregulate this selection process. Recently, Cyster and Goodnow (39Cyster J.G. Goodnow C.C. Immunity. 1995; 2: 13-24Abstract Full Text PDF PubMed Scopus (349) Google Scholar) have provided evidence that such a mechanism may indeed be operative.BCR-induced protein tyrosine kinase activation results in tyrosine phosphorylation of several accessory molecules, including CD5(23Lankester A.C van Schijndel G.M.W. van Noesel C.J.M. van Lier R.A.W. Eur. J. Immunol. 1994; 24: 812-816Crossref PubMed Scopus (101) Google Scholar), CD19(40Tuveson D.A. Carter R.H. Soltoff S.P. Fearon D.T. Science. 1993; 260: 986-989Crossref PubMed Scopus (281) Google Scholar), and CD22(31Schulte R.J. Campbell M.A. Fischer W.H. Sefton B.M. Science. 1992; 258: 1001-1004Crossref PubMed Scopus (120) Google Scholar), creating potential binding sites for SH2 domain-containing proteins. Indeed, it has been shown that tyrosine-phosphorylated CD19 serves as a specific and preferential binding site for the 85-kDa subunit of phosphatidylinositol 3-kinase (40Tuveson D.A. Carter R.H. Soltoff S.P. Fearon D.T. Science. 1993; 260: 986-989Crossref PubMed Scopus (281) Google Scholar). Our present finding that CD22 specifically recruits HCP provides further support for this function of accessory molecules. Thus, accessory molecules appear to have a dual function. They have the capacity to cooperate with the BCR at the extracellular level in the process of antigen recognition(41van Noesel C.J.M. Lankester A.C. van Lier R.A.W. Immunol. Today. 1993; 14: 8-11Abstract Full Text PDF PubMed Scopus (76) Google Scholar, 42Law C.L. Sidorenko S.V. Clark E.A. Immunol. Today. 1994; 15: 442-449Abstract Full Text PDF PubMed Scopus (6) Google Scholar). In addition, they provide the BCR with molecular substrates to couple to specific intracellular activation pathways. INTRODUCTIONAntigen receptor-mediated B cell activation critically depends on the regulated activities of both protein tyrosine kinases and protein tyrosine phosphatases. Early after BCR 1The abbreviations used are: BCRB cell antigen receptorHCPhematopoietic cell phosphatasemAbmonoclonal antibodyPAGEpolyacrylamide gel electrophoresisILinterleukinCLBCentral Laboratory of the Netherlands Red Cross Blood Transfusion Service. cross-linking a large number of cellular proteins become phosphorylated on tyrosine residues(1Pleiman C.M. D'Ambrosio D. Cambier J.C. Immunol. Today. 1994; 15: 393-399Abstract Full Text PDF PubMed Scopus (29) Google Scholar). This change in phosphorylation status of cellular proteins has two potential consequences. First, it may alter the enzymatic activity of certain proteins (e.g. PLCγ (2Padeh S. Levitsky A. Gazit A. Mills G.B. Roifman C.M. J. Clin. Invest. 1991; 87: 1114-1118Crossref PubMed Scopus (61) Google Scholar)). Second, the induction of tyrosine phosphorylation provides a mechanism to accomplish specific interactions with SH2 domain-containing proteins and can result in an altered subcellular distribution of proteins or protein complexes(3Koch C.A. Anderson D. Moran M.F. Ellis C. Pawson T. Science. 1991; 252: 668-674Crossref PubMed Scopus (1429) Google Scholar). It has been shown previously that two types of PTK are physically and functionally associated with the BCR. These include the src family members lyn, fyn, blk, and lck(4Yamanashi Y. Kakiuchi T. Mizuguchi J. Yamamoto T. Toyoshima K. Science. 1991; 251: 192-194Crossref PubMed Scopus (335) Google Scholar, 5Burkhardt A.L. Brunswick M. Bolen J.B. Mond J.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7410-7414Crossref PubMed Scopus (354) Google Scholar) and the ZAP70-related PTK syk(6Hutchcroft J.E. Harrison M.L. Geahlen R.L. J. Biol. Chem. 1991; 266: 14846-14849Abstract Full Text PDF PubMed Google Scholar, 7Hutchcroft J.E. Harrison M.L. Geahlen R.L. J. Biol. Chem. 1992; 267: 8613-8619Abstract Full Text PDF PubMed Google Scholar, 8Kolanus W. Romeo C. Seed B. Cell. 1993; 74: 171-183Abstract Full Text PDF PubMed Scopus (301) Google Scholar, 9Law C.L. Sidorenko S.P. Chandran K.A. Draves K.E Chan A.C. Weiss A. Edelhoff S. Disteche C.M. Clark E.A. J. Biol. Chem. 1994; 269: 12310-12319Abstract Full Text PDF PubMed Google Scholar).In contrast to the considerable number of protein tyrosine kinase that are known to be involved in BCR signaling, studies on the contribution of protein tyrosine phosphatase have so far been restricted to the CD45 protein. Expression of CD45 is required for BCR signaling, because BCR-induced tyrosine phosphorylation is severely affected in B cells lacking CD45(10Justement L.B. Campbell K.S. Chien N.C. Cambier J.C. Science. 1991; 252: 1839-1842Crossref PubMed Scopus (244) Google Scholar). The recent observation that CD45 may be physically associated with the BCR supports this notion(11Brown V.K. Ogle E.W. Burkhardt A.L. Rowley R.B. Bolen J.B. Justement L.B. J. Biol. Chem. 1994; 269: 17238-17244Abstract Full Text PDF PubMed Google Scholar). A potential role for a second class of protein tyrosine phosphatase was suggested by the recent identification of the intracellular protein tyrosine phosphatase 1C-hematopoietic cell phosphatase (HCP) (12Yi T. Cleveland J.L. Ihle J.N. Mol. Cell. Biol. 1992; 12: 836-846Crossref PubMed Scopus (306) Google Scholar) and Syp (protein tyrosine phosphatase 1D)(13Freeman R.M. Plutzky Jr., J. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11239-11243Crossref PubMed Scopus (326) Google Scholar). HCP is mainly expressed in cells of hematopoietic origin, whereas Syp is ubiquitously expressed. Both protein tyrosine phosphatases are characterized by the presence of two SH2 domains, which provide them with the capacity to become recruited toward tyrosine-phosphorylated substrates(14Yi T. Ihle J.N. Mol. Cell. 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