The Src-homology domain 2 (SH2)-containing cytoplasmic tyrosine phosphatase, SHP-1 (SH2-containing protein tyrosine phosphatase-1), interacts with several B cell surface and intracellular signal transduction molecules through its SH2 domains. Mice with the motheaten and viable motheaten mutations are deficient in SHP-1 and lack most mature B cells. To define the role of SHP-1 in mature B cells, we expressed phosphatase-inactive SHP-1 (C453S) in a mature B cell lymphoma line. SHP-1 (C453S) retains the ability to bind to both substrates and appropriate tyrosine-phosphorylated proteins and therefore can compete with the endogenous wild-type enzyme. We found that B cells expressing SHP-1 (C453S) demonstrated enhanced and prolonged tyrosine phosphorylation of proteins with molecular masses of 110, 70, and 55–60 kDa after stimulation with anti-mouse IgG. The tyrosine kinase Syk was hyperphosphorylated and hyperactive in B cells expressing SHP-1 (C453S). SHP-1 and Syk were coimmunoprecipitated from wild-type K46 cells, K46 SHP-1 (C453S) cells, and splenic B cells, and SHP-1 dephosphorylated Syk. Cells expressing SHP-1 (C453S) showed increased Ca2+ mobilization, extracellular signal-regulated kinase activation, and homotypic adhesion after B cell Ag receptor engagement. Thus, SHP-1 regulates multiple early and late events in B lymphocyte activation.

Bcell development and activation are regulated by tyrosine phosphorylation. B cell Ag receptor (BCR)3 engagement by Ag or cross-linking Ab results in the activation of src-family tyrosine kinases including p53/56lyn, p59fyn, and p55blk1, 2 and Bruton’s tyrosine kinase (Btk) 1, 3 The Ig-associated membrane proteins, Igα and Igβ, are phosphorylated at tyrosine residues within their immunoreceptor tyrosine-based activation motifs (ITAMs) 2 . These serve as a docking site for the Src-homology domain 2 (SH2) domains of p72syk (Syk), thereby localizing and activating the Syk tyrosine kinase 4, 5 . Tyrosine phosphorylation, by one or more of these kinases, may regulate downstream signaling by phosphatidylinositol-3 kinase, phospholipase C-γ1 and 2, and the Ras-Raf-MAP kinase pathway 2 . The balance of tyrosine phosphorylation by protein tyrosine kinases and protein tyrosine phosphatases (PTPases) is essential throughout B cell development. Mice lacking one or more hematopoietic tyrosine kinase or PTPase are severely impaired in B cell differentiation and function 3, 6, 7 

The SH2-containing protein tyrosine phosphatase, SHP-1 (SH2-containing protein tyrosine phosphatase-1), is a cytoplasmic PTPase with two amino-terminal SH2 domains and is expressed predominantly in cells of hematopoietic origin 8 . SHP-1-deficient mice (motheaten or viable motheaten (mev)) suffer from hematological, immunological, and inflammatory abnormalities 9, 10, 11, 12, 13 . Various levels of SHP-1 have been observed in the B cell-rich areas of the germinal center, suggesting a role for SHP-1 in the critical proliferative, differentiation, and selective events that occur there 14 . However, the B cell defect in SHP-1-deficient mice is first evident much earlier in B cell differentiation and may be at least partially due to selective pressures imposed by inflammatory bone marrow macrophages or other SHP-1-deficient cell types 7 . The use of bone marrow chimeric animals demonstrates that SHP-1-deficient (mev) B cells are altered in both development and activation, with skewing toward the development of B-1 B cells, down-regulation of the BCR, and increased Ca2+ mobilization after BCR engagement 7 .

SHP-1 associates with the BCR in resting B cells and dissociates rapidly after BCR stimulation 15 . SHP-1 may also regulate B cell activation by inducible associations with other transmembrane molecules such as CD22 16, 17, 18 and, possibly, FcγRIIB1 19 . Furthermore, SHP-1 is reported to associate with cytoplasmic signaling molecules including Vav, Grb2, mSos, and SLP-76 20, 21 . Tyrosine-phosphorylated peptide sequences, termed immunoreceptor tyrosine-based inhibitory motifs (ITIMs), can bind to the SH2 domains of SHP-1 and activate SHP-1 catalytic activity 22 . ITIMs with a consensus sequence of (I/V)X(p)YXXL have been identified in FcγRIIB1, CD22, the NK cell inhibitory receptor, and the erythropoietin (EPO) and IL-3 receptors 22, 23 . ITIMs may target SHP-1 catalytic activity to nearby phosphotyrosine residues. SHP-1 associated with the tyrosine-phosphorylated EPO receptor may directly dephosphorylate and inactivate the tyrosine kinase Jak2 24, 25 Similarly, constitutive association of SHP-1 with the αβ IFN receptor may permit SHP-1 to regulate the activity of Jak1 and/or Stat1α 26 . It has recently been demonstrated that SHP-1 can dephosphorylate and inactivate the tyrosine kinase ZAP-70 27 . These data demonstrate that SHP-1 may regulate a variety of responses in T cells, B cells, NK cells, and erythroid precursors by dephosphorylating signaling molecules associated with membrane receptors. However, other enzymes including the related PTPase, SHP-2, and the polyphosphate inositol phosphatase, SHIP (SH2-containing inositol phosphatase), can also bind to ITIM sequences on inhibitory receptors 28, 29, 30 . The presence of an ITIM sequence does not by itself indicate that a receptor’s function is mediated by SHP-1.

To better define the role of SHP-1 in the activation of mature B cells, we have expressed a catalytically inactive form of SHP-1 in K46, a murine B cell line with a mature (membrane IgG) phenotype 31 . Our results indicate that SHP-1 affects proximal and late events in BCR signaling and identify several molecules in which the tyrosine phosphorylation state is affected by SHP-1.

The murine K46 B lymphoma cell line (IgG2a, κ) 31 was a gift of Dr. L. Justement (University of Alabama, Birmingham, AL) and was maintained in Iscove’s modified DMEM, supplemented with 10% heat-inactivated FCS (HyClone, Logan, UT), 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml gentamicin, and 1 mM sodium pyruvate. Transfected clones were selected and grown continuously in 1.25 mg/ml G418 (Life Technologies, Grand Island, NY). Splenocytes were prepared from C57BL/6 mice.

A cysteine to serine substitution (C453S) in the active site of SHP-1 that ablates catalytic activity was previously described 32 . A c-myc epitope was appended by PCR to the C terminus to distinguish overexpressed SHP-1 (C453S) from endogenous SHP-1. The construct was cloned into the expression vector BSRαEN 33 . SHP-1 (C453S) BSRαEN or control vector was electroporated into K46 cells 31 . Expression of transfected SHP-1 (C453S) was confirmed by immunoblotting for the overexpressed SHP-1 and for the c-myc epitope (Fig. 1,A). Clones with high levels of SHP-1 (C453S) expression and unchanged levels of membrane Ig expression (Fig. 1 B) were chosen for further analysis.

Rabbit anti-mouse SHP-1 antisera were developed by immunization with either the purified SH2 or catalytic domains of murine SHP-1. Rabbit anti-Syk, specific for residues 260–370, was a gift of Dr. J. Bolen (DNAX, Palo Alto, CA; 34 ; anti-Syk antiserum recognizing the 28 C-terminal residues of Syk was a gift of Dr. R. Geahlen (Purdue University, West Lafayette, IN; 35 . Rabbit antisera for mouse Syk and horseradish peroxidase (HRP)-conjugated 4G10 antiphosphotyrosine were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-active MAP kinase rabbit antiserum was purchased from Promega (Madison, WI). FITC-conjugated goat anti-mouse IgG was purchased from Jackson ImmunoResearch (West Grove, PA).

K46 cells were resuspended at 5 × 106 to 2 × 107/ml in PBS for experiments. Stimulation conditions were as reported by others 35, 36 . Cells were stimulated at 37°C with 0.1–30 μg/ml goat anti-mouse IgG (Jackson ImmunoResearch) for 0.5–30 min, as indicated. The standard stimulation conditions were 10 μg/ml Ab for 5 min. For assays of extracellular signal-regulated kinase (ERK) activation, cells were stimulated for 2 min with 20 μg/ml goat anti-mouse IgG as described 37, 38 . After stimulation, cells were rapidly pelleted at 4°C and lysed in ice-cold Nonidet P-40 lysis buffer unless otherwise indicated. Nonidet P-40 lysis buffer contained 150 mM NaCl, 1% Nonidet P-40, and 50 mM Tris-HCl (pH 8.0). RIPA lysis buffer contained 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, and 50 mM Tris-HCl (pH 8.0). Lysis buffers were supplemented with 21 μg/ml aprotinin, 2 mM leupeptin, 1 mM phenylmethylsulfonylfluoride, 10 μg/ml soybean trypsin inhibitor, 5 mM iodoacetamide, 0.4 mM sodium orthovanadate, and 10 mM sodium fluoride (all inhibitors purchased from Sigma, St. Louis, MO). SHP-1 and Syk immunoprecipitations were performed in the presence of 1 mg/ml chicken OVA to reduce background. Normal murine C57BL/6 splenocytes were brought to 108/ml in PBS and stimulated with 5 mM pervanadate as described 28 .

Lysates were precleared on ice with Pansorbin cells (Calbiochem, San Diego, CA). Equal amounts of protein as determined by bicinchoninic acid (BCA) assay (Pierce, Rockford, IL) were analyzed by SDS-PAGE and immunoblotting. Rabbit antisera were immunoprecipitated with protein A-conjugated Sepharose beads (Sigma) and detected with HRP-conjugated protein A (Boehringer Mannheim, Indianapolis, IN). mAbs were precipitated with protein G-conjugated Sepharose beads (Boehringer Mannheim) and detected with HRP-conjugated goat anti-mouse IgG (Caltag, South San Francisco, CA).

Prewarmed cells (2 × 107/ml) were stimulated with 1 μg/ml avidin with or without 10 μg/ml biotinylated goat anti-mouse IgG (Jackson ImmunoResearch) for 2 min at 37°C. Cells were pelleted and lysed with ice-cold Nonidet P-40 lysis buffer containing 5 mM EDTA, 1 mg/ml OVA, 4 mM leupeptin, 1 μM pepstatin A, 10 μg/ml soybean trypsin inhibitor, 10 mM sodium fluoride, 10 mM sodium molybdate, and 200 μM sodium vanadate. After washing Syk immunoprecipitations three times in lysis buffer, the immunoprecipitations were split: one half was used for an immunoblot to control for loading and the other half was used in a Syk kinase assay. The Syk kinase assay was performed as previously described 39 . Briefly, immunoprecipitates were washed once in 10 mM Tris (pH 7.4), 0.5 M LiCl, and twice in kinase buffer (10 mM Tris (pH 7.4), 10 mM MgCl2). Kinase assays were then performed in 25 μl of kinase buffer supplemented with 10 μCi [γ-32P]ATP and 1 μg glutathione S-transferase (GST)-Band 3 (produced as described in 40 . Kinase assays were incubated at room temperature for 5 min and stopped by the addition of Laemmli loading buffer and boiling for 5 min. After separation by SDS-PAGE gel, proteins were transferred to nitrocellulose and exposed for autoradiography and PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA).

A fusion protein of GST-Syk was expressed in Sf9 insect cells in the presence or absence of SHP-1. Sf9 cells were lysed in lysis buffer, and cell lysates were tumbled with glutathione agarose for 1 h. Agarose beads were washed three times in lysis buffer, and GST-Syk was eluted by boiling in SDS-PAGE sample buffer. After SDS-PAGE, samples were immunoblotted sequentially with anti-phosphotyrosine and anti-Syk.

Calcium mobilization was studied with the calcium-sensitive dyes, Fluo-3 AM and Fura Red AM (Molecular Probes, Eugene, OR), as described 41 . Briefly, cells (5 × 106/ml) were loaded with 3 μM Fluo 3 AM and 6 μM Fura Red AM for 30 min at 30°C. Labeled cells were washed and resuspended at 1–2 × 106 cells/ml in Iscove’s modified DMEM supplemented with 10% FCS. Cells were prewarmed to 37°C before analysis on a FACSCaliber (Becton Dickinson Immunocytometry Systems, San Jose, CA). Fluo-3 and Fura Red fluorescence data were collected over time from viable cells, selected on forward and orthogonal scatter profile. Cells were stimulated with 3, 10, or 30 μg/ml goat anti-mouse IgG, as indicated. Ratiometric data were analyzed and graphed using FlowJo software (Tree Star, San Carlos, CA).

K46 or K46 SHP-1 (C453S) were cultured at 104 cells/well in a final volume of 200 μl/well in Corning/Costar (Corning, NY) tissue culture-treated 96-well flat-bottom trays. Cells were unstimulated or stimulated with goat anti-mouse IgG (0.1–10 μg/ml) at 37°C for 18–20 h. Images of undisturbed cultures were acquired on an inverted microscope with a cooled charged-coupled device (CCD) camera (Photometrics, Tucson, AZ) using IP Lab software (Signal Analytics, Vienna, VA).

The public domain image analysis program, NIH Image, was used for densitometric analysis of immunoblots and for measurement of B cell clusters. NIH Image was developed at the National Institutes of Health and is available on the Internet (http://rsb.info.nih.gov/nih-image/).

To define the role of SHP-1 in the activation of mature B cells, we overexpressed SHP-1 (C453S) in K46 B lymphoma cells (Fig. 1). A single amino acid substitution in the active site of SHP-1 (C453S) ablates phosphatase activity 32 . This enzymatically dead form of SHP-1 is incapable of dephosphorylating ITIM sequences and binds to ITIM sequences for significantly longer periods of time, preventing the endogenous phosphatase from inhibiting signal transduction (Julie Blasioli and M.L.T., unpublished data). Therefore, it is likely that this mutation functions as an efficient dominant-negative mutation. SHP-1 (C453S) was modified with a C-terminal c-myc epitope. An expression construct encoding the c-myc epitope tagged SHP-1 (C453S) was electroporated into K46 cells. Expression of transfected SHP-1 (C453S) was confirmed by immunoblotting for the overexpressed SHP-1 and for the c-myc epitope (Fig. 1,A). Clones with SHP-1 (C453S) expression 2- to 3-fold over endogenous SHP-1 levels plus unchanged levels of membrane Ig expression (Fig. 1 B) were chosen for further analysis.

We tested the effects of SHP-1 (C453S) overexpression on protein tyrosine phosphorylation in B cells stimulated by anti-BCR Ab. K46 B cells (5 × 106–107/ml) were activated with intact goat anti-mouse IgG for varying times at 37°C, and equal amounts of total cell lysate protein were analyzed for tyrosine phosphorylation. When compared with K46 cells, K46 (C453S) cells demonstrated increased protein tyrosine phosphorylation, evident at many doses of BCR stimulation (Fig. 2,A). This increase in phosphotyrosine content was observed within 30–60 s after stimulation and persisted for at least 10 min (Fig. 2,B). Some proteins remained hyperphosphorylated for at least 30 min. Some proteins were hyperphosphorylated even in unstimulated cells expressing SHP-1 (C453S) (Fig. 2, A and B). The degree of hyperphosphorylation correlated with the level of expression of SHP-1 (C453S) (Fig. 2 B and data not shown). Tyrosine phosphorylation of 55- to 60-kDa, 70-kDa, and 110-kDa proteins was increased at all doses of stimulus and at all time points. SHP-1 (C453S) expression also resulted in increased tyrosine phosphorylation of a similar set of protein bands in K46 cells stimulated with pervanadate (data not shown). These proteins may show increased tyrosine phosphorylation due to increased kinase activity or decreased dephosphorylation by SHP-1. Alternatively, these proteins may be protected from other phosphatases by binding the SH2 domains of SHP-1 (C453S). To examine this issue, we analyzed the association of Syk with SHP-1.

Although SHP-1 is reported to interact with a number of cell-surface and cytoplasmic signaling proteins, few have been directly shown to be targets for dephosphorylation by SHP-1. SHP-1 has been shown to regulate ZAP-70 and certain JAK tyrosine kinases 24, 26, 27 . Therefore, we examined the phosphorylation state of the ZAP-70-related tyrosine kinase, Syk, in two K46 clones overexpressing SHP-1 (C453S). Syk immunoprecipitates were resolved by SDS-PAGE and probed with anti-phosphotyrosine (Fig. 3,A, top) and anti-Syk (Fig. 3,A, bottom). Syk, immunoprecipitated from SHP-1 (C453S) mutant cells, demonstrated increased tyrosine phosphorylation compared with that from control K46 cells. When corrected for the amount of Syk protein in the anti-Syk immunoblot (Fig. 3,A, bottom), tyrosine phosphorylation was 4.5- to 12-fold higher in Syk immunoprecipitates from activated SHP-1 (C453S) mutant cells than from control cells (Fig. 3,A). Immunoprecipitation kinases assays were performed to determine whether the increase in Syk tyrosine phosphorylation resulted in altered catalytic activity. Syk tyrosine kinase activity was increased in both resting cells and cells stimulated with anti-IgG (Fig. 3 B).

SHP-1 associates with and regulates ZAP-70 in activated T cells 27 . To examine whether SHP-1 associates with Syk in B cells, Syk was immunoprecipitated from either K46 or K46 SHP-1 (C453S) cells and immunoblot analysis was performed (Fig. 4, A and B). SHP-1 was detected in Syk immunoprecipitates from resting K46 cells as well as from activated cells (Fig. 4,A). This result was observed with two different anti-Syk antisera, directed against the unique interdomain region of Syk 34 or the C-terminal 28 amino acids of Syk 35 . Surprisingly, SHP-1 coimmunoprecipitated with Syk from either resting or activated K46 cells (Fig. 4,B). This result was observed both with an anti-SHP-1 antiserum directed against the two SH2 domains of SHP-1 and with one directed against the catalytic domain of SHP-1. SHP-1 association with Syk was not substantially altered in the lysates of cells expressing SHP-1 (C453S). In resting K46 cells, Syk does not appear to be tyrosine phosphorylated. Therefore, this result suggests that the interaction between Syk and SHP-1 may not be phosphotyrosine-dependent in this instance. Because the constitutive association of Syk and SHP-1 was unexpected, we decided to examine whether these two enzymes were constitutively associated in primary B cells. In contrast, coimmunoprecipitation of SHP-1 with Syk immunoprecipitation from murine splenic B cells requires stimulation (Fig. 4 C), demonstrating that the association is inducible. The molecular basis for the constitutive association in K46 cells and inducible association in splenic B cells is unknown. However, it is possible that the transformed state of K46 results in increased basal activation, potentially affecting the steady-state association among Syk, SHP-1, and other proteins. One potential mechanism by which Syk and SHP-1 could associate is through the formation of a tri-molecular complex with CD22. However, coexpression of Syk, SHP-1, and CD22 in HeLa cells did not result in the formation of a tri-molecular complex despite appropriate phosphorylation of CD22 and the association with SHP-1 (D.R.P., Silke Paust, and M.L.T., unpublished data). We also could not demonstrate a direct association of SHP-1 and Syk in vitro or by coexpression. Thus, the kinase and phosphatase may associate through a novel mechanism.

That SHP-1 and Syk associate in B cells suggests that as reported for SHP-1 and ZAP-70 27 , these enzymes regulate each other. To confirm that the association SHP-1 and Syk is physiologically significant, we examined whether SHP-1 could dephosphorylate Syk. A GST-Syk fusion protein was expressed in Sf9 cells in the presence or absence of SHP-1. GST-Syk purified from cells expressing SHP-1 was decreased in phosphorylation when compared with GST-Syk purified from cells that did not express SHP-1 (Fig. 4 D). Furthermore, phosphorylated recombinant Syk protein stimulated SHP-1 phosphatase activity in vitro (data not shown).

BCR-stimulated Ca2+ mobilization and ERK phosphorylation were measured to assess later changes in signal transduction. Compared with parental cells, K46 cells expressing SHP-1 (C453S) demonstrated more pronounced increases in cytoplasmic Ca2+ after stimulation with all concentrations of anti-BCR Abs examined (Fig. 5). The kinetics of Ca2+ mobilization were accelerated and the fraction of responding cells was increased among K46 cells expressing SHP-1 (C453S). Similarly, BCR cross-linking resulted in greater MAP kinase activation as measured by ERK phosphorylation in cells expressing SHP-1 (C453S) (Fig. 6). Active ERK was not detected in either resting wild-type K46 cells or resting SHP-1 (C453S) transfectants. However, when cells were treated with goat anti-mouse IgG, active ERK levels were an average (n = 3) of 2.8-fold higher in K46 cells expressing SHP-1 (C453S). These results support the idea that SHP-1 (C453S) affects early events in the BCR signal transduction cascade such as Syk activation.

The experiments detailed above show that SHP-1 (C453S) alters protein tyrosine phosphorylation in K46 B cells during the first 30 min after stimulation of the BCR. Because the transformed cell lines used in this assay proliferate regardless of stimulation, it is not practical to measure the effects of dominant-negative SHP-1 on BCR-stimulated proliferation. Adhesion was measure as a parameter of late changes in activation. SHP-1 (C453S) permitted a dramatic increase in homotypic adhesion of BCR-stimulated cells after overnight stimulation with anti-BCR Abs (Fig. 7). Although control K46 cells formed small aggregates after overnight stimulation (Fig. 7,B), cells expressing SHP-1 (C453S) formed aggregates 3.5–7 times larger (Fig. 7 D). In both control and mutant cells, aggregation was increased as the dose of goat anti-mouse IgG was increased from 0.1 to 10 μg/ml. Mutant cells showed enhanced aggregation compared with control cells at all doses of stimulus tested.

SHP-1 plays a central regulatory role in B lymphocyte development and activation. SHP-1 may regulate the threshold for B cell activation through its association with the BCR in unstimulated B cells 15 . After BCR engagement, SHP-1 is recruited to CD22 17, 18, 42 and FcγRIIB1 19 and may contribute to the regulation of B cell activation by these membrane receptors. B cell differentiation is severely impaired in the absence of SHP-1 activity 7 . Motheaten B cells demonstrate increased B cell activation, as measured by Ca2+ mobilization 7 and proliferation 15 . It is possible that the B-1 lymphocytes that preferentially survive in the motheaten mouse have a different activation program. To separate the role of SHP-1 in B cell activation from its role in B lymphocyte differentiation, it is essential to interrupt its function specifically in mature B cells without subjecting these cells to the developmental abnormalities of the motheaten mouse.

We examined the effects of dominant-negative SHP-1 on activation of the B lymphoma line, K46, by anti-BCR Abs. The catalytically inactive SHP-1 (C453S) can compete with endogenous wild-type SHP-1 for association with other signaling molecules and for access to substrates and thus can serve as a dominant-negative mutation. Our results demonstrate that expression of dominant-negative SHP-1 in mature B cells causes increased tyrosine phosphorylation of a number of proteins after BCR engagement (Fig. 2). These proteins include the tyrosine kinase Syk (Fig. 3), which is a substrate for dephosphorylation by SHP-1 (Fig. 4,D). We also observed an association of SHP-1 with Syk in splenic B cells and in wild-type or SHP-1 (C453S) K46 cells (Fig. 4). That SHP-1 (C453S) altered early tyrosine kinase activity is supported by the increased changes in early downstream events in BCR signal transduction, such as Ca2+ mobilization (Fig. 5) and ERK phosphorylation (Fig. 6). Longer-term events are also affected by SHP-1. A full day after stimulation by goat anti-mouse IgG, K46 cells expressing SHP-1 (C453S) show increased homotypic adhesion (Fig. 7). Recent data demonstrate that CD45 regulates integrin-mediated adhesion in lymphocytes 43, 44 and macrophages 45 . Together, these results point to an emerging role for protein tyrosine phosphatases in the regulation of lymphocyte adhesion.

These results support the hypothesis that Syk is negatively regulated by SHP-1 in B cells. K46 B cells expressing SHP-1 (C453S) showed increased Syk tyrosine phosphorylation and tyrosine kinase activity (Fig. 3). In addition, Syk was dephosphorylated when coexpressed with SHP-1 in insect cells (Fig. 4,D). This finding adds Syk to the growing list of receptor-associated tyrosine kinases that are regulated by SHP-1 24, 25, 27, 46 . Furthermore, we found that SHP-1 and Syk associate physically both in wild-type K46 B cells and in K46 expressing SHP-1 (C453S) (Fig. 4). A complex containing both SHP-1 and Syk could be immunoprecipitated from K46 cells with various antisera specific for either enzyme. This complex likely contained a relatively small fraction of each enzyme in K46 B cells, and was most clearly identified when SHP-1 or Syk was immunoprecipitated from limiting numbers of cells. SHP-1 may interact with Syk by a mechanism independent of the binding of the SH2 domains of SHP-1 to phosphorylated tyrosine residues in Syk. Thus, BCR engagement and subsequent Syk tyrosine phosphorylation did not alter Syk and SHP-1 association in K46 B cells. This finding is consistent with the observation that a functional interaction between SHP-1 and Jak2 requires neither functional SHP-1 SH2 domains, nor tyrosine phosphorylation of Jak2 25 . In contrast, the association was increased by pervanadate stimulation in splenic B cells. The association may be mediated by an adapter protein expressed in B cells, but not other cell types (Fig. 4; D.R.P. and M.L.T., unpublished data). The SH2 domains of SHP-1 may bind to regulatory ITIMs while other interactions mediate a direct association between SHP-1 and its substrates.

These results support a model in which SHP-1 is recruited to tyrosine-phosphorylated transmembrane receptors, then acts to regulate the tyrosine phosphorylation of signal transduction molecules associated with or downstream of the transmembrane receptors. If SHP-1 dephosphorylates and inactivates signaling molecules associated with CD19 or CD22, this could reduce the tyrosine phosphorylation of the transmembrane receptors indirectly by inactivating those tyrosine kinases that are responsible for phosphorylating them. Evidence for such an indirect mechanism has been reported in SHP-1 regulation of signal transduction through the EPO receptor 25 . Similarly, SHP-1 recruited to the phosphorylated NK cell inhibitory may directly dephosphorylate downstream molecules including ZAP-70 and phospholipase C-γ2 47 

These results do not rule out SHP-1 in the regulation of other tyrosine kinases. Src-family tyrosine kinases including Lyn are differentially phosphorylated in cells expressing SHP-1 C453S (Fig. 2; L.B.D., Y.T.H., and M.L.T., unpublished data). We have not seen evidence for changes in phosphorylation of the tyrosine kinase Btk in SHP-1 (C453S) expressing cells, because the 77-kDa Btk does not comigrate with any of the differentially phosphorylated bands observed in Fig. 2 (data not shown). The possibility that SHP-1 and Btk may not act on the same pathways is suggested by the fact that Btk mutant mice lack B-1 B cells 3 , a subset that is selectively retained in mice with the SHP-1 mutations motheaten and viable motheaten7 .

At least four different mechanisms could account for the increased tyrosine phosphorylation of specific cellular proteins in B cells expressing catalytically inactive SHP-1. Some of these proteins may be targets for dephosphorylation by SHP-1. Syk is a candidate for such direct regulation by virtue of its association with SHP-1 in vivo, its dephosphorylation in cells coexpressing SHP-1, and because SHP-1 dephosphorylates the related kinase, ZAP-70 27 . Second, the kinases that phosphorylate these proteins may be targets for regulation by SHP-1. In this regard, SHP-1 selectively regulates the level of EPO receptor tyrosine phosphorylation by Jak2, an enzyme regulated by SHP-1, but not by c-Fes, an enzyme not known to be regulated by SHP-1 25 . Third, catalytically inactive SHP-1, expressed in excess, could bind to phosphotyrosine via its SH2 domains, denying access to phosphorylated substrates by endogenous SHP-1 as well as other endogenous phosphatases. Finally, the active site of catalytically inactive SHP-1 may stably bind to substrates and prevent endogenous, functional phosphatases from dephosphorylating these substrates. In these studies, we allowed SHP-1 and other phosphatases to dephosphorylate their substrates in intact cells before lysis. This maintains normal cellular structures and protein-protein interactions, thereby permitting SHP-1 to act on its normal substrates. SHP-1 may exert its regulatory effects on B cell development and activation by changing the threshold for activation through these receptors and nonreceptor tyrosine kinases.

We thank Drs. J. Bolen, A. Chan, and R. Gaehlen for generous gifts of reagents. We thank Dr. M. Dustin for critical reading of the manuscript and for assistance with microscopy.

1

This work was supported in part by National Institutes of Health Grant R01GM56455 and by the Humans Frontiers Science Program. L.B.D. was supported by National Institutes of Health Training Grant 5T32 AI07163. M.L.T. and A.C.C. are investigators of the Howard Hughes Medical Institute.

3

Abbreviations used in this paper: BCR, B cell Ag receptor; Btk, Bruton’s tyrosine kinase; EPO, erythropoietin; GST, glutathione S-transferase; HRP, horseradish peroxidase; ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-base inhibitory motif; mev, viable motheaten, PTPase, protein tyrosine phosphatase; SH2, Src-homology domain 2; SHP-1, SH2-containing protein tyrosine phosphatase-1; Syk, p72syk; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein.

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