©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Hematopoietic Cell Phosphatase Is Recruited to CD22 following B Cell Antigen Receptor Ligation (*)

(Received for publication, March 13, 1995; and in revised form, May 9, 1995)

Arjan C. Lankester (§) Gijs M. W. van Schijndel René A. W. van Lier

From the Central Laboratory of the Blood Transfusion Service of The Netherlands Red Cross and Laboratory for Experimental and Clinical Immunology, University of Amsterdam, Amsterdam, The Netherlands

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Antigen receptor-mediated B cell activation critically depends on the regulated activities of both protein tyrosine kinases and protein tyrosine phosphatases. Early after BCR (^1)cross-linking a large number of cellular proteins become phosphorylated on tyrosine residues(1) . This change in phosphorylation status of cellular proteins has two potential consequences. First, it may alter the enzymatic activity of certain proteins (e.g. PLC (2) ). 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(3) . 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(4, 5) and the ZAP70-related PTK syk(6, 7, 8, 9) .

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(10) . The recent observation that CD45 may be physically associated with the BCR supports this notion(11) . 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) (12) and Syp (protein tyrosine phosphatase 1D)(13) . 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(14, 15) . Interestingly, Motheaten mice and viable Motheaten mice, which do not express or express aberrant forms of HCP protein, respectively(16, 17) , 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(18, 19) . Clinically, Motheaten mice suffer from severe autoimmune diseases and severe combined immunodeficiency syndromes(20) . 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 METHODS

Cells

The 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 (21, 22) .

Antibodies

The 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 H chain mAb (TA-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 Blotting

Intact 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(22) . 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(23) . 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 Activation

The cells were washed twice in Hepes solution (132 mM NaCl, 6 mM KCl, 1 mM MgSO(4), 1 mM CaCl(2), 1.2 mM K(2)HPO(4), 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 Fractionation

Following 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(24) .


RESULTS AND DISCUSSION

BCR Cross-linking Induces Tyrosine Phosphorylation of HCP and the Formation of a Multimolecular HCP Complex

Previous reports have indicated that HCP may serve as substrate for src family protein tyrosine kinase(25, 26) . 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 (◂) 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).


Figure 1: BCR cross-linking induces tyrosine phosphorylation of HCP and the formation of a multimolecular HCP complex. Following incubation with or without 5 µg/ml biotinylated µH chain mAb (CLB MH-15), Daudi cells were stimulated for 3 min in Hepes medium containing streptavidin. Subsequently, cells were lysed in Nonidet P-40 immunoprecipitation buffer, and after preclearing, the HCP proteins were isolated with HCP antibodies. The immunoprecipitates were separated on SDS-PAGE, transferred to nitrocellulose membranes, probed with phosphotyrosine mAb (RC20, upper panel), and visualized by enhanced chemiluminescence. The symbols represent: HCP (arrow), phosphoprotein 68-70 (arrow*), and phosphoprotein 130-135 (◂). Subsequently, the membrane was reprobed with HCP antibodies (lower panel). Four separate experiments gave similar results.



Subcellular Distribution of Activation-induced HCP Complexes

Several studies have demonstrated that HCP interacts with tyrosine-phosphorylated transmembrane receptors in an activation-dependent manner(14, 15, 27) . 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(22, 28) .


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.



Tyrosine-phosphorylated CD22 Acts as Membrane Target for HCP

In 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, ◂), 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(29, 30) . 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)(31) . 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(32) . 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.




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.



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(27, 29) . 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 beta-chain, c-fms and c-kit in vitro provides a precedent for this possibility(14, 15) . 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 beta-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(18, 20, 33) . 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(34) . Several studies have reported structural and functional differences between the BCR in CD5 and conventional B cells, respectively(23, 35, 36, 37, 38) . 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 (39) 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(23) , CD19(40) , and CD22(31) , 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 (40) . 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(41, 42) . In addition, they provide the BCR with molecular substrates to couple to specific intracellular activation pathways.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Central Laboratory of the Blood Transfusion Service of The Netherlands Red Cross, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands. Tel.: 205123275; Fax: 205123110.

(^1)
The abbreviations used are: BCR, B cell antigen receptor; HCP, hematopoietic cell phosphatase; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; IL, interleukin; CLB, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service.


ACKNOWLEDGEMENTS

We thank Prof. D. Roos and Dr. A. J. Verhoeven for critical reading of manuscript.

Note Added in Proof-After acceptance of the manuscript, similar data have been reported by Campbell, M. A., and Klinman, N. R. (1995) Eur. J. Immunol.25, 1573-1579 and Doody et al. (Doody, G. M., Justement, L. B., Delibrias, C. C., Matthews, R. J., Lin, J., Thomas, M. L., and Fearon, D. T.(1995) 269, 242-244).


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.