Activation Syk Tyrosine Kinase Activity and B Cell Tyrosine ...

Expression of Dominant-Negative Src-Homology Domain 2-Containing Protein Tyrosine Phosphatase-1 Results in Increased Syk Tyrosine Kinase Activity and B Cell Activation1 Lynn B. Dustin,*† David R. Plas,* Jane Wong,*‡ Yonghong Tammy Hu,† Carlos Soto,† Andrew C. Chan,*‡ and Matthew L. Thomas2* 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 Ca21 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. The Journal of Immunology, 1999, 162: 2717–2724.

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cell 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 p55blk (1, 2) and Bruton’s tyrosine kinase (Btk) (1, 3) The Ig-associated membrane proteins, Iga and Igb, are phosphorylated at tyrosine residues within their immunoreceptor tyrosine-based activation motifs (ITAMs) (2). These serve as a docking site for the Srchomology 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-g1 and 2, and the Ras-Raf-MAP kinase pathway (2). The balance of tyrosine phosphorylation by protein tyrosine kinases Departments of *Pathology and Molecular Microbiology and ‡Medicine, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO 63110; and †Department of Molecular Microbiology and Immunology, St. Louis University School of Medicine, St. Louis, MO 63104 Received for publication June 15, 1998. Accepted for publication November 30, 1998. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 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. 2 Address correspondence and reprint requests to Dr. Matthew L. Thomas, Howard Hughes Medical Institute, Department of Pathology, Washington University School of Medicine, 660 S. Euclid Ave., Box 8118, St. Louis, MO 63110. E-mail address: [email protected] 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.

Copyright © 1999 by The American Association of Immunologists

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 (SH2containing 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 – 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 SHP1-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 Ca21 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 –18) and, possibly, FcgRIIB1 (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 FcgRIIB1, CD22, the NK cell inhibitory receptor, and the erythropoietin (EPO) and IL-3 receptors (22, 23). ITIMs may 0022-1767/99/$02.00

2718 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 ab IFN receptor may permit SHP-1 to regulate the activity of Jak1 and/or Stat1a (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 (SH2containing inositol phosphatase), can also bind to ITIM sequences on inhibitory receptors (28 –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.

Materials and Methods Cells The murine K46 B lymphoma cell line (IgG2a, k) (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 mg/ml streptomycin, 50 mg/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.

Construct and expression 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 BSRaEN (33). SHP-1 (C453S) BSRaEN 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. 1A). Clones with high levels of SHP-1 (C453S) expression and unchanged levels of membrane Ig expression (Fig. 1B) were chosen for further analysis.

Antibodies Rabbit anti-mouse SHP-1 antisera were developed by immunization with either the purified SH2 or catalytic domains of murine SHP-1. Rabbit antiSyk, specific for residues 260 –370, was a gift of Dr. J. Bolen (DNAX, Palo Alto, CA; Ref. 34); anti-Syk antiserum recognizing the 28 C-terminal residues of Syk was a gift of Dr. R. Geahlen (Purdue University, West Lafayette, IN; Ref. 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).

B cell stimulation K46 cells were resuspended at 5 3 106 to 2 3 107/ml in PBS for experiments. Stimulation conditions were as reported by others (35, 36). Cells were stimulated at 37°C with 0.1–30 mg/ml goat anti-mouse IgG (Jackson ImmunoResearch) for 0.5–30 min, as indicated. The standard stimulation conditions were 10 mg/ml Ab for 5 min. For assays of extracellular signalregulated kinase (ERK) activation, cells were stimulated for 2 min with 20 mg/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%

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FIGURE 1. Expression of SHP-1 (C453S) in K46 cells. A, Expression of SHP-1 (C453S) in transfected subclones. Total cell lysates were obtained from K46 cells and from representative clones transfected with SHP-1 (C453S), were normalized for protein content, resolved on SDSPAGE, and immunoblotted with rabbit antiserum specific for mouse SHP-1 or with 9E10, a mAb against the c-myc epitope tag. B, Membrane IgG expression on K46 cells, SHP-1 (C453S) clone 1, and SHP-1 (C453S) clone 2. Cells were stained with FITC-labeled goat anti-mouse IgG and analyzed by flow cytometry. SDS, and 50 mM Tris-HCl (pH 8.0). Lysis buffers were supplemented with 21 mg/ml aprotinin, 2 mM leupeptin, 1 mM phenylmethylsulfonylfluoride, 10 mg/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).

Immunoprecipitation and immunoblotting 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 Aconjugated 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).

Syk kinase assays Prewarmed cells (2 3 107/ml) were stimulated with 1 mg/ml avidin with or without 10 mg/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 mM pepstatin A, 10 mg/ml soybean trypsin inhibitor, 10 mM sodium fluoride, 10 mM sodium molybdate, and 200 mM 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 ml of kinase buffer supplemented with 10 mCi [g-32P]ATP and 1 mg glutathione S-transferase (GST)-Band 3 (produced as described in Ref. 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).

Analysis of Syk phosphorylation in vivo 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

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FIGURE 2. Increased tyrosine phosphorylation in K46 cells expressing SHP-1 (C453S). A, Dose-response changes in protein tyrosine phosphorylation in K46 cells (lanes labeled wt) and K46 cells expressing SHP-1 (C453S) (lanes labeled C453S) after BCR engagement. Cells were stimulated with the indicated concentrations of intact goat anti-mouse IgG at 37°C for 5 min. Stimulated cells were gently pelleted at 4°C and lysed in RIPA buffer. Total cell lysates normalized for protein content were subjected to SDSPAGE and immunoblotting with HRPcoupled 4G10 anti-phosphotyrosine Ab. B, Time course of changes in protein tyrosine phosphorylation in BCR-stimulated K46 or SHP-1 (C453S) cells. wt, K46 cells; C453S (1), clone 1; C453S (2), clone 2. Cells were stimulated with 10 mg/ml goat anti-mouse IgG at 37°C for the indicated times. Cells (t 5 0) were stimulated on ice for ,30 s. At the indicated times, cells were gently pelleted at 4°C and lysed in RIPA buffer. Cells were analyzed as described in A. The results shown are representative of eight experiments.

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 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 3 106/ml) were loaded with 3 mM Fluo 3 AM and 6 mM Fura Red AM for 30 min at 30°C. Labeled cells were washed and resuspended at 1–2 3 106 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 mg/ml goat anti-mouse IgG, as indicated. Ratiometric data were analyzed and graphed using FlowJo software (Tree Star, San Carlos, CA).

(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. 1A). Clones with SHP-1 (C453S) expression 2to 3-fold over endogenous SHP-1 levels plus unchanged levels of membrane Ig expression (Fig. 1B) were chosen for further analysis.

Homotypic adhesion K46 or K46 SHP-1 (C453S) were cultured at 104 cells/well in a final volume of 200 ml/well in Corning/Costar (Corning, NY) tissue culturetreated 96-well flat-bottom trays. Cells were unstimulated or stimulated with goat anti-mouse IgG (0.1–10 mg/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).

Analysis 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/).

Results Overexpression of SHP-1 (C453S) in K46 B cells 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

Dominant-negative SHP-1 increases BCR-stimulated tyrosine phosphorylation 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 3 106–107/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. 2A). This increase in phosphotyrosine content was observed within 30 – 60 s after stimulation and persisted for at least 10 min (Fig. 2B). 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. 2B and data not shown). Tyrosine phosphorylation of 55- to 60-kDa,

2720

B CELL RECEPTOR NEGATIVE REGULATION precipitation 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. 3B). SHP-1 interacts with Syk in B cells

FIGURE 3. Expression of SHP-1 (C453S) increases Syk tyrosine phosphorylation and kinase activity. A, Hyperphosphorylation of Syk in SHP-1 (C453S) transfected cells. Syk was immunoprecipitated from K46 cells (wt) and from K46 cells expressing SHP-1 (C453S). Each lane shows Syk precipitated from 107 cells. The blot was probed with the antiphosphotyrosine mAb 4G10 (top), then stripped and reprobed with antiSyk antiserum (bottom). B, Kinase activity. Syk was immunoprecipitated from 2 3 107 resting or anti-IgG-stimulated cells, then assayed for kinase activity as described in Materials and Methods. Kinase reactions were divided in half and samples were evaluated for Band 3 phosphorylation by PhosphorImager analysis (top) and for Syk enzyme levels by immunoblotting (bottom).

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. Dominant-negative SHP-1 is associated with increased Syk tyrosine phosphorylation and kinase activity Although SHP-1 is reported to interact with a number of cellsurface 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. 3A, top) and anti-Syk (Fig. 3A, 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. 3A, bottom), tyrosine phosphorylation was 4.5to 12-fold higher in Syk immunoprecipitates from activated SHP-1 (C453S) mutant cells than from control cells (Fig. 3A). Immuno-

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. 4A). 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. 4B). 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. 4C), 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. Syk is a substrate for SHP-1 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. 4D). Furthermore, phosphorylated recombinant Syk protein stimulated SHP-1 phosphatase activity in vitro (data not shown). Downstream alterations in BCR signal transduction in cells expressing SHP-1 (C453S) BCR-stimulated Ca21 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 Ca21 after stimulation with all concentrations of anti-BCR Abs examined (Fig. 5). The kinetics of Ca21 mobilization were accelerated and

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FIGURE 4. SHP-1 and Syk associate in B cells. A, Syk immunoprecipitates analyzed for the presence of Syk (top) and SHP-1 (bottom). Syk was immunoprecipitated with antiserum specific for the interdomain region of Syk (residues 260 –370); NRS (normal rabbit serum immunoprecipitate). Cells were unstimulated (2) or were stimulated (1) with 10 mg/ml goat anti-mouse IgG for 5 min at 37°C. Immunoprecipitates from 5 3 106 cells/lane were resolved on SDS-PAGE and analyzed by immunoblotting with antiserum raised against full length Syk. The blot was stripped and reprobed with antiserum specific for the SH2 domains of SHP-1. B, SHP-1 immunoprecipitates were analyzed for the presence of Syk (top) and SHP-1 (bottom). Lysates of either unstimulated or anti-IgG-stimulated cells (107 cells/lane) were subjected to immunoprecipitation with antiserum specific for the SH2 domains of SHP-1. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted with antiserum specific for full length Syk. The blot was stripped and reprobed with antiserum specific for SHP-1. C, Association of SHP-1 and Syk in splenic B cells. Splenocytes were stimulated with 5 mM pervanadate or were left untreated. SHP-1 or Syk was immunoprecipitated from 2 3 107 cells, resolved on SDS-PAGE, and analyzed by immunoblotting with anti SHP-1, anti-Syk, or anti- phosphotyrosine Abs, as indicated. D, Effects of SHP-1 on Syk tyrosine phosphorylation. GST-Syk was expressed alone (lane 1) or with SHP-1 (lane 2) in Sf9 insect cells. Cells were lysed and GST-Syk was purified by binding to glutathione-Sepharose beads. GST-Syk was resolved on SDS-PAGE and immunoblotted sequentially for phosphotyrosine (top) and Syk (bottom).

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 5 3) of 2.8-fold higher in K46 cells expressing SHP-1 (C453S).

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FIGURE 6. Effects of SHP-1 (C453S) expression on MAP kinase activation. K46 cells (lane 1-2) or K46 cells expressing SHP-1 (C453S) (lanes 3-6) were unstimulated (2) or stimulated with 20 mg/ml goat anti-mouse IgG (1) for 2 min at 37°C. Stimulated cells were gently pelleted at 4°C and lysed. Total cell lysates, normalized for protein content, were subjected to SDS-PAGE and immunoblotted with anti-ACTIVE ERK antiserum (top). Total protein was visualized in a duplicate gel by Coomassie Brilliant Blue staining (bottom). Data are representative of three independent experiments. FIGURE 5. Expression of SHP-1 (C453S) alters Calcium mobilization. A–F, K46 cells (A–C) or K46 cells expressing SHP-1 (C453S) (D–F) were loaded with Ca21-sensitive fluorescent dyes, Fluo-3 and Fura Red (41). Cells were equilibrated at 37°C and immediately analyzed by flow cytometry. Data were collected from resting cells for the first 10 s. Cells were then stimulated by addition of goat anti-mouse IgG to a final concentration of 3 mg/ml (A and D), 10 mg/ml (B and E), or 30 mg/ml (C and F) and data were collected for an additional 90 s. Graphs show changes in the ratio of Fluo-3 to Fura Red fluorescence in response to increased cytoplasmic Ca21 in individual cells over time. G–I, Graphs show the percentage of cells with a Fluo-3/Fura Red ratio greater than that of 95% of resting cells when stimulated with (G) 3, (H) 10, or (I) 30 mg/ml goat anti-mouse IgG. Data are representative of five independent experiments.

These results support the idea that SHP-1 (C453S) affects early events in the BCR signal transduction cascade such as Syk activation. Increased activation-induced aggregation of cells expressing SHP-1 (C453S)

(17, 18, 42) and FcgRIIB1 (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 Ca21 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 dominantnegative mutation. Our results demonstrate that expression of

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. 7B), cells expressing SHP-1 (C453S) formed aggregates 3.5–7 times larger (Fig. 7D). In both control and mutant cells, aggregation was increased as the dose of goat anti-mouse IgG was increased from 0.1 to 10 mg/ml. Mutant cells showed enhanced aggregation compared with control cells at all doses of stimulus tested.

Discussion 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

FIGURE 7. Homotypic aggregation of K46 cells expressing SHP-1 (C453S). Photographs show control K46 cells (A and B) or cells overexpressing SHP-1 (C453S) (C and D). Cells were plated at 104/well in 96well flat-bottom plates. Cells were left unstimulated (A and C) or were treated with 10 mg/ml goat anti-mouse IgG (B and D). Undisturbed aggregates were photographed in the tissue culture wells after 20 h of incubation.

The Journal of Immunology 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. 4D). 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 Ca21 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. 4D). 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-g2 (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

2723 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 motheaten (7). 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.

Acknowledgments 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.

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