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The Journal of Immunology, 2000, 164: 631-638.
Copyright © 2000 by The American Association of Immunologists

Effects of Src Homology Domain 2 (SH2)-Containing Inositol Phosphatase (SHIP), SH2-Containing Phosphotyrosine Phosphatase (SHP)-1, and SHP-2 SH2 Decoy Proteins on Fc{gamma}RIIB1-Effector Interactions and Inhibitory Functions1

Kazuhiro Nakamura2, Anne Brauweiler and John C. Cambier3

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Coaggregation of Fc{gamma}RIIB1 with B cell Ag receptors (BCR) leads to inhibition of BCR-mediated signaling via recruitment of Src homology domain 2 (SH2)-containing phosphatases. In vitro peptide binding experiments using phosphotyrosine-containing sequences derived from the immunoreceptor tyrosine-based inhibitory motif (ITIM) known to mediate Fc{gamma}RIIB1 effects suggest that the receptor uses SH2-containing inositol phophatase (SHIP) and SH2-containing phophotyrosine phosphatase (SHP)-1, as well as SHP-2 as effectors. In contrast, coimmunoprecipitation studies of receptor-effector associations suggest that the predominant Fc{gamma}RIIB1 effector protein is SHIP. However, biologically significant interactions may be lost in such studies if reactants’ dissociation rates (Kd) are high. Thus, it is unclear to what extent these assays reflect the relative recruitment of SHIP, SHP-1, and SHP-2 to the receptor in vivo. As an alternative approach to this question, we have studied the effects of ectopically expressed SHIP, SHP-1, or SHP-2 SH2-containing decoy proteins on Fc{gamma}RIIB1 signaling. Results demonstrate the SHIP is the predominant intracellular ligand for the phosphorylated Fc{gamma}RIIB1 ITIM, although the SHP-2 decoy exhibits some ability to bind Fc{gamma}RIIB1 and block Fc receptor function. The SHIP SH2, while not affecting Fc{gamma}RIIB1 tyrosyl phosphorylation, blocks receptor-mediated recruitment of SHIP, SHIP phosphorylation, recruitment of p52 Shc, phosphatidylinositol 3,4,5-trisphosphate hydrolysis, inhibition of mitogen-activated protein kinase activation, and, albeit more modestly, Fc{gamma}RIIB1 inhibition of Ca2+ mobilization. Taken together, results implicate ITIM interactions with SHIP as a major mechanism of Fc{gamma}RIIB1-mediated inhibitory signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Aggregation of B cell Ag receptor (BCR)4 with Ag triggers transduction of signals via a rapid increase in tyrosyl phosphorylation of the receptor and many downstream effector proteins, activation of phosphatidylinositol 3-kinase (PI 3K), generation of inositol polyphosphates, sustained Ca2+ mobilization, and mitogen-activated protein (MAP) kinase activation (1, 2, 3). Coaggregation of the low-affinity Fc receptor, Fc{gamma}RIIB1, with BCR, which occurs in physiological conditions as a consequence of Ag-specific B cell interactions with IgG-containing immune complexes, leads to inhibition of certain BCR-coupled signaling pathways, terminating blastogenesis, cell proliferation, and Ab production (4, 5, 6, 7). The initial step in inhibitory signaling by Fc{gamma}RIIB1 is phosphorylation of a tyrosine residue found in the immunoreceptor tyrosine-based inhibitory motif (ITIM) present in the receptor’s cytoplasmic domain (8, 9). Phosphorylation of the ITIM upon BCR-Fc{gamma}RIIB1 coaggregation results in the recruitment of the Src homology domain 2 (SH2)-containing molecules to phosphorylated ITIM (pITIM) in Fc{gamma}RIIB1. These SH2-containing molecules presumably mediate inhibitory signaling through Fc{gamma}RIIB1. Candidate effector molecules, SH2-containing phosphotyrosine phosphatase (SHP)-1 (10) and SHP-2 (11), and an SH2-containing phosphatidylinositol-3,4,5-trisphosphate (PIP3) 5'-phosphatase (SHIP) (12, 13, 14, 15) have been shown to bind to pITIM peptides derived from Fc{gamma}RIIB1. SHP-1 and SHP-2 are closely related intracellular PTPs characterized by content of tandem SH2 domains at their N termini followed by a single catalytic domain and unique region (16), whereas SHIP is a cytosolic protein composed of a single SH2 domain at its N terminus, a catalytic domain, two phosphotyrosine binding domain (PTB) consensus sequences, and several putative SH3 interacting motifs at the C terminus (17, 18, 19). Although studies with chimeric receptors and targeted gene disruption indicate that both SHP-1 (10, 20) and SHIP (21, 22, 23) can mediate the inhibitory Fc{gamma}RIIB1 signal, little is known about the function of SHP-2 in this paradigm. SHP-1 appears to mediate inhibitory signaling via its phosphatase activity (10, 20). SHIP cleaves the 5'-phosphate from PIP3 and inositol 1,3,4,5-tetrakisphosphate (17, 18, 19). A drastic reduction of PIP3 by SHIP upon Fc{gamma}RIIB1 coaggregation with BCR has been shown to inhibit BCR-mediated Ca2+ mobilization (24, 25). In addition to its catalytic activity, SHIP functions as an adaptor molecule by binding Shc (17, 18, 19), which couples the BCR to the Grb2/Sos/Ras activation pathway (26), leading to MAP kinase activation in B cells (27, 28). SHIP interaction with p62dok, a RasGAP binding protein, has also been demonstrated.5 SHIP may mediate inhibition of the Ras signaling pathway via its tyrosyl phosphorylation and its association with Shc and p62dok (29, 30).5

Among SHIP, SHP-1, and SHP-2 molecules, SHIP is the most predominant molecule detected in Fc{gamma}RIIB1 immunoprecipitates prepared from cells in which BCR and Fc{gamma}RIIB1 have been coaggregated (12, 21, 31, 32, 33). Association of SHP-1 (10, 34) and SHP-2 (35) with Fc{gamma}RIIB1 is also detectable. This trend is consistent with results of surface plasmon resonance analysis that documented specific binding of these molecules SH2 domain(s) to the phosphorylated Fc{gamma}RIIB1 ITIM (pITIM) (36). However, the relative occurrence of pITIM interactions with these SH2-containing molecules in vivo during signaling is not well clarified. Thus, to study the role of the interaction between phosphorylated Fc{gamma}RIIB1 and these three SH2-containing molecules in Fc{gamma}RIIB1-mediated inhibitory signaling, we introduced FLAG-tagged SHIP SH2 domain, SHP-1 (SH2)2 domain, and SHP-2 (SH2)2 domain constructs into a mouse B cell lymphoma using a retroviral gene transfer system (37, 38) and analyzed their effects on Fc{gamma}RIIB1 signaling. Among these SH2 domain(s) constructs, only FLAG-SHIP SH2 domain bound efficiently to Fc{gamma}RIIB1 and markedly inhibited the interaction of endogenous SHIP with phosphorylated Fc{gamma}RIIB1, functioning as a competitive inhibitor. This inhibition was correlated with normalization of BCR-mediated signaling in terms of PIP3 production, Ca2+ mobilization, and MAP kinase activation. FLAG-SHP-2 (SH2)2 domain weakly interacted with phosphorylated Fc{gamma}RIIB1 and showed much less capacity to modulate Fc{gamma}RIIB1-mediated inhibitory signaling. FLAG-SHP-1 (SH2)2 domain had no detectable binding activity or functional effect. Taken together, our data demonstrate that among FLAG-SH2 domain(s) derived from three SH2-containing molecules, only SH2 domain of SHIP functions as an efficient competitive inhibitor of SHIP recruitment to Fc{gamma}RIIB1 and blocks Fc{gamma}RIIB1-mediated inhibitory signaling in vivo, indicating the interaction of SHIP with pITIM is an initial and crucial step 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 cells (39) and Phoenix cells (a gift of Dr. Garry P. Nolan, Stanford University), a derivative of the 293T cell line, 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. Rabbit Abs to mouse SHIP (35) and mouse Fc{gamma}RIIB1 (35) were prepared and affinity purified before use. Anti-FLAG (M5) Ab was obtained from Sigma (St. Louis, MO). Anti-phosphotyrosine Ab (Ab-2) was obtained from Oncogene Science (Manhasset, NY). Anti-phospho-p44/p42 MAP kinase Ab was obtained from New England Biolabs (Beverly, MA). Anti-Shc and anti-p44 and -p42 MAP kinase Abs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); HRP-conjugated rat anti-mouse IgG1, HRP-conjugated protein A, and intact and F(ab')2 rabbit anti-mouse IgG (H+L) (RAMIG) were purchased from Zymed (San Francisco, CA).

Constructs

For the generation of FLAG fusion proteins containing the SH2 domain(s) of SHIP, SHP-2, and SHP-1, PCR was used to amplify mouse cDNAs encoding either single or double SH2 domain(s). To prepare primer pairs for the PCR, restriction enzyme site plus Kozak sequence followed by FLAG sequence and stop codon plus restriction enzyme site were designed to be located before and after SH2 domain(s) coding sequence, respectively. FLAG-SHIP SH2 contains amino acid residues 1–114 in SHIP, FLAG-SHP-2 (SH2)2 contains amino acid residues 1–249 in SHP-2, and FLAG-SHP-1 (SH2)2 contains amino acid residues 1–241 in SHP-1. After digesting with the appropriate restriction enzymes, the resulting fragments were ligated into the same restriction enzyme cut pMXI-egfp vector (a gift of Dr. Alice Mui, DNAX, Palo Alto, CA). The FLAG-SH2 constructs were placed 5' from the internal ribosomal entry site, while enhanced green fluorescence protein (GFP) was encoded 3' from this site. Details of these constructions are available upon request.

Retroviral infection and cell sorting to isolate FLAG-SH2 domain(s)-expressing cells

Amphotropic Phoenix cells were used as packaging cells for the retrovial gene transfer system (37, 38). Prepared pMXI-egfp vectors were transfected into Phoenix cells using effectine transfection reagent (Qiagen, Valencia, CA). Two days after transfection, the supernatants were collected, diluted with the same volume of fresh complete IMDM, and polybrene was added into this diluted infection medium at 8 µg/ml. The infection mixtures were added onto the A20 cells seeded on 12-well plates, and the plates were centrifuged at 1000 x g for 1 h at room temperature. The cells were incubated at 32°C overnight and then transferred to 10-cm dishes with fresh complete IMDM. Typically, 30–60% of cells were GFP-positive following exposure to virus, indicating that this proportion of cells was infected. After expansion, cells were sorted twice based on GFP expression using an Epics Elite cell sorter (Miami, FL). The sorted cells were propagated before use in the experiments. Immediately before use, cells were analyzed by flow microfluorometry to assure comparable expression of GFP (see Fig. 1Go).



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  		FIGURE 1. Expression of GFP (A) and FLAG-tagged SH2 domain(s) (B) in SHIP, SHP-2, and SHP-1 SH2 domain(s)-expressing A20 cells. FLAG-SHIP SH2, -SHP-2 (SH2)2, or -SHP-1 (SH2)2 domain cDNA inserted in multicloning site of pMXI-egfp vector or pMXI-egfp vector alone was introduced into A20 cells as described in Materials and Methods. A, GFP expression in the inserted A20 cells detected by flow cytometery. Gray and black lines illustrate the fluorescence intensity of A20 cells and FLAG-SH2 domain(s)- or vector-alone-expressing cells, respectively. B, Anti-FLAG immunoblots of SDS-PAGE-fractionated aliquots (1 x 106 cell equivalent) from the cells. The aliquots were from the cell lysates prepared for Fig. 2Go experiment. The lysates from the nonstimulated cells (-) and the cells stimulated with BCR-Fc{gamma}RIIB1 cross-linking Ab (20 µg/ml) (I) for 1 min were fractionated using SDS-PAGE, transferred, and analyzed using anti-FLAG immunoblotting. Bottom panel shows anti-SHIP immunoblots of upper membrane to confirm equal protein amount loading in each lane.

 
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, they were stimulated with intact or F(ab')2 RAMIG for the period indicated. After stimulation, the cells were washed three times with ice-cold PBS and lysed with solubilizing buffer (1% Triton-X, 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.

Immunoprecipitation and immunoblotting analysis

To confirm the expression of the SHIP, Fc{gamma}RIIB1, and FLAG-SH2 domains in the cells, lysates were subjected to immunoprecipitation with anti-SHIP, -Fc{gamma}RIIB1, or -FLAG Ab. Immune complexes were collected with protein A Sepharose beads (Pharmacia, Piscataway, NJ) (for SHIP and Fc{gamma}RIIB1 immunoprecipitation) or protein G Sepharose beads (for FLAG immunoprecipitation), separated by 8% SDS-PAGE, and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). After blocking, PVDF membranes were blotted with anti-phosphotyrosine or -FLAG Ab and HRP-conjugated rat anti-mouse IgG1 using the enhanced chemiluminescence Western blotting detection system (Amersham, Buckinghamshire, U.K.). In some cases, after blotting with anti-phosphotyrosine or -FLAG Ab, membranes were stripped to remove the Ab and subject to sequential blotting with other Abs. The membranes were incubated with the anti-SHIP, -Fc{gamma}RIIB1, -Shc, -p44/p42 MAP kinase, -phospho-p44/p42 MAP kinase Ab followed by incubation with HRP-conjugated protein A.

Measurement of PIP3

As described previously (40), cells were harvested, incubated at 107 cells/ml in low phosphate medium with 0.5 mCi/ml [32P]orthophosphate for 1 h, and washed. 32P-labeled cells were stimulated with intact or F(ab')2 RAMIG for the indicated period and lysed with acidified methanol/chloroform (2:1 v/v). Lipids were extracted, deacylated with methanol/25% methylamine/n-butanol (45.7:42.8:11.4 v/v/vol), and HPLC was used to fractionate deacylated phosphoinositides. The fractions containing PIP3 were collected and quantitated radiometrically.

Flow cytometric analysis of Ca2+ mobilization

Intracellular free calcium concentration ([Ca2+]i) was determined by preloading the cells with Indo-1 AM (Molecular Probes, Eugene, OR) and monitoring with 400 and 490 fluorescence by flow cytometry (model 50H; Ortho Diagnostic Systems, MA) as previously described (41). The mean [Ca2+] i was evaluated with an appended data acquisition system and MultiTIME software (Phoenix Flow Systems, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The expression of FLAG-SH2 domain(s)-containing proteins in A20 B lymphoma cells

SHIP (17, 18, 19) SHP-1 and -2 molecules contain SH2 domains that function to localize and regulate enzymes (16). To compare the ability of the SH2 domain(s) of these three molecules to inhibit Fc{gamma}RIIB1 function in B cells, FLAG-tagged SHIP SH2 domain, SHP-1 (SH2)2 domain, and SHP-2 (SH2)2 domain cDNAs were prepared and expressed in the mouse B cell lymphoma cell line, A20 (mIgG2a (+) and Fc{gamma}RIIB1 (+)). GFP expression-matched populations were selected by cell sorting and expanded (Fig. 1GoA). To further confirm the expression of FLAG-SH2 domain(s) fusion proteins in A20 cells, total cell lysates from these gene-transferred cells were analyzed with anti-FLAG immunoblotting (Fig. 1GoB). The expression levels of FLAG fusion protein were equal in FLAG-SHIP SH2 domain and -SHP-2 (SH2)2 domain-containing cells, and 40% less in FLAG-SHP-1 (SH2)2 domain-containing cells. Moreover, we confirmed that the expression of mouse IgG and Fc{gamma}RIIB1 by these three FLAG-SH2 domain(s)-expressing populations was the same as the parent A20 cells (data not shown). These populations were used for the subsequent experiments.

The FLAG-SHIP SH2 domain binds to tyrosyl-phosphorylated Fc{gamma}RIIB1 in vivo

To assess the binding of FLAG-SH2 domain(s) to Fc{gamma}RIIB1, and the effect of these constructs on Fc{gamma}RIIB1 tyrosyl phosphorylation, we immunoprecipitated Fc{gamma}RIIB1 from the FLAG-SH2 domain(s)-expressing cells following stimulation with intact (I) RAMIG H and L chains. This Ab coaggregates BCR-Fc{gamma}RIIB1. Immunoprecipitates were fractionated and immunoblotted with anti-phosphotyrosine, -FLAG, and -Fc{gamma}RIIB1 Abs (Fig. 2GoA). The tyrosyl phosphorylation of Fc{gamma}RIIB1 upon BCR-Fc{gamma}RIIB1 coaggregation was detected in all FLAG-SH2 domain(s)-expressing cells as well as vector alone cells. The tyrosyl phosphorylation of Fc{gamma}RIIB1 in FLAG-SH2 domain(s)-expressing cells was equivalent and slightly stronger than the Fc{gamma}RIIB1 phosphorylation in vector alone cells (Fig. 2GoA, middle panel). Analysis of phosphorylation at later time points revealed no significant effects of the decoy proteins on Fc{gamma}RIIB1 phosphorylation (data not shown). Despite comparable tyrosyl phosphorylation, the pattern of proteins that coimmunoprecipitated with Fc{gamma}RIIB1 was affected by the expression of SH2 domain constructs. As shown in Fig. 2GoA, top panel, FLAG-SHIP SH2 domain and much less -SHP-2 (SH2)2 domain, but no -SHP-1 (SH2)2 domain, were detected in Fc{gamma}RIIB1 immunoprecipitates from BCR-Fc{gamma}RIIB1 coaggregated cells. To further assess the interaction between FLAG-SH2 domain(s) and Fc{gamma}RIIB1, anti-FLAG immunoprecipitation was undertaken (Fig. 2GoB). Basal binding of Fc{gamma}RIIB1 to FLAG-SHIP SH2 domain was faintly detected, and it was dramatically enhanced by BCR-Fc{gamma}RIIB1 coaggregation correlating with Fc{gamma}RIIB1 phosphorylation. In FLAG-SHP-2 (SH2)2 domain-expressing cells, much less, though significant, Fc{gamma}RIIB1 was detected in anti-FLAG immunoprecipitates, and this was only seen upon BCR-Fc{gamma}RIIB1 coaggregation. By contrast, Fc{gamma}RIIB1 was not detectable in FLAG-SHP-1 (SH2)2 domain-expressing cells even after the BCR-Fc{gamma}RIIB1 coaggregation. These data demonstrate that FLAG-SHIP SH2 domain and to a much lesser extent the FLAG-SHP-2 (SH2)2 domain are capable of occupying the pITIM of Fc{gamma}RIIB1 in vivo, suggesting that SHIP is the predominant Fc{gamma}RIIB1 binding molecule in vivo. However, it seems likely that following receptor coaggregation a significant proportion of pITIMs bind SHP-2.



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  		FIGURE 2. The recruitment of FLAG-tagged SH2 domain(s) to Fc{gamma}RIIB1 upon BCR-Fc{gamma}RIIB1 coaggregation and its effect on Fc{gamma}RIIB1 phosphorylation. The indicated FLAG-SH2 domain(s) construct or vector-alone-expressing A20 cells were cultured without stimulus (-) or stimulated with intact (20 µg/ml) RAMIG (I) for 1 min at 37°C. The cells were lysed as described in Materials and Methods. A, Fc{gamma}RIIB1(1 x 108 cells) was immunoprecipitated with anti-Fc{gamma}RIIB Ab. Fc{gamma}RIIB1 immunoprecipitates were fractionated using SDS-PAGE and transferred on PVDF membrane. The membrane was sequentially blotted with anti-FLAG, -phosphotyrosine, and -Fc{gamma}RIIB1 Abs. The top panel was blotted with anti- FLAG Ab. Middle and bottom panels were blotted with anti-phosphotyrosine and anti-Fc{gamma}RIIB1, respectively. B, FLAG-SH2 domain(s) proteins in the cell lysates (1 x 108 cells) were immunoprecipitated with anti-FLAG Ab. The immunoprecipitates were fractionated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with anti-Fc{gamma}RIIB1. Results shown in this and subsequent figures are in each case representative of three replicate experiments

 
The FLAG-SHIP SH2 domain blocks Fc{gamma}RIIB1-mediated SHIP tyrosyl-phosphorylation and its association with Fc{gamma}RIIB1 and Shc molecules

To study the effects of FLAG-SH2 domain(s) on endogenous SHIP phosphorylation and association with effector molecules, SHIP was immunoprecipitated from FLAG-SH2 domain(s)-expressing cells and analyzed by anti-phosphotyrosine, -Fc{gamma}RIIB1, and -Shc immunoblottings (Fig. 3Go). Upon BCR-Fc{gamma}RIIB1 coaggregation, endogenous SHIP was less tyrosyl-phosphorylated and associated with much less Fc{gamma}RIIB1 in FLAG-SHIP SH2 domain-expressing cells compared with other FLAG-(SH2)2 domain- or vector-alone-expressing cells (Fig. 3Go, top two panels). Because the level of tyrosyl phosphorylation of Fc{gamma}RIIB1 following BCR-Fc{gamma}RIIB1 coaggregation in FLAG-SHIP SH2 domain-expressing cells was not weaker than the control cells (Fig. 2GoA), we concluded that the occupancy of pITIM in Fc{gamma}RIIB1 by FLAG-SHIP SH2 domain blocked the binding of phosphorylated Fc{gamma}RIIB1 to endogenous SHIP. This resulted in reduced SHIP tyrosyl phosphorylation, presumably due to the reduced proximity of SHIP molecules to BCR-activated tyrosine kinase(s). By contrast, neither FLAG-SHP-2 (SH2)2 domains nor -SHP-1 (SH2)2 domains inhibited the tyrosyl phosphorylation of SHIP or its association to Fc{gamma}RIIB1. The association of SHIP with Fc{gamma}RIIB1 was somewhat enhanced in these two FLAG-(SH2)2 domain-expressing cells. This may be due to the effect of marginally increased tyrosyl phosphorylation of Fc{gamma}RIIB1 in FLAG-SH2 domain(s)-expressing cells (Figs. 2GoA and 3). These data indicate that FLAG-SHIP SH2 domain blocks the recruitment of endogenous SHIP molecules to phosphorylated Fc{gamma}RIIB1.



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  		FIGURE 3. The effect of FLAG-tagged SH2 domain(s) on SHIP phosphorylation and its association with other proteins. Following stimulation with intact (20 µg/ml) (I) or F(ab')2 (12 µg/ml) (F) RAMIG for 1 min at 37°C, the indicated FLAG-SH2 domain(s) constructs or vector-alone-expressing A20 cells (1 x 108 cells) were solubilized. SHIP was immunoprecipitated with anti-SHIP Ab, fractionated by SDS-PAGE, and transferred on PVDF membrane. The membrane was sequentially blotted with anti-phosphotyrosine (top panel), -Fc{gamma}RIIB1 (second panel), -Shc (third panel), and -SHIP (bottom panel) Abs.

 
It was previously shown that BCR-Fc{gamma}RIIB1 coaggregation leads to SHIP association with Shc (29). Interestingly, we found that p46 Shc is bound to SHIP in resting cells (Fig. 3Go, third panel from top). In all FLAG-SH2 domain(s)-expressing cells, as well as in vector-alone-expressing cells, BCR aggregation and BCR-Fc{gamma}RIIB1 coaggregation led to dissociation of about 50% and >80% of p46 Shc molecules, respectively. Conversely, BCR aggregation induced the association of p52 Shc with SHIP, and the amount of p52 Shc in SHIP immunoprecipitates was further increased by coaggregation with Fc{gamma}RIIB1. In the same stimulation conditions, there was no significant difference in the amount of p46 Shc in SHIP immunoprecipitates among any FLAG-SH2 domain(s) and control vector-expressing cells. By contrast, in FLAG-SHIP SH2 domain-expressing cells and in FLAG-SHP-2 (SH2)2 domain-expressing cells, BCR-Fc{gamma}RIIB1 coaggregation decreased the amount of p52 Shc in SHIP immunoprecipitates to 25 and 60% of vector alone cells, whereas there was no difference in p52 Shc-SHIP association in SHP-1 (SH2)2 domain-expressing cells compared with vector-alone cells. These data indicate that the interaction of SHIP with p52 Shc is inhibited by FLAG-SHIP SH2 and -SHP-2 (SH2)2 domains. These results are correlated with decoy protein inhibition of SHIP phosphorylation, consistent with Shc PTB domain binding to SHIP via phosphotyrosines.

The FLAG-SHIP SH2 domain inhibits the hydrolysis of PIP3 following Fc{gamma}RIIB1 coaggregation with BCR

It has been hypothesized that levels of PIP3 in B cells are mainly balanced by the activity/location of PI 3K and SHIP. PIP3 is generated from phosphatidylinositol 4,5 biphosphate by PI 3K upon BCR stimulation (40). In the case of BCR-Fc{gamma}RIIB1 coaggregation, the elevation in PIP3 following BCR stimulation is rapidly terminated by the recruitment of SHIP to the plasma membrane by phosphorylated-Fc{gamma}RIIB1 (25). Thus, to assess the effect of FLAG-SH2 domain(s) on PIP3 hydrolysis by SHIP, PIP3 levels were compared following BCR aggregation alone or BCR-Fc{gamma}RIIB1 coaggregation among the FLAG-SH2 domain(s)-expressing cells (Fig. 4Go). In FLAG-SHIP SH2 domain-expressing cells, the normal complete loss of PIP3 product following receptor coaggregation was normalized nearly to that seen in BCR-stimulated cells. The decrease in PIP3 normally seen upon BCR-Fc{gamma}RIIB1 coaggregation was reduced modestly in FLAG-SHP-1 and -SHP-2 (SH2)2 domain expressing cells, i.e., up to 20 and 10% of PIP3 generated by BCR stimulation. Together with Figs. 2Go, A and B, and 3, these data indicate that FLAG-SHIP SH2 domain inhibits the recruitment of SHIP to phosphorylated Fc{gamma}RIIB1, consequently sustaining the high level of PIP3 created by BCR stimulation even in the presence of a normal phosphorylation of Fc{gamma}RIIB1. Blocking of the pITIM prevents Fc{gamma}RIIB1-mediated modulation of PIP3 levels.



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  		FIGURE 4. The effect of FLAG-tagged SH2 domain(s) on FCR-mediated PIP3 degradation. The indicated constructs or vector-alone-expressing A20 cells (1 x 107 cells/ml) were labeled with [32P]orthophosphate for 1 h. These cells were stimulated with intact (20 µg/ml) (I) or F(ab')2 (12 µg/ml) (F) RAMIG for 1 min at 37°C and immediately lysed with methanol/chloroform (2:1 ratio by volume). Phospholipids were extracted, deacylated, and fractionated by HPLC. The fractions containing PIP3 were quantitated by liquid scintillation counting.

 
The FLAG-SHIP SH2 domain blocks Fc{gamma}RIIB1-mediated inhibition of Ca2+ mobilization

It is known that the interaction of Fc{gamma}RIIB1 with SHIP plays a critical role in Fc{gamma}RIIB1-mediated inhibition of BCR-induced Ca2+ influx (21, 24). This reportedly reflects a PIP3 requirement for Btk (24, 25, 42) and phospholipase C {gamma} translocation (43) to the plasma membrane. To assess the effect of pITIM blockade on Fc{gamma}RIIB1-mediated inhibition of this event, we measured Ca2+ mobilization following stimulation of FLAG-SH2 domain(s)-expressing cells (Fig. 5Go). In FLAG-SHIP SH2 domain-expressing cells, the early phase of Ca2+ mobilization after BCR-Fc{gamma}RIIB1 coaggregation was longer than in cells expressing vector alone, and the late-phase Ca2+ response was significantly elevated and similar to that induced by BCR aggregation alone. Following BCR-Fc{gamma}RIIB1 coaggregation, FLAG-SHP-2 and -SHP-1 (SH2)2 domain-expressing cells also showed a longer early phase compared with cells expressing vector alone, but the Fc{gamma}RIIB1-dependent suppression of late-phase Ca2+ mobilization was similar to control cells. The data indicate that the FLAG-SHIP SH2 domain is capable of partially extinguishing the pITIM-mediated inhibitory effect of Fc{gamma}RIIB1 on BCR-mediated Ca2+ mobilization.



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  		FIGURE 5. The effect of FLAG-tagged SH2 domain(s) on Fc{gamma}RIIB1-mediated inhibition of Ca2+ mobilization. The indicated construct-expressing A20 cells (1 x 106/ml/sample) were loaded with Indo-1 AM and stimulated with either intact (20 µg/ml) or F(ab')2 (12 µg/ml) RAMIG. Shown is the mean intracellular free Ca2+ concentration ([Ca2+]i) of the entire population over time based on analysis of ~900 cells/second.

 
The effects of FLAG-SH2 domain(s) on Fc{gamma}RIIB1-dependent inhibition of MAP kinase phosphorylation

It has been reported that BCR stimulation activates MAP kinase(s) and that this MAP kinase(s) activation is prematurely terminated by Fc{gamma}RIIB1 coaggregation with the BCR (23, 30, 44). The activation of MAP kinase(s) is known to be dependent on its phosphorylation so that, in this experiment, MAP kinase activity was evaluated based on an increase in the phosphorylation, as well as upper mobility shift in a separating gel (45). To see the effects of FLAG-SH2 domain(s) constructs in MAP kinase phosphorylation, total cell lysates were prepared from stimulated FLAG-SH2 domain(s)-expressing cells and analyzed with anti-phospho-p44/42 MAP kinase and -p44/42 MAP kinase Abs (Fig. 6Go). In the control cells, BCR stimulation induced both p44 and p42 MAP kinase phosphorylation and a correlated mobility shift of p44/p42 MAP kinase was detected. Both the phosphorylation and mobility shift induced by BCR stimulation were inhibited by the coaggregation with Fc{gamma}RIIB1. In FLAG-SHIP SH2 domain-expressing cells, the inhibition of both MAP kinase phosphorylation and mobility shift of p44/p42 molecules induced by Fc{gamma}RIIB1 coaggregation was almost completely blocked. The FLAG SHP-2 SH2 domain had a similar but more modest effect. In contrast, the FLAG-SHP-1 (SH2)2 domain had no effect on Fc{gamma}RIIB1-mediated inhibition of MAP kinase activation. These results are consistent with previous findings that Fc{gamma}RIIB1 inhibition of extracellular signal-related kinase phosphosphorylation involves SHIP and confirm that this effect is mediated by the ITIM (23).



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  		FIGURE 6. The effect of FLAG-tagged SH2 domain(s) on Fc{gamma}RIIB1-mediated inhibition of p44/p42 MAP kinase phosphorylation. The indicated construct-expressing A20 cells (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 (1 x 106 cell equivalent) were fractionated by SDS-PAGE, transferred, and analyzed by anti-phospho-p44/p42 MAP kinase immunoblotting (upper panel). The lower panel shows an anti-p44/p42 MAP kinase immunoblot generated after stripping the upper blot.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Fc{gamma}RIIB1 is a member of a growing family of inhibitory receptors characterized by content of a cytoplasmic sequence motif termed the ITIM (I/VxYxxL/V) (6). The ITIM contains all structural information needed for transduction of inhibitory signals (9). While the phosphorylated Fc{gamma}RIIB1 ITIM binds tyrosine phosphatases SHP-1 (10) and SHP-2 (11), and the inositol 5' phosphatase SHIP (12, 13, 14, 15) in vitro, and, under certain circumstances, coimmunoprecipitates with all of these molecules (10, 12, 21, 31, 32, 33, 34, 35), only SHIP appears necessary for the manifestations of inhibitory signaling documented to date (21, 22, 23). All other members of the ITIM-containing receptor family require SHP-1, not SHIP, for inhibitory signaling (6). Finally, Fc{gamma}RIIB receptor chimeras containing either SHIP or SHP-1 cytoplasmic domains can mediate inhibition of BCR signaling (21). Based on these findings, one would predict that both SHIP and SHP-1 could mediate Fc{gamma}RIIB1 signals.

In an effort to resolve these apparent inconsistencies and further explore the function of the ITIM and its effectors, we assessed the ability of ectopically expressed SHIP, SHP-1 and SHP-2 SH2 domain decoy proteins to bind Fc{gamma}RIIB1 in vivo and to modulate Fc{gamma}RIIB1 signaling. These experiments were designed to 1) establish the relative ability of these proteins to associate with, and thus function as, effectors of Fc{gamma}RIIB1, and 2) provide independent verification of the downstream targets of the receptor’s ITIM in BCR signaling cascades. Results demonstrate that the SHIP SH2 domain binds efficiently to phosphorylated Fc{gamma}RIIB1 in vivo; a finding consistent with its reported ~100 nM binding affinity (Ko) defined by surface plasmon resonance analysis (36). Thus, binding of SHIP to Fc{gamma}RIIB1 in vivo does not require regions outside its SH2 domain, such as C-terminal proline-rich regions that bind Grb2 (18). Although to a much lessor degree, decoy proteins containing both SHP-2 SH2 domains also bound Fc{gamma}RIIB1 in vivo, while SHP-1 (SH2)2 did not bind detectably. These findings are consistent with the affinity hierarchy of SHIP > SHP-2 > SHP-1 SH2 binding to Fc{gamma}RIIB1 pITIM peptides (36).

Despite the apparent efficiency of SHIP SH2 binding to phosphorylated Fc{gamma}RIIB1, expression of the fusion protein did not enhance phosphorylation of the receptor significantly as would be expected if the SH2 shielded ITIM phosphotyrosines from phosphatases (Fig. 2GoA). This could result from the high off-rate of the SHIP SH2-pITIM, or insufficient expression of the SH2 to occupy most pITIMs. The latter seems unlikely because sufficient SH2 was expressed to block >70% of phosphorylated Fc{gamma}RIIB-endogenous SHIP interaction. These data indicate, consistent with conclusions reached using other approaches, that SHIP is likely to be the most biologically significant Fc{gamma}RIIB1 effector among SHIP, SHP-1, and SHP-2. However, one cannot exclude that inter- and intramolecular interactions involving other domains of these molecules may modulate their binding, compromising this interpretation. For example, it is hypothesized that interaction of the SHIP C-terminal proline-rich region with Grb2 enhances SHIP binding to the phosphorylated Fc{gamma}RIIB16 because the Grb2 SH2 domain appears to bind phosphorylated Y326 of Fc{gamma}RIIB1 cytoplasmic tail. The resultant bidentate interaction would further enhance SHIP avidity for phosphorylated receptors. There is no evidence that non-SH2 determinants in either SHP-2 or SHP-1 modulate their binding to Fc{gamma}RIIB.

Consistent with a major role for the ITIM in mediating Fc{gamma}RIIB1 inhibitory function, the SHIP SH2 decoy blocks a number of effects of the receptor. It blocked Fc{gamma}RIIB1 binding to endogenous SHIP, supporting the criticality of the SH2 for formation of detectable SHIP-phosphorylated Fc{gamma}RIIB1 complexes. Consistent with blocking of SHIP binding, the decoy protein inhibited Fc{gamma}RIIB1-mediated hydrolysis of PIP3, and reduction of late-phase calcium mobilization and MAP kinase phosphorylation. These findings are consistent with PIP3 requirements for optimal Btk and phospholipase C {gamma} activation, and subsequent Ca2+ mobilization (42, 43), and pITIM targeting of PIP3 by recruitment of SHIP (24, 25). Effects on MAP kinase activation are consistent with the possibility that pITIM recruitment of SHIP leads to recruitment of Shc and p62dok to SHIP, resulting in the inhibition of the Ras pathway (29, 30).5 Consistent with their in vivo binding activity, the SHP-2 (SH2)2 modestly inhibited Fc{gamma}R effects on Ca2+ mobilization and MAP kinase phosphorylation, while SHP-1 (SH2)2 had no effect. These findings support the conclusion that SHP-2 may play a minor, but significant role in Fc{gamma}RIIB1 signaling.

One of the most surprising findings of this study is the observation that SHIP associates with p46 Shc in unstimulated B cells, and the association is reduced following BCR signaling and lost following BCR-Fc{gamma}RIIB1 coaggregation (Fig. 3Go). Conversely, BCR signaling and BCR-Fc{gamma}RIIB1 coaggregation leads to increased binding to p52 Shc. These Shc isoforms differ in N-terminal sequence just before or within the PTB domain; the domain that mediates Shc binding to phosphorylated SHIP (46, 47, 48). It is tempting to speculate that the PTB domain in p46 Shc preferentially binds a nonphosphorylated (NPxY) site in SHIP. Phosphorylation of this site may reduce its affinity for the p46 Shc PTB domain and increase its affinity for the canonical p52 Shc PTB domain. The functional consequence of this switch is unknown. Studies are in progress to better define the basis of this interesting phenomenon and its relationship to SHIP and ITIM function.


   Acknowledgments
 
We thank Dr. John C. Stolpa and Sara J. Famiglietti for technical assistance and Natalie C. Arnold for secretarial assistance.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health. J.C.C. is Ida and Cecil Green Professor of Cell Biology. K.N. is supported by the Ryoichi Naito Foundation for Medical Research. Back

2 Current address: Department of Immunology and Immunopathology, Kagawa Medical University, Kagawa, 761-0793, Japan. Back

3 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 Street, Denver, CO 80206. E-mail address: Back

4 Abbreviations used in this paper: BCR, B cell Ag receptor; PI 3K, phosphatidylinositol 3-kinase; MAP, mitogen-activated protein; ITIM, immunoreceptor tyrosine-based inhibitory motif; pITIM, phosphorylated ITIM; SH2, Src homology domain 2; PTP, protein tyrosine phosphatase; SHP, SH2-containing phophotyrosine phosphatase; PTB, phosphotyrosine binding domain; PIP3, phosphatidylinositol 3,4,5-trisphosphate; SHIP, SH2-containing inositol phosphatase; GFP, enhanced green fluorescent protein; RAMIG, rabbit anti-mouse IgG; PVDF, polyvinylidene difluoride. Back

5 I. Tamir, J. C. Stolpa, C. Helgason, R. K. Humphries, K. Nakamura, P. Bruhns, M. Daeron, and J. C. Cambier. 1999. The RasGAP-binding protein p62dok is a mediator of inhibitory Fc{gamma}RIIb signals in B cells. Submitted for publication. Back

6 D. C. Fong, A. Brauweiler, S. A. Minskoff, P. Bruhns, I. Tamir, I. Mellman, M. Daeron, and J. C. Cambier. 1999. Mutational analysis reveals multiple distinct sites within Fc{gamma}RIIb that function in inhibitory signaling. Submitted for publication. Back

Received for publication August 5, 1999. Accepted for publication October 28, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cambier, J. C., C. M. Pleiman, M. R. Clark. 1994. Signal transduction by the B cell antigen receptor and its coreceptors. Annu. Rev. Immunol. 12:457.[Medline]
  2. Gold, M. R., A. L. DeFranco. 1994. Biochemistry of B lymphocyte activation. Adv. Immunol. 55:221.[Medline]
  3. Campbell, K. S.. 1999. Signal transduction from the B cell antigen-receptor. Curr. Opin. Immunol. 11:256.[Medline]
  4. Chan, P. L., N. R. Sinclair. 1971. Regulation of the immune response. V. An analysis of the function of the Fc portion of antibody in suppression of an immune response with respect to interaction with components of the lymphoid system. Immunology 21:967.[Medline]
  5. Phillips, N. E., D. C. Parker. 1983. Fc-dependent inhibition of mouse B cell activation by whole anti-µ antibodies. J. Immunol. 130:602.[Abstract]
  6. Cambier, J. C.. 1997. Inhibitory receptors abound?. Proc. Natl. Acad. Sci. USA 94:5993.[Free Full Text]
  7. Coggeshall, K. M.. 1998. Inhibitory signaling by B cell Fc{gamma}RIIb. Curr. Opin. Immunol. 10:306.[Medline]
  8. Amigorena, S., C. Bonnerot, J. R. Drake, D. Choquet, W. Hunziker, J. G. Guillet, P. Webster, C. Sautes, I. Mellman, W. H. Fridman. 1992. Cytoplasmic domain heterogeneity and functions of IgG Fc receptors in B lymphocytes. Science 256:1808.[Abstract/Free Full Text]
  9. Muta, T., T. Kurosaki, Z. Misulovin, M. Sanchez, M. C. Nussenzweig, J. V. Ravetch. 1994. A 13-amino-acid motif in the cytoplasmic domain of Fc{gamma}RIIB modulates B-cell receptor signalling. Nature 368:70.[Medline]
  10. D’Ambrosio, D., K. L. Hippen, S. A. Minskoff, I. Mellman, G. Pani, K. A. Siminovitch, J. C. Cambier. 1995. Recruitment and activation of PTP1C in negative regulation of antigen receptor signaling by Fc{gamma}RIIB1. Science 268:293.[Abstract/Free Full Text]
  11. Olcese, L., P. Lang, F. Vely, A. Cambiaggi, D. Marguet, M. Blery, K. L. Hippen, R. Biassoni, A. Moretta, L. Moretta, J. C. Cambier, E. Vivier. 1996. Human and mouse killer-cell inhibitory receptors recruit PTP1C and PTP1D protein tyrosine phosphatases. J. Immunol. 156:4531.[Abstract]
  12. Ono, M., S. Bolland, P. Tempst, J. V. Ravetch. 1996. Role of the inositol phosphatase SHIP in negative regulation of the immune system by the receptor Fc{gamma}RIIB. Nature 383:263.[Medline]
  13. D’Ambrosio, D., D. C. Fong, J. C. Cambier. 1996. The SHIP phosphatase becomes associated with Fc{gamma}RIIB1 and is tyrosine phosphorylated during ‘negative’ signaling. Immunol. Lett. 54:77.[Medline]
  14. Fong, D. C., O. Malbec, M. Arock, J. C. Cambier, W. H. Fridman, M. Daeron. 1996. Selective in vivo recruitment of the phosphatidylinositol phosphatase SHIP by phosphorylated Fc{gamma}RIIB during negative regulation of IgE- dependent mouse mast cell activation. Immunol. Lett. 54:83.[Medline]
  15. Tridandapani, S., T. Kelley, M. Pradhan, D. Cooney, L. B. Justement, K. M. Coggeshall. 1997. Recruitment and phosphorylation of SH2-containing inositol phosphatase and Shc to the B-cell Fc{gamma} immunoreceptor tyrosine-based inhibition motif peptide motif. Mol. Cell. Biol. 17:4305.[Abstract]
  16. Tonks, N. K., B. G. Neel. 1996. From form to function: signaling by protein tyrosine phosphatases. Cell 87:365.[Medline]
  17. Damen, J. E., L. Liu, P. Rosten, R. K. Humphries, A. B. Jefferson, P. W. Majerus, G. Krystal. 1996. The 145-kDa protein induced to associate with Shc by multiple cytokines is an inositol tetraphosphate and phosphatidylinositol 3,4,5-triphosphate 5-phosphatase. Proc. Natl. Acad. Sci. USA 93:1689.[Abstract/Free Full Text]
  18. Kavanaugh, W. M., D. A. Pot, S. M. Chin, M. Deuter-Reinhard, A. B. Jefferson, F. A. Norris, F. R. Masiarz, L. S. Cousens, P. W. Majerus, L. T. Williams. 1996. Multiple forms of an inositol polyphosphate 5-phosphatase form signaling complexes with Shc and Grb2. Curr. Biol. 6:438.[Medline]
  19. Lioubin, M. N., P. A. Algate, S. Tsai, K. Carlberg, A. Aebersold, L. R. Rohrschneider. 1996. p150Ship, a signal transduction molecule with inositol polyphosphate-5-phosphatase activity. Genes Dev. 10:1084.[Abstract/Free Full Text]
  20. Pani, G., M. Kozlowski, J. C. Cambier, G. B. Mills, K. A. Siminovitch. 1995. Identification of the tyrosine phosphatase PTP1C as a B cell antigen receptor-associated protein involved in the regulation of B cell signaling. J. Exp. Med. 181:2077.[Abstract/Free Full Text]
  21. Ono, M., H. Okada, S. Bolland, S. Yanagi, T. Kurosaki, J. V. Ravetch. 1997. Deletion of SHIP or SHP-1 reveals two distinct pathways for inhibitory signaling. Cell 90:293.[Medline]
  22. Gupta, N., A. M. Scharenberg, D. N. Burshtyn, N. Wagtmann, M. N. Lioubin, L. R. Rohrschneider, J. P. Kinet, E. O. Long. 1997. Negative signaling pathways of the killer cell inhibitory receptor and Fc{gamma}RIIb1 require distinct phosphatases. J. Exp. Med. 186:473.[Abstract/Free Full Text]
  23. Liu, Q., A. J. Oliveira-Dos-Santos, S. Mariathasan, D. Bouchard, J. Jones, R. Sarao, I. Kozieradzki, P. S. Ohashi, J. M. Penninger, D. J. Dumont. 1998. The inositol polyphosphate 5-phosphatase SHIP is a crucial negative regulator of B cell antigen receptor signaling. J. Exp. Med. 188:1333.[Abstract/Free Full Text]
  24. Bolland, S., R. N. Pearse, T. Kurosaki, J. V. Ravetch. 1998. SHIP modulates immune receptor responses by regulating membrane association of Btk. Immunity 8:509.[Medline]
  25. Scharenberg, A. M., O. El-Hillal, D. A. Fruman, L. O. Beitz, Z. Li, S. Lin, I. Gout, L. C. Cantley, D. J. Rawlings, J. P. Kinet. 1998. Phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3)/Tec kinase-dependent calcium signaling pathway: a target for SHIP-mediated inhibitory signals. EMBO J. 17:1961.[Medline]
  26. Saxton, T. M., I. van Oostveen, D. Bowtell, R. Aebersold, M. R. Gold. 1994. B cell antigen receptor cross-linking induces phosphorylation of the p21ras oncoprotein activators SHC and mSOS1 as well as assembly of complexes containing SHC, GRB-2, mSOS1, and a 145-kDa tyrosine-phosphorylated protein. J. Immunol. 153:623.[Abstract]
  27. Sarmay, G., G. Koncz, J. Gergely. 1996. Human type II Fc{gamma} receptors inhibit B cell activation by interacting with the p21(ras)-dependent pathway. J. Biol. Chem. 271:30499.[Abstract/Free Full Text]
  28. Hashimoto, A., H. Okada, A. Jiang, M. Kurosaki, S. Greenberg, E. A. Clark, T. Kurosaki. 1998. Involvement of guanosine triphosphatases and phospholipase C-{gamma}2 in extracellular signal-regulated kinase, c-Jun NH2-terminal kinase, and p38 mitogen-activated protein kinase activation by the B cell antigen receptor. J. Exp. Med. 188:1287.[Abstract/Free Full Text]
  29. Chacko, G. W., S. Tridandapani, J. E. Damen, L. Liu, G. Krystal, K. M. Coggeshall. 1996. Negative signaling in B lymphocytes induces tyrosine phosphorylation of the 145-kDa inositol polyphosphate 5-phosphatase, SHIP. J. Immunol. 157:2234.[Abstract]
  30. Tridandapani, S., G. W. Chacko, J. R. Van Brocklyn, K. M. Coggeshall. 1997. Negative signaling in B cells causes reduced Ras activity by reducing Shc-Grb2 interactions. J. Immunol. 158:1125.[Abstract]
  31. Kiener, P. A., M. N. Lioubin, L. R. Rohrschneider, J. A. Ledbetter, S. G. Nadler, M. L. Diegel. 1997. Co-ligation of the antigen and Fc receptors gives rise to the selective modulation of intracellular signaling in B cells: regulation of the association of phosphatidylinositol 3-kinase and inositol 5'-phosphatase with the antigen receptor complex. J. Biol. Chem. 272:3838.[Abstract/Free Full Text]
  32. Nadler, M. J. S., B. Chen, J. S. Anderson, H. H. Wortis, B. G. Neel. 1997. Protein-tyrosine phosphatase SHP-1 is dispensable for Fc{gamma}RIIB-mediated inhibition of B cell antigen receptor activation. J. Biol. Chem. 272:20038.[Abstract/Free Full Text]
  33. Tridandapani, S., M. Pradhan, J. R. LaDine, S. Garber, C. L. Anderson, K. M. Coggeshall. 1999. Protein interactions of Src homology 2 (SH2) domain-containing inositol phosphatase (SHIP): association with Shc displaces SHIP from Fc{gamma}RIIb in B cells. J. Immunol. 162:1408.[Abstract/Free Full Text]
  34. Sato, K., A. Ochi. 1998. Superclustering of B cell receptor and Fc{gamma}RIIB1 activates Src homology 2-containing protein tyrosine phosphatase-1. J. Immunol. 161:2716.[Abstract/Free Full Text]
  35. Nakamura, K., J. C. Cambier. 1998. B cell antigen receptor (BCR)-mediated formation of a SHP-2-pp120 complex and its inhibition by Fc{gamma}RIIB1-BCR coligation. J. Immunol. 161:684.[Abstract/Free Full Text]
  36. Famiglietti, S. J., K. Nakamura, J. C. Cambier. 1999. Unique features of SHIP, SHP-1, and SHP-2 binding to Fc{gamma}RIIb revealed by surface plasmon resonance analysis. Immunol. Lett. 68:35.[Medline]
  37. Nolan, G. P., A. R. Shatzman. 1998. Expression vectors and delivery systems. Curr. Opin. Biotechnol. 9:447.[Medline]
  38. Pear, W. S., G. P. Nolan, M. L. Scott, D. Baltimore. 1993. Production of high-titer helper-free retroviruses by transient transfection. Proc. Natl. Acad. Sci. USA 90:8392.[Abstract/Free Full Text]
  39. Kim, K. J., C. Kanellopoulos-Langevin, R. M. Merwin, D. H. Sachs, R. Asofsky. 1979. Establishment and characterization of BALB/c lymphoma lines with B cell properties. J. Immunol. 122:549.[Abstract/Free Full Text]
  40. Gold, M. R., R. Aebersold. 1994. Both phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase products are increased by antigen receptor signaling in B cells. J. Immunol. 152:42.[Abstract]
  41. Hippen, K. L., A. M. Buhl, D. D’Ambrosio, K. Nakamura, C. Persin, J. C. Cambier. 1997. Fc{gamma}RIIB1 inhibition of BCR-mediated phosphoinositide hydrolysis and Ca2+ mobilization is integrated by CD19 dephosphorylation. Immunity 7:49.[Medline]
  42. Fluckiger, A. C., Z. Li, R. M. Kato, M. I. Wahl, H. D. Ochs, R. Longnecker, J. P. Kinet, O. N. Witte, A. M. Scharenberg, D. J. Rawlings. 1998. Btk/Tec kinases regulate sustained increases in intracellular Ca2+ following B-cell receptor activation. EMBO J. 17:1973.[Medline]
  43. Falasca, M., S. K. Logan, V. P. Lehto, G. Baccante, M. A. Lemmon, J. Schlessinger. 1998. Activation of phospholipase C {gamma} by PI 3-kinase-induced PH domain-mediated membrane targeting. EMBO J. 17:414.[Medline]
  44. Chan, V. W., F. Meng, P. Soriano, A. L. DeFranco, C. A. Lowell. 1997. Characterization of the B lymphocyte populations in Lyn-deficient mice and the role of Lyn in signal initiation and down-regulation. Immunity 7:69.[Medline]
  45. Gold, M. R., J. S. Sanghera, J. Stewart, S. L. Pelech. 1992. Selective activation of p42 mitogen-activated protein (MAP) kinase in murine B lymphoma cell lines by membrane immunoglobulin cross-linking: evidence for protein kinase C-independent and -dependent mechanisms of activation. Biochem. J. 287:269.
  46. van der Geer, P., S. Wiley, V. K. Lai, J. P. Olivier, G. D. Gish, R. Stephens, D. Kaplan, S. Shoelson, T. Pawson. 1995. A conserved amino-terminal Shc domain binds to phosphotyrosine motifs in activated receptors and phosphopeptides. Curr. Biol. 5:404.[Medline]
  47. Kavanaugh, W. M., L. T. Williams. 1994. An alternative to SH2 domains for binding tyrosine-phosphorylated proteins. Science 266:1862.[Abstract/Free Full Text]
  48. Blaikie, P., D. Immanuel, J. Wu, N. Li, V. Yajnik, B. Margolis. 1994. A region in Shc distinct from the SH2 domain can bind tyrosine-phosphorylated growth factor receptors. J. Biol. Chem. 269:32031.[Abstract/Free Full Text]



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