The SH2 Domain-containing Inositol 5'-Phosphatase (SHIP) Recruits the p85 Subunit of Phosphoinositide 3-Kinase during Fcgamma RIIb1-mediated Inhibition of B Cell Receptor Signaling*

Neetu GuptaDagger , Andrew M. Scharenberg§, David A. Frumanparallel , Lewis C. Cantley, Jean-Pierre Kinet§, and Eric O. LongDagger **

From the Dagger  Laboratory of Immunogenetics, NIAID, National Institutes of Health, Rockville, Maryland 20852-1727 and the Laboratories of § Allergy and Immunology and  Signal Transduction, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215

    ABSTRACT
Top
Abstract
Introduction
References

Coligation of Fcgamma RIIb1 with the B cell receptor (BCR) or Fcepsilon RI on mast cells inhibits B cell or mast cell activation. Activity of the inositol phosphatase SHIP is required for this negative signal. In vitro, SHIP catalyzes the conversion of the phosphoinositide 3-kinase (PI3K) product phosphatidylinositol 3,4,5-trisphosphate (PIP3) into phosphatidylinositol 3,4-bisphosphate. Recent data demonstrate that coligation of Fcgamma RIIb1 with BCR inhibits PIP3-dependent Btk (Bruton's tyrosine kinase) activation and the Btk-dependent generation of inositol trisphosphate that regulates sustained calcium influx. In this study, we provide evidence that coligation of Fcgamma RIIb1 with BCR induces binding of PI3K to SHIP. This interaction is mediated by the binding of the SH2 domains of the p85 subunit of PI3K to a tyrosine-based motif in the C-terminal region of SHIP. Furthermore, the generation of phosphatidylinositol 3,4-bisphosphate was only partially reduced during coligation of BCR with Fcgamma RIIb1 despite a drastic reduction in PIP3. In contrast to the complete inhibition of Tec kinase-dependent calcium signaling, activation of the serine/threonine kinase Akt was partially preserved during BCR and Fcgamma RIIb1 coligation. The association of PI3K with SHIP may serve to activate PI3K and to regulate downstream events such as B cell activation-induced apoptosis.

    INTRODUCTION
Top
Abstract
Introduction
References

Coengagement of Fcgamma RIIb1 with the B cell receptor (BCR)1 by an immune complex consisting of antigen and a specific antibody provides a feedback mechanism for the down-regulation of B cell activation (1, 2). A distinct effect of BCR/Fcgamma RIIb1 coligation is the loss of sustained calcium influx and a selective reduction in the tyrosine phosphorylation of certain proteins (3-7). The molecular events responsible for this phenotype are not clearly understood.

Cocross-linking of BCR with Fcgamma RIIb1 results in recruitment of SHIP (SH2 domain-containing inositol-polyphosphate 5'-phosphatase) to the immunoreceptor tyrosine-based inhibition motif present in the cytoplasmic tail of Fcgamma RIIb1 (8). Two approaches provided evidence for a functional requirement for SHIP during Fcgamma RIIb1-mediated inhibitory signaling. Ectopic expression of a chimeric KIR/Fcgamma RIIb1 protein, containing the extracellular and transmembrane regions of KIR and the cytoplasmic tail of Fcgamma RIIb1, in natural killer cells inhibited the lysis of target cells bearing the HLA class I ligand for the extracellular KIR portion of the chimeric receptor (9). Coexpression of a dominant-negative mutant of SHIP, but not the tyrosine phosphatase Shp-1, reverted the inhibitory signal delivered by Fcgamma RIIb1 in natural killer cells. Conversely, dominant-negative Shp-1, but not SHIP, reverted the negative signal mediated by KIR (9). The second approach made use of chicken DT40 B cells in which the SHIP or Shp-1 genes had been deleted by targeted homologous recombination (10). Fcgamma RIIb1dependent inhibition was lost in the absence of SHIP, but remained intact in cells lacking Shp-1 (10).

SHIP is a 145-kDa cytosolic protein that contains a single SH2 domain, a catalytic region that bears significant homology to inositol 5'-phosphatases, and several binding sites for other signaling proteins in its C-terminal region (11-13). SHIP interacts with Shc (14), which couples proximal signaling to the Grb2/Sos/Ras activation pathway. SHIP tyrosine phosphorylation and association with Shc increases upon BCR/Fcgamma RIIb1 coligation (14). It was proposed that SHIP inhibits the BCR activation signal by competing with Grb2 for binding to Shc, thereby breaking the Ras signaling pathway (15).

BCR ligation leads to phosphorylation of the tyrosines at positions 484 and 515 in CD19, which then recruit and activate phosphoinositide 3-kinase (PI3K) (16, 17). Coligation of BCR with Fcgamma RIIb1 leads to initial phosphorylation of CD19, followed by its rapid dephosphorylation (6, 7). One model proposes that a tyrosine phosphatase, such as Shp-1, dephosphorylates CD19, thereby blocking BCR-mediated activation by preventing PI3K activation (7). However, CD19 dephosphorylation cannot always account for Fcgamma RIIb1-mediated negative signaling because such signaling operates in mast cells and natural killer cells that do not express CD19 (8, 9). Furthermore, dephosphorylation by Shp-1 is not required for Fcgamma RIIb1-mediated inhibition (8-10, 18).

In vitro, SHIP cleaves the 5'-phosphate from phosphatidylinositol 3,4,5-trisphosphate (PIP3) and inositol 1,3,4,5-tetrakisphosphate to give rise to phosphatidylinositol 3,4-bisphosphate (PIP2) and inositol 1,3,4-trisphosphate, respectively (12). Unlike other 5'-phosphatases, SHIP preferentially utilizes substrates that are phosphorylated on the D3 position of the inositol ring, thereby linking its activity to the PI3K pathway. Coengagement of Fcgamma RIIb1 with BCR leads to a drastic reduction of cellular PIP3 at any time point of cross-linking as detected by thin-layer chromatography (19). PIP3 may either not be produced because of inactivation of PI3K, as proposed in the CD19 dephosphorylation model, or be rapidly turned over. Recruitment of SHIP by Fcgamma RIIb1 may serve to achieve a rapid conversion of PIP3 to PIP2. Therefore, the possibility of a physical association between SHIP and PI3K was investigated. Coengagement of BCR with Fcgamma RIIb1 resulted in a tyrosine phosphorylation-dependent recruitment of the p85 subunit of PI3K to SHIP. This interaction is mediated by direct binding of the SH2 domain of PI3K to a signature motif in the C-terminal region of SHIP. In addition, production of PIP2 and activation of Akt (also called protein kinase B) were observed during BCR/Fcgamma RIIb1 coengagement.

    EXPERIMENTAL PROCEDURES

Cells, Antibodies, and Other Reagents-- The B cell line A20 was maintained in RPMI 1640 medium with 10% fetal bovine serum, 2 mM glutamine, and 50 µM beta -mercaptoethanol. NIH 3T3 cells were grown in Dulbecco's modified Eagle's medium with 10% calf serum and 2 mM glutamine. F(ab')2, intact rabbit anti-mouse IgG, and peroxidase-conjugated goat anti-rabbit IgG were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Anti-PI3K p85 and p110 subunit antibodies, unconjugated and biotin-conjugated anti-phosphotyrosine 4G10 antibodies, and a glutathione S-transferase (GST) fusion protein of the PI3K p85 C-terminal SH2 domain were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Recombinant GST protein and wortmannin were obtained from Sigma. Antibodies against Akt and phospho-Akt (specific for phosphoserine 473) were from New England Biolabs Inc. (Beverly, MA), and anti-Flag antibody (M2) was from Eastman Kodak Co. A rabbit antiserum against the peptide sequence VPACGVSSLNEMINP in the C-terminal region of SHIP was generated (Research Genetics, Huntsville, AL). Peroxidase conjugates of streptavidin and sheep anti-mouse IgG were from Amersham Pharmacia Biotech.

Deletion Mutants of SHIP and Recombinant Vaccinia Virus Production-- Different deletion mutants of SHIP were obtained from M. Lioubin and L. Rohrschneider (Fred Hutchinson Cancer Research Center, Seattle, WA). The N-terminal SH2 domain is designated as n, the catalytic domain as cat, and the C-terminal region following the catalytic domain as c. Thus, the truncated mutants contain different combinations of n, cat, and c regions, i.e. ncat, nc, and catc. SHIPncat has amino acids 5-866; SHIPnc has a deletion in the catalytic domain corresponding to amino acids 500-809 and a replacement with amino acids EF arising from an EcoRI site located at the site of deletion; and SHIPcatc contains amino acids 174-1190. All constructs have a Flag tag followed by a NotI site at the amino terminus, which adds amino acids MGDYKDDDDKRPH onto the amino terminus of each. The cDNAs were cloned into plasmid pSCF4, a modified pSC65 plasmid (a gift of B. Moss), which contains a Kozak sequence and a Flag sequence followed by a multiple cloning site. Recombinant vaccinia viruses were generated and amplified as described (20).

A20 Cell Stimulation, Lysis, and Immunoprecipitation-- A20 cells (2 × 107) were washed twice with serum-free Iscove's medium, resuspended, and incubated with F(ab')2 fragment or intact rabbit anti-mouse IgG for the indicated times at 37 °C. Stimulation was stopped by addition of cold Dulbecco's phosphate-buffered saline (DPBS) and by rapid centrifugation of the cells in a Picofuge (Stratagene, La Jolla, CA). The cells were lysed in Tris-buffered saline, pH 8.0, containing 0.5% Triton X-100, 5 mM EDTA, 2 mM iodoacetamide, 5 µg/ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium metavanadate, and 10 mM sodium fluoride. Lysates were immunoprecipitated with the indicated antibodies and protein G-agarose beads.

Vaccinia Virus Infection of NIH 3T3 Cells and Stimulation-- Recombinant vaccinia viruses encoding SHIPncat, SHIPnc, or SHIPcatc were used to infect NIH 3T3 cells as described (21). Briefly, NIH 3T3 cells (5 × 106) were infected in suspension at 5 plaque-forming units/cell with the indicated recombinant viruses in 2 ml of infection medium consisting of Dulbecco's modified Eagle's medium, 2 mM glutamine, 10 mM HEPES, and 0.5% bovine serum albumin for 3.5 h at 37 °C. The cells were washed once with DPBS and incubated in 1 ml of DPBS or pervanadate solution (10 mM H2O2 + 0.1 mM sodium metavanadate in DPBS) for 15 min at 37 °C. Subsequently, the cells were washed with cold DPBS and lysed, and the lysates were used for immunoprecipitation as described above.

Synthetic Peptides and Agarose Beads-- Synthetic peptides corresponding to amino acid sequences in the C-terminal region of SHIP and in the PI3K-binding motif in CD19 (SLGSQS(pY)EDMRG) were purchased from Quality Controlled Biochemicals (Hopkinton, MA). The SHIP peptides used were EMINPNYIGMGP, EMINPN(pY)IGMGP, and EMINPN(pY)IGRGP. All peptides were synthesized with an N-terminal biotin tag for coupling with streptavidin-agarose beads. The peptides were dissolved at 0.1 mg/ml in PBS, pH 7.4, and incubated with streptavidin-agarose beads (1-ml packed volume) overnight at 4 °C. The beads were washed four times with PBS, pH 7.4, and suspended in 1 ml of PBS. Lysates of unstimulated A20 cells were prepared as described above and incubated with 100 µl of the above peptide-streptavidin-agarose conjugate overnight at 4 °C. Beads were washed and boiled with SDS-PAGE sample buffer, and the bound material was separated by SDS-PAGE and subjected to silver staining or immunoblotting.

Western and Far Western Blotting-- Immunoprecipitates were separated on SDS-polyacrylamide gels and transferred to Immobilon P membranes. The blots were probed with the indicated antibodies and developed using the ECL detection reagents from Amersham Pharmacia Biotech. In the far Western blotting procedure, membranes were overlaid with 4 µg/ml recombinant GST protein or GST fused to the C-terminal SH2 domain of PI3K p85 in phosphate-buffered saline containing 5% bovine serum albumin, 0.1% Tween 20, and 1 mM dithiothreitol. The membranes were washed with buffer without dithiothreitol, reblocked, and incubated with rabbit polyclonal anti-GST antibodies. After washing, the membranes were incubated with peroxidase-conjugated goat anti-rabbit IgG and developed with ECL reagents.

Phosphoinositide Analysis-- A20 cells were labeled with 32P and stimulated as described above. This was followed by extraction and deacylation of lipids and high performance liquid chromatography (HPLC) analysis of the glycerophosphoinositol head groups (22, 23).

    RESULTS

Recruitment of PI3K to Tyrosine-phosphorylated SHIP upon Coligation of the B Cell Receptor with Fcgamma RIIb1-- A20 cells were stimulated with F(ab')2 or intact anti-IgG antibodies, and immunoprecipitates of PI3K were resolved by SDS-PAGE and probed for associated phosphotyrosine-containing proteins by Western blotting. A distinct phosphoprotein band migrating at ~145 kDa coimmunoprecipitated with PI3K as early as 5 s after stimulation with intact antibody, but not with the F(ab')2 antibody (Fig. 1A). Probing with anti-SHIP antiserum revealed the presence of SHIP at that position (Fig. 1B). To test whether SHIP was directly associated with PI3K or whether it was immunoprecipitated as part of the receptor complex by the stimulating intact anti-Ig antibody, the lysates were incubated with protein G-agarose beads alone prior to SDS-PAGE and Western blotting with anti-SHIP antibodies. Under those conditions, no 145-kDa band was seen in the protein G precipitates (data not shown). Cocross-linking of BCR with Fcgamma RIIb1 through an intact IgG also enhanced the level of p85 in immunoprecipitates of SHIP (data not shown). Thus, coligation of BCR with Fcgamma RIIb1 leads to the recruitment of PI3K to SHIP.


View larger version (79K):
[in this window]
[in a new window]
 
Fig. 1.   Association of PI3K with tyrosine-phosphorylated SHIP upon coligation of the B cell receptor with Fcgamma RIIb1. A20 cells were unstimulated (N) or stimulated for 5, 15, 30, 60, or 180 s with F(ab')2 (F) or intact (I) anti-mouse IgG (Anti-mIgG). Lysates were immunoprecipitated with anti-p85 antibodies. Immunoprecipitates were fractionated on a 7.5% SDS-polyacrylamide gel and Western-blotted with monoclonal anti-phosphotyrosine antibody 4G10 (A) or rabbit polyclonal anti-SHIP antiserum (B).

Tyrosine phosphorylation of SHIP upon BCR/Fcgamma RIIb1 coligation exceeds that obtained by cross-linking BCR alone (8, 14). Therefore, PI3K association with SHIP observed during coligation could be mediated by the binding of PI3K SH2 domains to phosphorylated tyrosine residues in SHIP. The p85 subunit of PI3K has two SH2 domains, one each at the N and C termini. The phosphotyrosine-containing motif recognized by these two domains includes a pYXXM sequence for the C-terminal SH2 domain and a more stringent pY(I/V/L)XM sequence for the N-terminal SH2 domain (24). A GST fusion protein of the C-terminal SH2 domain was used to test for direct binding to SHIP. A20 cells were stimulated with F(ab')2 or intact antibodies, and either SHIP or phosphotyrosine-containing proteins were immunoprecipitated from the lysates. In both cases, the GST-p85 SH2 fusion protein bound in a far Western blot to a protein of 145 kDa present in lysates of A20 cells stimulated with intact antibody (Fig. 2A, Expts. 1 and 2). Thus, p85 can bind directly to SHIP and to a tyrosine-phosphorylated protein that comigrated with SHIP on SDS-PAGE. GST alone did not bind SHIP under the same conditions (Fig. 2B), but it reacted with two nonspecific bands migrating at ~135 and 140 kDa in anti-SHIP immunoprecipitates of both unstimulated and F(ab')2- and intact anti-Ig-stimulated cell lysates. The presence of SHIP in the anti-phosphotyrosine and anti-SHIP immunoprecipitates is shown in Fig. 2C. Increased tyrosine phosphorylation of SHIP under conditions of BCR and Fcgamma RIIb1 coligation is evident. Direct binding of the PI3K SH2 domain to SHIP by far Western blotting was also greater after receptor coligation than after cross-linking BCR alone.


View larger version (65K):
[in this window]
[in a new window]
 
Fig. 2.   The SH2 domain of PI3K directly binds to tyrosine-phosphorylated SHIP upon Fcgamma RIIb1 engagement. A20 cells were either unstimulated (N) or stimulated with F(ab')2 (F) or intact (I) anti-mouse IgG (Anti-mIgG) for 5 s, and lysates were immunoprecipitated with anti-phosphotyrosine (alpha pY) or anti-SHIP (alpha SHIP) antibodies. Immunoprecipitates (IP) were subjected to 7.5% SDS-PAGE and far Western blotting with GST-PI3K p85 C-terminal SH2 domain fusion protein (A) and recombinant GST protein (B) or to Western blotting with anti-SHIP antiserum (C).

The SH2 Domain of PI3K Binds to the C-terminal Region of SHIP-- The SHIP cDNA was broadly divided into three regions encoding the SH2 domain designated as n, the central catalytic region containing the sequences conserved in several 5'-phosphatases designated as cat, and the C-terminal region designated as c (which contains sites for interaction with the PTB domains of Shc (12, 25) and multiple prolines that interact with Grb2 (11)). Deletion mutants containing different combinations of these three domains (Fig. 3), namely ncat (~105 kDa), nc (~110 kDa), and catc (~120 kDa), were inserted into recombinant vaccinia viruses and tested for their ability to bind PI3K.


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 3.   Schematic representation of different deletion mutants of SHIP. See "Experimental Procedures" for details.

The deletion mutants were expressed in NIH 3T3 fibroblasts, immunoprecipitated following a stimulation with pervanadate, and subjected to far Western blotting with the GST-p85 SH2 fusion protein. All three mutants were tyrosine-phosphorylated upon pervanadate treatment (Fig. 4A), but only the nc and catc mutants bound the SH2 domain of PI3K (Fig. 4B). As these two molecules share only the C-terminal sequence of SHIP, the binding site must be in that region. The level of expression of all three deletion mutants was comparable (Fig. 4C). The deletion mutants ncat and nc also coimmunoprecipitated a protein at 52 kDa upon pervanadate stimulation (Fig. 4A), which could be the Shc adaptor protein associated with the N-terminal SH2 domain of SHIP.


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 4.   The SH2 domain of PI3K binds to the C terminus of SHIP. NIH 3T3 cells were either uninfected (first and second lanes) or infected with 5 plaque-forming units/cell of Vac-SHIPncat (third and fourth lanes), Vac-SHIPnc (fifth and sixth lanes), or Vac-SHIPcatc (seventh and eighth lanes) for 4 h at 37 °C. The cells were washed once with DPBS, stimulated (+) or not (-) with pervanadate for 15 min at 37 °C, washed twice with DPBS, and lysed. Immunoprecipitates of Flag-tagged SHIP deletion mutants (A and B) and total lysates (C) were separated by 7.5% SDS-PAGE and subjected to Western blotting with anti-phosphotyrosine antibody 4G10 (A) and to far Western blotting with GST-PI3K SH2 domain fusion protein (B) and anti-Flag antibody (C).

A SHIP Phosphotyrosine Peptide Binds to the p85/p110 Subunits of PI3K in Lysates of A20 Cells-- Amino acids 917-920 (YIGM) in the C-terminal region of SHIP correspond to a perfect motif for binding the N- and C-terminal SH2 domains of PI3K (24). The tyrosine at position 917 is phosphorylated upon BCR/Fcgamma RIIb1 coligation (25). Twelve-amino acid-long peptides containing SHIP sequence 917-920 were synthesized with either unphosphorylated (YIGM) or phosphorylated (pYIGM) Tyr-917. Another phosphorylated peptide carrying the substitution M920R (pYIGR) and a phosphopeptide corresponding in sequence to the C-terminal PI3K-binding motif in CD19 (SLGSQSYEDMRG) were also synthesized for negative and positive controls, respectively. All biotinylated peptides were coupled to streptavidin-agarose beads and used to pull down proteins in A20 lysates. Two proteins of ~85 and 110 kDa bound only to the CD19 peptide and the pYIGM peptide and not to the streptavidin-agarose beads alone or to pYIGR and unphosphorylated YIGM peptides (Fig. 5A). Immunoblotting with anti-PI3K antibodies revealed that these proteins comigrated with the p85 (Fig. 5B) and p110 (Fig. 5C) subunits of PI3K, respectively. Thus, the in vitro data shown in Figs. 4 and 5 suggest a possible mechanism by which SHIP binds PI3K upon B cell stimulation with intact anti-Ig antibodies or immune complexes.


View larger version (40K):
[in this window]
[in a new window]
 
Fig. 5.   A phosphopeptide corresponding to 12 amino acids in the C-terminal region of SHIP binds to the p85 and p110 subunits of PI3K in A20 lysates. A, A20 lysates (~80 × 106 cells) were incubated with streptavidin-agarose beads (lane 1) or with beads coupled to CD19 phosphopeptide (lane 2), YIGM peptide (lane 3), pYIGM peptide (lane 4), or pYIGR peptide (lane 5) overnight at 4 °C with end-to-end mixing. Bound proteins were separated by 7.5% SDS-PAGE and silver-stained. Lane M refers to molecular mass markers (in kilodaltons). B and C, A20 lysates (~10 × 106 cells) were treated similarly as described for A, but the separated proteins were sequentially immunoblotted using anti-p85 (B) and anti-p110 (C) antibodies.

Production of PIP2 and Akt Activation during Coligation of BCR with Fcgamma RIIb1-- A potential outcome of the association of SHIP with PI3K in A20 cells stimulated with intact anti-Ig is the efficient production of PIP2, provided that SHIP and PI3K retain their catalytic activities. To test this possibility, A20 cells were stimulated with F(ab')2 or intact antibodies for different times, and the total cellular levels of PIP2 and PIP3 were determined using a sensitive HPLC assay. Production of PIP2 upon Fcgamma RIIb1 coligation was approximately two-thirds of that upon BCR stimulation alone (Fig. 6, upper panel). In contrast, there was a marked inhibition of the PI3K product PIP3 at early time points and complete loss at sustained time points (Fig. 6, lower panel).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 6.   Generation of PIP2 and PIP3 upon BCR ligation and BCR coligation with Fcgamma RIIb1. A20 cells were either unstimulated or stimulated with F(ab')2 () or intact anti-mouse IgG (open circle ) for 1, 2, 5, or 10 min. Lipids were extracted, deacylated, and analyzed by HPLC. PIP2 (PI-3,4-P2; upper panel) and PIP3 (PI-3,4,5-P3; lower panel) levels are expressed as percentage of total phosphoinositide. The PIP2 data are presented as an average of n = 2 experiments.

Activation of the serine/threonine kinase Akt requires binding of its pleckstrin homology domain to membrane-bound phosphatidylinositides (26, 27). In particular, binding to PIP2 results in activation of Akt in vitro (28, 29). Full Akt activation requires sequential phosphorylation by two kinases, the second of which phosphorylates serine 473 in Akt after binding to PIP3 (30). We used phosphorylation of serine 473 as an indicator of Akt activation after signaling via BCR. A20 cells were stimulated with F(ab')2 or intact antibodies for 2, 5, or 10 min, and active Akt was immunoprecipitated and immunoblotted using antibodies specific for phosphoserine 473. Fig. 7A reveals a large increase in the activity of Akt, which was clearly diminished during coligation of BCR with Fcgamma RIIb1. To test whether PI3K activity is required for Akt activation upon B cell stimulation, two inhibitors, wortmannin and LY294002, were used. At low concentrations, these inhibitors block PI3K activity without affecting phosphoinositide 4-kinases (31). A20 cells were pretreated with wortmannin (Fig. 7B) or LY294002 (data not shown) and then stimulated with F(ab')2 or intact antibodies for 2 min. Both BCR- and BCR/Fcgamma RIIb1-induced Akt activities were completely lost upon inhibition of PI3K (Fig. 7B). Thus, PI3K activity persists during BCR/Fcgamma RIIb1 coligation and is required for Akt activation.


View larger version (43K):
[in this window]
[in a new window]
 
Fig. 7.   PI3K-dependent activation of the Ser/Thr kinase Akt upon B cell stimulation. A, A20 cells were either unstimulated (N) or stimulated with F(ab')2 (F) or intact (I) anti-mouse IgG (Anti-mIgG) for 2, 5, or 10 min. Cell lysates were immunoprecipitated with an anti-Ser(P)473 Akt antibody. Immunoprecipitates were subjected to 7.5% SDS-PAGE and Western blotting with the same antibody. B, A20 cells were either untreated (-) or pretreated with 100 nM wortmannin for 10 min (+) before stimulation with F(ab')2 (F) or intact (I) anti-mouse IgG for 2 min. Immunoprecipitates were analyzed as described for A. C, equal loading of protein was confirmed by blotting the lysates with an anti-Akt antibody. The data shown are representative of five different experiments.


    DISCUSSION

Coengagement of BCR with Fcgamma RIIb1 results in a diminished transient calcium flux and a loss of sustained calcium flux (3-5). The sustained calcium flux in BCR-triggered B cells requires activation of Btk, a member of the Tec kinase family that, in turn, activates phospholipase Cgamma (19, 32-34). Activation of Btk is dependent on the binding of its pleckstrin homology domain to PIP3 (19). A noticeable effect of Fcgamma RIIb1 coligation is a drastic reduction of PIP3, otherwise produced very rapidly upon BCR triggering (19). The loss of PIP3 could be due to a reduced PI3K activity and conversion to PIP2 by a non-rate-limiting SHIP, to an increased SHIP activity, or to a complete loss of PI3K activity. However, the production of the SHIP metabolite PIP2 suggests that PI3K remains active during Fcgamma RIIb1-mediated inhibition of the BCR activation signal. In addition, the direct association of PI3K with SHIP, demonstrated here, may serve to enhance the conversion of PIP3 to PIP2. Far Western blotting with the C-terminal SH2 domain of the p85 subunit of PI3K mapped the site of interaction to the C-terminal region of SHIP. Synthetic phosphopeptides that included sequences flanking tyrosine 917 of SHIP bound PI3K in cell lysates.

The inducible association of PI3K with tyrosine-phosphorylated SHIP described here is different from the constitutive association of PI3K with an unidentified PIP3 5-phosphatase activity in human platelets (35). The novel 5-phosphatase reported in that study is distinct from SHIP since its catalytic activity in vitro was limited to the substrate PIP3.

Production of PIP2 during BCR/Fcgamma RIIb1 coligation was consistently less than during BCR-mediated activation. This is probably due, in part, to a lower activity of PI3K and hence lower production of the SHIP substrate PIP3. As CD19 is dephosphorylated rapidly after BCR/Fcgamma RIIb1 coligation (6, 7), a major source of PI3K activation is lost. Recruitment of PI3K by tyrosine-phosphorylated SHIP may serve to compensate for this loss. However, SHIP is not absolutely required for PI3K activation in avian DT40 B cells because a sustained calcium signal was observed after BCR/Fcgamma RIIb1 coengagement in a SHIP-negative DT40 mutant cell (10). It is also possible that the rapid conversion of PIP3 by SHIP affects PI3K activation directly, or indirectly through a diminished PIP3-dependent activation of Ras (via Sos) (36, 37). To clearly address whether the catalytic activity of SHIP and/or PI3K is responsible for the observed pattern of PIP3 and PIP2 production, an inhibitor of SHIP phosphatase activity would be necessary.

PIP2 and PIP3 control the activation of Akt by recruiting the pleckstrin homology domains of Akt and of another serine/threonine kinase that phosphorylates Akt (26-30). Akt delivers an anti-apoptotic signal by phosphorylating the pro-apoptotic molecule BAD, a member of the Bcl-2 protein family (38, 39). Our data show residual activation of Akt during BCR/Fcgamma RIIb1 coligation as measured by Akt phosphorylation on serine 473. This remaining Akt activation is in contrast to the complete loss of the sustained calcium flux mediated by the PIP3-dependent Tec kinase Btk during BCR/Fcgamma RIIb1 coligation (19). The wortmannin sensitivity of Akt activation strongly suggests that PI3K activity is also retained. Although apoptosis of B cells after BCR/Fcgamma RIIb1 coligation can occur and may even exceed that observed after BCR-mediated activation (40), the SHIP/PI3K/Akt pathway described here may lead to at least some anti-apoptotic signal. An anti-apoptotic effect of SHIP after BCR/Fcgamma RIIb1 coligation has been suggested by the observation of increased apoptosis of DT40 cells deficient in SHIP and of DT40 cells expressing a mutant Fcgamma RIIb1 that fails to bind SHIP (10). A pro-apoptotic mediator that binds to Fcgamma RIIb1 was proposed to explain these observations (10). On the other hand, the reduced survival of DT40 cells expressing the mutated Fcgamma RIIb1 that fails to recruit SHIP may have been caused by the lack of SHIP-mediated PIP2 production and, in turn, by a reduced Akt-mediated survival signal.

In conclusion, this study demonstrates an association of the p85 subunit of PI3K with the inositol phosphatase SHIP in response to coligation of BCR with the inhibitory receptor Fcgamma RIIb1. PI3K activity and PIP2 production were not abrogated by Fcgamma RIIb1 ligation to BCR. We suggest that the physical association of SHIP and PI3K may provide a novel mode of PI3K activation and an enhanced conversion of PIP3 to PIP2.

    ACKNOWLEDGEMENTS

We thank M. Lioubin and L. Rohrschneider for deletion mutants of SHIP, L. Samelson for helpful suggestions, and M. Weston for technical assistance.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel Supported by a fellowship from the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation.

** To whom correspondence should be addressed: LIG, NIAID, NIH, Twinbrook II, 12441 Parklawn Dr., Rockville, MD 20852-1727. Tel.: 301-496-8266; Fax: 301-402-0259; E-mail: elong{at}nih.gov.

    ABBREVIATIONS

The abbreviations used are: BCR, B cell receptor; PI3K, phosphoinositide 3-kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PIP2, phosphatidylinositol 3,4-bisphosphate; GST, glutathione S-transferase; DPBS, Dulbecco's phosphate-buffered saline; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography.

    REFERENCES
Top
Abstract
Introduction
References
  1. Chan, P. L., and Sinclair, N. R. (1971) Immunology 21, 967-981[Medline] [Order article via Infotrieve]
  2. Phillips, N. E., and Parker, D. C. (1983) J. Immunol. 130, 602-606[Abstract/Free Full Text]
  3. Bijsterbosch, M. K., and Klaus, G. G. (1985) J. Exp. Med. 162, 1825-1836[Abstract]
  4. Wilson, H. A., Greenblatt, D., Taylor, C. W., Putney, J. W., Tsien, R. Y., Finkelman, F. D., and Chused, T. M. (1987) J. Immunol. 138, 1712-1718[Abstract/Free Full Text]
  5. Choquet, D., Partiseti, M., Amigorena, S., Bonnerot, C., Fridman, W. H., and Korn, H. (1993) J. Cell Biol. 121, 355-363[Abstract]
  6. Kiener, P. A., Lioubin, M. N., Rohrschneider, L. R., Ledbetter, J. A., Nadler, S. G., and Diegel, M. L. (1997) J. Biol. Chem. 272, 3838-3844[Abstract/Free Full Text]
  7. Hippen, K. L., Buhl, A. M., D'Ambrosio, D., Nakamura, K., Persin, C., and Cambier, J. C. (1997) Immunity 7, 49-58[Medline] [Order article via Infotrieve]
  8. Ono, M., Bolland, S., Tempst, P., and Ravetch, J. V. (1996) Nature 383, 263-266[CrossRef][Medline] [Order article via Infotrieve]
  9. Gupta, N., Scharenberg, A. M., Burshtyn, D. N., Wagtmann, N., Lioubin, M. N., Rohrschneider, L. R., Kinet, J.-P., and Long, E. O. (1997) J. Exp. Med. 186, 473-478[Abstract/Free Full Text]
  10. Ono, M., Okada, H., Bolland, S., Yanagi, S., Kurosaki, T., and Ravetch, J. V. (1997) Cell 90, 293-301[Medline] [Order article via Infotrieve]
  11. Lioubin, M. N., Algate, P. A., Tsai, S., Carlberg, K., Aebersold, R., and Rohrschneider, L. R. (1996) Genes Dev. 10, 1084-1095[Abstract]
  12. Damen, J. E., Liu, L., Rosten, P., Humphries, R. K., Jefferson, A. B., Majerus, P. W., and Krystal, G. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1689-1693[Abstract/Free Full Text]
  13. Kavanaugh, W. M., Pot, D. A., Chin, S. M., Deuter-Reinhard, M., Jefferson, A. B., Norris, F. A., Masiarz, F. R., Cousens, L. S., Majerus, P. W., and Williams, L. T. (1996) Curr. Biol. 6, 438-445[Medline] [Order article via Infotrieve]
  14. Chacko, G. W., Tridandapani, S., Damen, J. E., Liu, L., Krystal, G., and Coggeshall, K. M. (1996) J. Immunol. 157, 2234-2238[Abstract]
  15. Tridandapani, S., Kelley, T., Cooney, D., Pradhan, M., and Coggeshall, K. M. (1997) Immunol. Today 18, 424-427[CrossRef][Medline] [Order article via Infotrieve]
  16. Tuveson, D. A., Carter, R. H., Soltoff, S. P., and Fearon, D. T. (1993) Science 260, 986-989[Medline] [Order article via Infotrieve]
  17. Weng, W.-K., Jarvis, L., and LeBien, T. W. (1994) J. Biol. Chem. 269, 5241-5248[Abstract/Free Full Text]
  18. Nadler, M. J. S., Chen, B., Anderson, J. S., Wortis, H. H., and Neel, B. G. (1997) J. Biol. Chem. 272, 20038-20043[Abstract/Free Full Text]
  19. Scharenberg, A. M., El-Hillal, O., Fruman, D. A., Beitz, L. O., Li, Z. M., Lin, S. Q., Gout, I., Cantley, L. C., Rawlings, D. J., and Kinet, J.-P. (1998) EMBO J. 17, 1961-1972[Free Full Text]
  20. Earl, P. L., and Moss, B. (1988) in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K., eds), pp. 16.17.1-16.17.16, John Wiley & Sons, Inc., New York
  21. Scharenberg, A. M., Lin, S., Cuenod, B., Yamamura, H., and Kinet, J.-P. (1995) EMBO J. 14, 3385-3394[Abstract]
  22. Auger, K. R., Serunian, L. A., Soltoff, S. P., Libby, P., and Cantley, L. C. (1989) Cell 57, 167-175[Medline] [Order article via Infotrieve]
  23. Serunian, L. A., Auger, K. R., and Cantley, L. C. (1991) Methods Enzymol. 198, 78-87[Medline] [Order article via Infotrieve]
  24. Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J., Neel, B. G., Birge, R. B., Fajardo, J. E., Chou, M. M., Hanafusa, H., Schaffhausen, B., and Cantley, L. C. (1993) Cell 72, 767-778[Medline] [Order article via Infotrieve]
  25. Liu, L., Damen, J. E., Hughes, M. R., Babic, I., Jirik, F. R., and Krystal, G. (1997) J. Biol. Chem. 272, 8983-8988[Abstract/Free Full Text]
  26. Hemmings, B. A. (1997) Science 275, 628-630[Free Full Text]
  27. Stokoe, D., Stephens, L. R., Copeland, T., Gaffney, P. R., Reese, C. B., Painter, G. F., Holmes, A. B., McCormick, F., and Hawkins, P. T. (1997) Science 277, 567-570[Abstract/Free Full Text]
  28. Franke, T. F., Kaplan, D. R., Cantley, L. C., and Toker, A. (1997) Science 275, 628-630[Free Full Text]
  29. Klippel, A., Kavanaugh, W. M., Pot, D., and Williams, L. T. (1997) Mol. Cell. Biol. 17, 338-344[Abstract]
  30. Downward, J. (1998) Science 279, 673-674[Free Full Text]
  31. Okada, T., Sakuma, L., Fukui, Y., Hazeki, O., and Ui, M. (1994) J. Biol. Chem. 269, 3563-3567[Abstract/Free Full Text]
  32. Takata, M., and Kurosaki, T. (1996) J. Exp. Med. 184, 31-40[Abstract]
  33. Fluckiger, A.-C., Li, Z., Kato, R. M., Wahl, M. I., Ochs, H. D., Longnecker, R., Kinet, J.-P., Witte, O. N., Scharenberg, A. M., and Rawlings, D. J. (1998) EMBO J. 17, 1973-1985[Free Full Text]
  34. Bolland, S., Pearse, R. N., Kurosaki, T., and Ravetch, J. V. (1998) Immunity 8, 509-516[Medline] [Order article via Infotrieve]
  35. Jackson, S. P., Schoenwaelder, S. M., Matzaris, M., Brown, S., and Mitchell, C. A. (1995) EMBO J. 14, 4490-4500[Abstract]
  36. Chen, R. H., Corbalan-Garcia, S., and Bar-Sagi, D. (1997) EMBO J. 16, 1351-1359[Abstract/Free Full Text]
  37. Rameh, L. E., Arvidsson, A., Carraway, K. L., III, Couvillon, A. D., Rathbun, G., Crompton, A., VanRenterghem, B., Czech, M. P., Ravichandran, K. S., Burakoff, S. J., Wang, D. S., Chen, C. S., and Cantley, L. C. (1997) J. Biol. Chem. 272, 22059-22066[Abstract/Free Full Text]
  38. Yang, E., Zha, J., Jockel, J., Boise, L. H., Thompson, C. B., and Korsmeyer, S. J. (1995) Cell 80, 285-291[Medline] [Order article via Infotrieve]
  39. White, E. (1996) Genes Dev. 10, 1-15[CrossRef][Medline] [Order article via Infotrieve]
  40. Ashman, R. F., Peckham, D., and Stunz, L. L. (1996) J. Immunol. 157, 5-11[Abstract]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.