Hematopoietic Cell Phosphatase Is Recruited to CD22 following B Cell Antigen Receptor Ligation

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. 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 × 50 of the supernatant of and the of the sucrose of were and as indicated Verhoeven A.J. D. J. Biol. Chem. Full Text PDF PubMed Google AND of HCP and the of a HCP have indicated that HCP may serve as substrate for src family protein tyrosine U. K.S. D. Neel B.G. Mol. Cell. Biol. 1994; PubMed Scopus Google Scholar, T. T. Y. M. 1994; PubMed Scopus Google Scholar). the BCR is functionally and physically to of these src family protein tyrosine kinase we have BCR in tyrosine phosphorylation of HCP was isolated from and Daudi cells and was in Following activation, a tyrosine phosphorylated HCP protein was detected that with an molecular of This protein with antibodies additional tyrosine phosphorylated protein was visualized that HCP was with antibodies to HCP and the protein a tyrosine-phosphorylated protein with an molecular of was detected in immunoprecipitates BCR were obtained HCP was isolated from B cells not of HCP studies have that HCP with tyrosine-phosphorylated transmembrane in an 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, T. J. Ihle J.N. PubMed Google Scholar). the detected in immunoprecipitates constituents of the BCR complex that serve to HCP toward potential associated with the BCR complex. the immunoprecipitates were in HCP with distinct were in membrane and cytosolic of the HCP proteins in the cytosolic after activation a was In B cytosolic HCP proteins were phosphorylated on tyrosine and were associated with the a small of the HCP proteins was detected in the membrane In contrast to was in the cytosolic tyrosine phosphorylation of the HCP protein of the protein was detected in the membrane it can not be that the tyrosine phosphorylation of HCP proteins in the membrane fraction is these findings against a membrane of tyrosine phosphorylated HCP which to be the for Shc 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, van I. D. J. Immunol. 1994; Google HCP are in the membrane and cytosolic Daudi cells were for the indicated of as described in to subcellular fractions were by Next, HCP proteins were specifically from the precleared membrane and cytosolic fractions and in and Western blots are used as described in additional CD22 as for contrast to the the tyrosine-phosphorylated was detected in with the HCP protein that this the membrane of The that this was not detected in HCP from cells not suggested that this protein be a B transmembrane the B 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 CD22 to be a possible C. Draves Geahlen R.L. Clark E.A. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar, Eur. J. Immunol. 1993; PubMed Scopus Google Scholar). this and CD22 immunoprecipitates were isolated from Daudi and of was in In with CD22 was detected as a 135-kDa protein R.J. Campbell Science. 1992; PubMed Scopus Google Scholar). with the 135-kDa that both proteins at the same in the both and not of the of the immunoprecipitates with to analysis in blots that both CD22 and the 135-kDa protein were and at the same The molecular of with the protein of B. G. W. B. M. Scholar). evidence for the of CD22 with HCP was obtained CD22 isolated from B were in In with the subcellular a small of the of HCP protein was to with CD22 analysis indicated that of the cellular HCP may associate with CD22 The observation that in these the phosphotyrosine of CD22 is with that of isolated CD22 that most of the tyrosine-phosphorylated CD22 is by 135-kDa glycoprotein with HCP and CD22 were specifically isolated from lysates of Daudi cells activation as described in Subsequently, the immunoprecipitates were separated by either or after with and in Western additional is associated with tyrosine-phosphorylated HCP and CD22 were specifically isolated from lysates of Daudi cells activation as described in The immunoprecipitates were separated by and in Western blots Subsequently, the membrane was with HCP antibodies analysis indicated that HCP of the isolated HCP additional that HCP as a substrate for BCR-induced protein tyrosine kinase with the identification of tyrosine-phosphorylated CD22 as the specific for HCP the BCR provides the first direct evidence for a role of this cytoplasmic tyrosine phosphatase in BCR or the CD22 cytoplasmic the with or both SH2 of of these CD22 with the described that for the SH2 of T. J. Ihle J.N. PubMed Google Scholar, C. Draves Geahlen R.L. Clark E.A. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar). The of HCP via CD22 the BCR complex that or BCR associated phosphorylated proteins may serve as substrate for its tyrosine phosphatase activity. we to protein tyrosine phosphatase activity of HCP directed against BCR constituents in not the recent observation that HCP has the capacity to the receptor and in provides a for this 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 The between the receptor and HCP in HCP tyrosine phosphorylation and a induction of HCP tyrosine phosphorylation was detected and on its activation that tyrosine-phosphorylated HCP is preferentially in the cytosolic tyrosine phosphorylation of HCP the potential interactions with cytosolic proteins SH2 studies in Motheaten mice, which HCP protein, have the of HCP in B cell C.L. Schultz L.D. Unanue E.R. J. Immunol. 1978; 121: 2392-2404PubMed Google Scholar, L.D. Green M.C. J. Immunol. 1976; 116: 936-943PubMed Google Scholar, Schultz L.D. Unanue E.R. J. Immunol. 1978; 121: Google Scholar). Interestingly, most of the B cells in these mice to the which is to be for the of C.L. Schultz L.D. K. Science. 1986; PubMed Scopus Google Scholar). studies have and between the BCR in and B 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, S. M. L. M. Eur. J. Immunol. 1991; PubMed Scopus Google Scholar, Immunol. 1992; 4: PubMed Scopus Google Scholar, T. B. I. J. J. Eur. J. Immunol. 1992; PubMed Scopus Google Scholar, K.A. J. Immunol. 1986; Google Scholar). This may that BCR signals required for differentiation of the of HCP or that the presence of the BCR complex for this be that HCP is involved in the of B of HCP this and Full Text PDF PubMed Scopus Google Scholar) have evidence that a mechanism may be protein tyrosine kinase activation in tyrosine phosphorylation of including 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), S.P. Science. 1993; PubMed Scopus Google Scholar), and R.J. Campbell Science. 1992; PubMed Scopus Google Scholar), potential for SH2 domain-containing it has been shown that tyrosine-phosphorylated CD19 as a specific and for the of phosphatidylinositol 3-kinase S.P. Science. 1993; PubMed Scopus Google Scholar). that CD22 specifically HCP provides for this of molecules to have a have the capacity to with the BCR at the in the of antigen Noesel C.J.M. A.C. van Lier R.A.W. Immunol. Today. 1993; Full Text PDF PubMed Scopus Google Scholar, C.L. Sidorenko Clark E.A. Immunol. Today. 1994; 15: Full Text PDF PubMed Scopus Google Scholar). In provide the BCR with molecular to to specific intracellular activation 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