(Received for publication, March 13, 1995; and in revised form, May 9, 1995)
From the
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.
Antigen receptor-mediated B cell activation critically depends
on the regulated activities of both protein tyrosine kinases and
protein tyrosine phosphatases. Early after BCR ()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.
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.
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.
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 -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
-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.
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).