Activation Syk Tyrosine Kinase Activity and B Cell Tyrosine Phosphatase

Expression of Dominant-Negative Src-Homology Domain 2-Containing Protein Tyrosine Phosphatase-1 Results in Increased Syk Tyrosine Kinase Activity and B Cell This information is current as Activation of September 24, 2021. Lynn B. Dustin, David R. Plas, Jane Wong, Yonghong Tammy Hu, Carlos Soto, Andrew C. Chan and Matthew L. Thomas

J Immunol 1999; 162:2717-2724; ; Downloaded from http://www.jimmunol.org/content/162/5/2717

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 endog- Downloaded from enous 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 coimmunopre- cipitated 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. http://www.jimmunol.org/ The Journal of Immunology, 1999, 162: 2717–2724.

cell development and activation are regulated by tyrosine and protein tyrosine phosphatases (PTPases) is essential through- phosphorylation. B cell Ag receptor (BCR)3 engagement out B cell development. Mice lacking one or more hematopoietic by Ag or cross-linking Ab results in the activation of tyrosine kinase or PTPase are severely impaired in B cell differ- B lyn fyn blk src-family tyrosine kinases including p53/56 , p59 , and p55 entiation and function (3, 6, 7) (1, 2) and Bruton’s tyrosine kinase (Btk) (1, 3) The Ig-associated The SH2-containing protein tyrosine phosphatase, SHP-1 (SH2- membrane proteins, Ig␣ and Ig␤, are phosphorylated at tyrosine containing protein tyrosine phosphatase-1), is a cytoplasmic by guest on September 24, 2021 residues within their immunoreceptor tyrosine-based activation PTPase with two amino-terminal SH2 domains and is expressed motifs (ITAMs) (2). These serve as a docking site for the Src- predominantly in cells of hematopoietic origin (8). SHP-1-defi- syk homology domain 2 (SH2) domains of p72 (Syk), thereby lo- cient mice (motheaten or viable motheaten (mev)) suffer from he- calizing and activating the Syk tyrosine kinase (4, 5). Tyrosine matological, immunological, and inflammatory abnormalities (9– phosphorylation, by one or more of these kinases, may regulate 13). Various levels of SHP-1 have been observed in the B cell-rich downstream signaling by phosphatidylinositol-3 kinase, phospho- areas of the germinal center, suggesting a role for SHP-1 in the ␥ lipase C- 1 and 2, and the Ras-Raf-MAP kinase pathway (2). The critical proliferative, differentiation, and selective events that occur balance of tyrosine phosphorylation by protein tyrosine kinases 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 Departments of *Pathology and Molecular Microbiology and ‡Medicine, Howard least partially due to selective pressures imposed by inflammatory Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO bone marrow macrophages or other SHP-1-deficient cell types (7). 63110; and †Department of Molecular Microbiology and Immunology, St. Louis Uni- versity School of Medicine, St. Louis, MO 63104 The use of bone marrow chimeric animals demonstrates that SHP- v Received for publication June 15, 1998. Accepted for publication November 1-deficient (me ) B cells are altered in both development and ac- 30, 1998. tivation, with skewing toward the development of B-1 B cells, The costs of publication of this article were defrayed in part by the payment of page down-regulation of the BCR, and increased Ca2ϩ mobilization af- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ter BCR engagement (7). SHP-1 associates with the BCR in resting B cells and dissociates 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 rapidly after BCR stimulation (15). SHP-1 may also regulate B cell by National Institutes of Health Training Grant 5T32 AI07163. M.L.T. and A.C.C. are activation by inducible associations with other transmembrane investigators of the Howard Hughes Medical Institute. molecules such as CD22 (16–18) and, possibly, Fc␥RIIB1 (19). 2 Address correspondence and reprint requests to Dr. Matthew L. Thomas, Howard Hughes Medical Institute, Department of Pathology, Washington University School Furthermore, SHP-1 is reported to associate with cytoplasmic sig- of Medicine, 660 S. Euclid Ave., Box 8118, St. Louis, MO 63110. E-mail address: naling molecules including Vav, Grb2, mSos, and SLP-76 (20, 21). [email protected] Tyrosine-phosphorylated peptide sequences, termed immunore- 3 Abbreviations used in this paper: BCR, B cell Ag receptor; Btk, Bruton’s tyrosine ceptor tyrosine-based inhibitory motifs (ITIMs), can bind to the kinase; EPO, erythropoietin; GST, glutathione S-transferase; HRP, horseradish per- oxidase; ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunore- SH2 domains of SHP-1 and activate SHP-1 catalytic activity (22). ceptor tyrosine-base inhibitory motif; mev, viable motheaten, PTPase, protein tyrosine ITIMs with a consensus sequence of (I/V)X(p)YXXL have been phosphatase; SH2, Src-homology domain 2; SHP-1, SH2-containing protein tyrosine ␥ phosphatase-1; Syk, p72syk; ERK, extracellular signal-regulated kinase; MAP, mito- identified in Fc RIIB1, CD22, the NK cell inhibitory receptor, and gen-activated protein. the erythropoietin (EPO) and IL-3 receptors (22, 23). ITIMs may

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 2718 B CELL RECEPTOR NEGATIVE REGULATION 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 ␣␤ IFN receptor may permit SHP-1 to regulate the activity of Jak1 and/or Stat1␣ (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 (28–30). 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 FIGURE 1. Expression of SHP-1 (C453S) in K46 cells. A, Expression K46, a murine B cell line with a mature (membrane IgG) pheno- of SHP-1 (C453S) in transfected subclones. Total cell lysates were ob- Downloaded from type (31). Our results indicate that SHP-1 affects proximal and late tained from K46 cells and from representative clones transfected with events in BCR signaling and identify several molecules in which SHP-1 (C453S), were normalized for protein content, resolved on SDS- the tyrosine phosphorylation state is affected by SHP-1. PAGE, and immunoblotted with rabbit antiserum specific for mouse SHP-1 or with 9E10, a mAb against the c-myc epitope tag. B, Membrane IgG Materials and Methods 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 Cells http://www.jimmunol.org/ analyzed by flow cytometry. The murine K46 B lymphoma cell line (IgG2a, ␬) (31) was a gift of Dr. L. Justement (University of Alabama, Birmingham, AL) and was maintained SDS, and 50 mM Tris-HCl (pH 8.0). Lysis buffers were supplemented with in Iscove’s modified DMEM, supplemented with 10% heat-inactivated 21 ␮g/ml aprotinin, 2 mM leupeptin, 1 mM phenylmethylsulfonylfluoride, FCS (HyClone, Logan, UT), 2 mM L-glutamine, 100 U/ml penicillin, 100 10 ␮g/ml soybean trypsin inhibitor, 5 mM iodoacetamide, 0.4 mM sodium ␮g/ml streptomycin, 50 ␮g/ml gentamicin, and 1 mM sodium pyruvate. orthovanadate, and 10 mM sodium fluoride (all inhibitors purchased from Transfected clones were selected and grown continuously in 1.25 mg/ml Sigma, St. Louis, MO). SHP-1 and Syk immunoprecipitations were per- G418 (Life Technologies, Grand Island, NY). Splenocytes were prepared formed in the presence of 1 mg/ml chicken OVA to reduce background. from C57BL/6 mice. Normal murine C57BL/6 splenocytes were brought to 108/ml in PBS and stimulated with 5 mM pervanadate as described (28). Construct and expression by guest on September 24, 2021 A cysteine to serine substitution (C453S) in the active site of SHP-1 that Immunoprecipitation and immunoblotting ablates catalytic activity was previously described (32). A c-myc epitope was appended by PCR to the C terminus to distinguish overexpressed Lysates were precleared on ice with Pansorbin cells (Calbiochem, San SHP-1 (C453S) from endogenous SHP-1. The construct was cloned into Diego, CA). Equal amounts of protein as determined by bicinchoninic acid the expression vector BSR␣EN (33). SHP-1 (C453S) BSR␣EN or control (BCA) assay (Pierce, Rockford, IL) were analyzed by SDS-PAGE and vector was electroporated into K46 cells (31). Expression of transfected immunoblotting. Rabbit antisera were immunoprecipitated with protein A- SHP-1 (C453S) was confirmed by immunoblotting for the overexpressed conjugated Sepharose beads (Sigma) and detected with HRP-conjugated SHP-1 and for the c-myc epitope (Fig. 1A). Clones with high levels of protein A (Boehringer Mannheim, Indianapolis, IN). mAbs were precipi- SHP-1 (C453S) expression and unchanged levels of membrane Ig expres- tated with protein G-conjugated Sepharose beads (Boehringer Mannheim) sion (Fig. 1B) were chosen for further analysis. and detected with HRP-conjugated goat anti-mouse IgG (Caltag, South San Francisco, CA). Antibodies Syk kinase assays Rabbit anti-mouse SHP-1 antisera were developed by immunization with 7 either the purified SH2 or catalytic domains of murine SHP-1. Rabbit anti- Prewarmed cells (2 ϫ 10 /ml) were stimulated with 1 ␮g/ml avidin with or Syk, specific for residues 260–370, was a gift of Dr. J. Bolen (DNAX, Palo without 10 ␮g/ml biotinylated goat anti-mouse IgG (Jackson ImmunoRe- Alto, CA; Ref. 34); anti-Syk antiserum recognizing the 28 C-terminal res- search) for 2 min at 37°C. Cells were pelleted and lysed with ice-cold idues of Syk was a gift of Dr. R. Geahlen (Purdue University, West Lafay- Nonidet P-40 lysis buffer containing 5 mM EDTA, 1 mg/ml OVA, 4 mM ette, IN; Ref. 35). Rabbit antisera for mouse Syk and horseradish peroxi- leupeptin, 1 ␮M pepstatin A, 10 ␮g/ml soybean trypsin inhibitor, 10 mM dase (HRP)-conjugated 4G10 antiphosphotyrosine were purchased from sodium fluoride, 10 mM sodium molybdate, and 200 ␮M sodium vanadate. Upstate Biotechnology (Lake Placid, NY). Anti-active MAP kinase rabbit After washing Syk immunoprecipitations three times in lysis buffer, the antiserum was purchased from Promega (Madison, WI). FITC-conjugated immunoprecipitations were split: one half was used for an immunoblot to goat anti-mouse IgG was purchased from Jackson ImmunoResearch (West control for loading and the other half was used in a Syk kinase assay. The Grove, PA). Syk kinase assay was performed as previously described (39). Briefly, immunoprecipitates were washed once in 10 mM Tris (pH 7.4), 0.5 M

B cell stimulation LiCl, and twice in kinase buffer (10 mM Tris (pH 7.4), 10 mM MgCl2). Kinase assays were then performed in 25 ␮l of kinase buffer supplemented K46 cells were resuspended at 5 ϫ 106 to 2 ϫ 107/ml in PBS for exper- with 10 ␮Ci [␥-32P]ATP and 1 ␮g glutathione S-transferase (GST)-Band 3 iments. Stimulation conditions were as reported by others (35, 36). Cells (produced as described in Ref. 40). Kinase assays were incubated at room were stimulated at 37°C with 0.1–30 ␮g/ml goat anti-mouse IgG (Jackson temperature for 5 min and stopped by the addition of Laemmli loading ImmunoResearch) for 0.5–30 min, as indicated. The standard stimulation buffer and boiling for 5 min. After separation by SDS-PAGE gel, proteins conditions were 10 ␮g/ml Ab for 5 min. For assays of extracellular signal- were transferred to nitrocellulose and exposed for autoradiography and regulated kinase (ERK) activation, cells were stimulated for 2 min with 20 PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA). ␮g/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 Analysis of Syk phosphorylation in vivo 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 A fusion protein of GST-Syk was expressed in Sf9 insect cells in the contained 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% presence or absence of SHP-1. Sf9 cells were lysed in lysis buffer, and cell The Journal of Immunology 2719

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 SDS- PAGE and immunoblotting with HRP- coupled 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), Downloaded from clone 2. Cells were stimulated with 10 ␮g/ml goat anti-mouse IgG at 37°C for the indicated times. Cells (t ϭ 0) were stimulated on ice for Ͻ30 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 re- http://www.jimmunol.org/ sults shown are representative of eight experiments.

lysates were tumbled with glutathione agarose for 1 h. Agarose beads were (C453S) ablates phosphatase activity (32). This enzymatically washed three times in lysis buffer, and GST-Syk was eluted by boiling in dead form of SHP-1 is incapable of dephosphorylating ITIM se- SDS-PAGE sample buffer. After SDS-PAGE, samples were immunoblot- quences and binds to ITIM sequences for significantly longer pe- ted sequentially with anti-phosphotyrosine and anti-Syk. riods of time, preventing the endogenous phosphatase from inhib- by guest on September 24, 2021 Calcium mobilization iting signal transduction (Julie Blasioli and M.L.T., unpublished Calcium mobilization was studied with the calcium-sensitive dyes, Fluo-3 data). Therefore, it is likely that this mutation functions as an ef- AM and Fura Red AM (Molecular Probes, Eugene, OR), as described (41). ficient dominant-negative mutation. SHP-1 (C453S) was modified Briefly, cells (5 ϫ 106/ml) were loaded with 3 ␮M Fluo 3 AM and 6 ␮M with a C-terminal c-myc epitope. An expression construct encod- Fura Red AM for 30 min at 30°C. Labeled cells were washed and resus- ing the c-myc epitope tagged SHP-1 (C453S) was electroporated pended at 1–2 ϫ 106 cells/ml in Iscove’s modified DMEM supplemented with 10% FCS. Cells were prewarmed to 37°C before analysis on a into K46 cells. Expression of transfected SHP-1 (C453S) was con- FACSCaliber (Becton Dickinson Immunocytometry Systems, San Jose, firmed by immunoblotting for the overexpressed SHP-1 and for the CA). Fluo-3 and Fura Red fluorescence data were collected over time from c-myc epitope (Fig. 1A). Clones with SHP-1 (C453S) expression 2- viable cells, selected on forward and orthogonal scatter profile. Cells were to 3-fold over endogenous SHP-1 levels plus unchanged levels stimulated with 3, 10, or 30 ␮g/ml goat anti-mouse IgG, as indicated. Ratiometric data were analyzed and graphed using FlowJo software (Tree of membrane Ig expression (Fig. 1B) were chosen for further Star, San Carlos, CA). analysis. Homotypic adhesion K46 or K46 SHP-1 (C453S) were cultured at 104 cells/well in a final Dominant-negative SHP-1 increases BCR-stimulated tyrosine volume of 200 ␮l/well in Corning/Costar (Corning, NY) tissue culture- phosphorylation treated 96-well flat-bottom trays. Cells were unstimulated or stimulated with goat anti-mouse IgG (0.1–10 ␮g/ml) at 37°C for 18–20 h. Images of We tested the effects of SHP-1 (C453S) overexpression on protein undisturbed cultures were acquired on an inverted microscope with a tyrosine phosphorylation in B cells stimulated by anti-BCR Ab. cooled charged-coupled device (CCD) camera (Photometrics, Tucson, AZ) K46 B cells (5 ϫ 106–107/ml) were activated with intact goat using IP Lab software (Signal Analytics, Vienna, VA). anti-mouse IgG for varying times at 37°C, and equal amounts of Analysis total cell lysate protein were analyzed for tyrosine phosphorylation. When compared with K46 cells, K46 (C453S) cells demon- The public domain image analysis program, NIH Image, was used for densitometric analysis of immunoblots and for measurement of B cell clus- strated increased protein tyrosine phosphorylation, evident at many ters. NIH Image was developed at the National Institutes of Health and is doses of BCR stimulation (Fig. 2A). This increase in phosphoty- available on the Internet (http://rsb.info.nih.gov/nih-image/). rosine content was observed within 30–60 s after stimulation and persisted for at least 10 min (Fig. 2B). Some proteins remained Results hyperphosphorylated for at least 30 min. Some proteins were hy- Overexpression of SHP-1 (C453S) in K46 B cells perphosphorylated even in unstimulated cells expressing SHP-1 To define the role of SHP-1 in the activation of mature B cells, we (C453S) (Fig. 2, A and B). The degree of hyperphosphorylation overexpressed SHP-1 (C453S) in K46 B lymphoma cells (Fig. 1). correlated with the level of expression of SHP-1 (C453S) (Fig. 2B A single amino acid substitution in the active site of SHP-1 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 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 coimmunoprecipi- tated 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 Downloaded from 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 phosphoty- FIGURE 3. Expression of SHP-1 (C453S) increases Syk tyrosine phos- rosine-dependent in this instance. Because the constitutive associ- phorylation and kinase activity. A, Hyperphosphorylation of Syk in SHP-1 ation of Syk and SHP-1 was unexpected, we decided to examine http://www.jimmunol.org/ (C453S) transfected cells. Syk was immunoprecipitated from K46 cells whether these two enzymes were constitutively associated in pri- (wt) and from K46 cells expressing SHP-1 (C453S). Each lane shows mary B cells. In contrast, coimmunoprecipitation of SHP-1 with Syk precipitated from 107 cells. The blot was probed with the anti- Syk immunoprecipitation from murine splenic B cells requires phosphotyrosine mAb 4G10 (top), then stripped and reprobed with anti- Syk antiserum (bottom). B, Kinase activity. Syk was immunoprecipitated stimulation (Fig. 4C), demonstrating that the association is induc- from 2 ϫ 107 resting or anti-IgG-stimulated cells, then assayed for kinase ible. The molecular basis for the constitutive association in K46 activity as described in Materials and Methods. Kinase reactions were cells and inducible association in splenic B cells is unknown. How- divided in half and samples were evaluated for Band 3 phosphorylation by ever, it is possible that the transformed state of K46 results in PhosphorImager analysis (top) and for Syk enzyme levels by immunoblot- increased basal activation, potentially affecting the steady-state as- ting (bottom). sociation among Syk, SHP-1, and other proteins. One potential by guest on September 24, 2021 mechanism by which Syk and SHP-1 could associate is through the formation of a tri-molecular complex with CD22. However, 70-kDa, and 110-kDa proteins was increased at all doses of stim- coexpression of Syk, SHP-1, and CD22 in HeLa cells did not result ulus and at all time points. SHP-1 (C453S) expression also resulted in the formation of a tri-molecular complex despite appropriate in increased tyrosine phosphorylation of a similar set of protein phosphorylation of CD22 and the association with SHP-1 (D.R.P., bands in K46 cells stimulated with pervanadate (data not shown). Silke Paust, and M.L.T., unpublished data). We also could not These proteins may show increased tyrosine phosphorylation due demonstrate a direct association of SHP-1 and Syk in vitro or by to increased kinase activity or decreased dephosphorylation by coexpression. Thus, the kinase and phosphatase may associate SHP-1. Alternatively, these proteins may be protected from other through a novel mechanism. phosphatases by binding the SH2 domains of SHP-1 (C453S). To examine this issue, we analyzed the association of Syk with Syk is a substrate for SHP-1 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 Dominant-negative SHP-1 is associated with increased Syk confirm that the association SHP-1 and Syk is physiologically sig- tyrosine phosphorylation and kinase activity nificant, we examined whether SHP-1 could dephosphorylate Syk. Although SHP-1 is reported to interact with a number of cell- A GST-Syk fusion protein was expressed in Sf9 cells in the pres- surface and cytoplasmic signaling proteins, few have been directly ence or absence of SHP-1. GST-Syk purified from cells expressing shown to be targets for dephosphorylation by SHP-1. SHP-1 has SHP-1 was decreased in phosphorylation when compared with been shown to regulate ZAP-70 and certain JAK tyrosine kinases GST-Syk purified from cells that did not express SHP-1 (Fig. 4D). (24, 26, 27). Therefore, we examined the phosphorylation state of Furthermore, phosphorylated recombinant Syk protein stimulated the ZAP-70-related tyrosine kinase, Syk, in two K46 clones over- SHP-1 phosphatase activity in vitro (data not shown). expressing SHP-1 (C453S). Syk immunoprecipitates were resolved by SDS-PAGE and probed with anti-phosphotyrosine (Fig. Downstream alterations in BCR signal transduction in cells 3A, top) and anti-Syk (Fig. 3A, bottom). Syk, immunoprecipitated expressing SHP-1 (C453S) from SHP-1 (C453S) mutant cells, demonstrated increased tyro- BCR-stimulated Ca2ϩ mobilization and ERK phosphorylation sine phosphorylation compared with that from control K46 cells. were measured to assess later changes in signal transduction. Com- When corrected for the amount of Syk protein in the anti-Syk pared with parental cells, K46 cells expressing SHP-1 (C453S) immunoblot (Fig. 3A, bottom), tyrosine phosphorylation was 4.5- demonstrated more pronounced increases in cytoplasmic Ca2ϩ af- to 12-fold higher in Syk immunoprecipitates from activated SHP-1 ter stimulation with all concentrations of anti-BCR Abs examined (C453S) mutant cells than from control cells (Fig. 3A). Immuno- (Fig. 5). The kinetics of Ca2ϩ mobilization were accelerated and The Journal of Immunology 2721 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

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 (Ϫ) or were stimulated (ϩ) with 10 ␮g/ml goat anti-mouse IgG for 5 min at 37°C. Immunoprecipitates from 5 ϫ 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 ϫ 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 was not detected in either resting wild-type K46 cells or resting expressing SHP-1 (C453S). Similarly, BCR cross-linking resulted SHP-1 (C453S) transfectants. However, when cells were treated in greater MAP kinase activation as measured by ERK phosphor- with goat anti-mouse IgG, active ERK levels were an average (n ϭ ylation in cells expressing SHP-1 (C453S) (Fig. 6). Active ERK 3) of 2.8-fold higher in K46 cells expressing SHP-1 (C453S). 2722 B CELL RECEPTOR NEGATIVE REGULATION

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 (Ϫ) or stimulated with 20 ␮g/ml goat anti-mouse IgG (ϩ) 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 Downloaded from 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 2ϩ loaded with Ca -sensitive fluorescent dyes, Fluo-3 and Fura Red (41). (17, 18, 42) and Fc␥RIIB1 (19) and may contribute to the regu- Cells were equilibrated at 37°C and immediately analyzed by flow cytom- lation of B cell activation by these membrane receptors. B cell http://www.jimmunol.org/ etry. Data were collected from resting cells for the first 10 s. Cells were differentiation is severely impaired in the absence of SHP-1 activ- then stimulated by addition of goat anti-mouse IgG to a final concentration ␮ ␮ ␮ ity (7). Motheaten B cells demonstrate increased B cell activation, of 3 g/ml (A and D), 10 g/ml (B and E), or 30 g/ml (C and F) and data 2ϩ were collected for an additional 90 s. Graphs show changes in the ratio of as measured by Ca mobilization (7) and proliferation (15). It is Fluo-3 to Fura Red fluorescence in response to increased cytoplasmic Ca2ϩ possible that the B-1 lymphocytes that preferentially survive in the in individual cells over time. G–I, Graphs show the percentage of cells with motheaten mouse have a different activation program. To separate a Fluo-3/Fura Red ratio greater than that of 95% of resting cells when the role of SHP-1 in B cell activation from its role in B lymphocyte stimulated with (G)3,(H) 10, or (I)30␮g/ml goat anti-mouse IgG. Data differentiation, it is essential to interrupt its function specifically in are representative of five independent experiments.

mature B cells without subjecting these cells to the developmental by guest on September 24, 2021 abnormalities of the motheaten mouse. We examined the effects of dominant-negative SHP-1 on acti- These results support the idea that SHP-1 (C453S) affects early vation of the B lymphoma line, K46, by anti-BCR Abs. The cat- events in the BCR signal transduction cascade such as Syk alytically inactive SHP-1 (C453S) can compete with endogenous activation. wild-type SHP-1 for association with other signaling molecules and for access to substrates and thus can serve as a dominant- Increased activation-induced aggregation of cells expressing negative mutation. Our results demonstrate that expression of SHP-1 (C453S) 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 param- eter of late changes in activation. SHP-1 (C453S) permitted a dra- matic 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 ␮g/ml. Mutant cells showed enhanced aggregation compared with control cells at all doses of stimulus tested.

Discussion FIGURE 7. Homotypic aggregation of K46 cells expressing SHP-1 (C453S). Photographs show control K46 cells (A and B) or cells overex- SHP-1 plays a central regulatory role in B lymphocyte develop- pressing SHP-1 (C453S) (C and D). Cells were plated at 104/well in 96- ment and activation. SHP-1 may regulate the threshold for B cell well flat-bottom plates. Cells were left unstimulated (A and C) or were activation through its association with the BCR in unstimulated B treated with 10 ␮g/ml goat anti-mouse IgG (B and D). Undisturbed aggre- cells (15). After BCR engagement, SHP-1 is recruited to CD22 gates were photographed in the tissue culture wells after 20 h of incubation. The Journal of Immunology 2723 dominant-negative SHP-1 in mature B cells causes increased ty- Btk does not comigrate with any of the differentially phosphory- rosine phosphorylation of a number of proteins after BCR engage- lated bands observed in Fig. 2 (data not shown). The possibility ment (Fig. 2). These proteins include the tyrosine kinase Syk (Fig. that SHP-1 and Btk may not act on the same pathways is suggested 3), which is a substrate for dephosphorylation by SHP-1 (Fig. 4D). by the fact that Btk mutant mice lack B-1 B cells (3), a subset that We also observed an association of SHP-1 with Syk in splenic B is selectively retained in mice with the SHP-1 mutations motheaten cells and in wild-type or SHP-1 (C453S) K46 cells (Fig. 4). That and viable motheaten (7). SHP-1 (C453S) altered early tyrosine kinase activity is supported At least four different mechanisms could account for the in- by the increased changes in early downstream events in BCR sig- creased tyrosine phosphorylation of specific cellular proteins in B nal transduction, such as Ca2ϩ mobilization (Fig. 5) and ERK cells expressing catalytically inactive SHP-1. Some of these pro- phosphorylation (Fig. 6). Longer-term events are also affected by teins may be targets for dephosphorylation by SHP-1. Syk is a SHP-1. A full day after stimulation by goat anti-mouse IgG, K46 candidate for such direct regulation by virtue of its association cells expressing SHP-1 (C453S) show increased homotypic adhe- with SHP-1 in vivo, its dephosphorylation in cells coexpressing sion (Fig. 7). Recent data demonstrate that CD45 regulates inte- SHP-1, and because SHP-1 dephosphorylates the related kinase, grin-mediated adhesion in lymphocytes (43, 44) and macrophages ZAP-70 (27). Second, the kinases that phosphorylate these pro- (45). Together, these results point to an emerging role for protein teins may be targets for regulation by SHP-1. In this regard, SHP-1 tyrosine phosphatases in the regulation of lymphocyte adhesion. selectively regulates the level of EPO receptor tyrosine phosphor- These results support the hypothesis that Syk is negatively reg- ylation by Jak2, an enzyme regulated by SHP-1, but not by c-Fes, ulated by SHP-1 in B cells. K46 B cells expressing SHP-1 (C453S) an enzyme not known to be regulated by SHP-1 (25). Third, cat- showed increased Syk tyrosine phosphorylation and tyrosine ki- alytically inactive SHP-1, expressed in excess, could bind to phos- Downloaded from nase activity (Fig. 3). In addition, Syk was dephosphorylated when photyrosine via its SH2 domains, denying access to phosphory- coexpressed with SHP-1 in insect cells (Fig. 4D). This finding adds lated substrates by endogenous SHP-1 as well as other endogenous Syk to the growing list of receptor-associated tyrosine kinases that phosphatases. Finally, the active site of catalytically inactive SHP-1 are regulated by SHP-1 (24, 25, 27, 46). Furthermore, we found may stably bind to substrates and prevent endogenous, functional that SHP-1 and Syk associate physically both in wild-type K46 B phosphatases from dephosphorylating these substrates. In these cells and in K46 expressing SHP-1 (C453S) (Fig. 4). A complex studies, we allowed SHP-1 and other phosphatases to dephosphor- http://www.jimmunol.org/ containing both SHP-1 and Syk could be immunoprecipitated from ylate their substrates in intact cells before lysis. This maintains K46 cells with various antisera specific for either enzyme. This normal cellular structures and protein-protein interactions, thereby complex likely contained a relatively small fraction of each en- permitting SHP-1 to act on its normal substrates. SHP-1 may exert zyme in K46 B cells, and was most clearly identified when SHP-1 its regulatory effects on B cell development and activation by or Syk was immunoprecipitated from limiting numbers of cells. changing the threshold for activation through these receptors and SHP-1 may interact with Syk by a mechanism independent of the nonreceptor tyrosine kinases. 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 Acknowledgments by guest on September 24, 2021 K46 B cells. This finding is consistent with the observation that a We thank Drs. J. Bolen, A. Chan, and R. Gaehlen for generous gifts of functional interaction between SHP-1 and Jak2 requires neither reagents. We thank Dr. M. Dustin for critical reading of the manuscript and functional SHP-1 SH2 domains, nor tyrosine phosphorylation of for assistance with microscopy. Jak2 (25). In contrast, the association was increased by pervanadate stimulation in splenic B cells. The association may be medi- References ated by an adapter protein expressed in B cells, but not other cell 1. Saouaf, S. J., S. Mahajan, R. B. Rowley, S. A. Kut, J. Fargnoli, A. L. Burkhardt, types (Fig. 4; D.R.P. and M.L.T., unpublished data). The SH2 S. Tsukada, O. N. Witte, and J. B. Bolen. 1994. 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