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The Journal of Immunology, 1998, 161: 684-691.
Copyright © 1998 by The American Association of Immunologists

B Cell Antigen Receptor (BCR)-Mediated Formation of a SHP-2-pp120 Complex and Its Inhibition by Fe{gamma}RIIB1-BCR Coligation1

Kazuhiro Nakamura and John C. Cambier2

Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center, and Department of Immunology, University of Colorado Health Science Center, Denver, CO 80206


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Accumulating evidence indicates that the Src homology 2-containing tyrosine phosphatase 2 (SHP-2) plays an important role in signal transduction through receptor tyrosine kinase and cytokine receptors. In most models, SHP-2 appears to be a positive mediator of signaling. However, coligation of Fc{gamma}RIIB1 with B cell Ag receptors (BCR) inhibits BCR-mediated signaling by a mechanism that may involve recruitment of phosphatases SHP-1, SHP-2, and the SH2 containing inositol 5'phosphatase (SHIP) to the phosphorylated Fc{gamma}RIIB1 immunoreceptor tyrosine-based inhibitory motif. The role of SHP-2 in BCR-mediated cell activation and in Fc{gamma}RIIB1-mediated inhibitory signaling is unclear. In this study we assessed the association of SHP-2 with phosphotyrosine-containing cellular protein(s) before and after stimulation through these receptors. BCR stimulation induced the association of SHP-2 with a single major tyrosyl-phosphorylated molecule (pp120) that had an apparent molecular mass of 120 kDa. Coligation of Fc{gamma}RIIB1 with BCR led to a rapid decrease in SHP-2 association with pp120. Analysis of the subcellular localization of pp120 showed that the complex of SHP-2 and tyrosyl-phosphorylated p120 occurs predominantly in the cytosol. Furthermore, the binding of the two molecules was mediated by the interaction of tyrosyl-phosphorylated p120 with the SHP-2 N-terminal SH2 domain. These findings indicate that SHP-2 and pp120 function in BCR signaling, and this function may be inhibited by Fc{gamma}RIIB1 signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Protein tyrosine phosphatases (PTPs)3 can participate in both activation and inactivation of tyrosine kinase-mediated signaling pathways. For example, despite structural similarities, a member of the Src homology domain 2 (SH2)-containing PTP subfamily that consists of the mammalian proteins SHP-1 and SHP-2 and Drosophila Csw, the putative homologue of SHP-2, have unique and opposite roles in intracellular signaling pathways. The members of this nontransmembrane PTP family are characterized by two tandem SH2 domains at their N-termini and a single tyrosine phosphatase domain. SHP-1 (also named SHPTP1, PTP1C, HCP, or SHP) (1) is predominantly expressed in hemopoietic cells, where it functions as a negative regulator of intracellular signaling pathways (2). By contrast, SHP-2 (also named SHPTP2, PTP1D, Syp, PTP2C, and SHPTP3) (1) is expressed ubiquitously (3, 4, 5, 6) and plays a positive signaling role in responses to growth factors such as platelet-derived growth factor (PDGF) (5, 6, 7), epidermal growth factor (EGF) (5, 6), insulin (8), and insulin-like growth factor (9). Genetic ablation of SHP-2 is lethal. It has been shown that a catalytically inactive SHP-2, presumably acting as dominant negative, inhibits these growth factor-induced mitogenic responses (10, 11, 12, 13, 14, 15). SHP-2 SH2 domains bind to tyrosyl-phosphorylated growth factor receptors in response to growth factor stimulation. The deletion mutation of the N-terminal SH2 domain of SHP-2 severely compromises its biologic function (16, 17). Moreover, the C-terminal tyrosine residue(s) in SHP-2 can be phosphorylated upon PDGF or EGF stimulation (5, 6), providing a docking site(s) for the SH2-containing molecule, Grb2 (18, 19, 20). Thus, in addition to its role as a phosphatase, SHP-2 may function as an adaptor protein to recruit downstream signaling molecules. Genetic studies of csw (21) suggests that Csw functions either downstream of Ras or in a parallel pathway to the Ras in the sevenless receptor tyrosine kinase pathway (22).

To further investigate the function of SHP-2, it is critical to identify SHP-2 substrates and associated molecules that may function as its effectors or regulators. A 120-kDa protein that is hyperphosphorylated upon insulin stimulation binds to SH2 domains of a catalytically inactive SHP-2 construct (23). In addition, a tyrosyl-phosphorylated 115-kDa molecule has been shown to bind to SHP-2 upon insulin (24, 25) or EGF (26) stimulation, although, in the case of insulin stimulation, SHP-2 SH2 domains were not involved in the binding (24). It is likely that these molecules are members of the SIRP family. SIRPs are transmembrane proteins that become tyrosyl phosphorylated upon growth factor stimulation and bind SHP-2 (27, 28). When overexpressed, SIRP{alpha}1 is tyrosyl phosphorylated and inhibits the response to growth factor stimulation (28). Thus, SIRPs appear to play an important regulatory role in signaling through growth factor receptors. In Drosophila, Daughter of sevenless (Dos) contains an N-terminal PH domain, a polyproline motif, and 10 potential tyrosyl phosphorylation sites, and is reported to be a substrate for Csw (29, 30). It is proposed that Dos dephosphorylated by Csw participates in the sevenless receptor signaling pathway.

SHP-2 has also been shown to be involved in signaling by cytokine receptors. SHP-2 binds intracellular signaling molecules, including JAK2 (31, 32, 33), Grb2 (34, 35, 36), phosphatidylinositol 3-kinase (35), and SHIP (37) in response to cytokine stimulation. In addition, a 100-kDa cytosolic molecule (38, 39) and a 135-kDa transmembrane molecule (39) are reported to bind to SHP-2 dependent and independent of cytokine stimulation, respectively. These experiments indicate that SHP-2 is capable of binding different sets of effector molecules or substrates before and after stimulation.

In B cells, coligation of B cell low affinity receptors for IgG, Fc{gamma}RIIB1, with B cell Ag receptor (BCR) inhibits many BCR-mediated biologic responses (40, 41). A tyrosine residue in immunoreceptor tyrosine-based inhibitory motif (ITIM) of Fc{gamma}RIIB1 is phosphorylated upon BCR-Fc{gamma}RIIB1 coligation, resulting in the recruitment of SH2-containing molecules to Fc{gamma}RIIB1. These SH2-containing molecules presumably mediate the inhibitory signal through Fc{gamma}RIIB1 (42, 43). As candidate molecules, PTPs, SHP-1 (44), SHP-2 (45), and a phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase, SHIP (46, 47, 48), were demonstrated to bind in vitro to phosphorylated ITIM peptides derived from Fc{gamma}RIIB1. Although studies with chimeric receptors indicate that SHP-1 (44, 49, 50) and SHIP (50, 51, 52, 53, 54) can mediate the inhibitory Fc{gamma}RIIB1 signal, little is known about the function of SHP-2 in this paradigm.

In the present study, we describe the association of a novel 120-kDa molecule (pp120) with SHP-2 in B cells. BCR stimulation appears to induce the tyrosyl phosphorylation of p120, resulting in its interaction with SHP-2 via its N-terminal SH2 domain. Formation of the SHP-2-pp120 complex induced by BCR stimulation is inhibited by the coligation of Fc{gamma}RIIB1. Our results suggest that pp120 acts as a positively functioning intermediary in BCR-mediated signaling and that the dephosphorylation of pp120 may be an important event in Fc{gamma}RIIB1-mediated inhibitory signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and reagents

The murine B lymphoma cell lines A20 (Fc{gamma}RIIB1 positive) (55), IIA1.6 (Fc{gamma}RIIB1 negative variant from A20) (56), and K46µ (57) were cultured in IMDM with 5% heat-inactivated FCS (HyClone, Logan, UT), 50 U/ml penicillin, and 50 µg/ml streptomycin at 37°C with 7% CO2. Splenic B cells were isolated by depletion of T cells using Ab- and complement-mediated lysis, and high density B cells ({rho} > 1.066) were prepared by discontinuous density gradient centrifugation using Percoll (58). Rabbit Abs to human SHP-2, mouse SHIP, mouse Fc{gamma}RIIB1, mouse Lyn, and mouse CD19 were prepared using the following immunogens and affinity purified before use. Glutathione-S-transferase (GST) fusion proteins containing partial sequences (C-terminal 44 amino acids of SHP-2 (59), amino acid residues 909 to 959 of SHIP, the entire cytoplasmic domain of Fc{gamma}RIIB1, amino acid residues 1 to 131 of Lyn, and amino acid residues 411 to 547 of CD19) were prepared and cleaved to remove GST before immunization. The rat anti-mouse Fc{gamma}RIIIA/IIB 2.4G2 mAb was affinity purified from 2.4G2 hybridoma culture supernatants using protein G-Sepharose (Pharmacia, Piscataway, NJ). Anti-phosphotyrosine Ab (Ab-2) was obtained from Oncogene Science (Manhasset, NY). Horseradish peroxidase (HRP)-conjugated rat anti-mouse IgG1, HRP-conjugated protein A, and intact and F(ab')2 rabbit anti-mouse IgG (H+L) and rat anti-mouse IgG (H+L) were purchased from Zymed (San Francisco, CA) and Jackson ImmunoResearch (West Grove, PA), respectively. Hydrogen peroxide (H2O2) and sodium orthovanadate (Na3VO4) were obtained from Sigma (St. Louis, MO). V8 protease was purchased from ICN (Aurora, OH).

Constructs

For the generation of GST fusion proteins containing the SH2 domains of SHP-2, PCR was used to amplify cDNAs encoding either single or double SH2 domains using the following primer pairs: GST-N SH2: 5' primer, 5'-CGGAATTCATGACATCGCGGAGATGGTTTCAC; 3' primer, 5'-TTTTCCTTTTGCGGCCGCCTTTCAGAGGTAGGGTCTGC; GST-C SH2: 5' primer, 5'-CGGGATCCCTACCTCTGAAAGG; 3' primer, 5'-GGAATTCCCTTCCCAAAAGCCCTG; and GST-N/C (SH2)2: 5' primer, 5'-CGGGATCCCCATGACATCGCGGAGA; 3' primer, same as GST-C SH2 3' primer. The underlined nucleotides show EcoRI and NotI sites (for GST-N SH2) and BamHI and EcoRI sites (for GST-C SH2 and GST-N/C (SH2)2). After digesting with the above restriction enzymes, the resulting fragments were ligated into EcoRI/NotI-cut pGEX-5X-1 (Pharmacia) or BamHI/EcoRI-cut pGEX-3X. All fusion proteins were produced in bacteria, purified with glutathione-Sepharose beads (Pharmacia), and cleaved with factor Xa (Boehringer Mannheim, Indianapolis, IN). Cleaved proteins were coupled to cyanogen bromide-activated Sepharose beads (Pharmacia).

B cell stimulation and cell lysis

The cells were washed with IMDM three times and resuspended in IMDM. After prewarming at 37°C for 10 min, the cells were stimulated with intact or F(ab')2 rabbit anti-mouse IgG (H+L) for the indicated period. In the study of primary B cells, the cells were preincubated with 2.4G2 mAb for 30 min before stimulating with rat anti-mouse IgG. In the case of pervanadate treatment, a mixed solution of 100 mM H2O2 and 30 mM sodium orthovanadate was preincubated for 10 min at room temperature, and then 10 µl of the mixture was added to 1 ml of cell suspension (5 x 107 cells/ml). After stimulation, the cells were washed three times with ice-cold PBS and lysed with solubilizing buffer (1% Triton X-100, 10 mM Tris (pH 7.5), 150 mM NaCl, 0.4 mM EDTA, 10 mM NaF, 2 mM Na3VO4, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml {alpha}1-antitrypsin, and 1 mM PMSF), and cleared supernatants were retained for further processing.

Subcellular fractionation

The cells were resuspended in ice-cold hypotonic solution (10 mM Tris (pH 7.5), 0.5 mM MgCl2, 2 mM Na3VO4 with protease inhibitors). The cell suspension was incubated on ice for 10 min and homogenized in a tight-fitting Dounce homogenizer (30 strokes; Kontes, Vineland, NJ). The tonicity of homogenized cells was restored to 0.15 M NaCl with tonicity restoration buffer (hypotonic solution containing 0.6 M NaCl). The suspension was centrifuged twice at 900 x g for 5 min each time to remove the nuclear fraction. The supernatant was centrifuged at 100,000 x g for 40 min to separate the S100 (supernatant) and P100 (pellet) fractions. The pellet fraction was solubilized in solubilizing buffer.

Immunoprecipitation and immunoblotting analysis

To analyze the contents of the SHP-2 or SHIP immunoprecipitates, the prepared lysates were incubated with anti-SHP-2 or anti-SHIP Ab. Immune complexes were collected with protein A-Sepharose beads (Pharmacia), separated by 8% SDS-PAGE, and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). To detect the binding of cellular proteins to SHP-2 SH2 domain(s), the lysates were incubated with SH2 domain(s)-conjugated cyanogen bromide beads, and adsorbates were fractionated and transferred as described above. After blocking, PVDF membranes were blotted with anti-phosphotyrosine Ab and HRP-conjugated rat anti-mouse IgG1 using the ECL Western blotting detection system (Amersham, Aylesbury, U.K.). In some cases, after blotting with anti-phosphotyrosine Ab, membranes were stripped to remove the Ab and subjected to sequential blotting with anti-SHP-2, -SHIP, -Lyn, or -CD19 Ab. The membranes were incubated with the anti-SHP-2, -SHIP, -Lyn, or -CD19 Ab followed by incubation with HRP-conjugated protein A.

Phosphopeptide mapping using V8 protease

SHP-2 immunoprecipitates and SHP-2 N/C-(SH2)2 domain binding molecules were prepared from pervanadate-treated cells in duplicate. After separating these two sets by 8% SDS-PAGE individually, one gel was stained using a Zinc Stain and Destain Kit (Bio-Rad, Hercules, CA), and the pieces containing the bands corresponding to tyrosyl-phosphorylated molecules detected in immunoblotting of the other gel were excised and destained. V8 protease (500 ng/sample) was added to the destained gel pieces, and these samples were subjected to the second SDS-PAGE (13%). V8 protease digestion was conducted in the stacking gel for 30 min. Detection of tyrosyl phosphopeptides was performed as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Association of tyrosyl-phosphorylated p120 with SHP-2 in B cells

To detect the SHP-2-associated proteins, we performed the anti-phosphotyrosine blotting on fractionated SHP-2 immunoprecipitates derived from various B cell lines. Following BCR stimulation of the mouse B lymphoma cell line, A20, a single tyrosyl-phosphorylated protein around 120 kDa (pp120) was detected in SHP-2 immunoprecipitates (Fig. 1GoA). Analysis using high percentage acrylamide gel to detect molecules in the lower molecular mass range (<50 kDa) revealed no additional SHP-2-associated phosphoproteins detectable under these circumstances (data not shown). The phosphorylation of p120 was seen variably in resting cells and increased upon intact or F(ab')2 rabbit anti-mouse IgG (H+L) (RAMIG) stimulation. Ligation of BCR alone using F(ab')2 RAMIG induced a stronger increase in pp120-SHP-2 association than BCR-Fc{gamma}RIIB1 co-cross-linking. By contrast, the tyrosyl phosphorylation of SHP-2 itself was not detected in either nonstimulated or BCR-stimulated cells. To confirm that binding of pp120 to SHP-2 is specific, SHIP immunoprecipitates were analyzed, because both SHIP (46, 47, 48) and SHP-2 (45) may be recruited to Fc{gamma}RIIB1 upon coligation of BCR and Fc{gamma}RIIB1. In SHIP immunoprecipitates, tyrosyl phosphorylation of SHIP was detected upon stimulation with intact RAMIG. In most experiments, although not the one shown here, F(ab')2 RAMIG stimulated much lower, but detectable, increases in tyrosyl phosphorylation of SHIP. However, we could not detect SHIP association with pp120, indicating that pp120 binds specifically to SHP-2.



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  		FIGURE 1. Detection of the interaction of pp120 with SHP-2 in B cells. A, A20 cells (1 x 108 cells/ml) were stimulated with intact (40 µg/ml; I) or F(ab')2 (24 µg/ml; F) RAMIG for 2 min at 37°C. The cells were lysed as described in Materials and Methods. SHP-2 and SHIP in the lysates (5 x 107 cells) were immunoprecipitated with anti-SHP-2 and anti-SHIP Abs, respectively. SHP-2 and SHIP immunoprecipitates were fractionated using SDS-PAGE, transferred, and analyzed using anti-phosphotyrosine immunoblotting (upper panel). Middle and lower panels show anti-SHP-2 or anti-SHIP immunoblotting after stripping the upper blots. B, Upper panel, Anti-phosphotyrosine blotting of SHP-2 immunoprecipitates from A20, IIA1.6, and K46µ cells (5 x 107 cells/ml) stimulated with intact (20 µg/ml; I) or F(ab')2 (12 µg/ml; F) RAMIG for 2 min at 37°C or treated with pervanadate (P.V.) for 10 min at 37°C. In the lower panel is shown anti-SHP-2 immunoblotting conducted after stripping the upper blots. C, Upper panel, Anti-phosphotyrosine blotting of SHP-2 immunoprecipitates (5 x 107 cells) from primary B cells (5 x 107 cells/ml) stimulated with intact rat anti-mouse IgG (20 µg/ml) in the absence or the presence of anti-Fc{gamma}RIIB mAb (2.4G2) for 2 min at 37°C. In the lower panel is shown anti-SHP-2 immunoblotting after stripping the upper blot.

 
To address whether the binding of pp120 to SHP-2 is a general feature in B cells, we analyzed SHP-2 immunoprecipitates from a Fc{gamma}RIIB1 negative variant of A20, IIA1.6 (Fig. 1GoB, center panel), and another mouse B lymphoma cell line, K46µ (Fig. 1GoB, right panel). In all B cell lines tested, we detected pp120 in SHP-2 immunoprecipitates upon BCR stimulation. In addition, when pervanadate, a mixture of H2O2 and Na3VO4, was used to induce massive tyrosyl phosphorylation of cellular proteins by inhibition of phosphatases, stronger tyrosyl phosphorylation of SHP-2-associated pp120 was seen. Following pervanadate treatment, SHP-2 was also tyrosyl phosphorylated (Fig. 1GoB, upper panel), probably leading to the upward mobility shift of SHP-2 seen in SDS-PAGE gels (Fig. 1GoB, lower panel). We next examined whether the binding of tyrosyl-phosphorylated p120 to SHP-2 occurred in primary B cells (Fig. 1GoC). In this study we used rat anti-mouse Fc{gamma}RIIB mAb (2.4G2) to block the coligation of BCR and Fc{gamma}RIIB1 before stimulating the cells with intact rat anti-mouse IgG instead of using F(ab')2 RAMIG. Upon both BCR ligation alone and BCR-Fc{gamma}RIIB1 coligation, several tyrosyl-phosphorylated molecules were detected in SHP-2 immunoprecipitates. Among these molecules, the most strongly tyrosyl-phosphorylated molecule was a species with molecular size comparable to that of pp120. These results show that in B cells, tyrosyl-phosphorylated p120 associates with SHP-2 upon BCR stimulation.

Interaction of pp120 with SHP-2 occurs rapidly upon BCR stimulation and is negatively regulated by Fc{gamma}RIIB1

In A20 cells, the association of SHP-2 with pp120 induced by F(ab')2 RAMIG was greater than that seen following stimulation with intact RAMIG (Fig. 1Go, A and B), whereas in IIA1.6 (an Fc{gamma}RIIB1-negative cell; Fig. 1GoB) and K46µ (Fc{gamma}RIIB1 negative, by immunofluorescence analysis; Fig. 1GoB) cells and primary B cells (in which Fc{gamma}RIIB1 is low, by immunofluorescence analysis; Fig. 1GoC), no difference was seen between BCR ligation alone and BCR-Fc{gamma}RIIB1 coligation. This suggested that Fc{gamma}RIIB1 ligation may inhibit the phosphorylation of p120 and/or its association with SHP-2. Alternatively, the kinetics of the response may be different. We therefore examined the kinetics of the interaction of pp120 with SHP-2 on BCR stimulation with or without coligation of Fc{gamma}RIIB1 in A20 cells (Fig. 2Go). Equivalent induction of association of pp120 with SHP-2 was detected 15 s following BCR stimulation alone or after BCR-Fc{gamma}RIIB1 coligation. However, after reaching the peak levels by 30 s, the association of pp120 with SHP-2 in BCR-Fc{gamma}RIIB1 coligated cells returned almost to the level in nonstimulated cells within 5 min. The association of pp120 with SHP-2 that occurred upon stimulation through BCR alone was more persistent. These results indicate that p120 phosphorylation and/or its association with SHP-2 occur rapidly and is a target of the Fc{gamma}RIIB1-mediated inhibitory signal.



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  		FIGURE 2. Time course of the interaction of pp120 with SHP-2. A20 cells (5 x 107 cells/ml) were stimulated with intact (20 µg/ml) or F(ab')2 (12 µg/ml) RAMIG for the period indicated (0–300 s) at 37°C. The cell lysates (5 x 107 cells) were subjected to immunoprecipitation with anti-SHP-2 Ab, and immunoprecipitates were fractionated and analyzed by anti-phosphotyrosine immunoblotting (upper panel). In the lower panel is shown anti-SHP-2 immunoblotting conducted after stripping the upper blot.

 
The SHP-2-pp120 complex occurs predominantly in the cytosolic fraction

Multiple molecules with molecular masses of approximately 100 to 120 kDa have been reported as SHP-2 binding proteins in other cellular models. SIRP family members (27, 28) were shown to be located in the membrane fraction consistent with their apparent glycosylation and content of a predicted membrane spanning region. Accordingly, the subcellular localization of pp120-SHP-2 complexes was examined biochemically. We prepared and fractionated A20 homogenates into membrane and cytosol fractions, and immunoprecipitated SHP-2 from each (Fig. 3GoA). To verify the effectiveness of the subfractionation procedure, a small aliquot collected from each fraction was subjected to anti-Lyn and anti-CD19 immunoblotting (Fig. 3GoB). Lyn is found in the membrane fraction due to its N-terminal lipid modification and association with membrane proteins. CD19, a glycoprotein expressed on B cells, is also detected in the membrane fraction. Upon both intact and F(ab')2 RAMIG stimulation, SHP-2 and the tyrosyl-phosphorylated p120-SHP-2 complex were found predominantly in the cytosol fraction (Fig. 3GoA). These observations indicate that SHP-2 is predominantly cytosolic in distribution and that the association of tyrosyl-phosphorylated p120 with SHP-2 occurs in the cytosol.



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  		FIGURE 3. Subcellular localization of SHP-2-pp120 complex. Following stimulation with intact (20 µg/ml; I) or F(ab')2 (12 µg/ml; F) RAMIG for 2 min at 37°C, A20 cells (5 x 107 cells) were either solubilized with detergent for preparing total cell lysate (whole cell lysate) or fractionated to S100 (cytosol) and P100 (membrane) fractions. A, SHP-2 was immunoprecipitated from each fraction, and immunoprecipitates were subjected to fractionation and immunoblotting with anti-phosphotyrosine Ab (upper panel). The lower panel shows anti-SHP-2 immunoblotting after stripping the upper blot. B, Anti-Lyn immunoblots of SDS-PAGE-fractionated aliquots (1 x 106 cell equivalent) from each fraction are shown. In the lower panel is shown anti-CD19 immunoblotting conducted after stripping the upper blot.

 
Interaction of pp120 with SHP-2 is mediated at least in part by SHP-2 SH2 domain(s)

SHP-2 contains two SH2 domains that can bind phosphorylated tyrosine residues. To address the role of SHP-2 SH2 domains in the binding of pp120, we prepared SHP-2 SH2 domains as GST fusion proteins. After stimulation of A20 cells with intact or F(ab')2 RAMIG or treatment with pervanadate, the cell lysates were prepared and incubated with cyanogen bromide-coupled SHP-2 double SH2 domain proteins (N/C-(SH2)2 domains). The matrix-bound proteins were eluted and analyzed by anti-phosphotyrosine immunoblotting (Fig. 4GoA). A tyrosyl-phosphorylated protein of a size equivalent to that seen in SHP-2 precipitates was detected even in nonstimulated cells, and its phosphorylation/binding activity was increased upon cell stimulation. The relative tyrosyl phosphorylation of the 120-kDa molecule found among SHP-2 N/C (SH2)2 binding proteins upon stimulation was similar to that of pp120 in SHP-2 immunoprecipitates. Interestingly, the amount of SHP-2 binding pp120 seen was greater in F(ab')2 RAMIG-stimulated cells than in intact RAMIG-stimulated cells. This indicated that the effect of Fc{gamma}RIIB1 coligation is on pp120, not on SHP-2.



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  		FIGURE 4. The binding of pp120 mediated by SHP-2 SH2 domain(s). A, A20 (5 x 107 cells/ml) were stimulated with intact (20 µg/ml; I) or F(ab')2 (12 µg/ml; F) RAMIG for 2 min at 37°C or treated with pervanadate (P.V.) for 10 min at 37°C. The cell lysates (5 x 107 cells) were incubated with anti-SHP-2 Ab plus protein A-Sepharose beads or immobilized SHP-2 N/C-(SH2)2 domains. The eluates from the matrix were fractionated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with anti-phosphotyrosine Ab (upper panel). Lower panel, Anti-SHP-2 immunoblotting after stripping the upper blot. B, A20 (1.5 x 108 cells/ml) were treated with pervanadate for 10 min at 37°C. The cell lysates (1.5 x 108 cells) were incubated with anti-SHP-2 Ab plus protein A-Sepharose beads or immobilized SHP-2 N/C-(SH2)2 domains. One-third of the eluates (5 x 107 cells) from the matrix were fractionated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with anti-phosphotyrosine Ab (left panel). Right panel, The rest of the eluates (1 x 108 cells) were fractionated by SDS-PAGE and stained with the zinc stain kit. The bands corresponding to tyrosyl-phosphorylated p120 (bands 1 and 2) and p62 (band 3) in the left panel were excised from the gel, rerun with V8 protease (500 ng/lane) in 13% SDS-PAGE, transferred to PVDF membrane, and subjected to anti-phosphotyrosine immunoblotting.

 
To confirm that these two species are the same molecule, we compared patterns of phosphotyrosine-containing peptides generated by V8 protease digestion (Fig. 4GoB). In pervanadate-treated A20 cells, the tyrosyl-phosphorylated 120-kDa molecule among N/C-(SH2)2 binding molecules showed the identical phosphopeptide mapping pattern as pp120 in SHP-2 immunoprecipitates. A distinct tyrosyl-phosphorylated molecule with a molecular mass of 62 kDa in N/C-(SH2)2 domains absorbates was used as a control for this experiment and showed a completely different peptide pattern from pp120. These data indicate that a tyrosyl-phosphorylated 120-kDa molecule detected in SHP-2 N/C-(SH2)2 domains absorbates is the same as pp120 in SHP-2 immunoprecipitates.

To further clarify the role of each SHP-2 SH2 domain in binding to pp120, we compared the binding of tyrosyl-phosphorylated proteins to SHP-2 N-terminal SH2 (N-SH2), C-terminal SH2 (C-SH2), and N/C-(SH2)2 domain fusion proteins (Fig. 5Go). In the analysis of IIA1.6 cells (Fig. 5GoA), pp120 was detected in N-SH2 and N/C-(SH2)2 domain(s) binding molecules upon both intact and F(ab')2 RAMIG stimulation. On the other hand, no tyrosyl-phosphorylated molecule around 120 kDa was seen in C-SH2 domain absorbates. This result suggests that the binding of pp120 to SHP-2 is mediated by SHP-2 N-SH2 domain or that prepared C-SH2 domain is not functional to trap any tyrosyl-phosphorylated molecules. To assess the role of individual SH2 domains in SHP-2 in detail, we performed the same analysis using A20 cells (Fig. 5GoB). A20 cells express Fc{gamma}RIIB1 on their surface abundantly. It has been reported that a tyrosine residue in ITIM motif of Fc{gamma}RIIB1 is phosphorylated upon BCR-Fc{gamma}RIIB1 coligation (43), and this phosphotyrosine may bind SHP-2 (45) as well as SHP-1 (44) and SHIP (46, 47, 48) via SH2 domain interactions. Consistent with these reports, we observed a diffuse tyrosyl-phosphorylated protein of about 50 kDa bound to both SHP-2 N- and C-SH2 domains upon coligation of BCR and Fc{gamma}RIIB1 (Fig. 5GoB, upper panel). Anti-Fc{gamma}RIIB1 blotting confirmed that the 50-kDa tyrosyl-phosphorylated protein was Fc{gamma}RIIB1 (Fig. 5GoB, lower panel). In contrast to Fc{gamma}RIIB1, the tyrosyl-phosphorylated p120 was detected in N-SH2 and N/C-(SH2)2 domain(s) adsorbates and not in C-SH2 domain adsorbates (Fig. 5GoB, upper panel). These results confirm the activity of the C-SH2 domain of SHP-2 and indicate that the interaction of pp120 with SHP-2 is mediated by the SHP-2 N-SH2 domain.



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  		FIGURE 5. The binding of tyrosyl-phosphorylated p120 to SHP-2 N-SH2 domain. A, IIA1.6 (5 x 107 cells/ml) were stimulated with intact (20 µg/ml; I) or F(ab')2 (12 µg/ml; F) RAMIG for 2 min at 37°C. The cell lysates (5 x 107 cells) were incubated with anti-SHP-2 Ab plus protein A-Sepharose beads or immobilized SHP-2 N-, C-, or N/C-SH2 domain(s). The eluates from the matrix were fractionated and analyzed by anti-phosphotyrosine immunoblotting (upper panel). The lower panel shows anti-SHP-2 immunoblotting after stripping the upper blot. B, A20 (5 x 107 cells/ml) were stimulated with intact (20 µg/ml; I) or F(ab')2 (12 µg/ml; F) RAMIG for 2 min at 37°C or treated with pervanadate (P.V.) for 10 min at 37°C. The cell lysates (5 x 107 cells) were incubated with immobilized SHP-2 N-, C-, or N/C-SH2 domain(s). The eluates from the matrix were fractionated and analyzed by anti-phosphotyrosine immunoblotting (upper panel). The lower panel shows anti-Fc{gamma}RIIB1 immunoblotting after stripping the upper blot.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Accumulating evidence shows a crucial role of SHP-2 in receptor tyrosine kinase and cytokine stimulation of various cellular responses (2). However, the involvement of SHP-2 in signaling through Ag receptors on immune cells is unclear. In this study we assessed the binding of cellular phosphoproteins to SHP-2 during BCR signaling. We have determined that only a single major tyrosyl phosphoprotein associates with SHP-2 following BCR ligation. This 120-kDa tyrosyl-phosphorylated protein forms a complex with SHP-2 that occurs predominantly in the cytosolic fraction. The association is mediated at least in part by the N-SH2 domain of SHP-2. Finally, the occurrence of the SHP-2-pp120 complex following BCR stimulation is attenuated by the coligation of the inhibitory receptor Fc{gamma}RIIB1 with the BCR. Our observations using B cell lines (Fig. 1Go, A and B) and primary B cells (Fig. 1GoC) indicate that the binding of tyrosyl-phosphorylated p120 to SHP-2 following BCR stimulation occurs in transformed as well as immediately ex vivo normal B cells.

The interaction of pp120 with SHP-2 may occur via several mechanisms. First, pp120 may be stably associated with SHP-2 and tyrosyl phosphorylated only upon BCR stimulation. Second, SHP-2 may bind to pp120 only after it is tyrosyl phosphorylated in response to BCR stimulation, and binding may be mediated by the phosphotyrosines. Third, pp120 may exist in a constitutively phosphorylated form and be recruited to SHP-2 upon signaling. The data using SHP-2 SH2 domains (Fig. 4Go) appear to support the second hypothesis, although we cannot exclude the possibility that other binding mechanisms are involved in the interaction of pp120 with SHP-2.

Some ligands for growth factor receptors (5, 6) or cytokine receptors (31, 32, 33, 34, 35, 36, 37, 60, 61) are reported to induce the tyrosyl phosphorylation of SHP-2. The phosphorylated tyrosine residue(s) in SHP-2 is capable of recruiting SH2-containing molecules such as Grb2, allowing the downstream molecules to interact with affector molecules. However, although phosphorylation of SHP-2 can be seen in B cells following pervanadate treatment (Fig. 1GoB), we have never observed Ag receptor-mediated phosphorylation of SHP-2. This difference may indicate that the role of SHP-2 in the BCR signaling pathway is distinct from its function when the interaction of ligand with surface receptor induces the tyrosyl phosphorylation of SHP-2.

Coligation of Fc{gamma}RIIB1 with BCR is known to inhibit BCR-mediated signal transduction (40, 41). It has been shown that SHP-1 (44), SHP-2 (45), and SHIP (46, 47, 48) can bind to phospho-ITIM in Fc{gamma}RIIB1, and both SHP-1 (44, 49, 50) and SHIP (50, 51, 52, 53, 54) can mediate inhibitory signaling of Fc{gamma}RIIB1 (50). The role of tyrosine phosphatases, SHP-1 and possibly SHP-2, in inhibitory signaling may be explained by their ability to dephosphorylate molecules involved in the relay of BCR signals (49, 52, 62, 63). In A20 cells that express high level of Fc{gamma}RIIB1, Fc{gamma}RIIB1 coligation decreases BCR-induced SHP-2 association with pp120 (Figs. 1GoB and 2), whereas BCR-induced SHP-2 association with pp120 is not affected when IIA1.6 (Fc{gamma}RIIB1-negative cell) and K46µ (low Fc{gamma}RIIB1 cell) cells and primary B cells (low Fc{gamma}RIIB1 cell) are stimulated with ligands that are competent to coligate BCR and Fc{gamma}RIIB1 (Fig. 1Go, B and C). Indeed, in a time-course experiment using A20 cells, the level of the pp120-SHP-2 complex seen upon BCR-Fc{gamma}RIIB1 coligation increases with normal kinetics, but then decreases prematurely compared with the BCR response (Fig. 2Go). These observations suggest that pp120-SHP-2 complex formation is one of the targets for negative signals transduced through Fc{gamma}RIIB1. The mechanism of the dephosphorylation/dissociation of pp120 is under investigation. However, it has been demonstrated that SHP-2 phosphatase activity is increased by the occupancy of both its SH2 domains by tyrosyl-phosphorylated protein(s) (64). Based on our analysis of SHP-2 SH2 domain(s) binding, pp120 and Fc{gamma}RIIB1 may bind to SHP-2 simultaneously (Fig. 5GoB). We hypothesize that double occupancy of SH2 domains with tyrosyl-phosphorylated Fc{gamma}RIIB1 and pp120 may stimulate SHP-2 catalytic activity, leading to the dephosphorylation of tyrosyl-phosphorylated p120.

Several reports have shown that molecules with a molecular size of approximately 90–120 kDa bind to SHP-2 in cells stimulated with growth factors or cytokines. SIRP family members with 90 to 120 kDa in size are reported to bind to SHP-2 in response to growth factor stimulation (27, 28). These are transmembrane glycoproteins with Ig-like domains. In contrast, the pp120-SHP-2 complex described here is predominantly detected in the cytosolic fraction (Fig. 3GoA), providing evidence that pp120 is not a SIRP member protein or another membrane protein. Furthermore, SHP-2 is also reported to bind to JAK2, a 120-kDa cytosolic tyrosine kinase that is tyrosyl phosphorylated in response to prolactin (31) or IL-11 (32) stimulation. To address whether pp120 is JAK2, SHP-2 immunoprecipitates isolated after BCR stimulation or pervanadate treatment were subjected to fractionation and JAK2 immunoblotting. JAK2 was not detectable in these SHP-2 immunoprecipitates (data not shown). Moreover, BCR stimulation did not induce the tyrosyl phosphorylation of JAK2 (data not shown). These results indicate that pp120 is not JAK2. Recently, it was reported that a 100-kDa cytosolic molecule associates with SHP-2 upon cytokine stimulation (38, 39) or constitutively associates with SHP-2 in Bcr-Abl-transformed cells (39). In this case, the binding is mediated by SHP-2 SH2 domains, and this 100-kDa molecule is a potential substrate for SHP-2. Although its biochemical properties are similar to those of our pp120, this 100-kDa molecule seems to be smaller than pp120. Furthermore, it was shown that in TCR-stimulated T cells, a tyrosyl-phosphorylated protein 110 to 120 kDa (65) or 105 kDa (26) in size associates with SHP-2. Unlike our pp120, however, the binding of the molecule to SHP-2 is mediated by SHP-2 C-terminal SH2 domain (65), indicating that pp120 is distinct. Dos is a 115-kDa cytosolic protein identified as a substrate for the Drosophila homologue of SHP-2, Csw (29, 30). Upon sevenless receptor stimulation, tyrosyl-phosphorylated Dos binds to Csw through its SH2 domains and acts as a substrate for Csw (29). Genetic analysis demonstrates that Dos functions upstream or independently of Ras in sevenless receptor signaling pathway (30). It is described that Gab-1, a distant mammalian homologue of Dos, binds to SHP-2 upon EGF and insulin stimulation (66). However, anti-Gab-1 Ab does not react with the pp120 in immunoblotting (data not shown).

In summary, our data demonstrate that in BCR-stimulated B cells, a 120-kDa tyrosyl-phosphorylated molecule (pp120) associates with SHP-2. Moreover, pp120 is rapidly dephosphorylated or dissociates following coligation of Fc{gamma}RIIB1 with BCR. These results suggest that pp120 may act as a positive signaling molecule of the BCR-mediated signaling pathway and that the dephosphorylation/dissociation of pp120 may be a mechanism by which Fc{gamma}RIIB1-mediated negative signals are integrated. Cloning of pp120 will be required to further clarify its role in the BCR signaling pathway.


   Acknowledgments
 
We thank Dr. Andrius Kazlauskas for providing GST fusion protein containing SHP-2 C terminus, Dr. Wayne Jensen and Ms. Sara Famiglietti for technical assistance, and Ms. Judy Franconi for secretarial assistance.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health, an Ida and Cecil Green Professorship of Cell Biology (to J.C.C.), and The Ryoichi Naito Foundation for Medical Research (to K.N.). Back

2 Address correspondence and reprint requests to Dr. John C. Cambier, Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206. E-mail address: Back

3 Abbreviations used in this paper: PTP, protein tyrosine phosphatase; SH2, Src homology domain 2; PDGF, platelet-derived growth factor; EGF, epidermal growth factor; SIRP, signal regulatory protein; Dos, daughter of sevenless; JAK2, Janus kinase-2; SHIP, SH2 containing inositol 5' phosphatase; BCR, B cell antigen receptor; ITIM, immunoreceptor tyrosine-based inhibitory motif; GST, glutathione-S-transferase; HRP, horseradish peroxidase; PVDF, polyvinylidene difluoride; RAMIG, rabbit anti-mouse immunoglobulin G; N-SH2, N-terminal Src homology domain 2; C-SH2, C-terminal Src homology domain 2. Back

Received for publication December 1, 1997. Accepted for publication March 18, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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J. Immunol., May 15, 1999; 162(10): 5917 - 5923.
[Abstract] [Full Text] [PDF]

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P. Bruhns, P. Marchetti, W. H. Fridman, E. Vivier, and M. Daeron
Differential Roles of N- and C-Terminal Immunoreceptor Tyrosine-Based Inhibition Motifs During Inhibition of Cell Activation by Killer Cell Inhibitory Receptors
J. Immunol., March 15, 1999; 162(6): 3168 - 3175.
[Abstract]</