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The Journal of Immunology, 1999, 162: 136-143.
Copyright © 1999 by The American Association of Immunologists

Signaling Through the EBV/C3d Receptor (CR2, CD21) in Human B Lymphocytes: Activation of Phosphatidylinositol 3-Kinase via a CD19-Independent Pathway1

Sylvie Bouillie, Monique Barel and Raymond Frade2

Immunochimie des Régulations Cellulaires et des Interactions Virales, Institut National de la Santé et de la Recherche Médicale, Unit 354, Centre INSERM, Hôpital Saint-Antoine, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We herein analyzed the regulation of phosphatidylinositol 3-kinase (PI 3-kinase) activity by CR2 activated on B lymphocyte cell surface. We demonstrated that CR2 activation triggered in vivo PI 3-kinase activity and interaction of PI 3-kinase p85 subunit with a tyrosine-phosphorylated p95 component. The specificity of PI 3-kinase activity was controlled using wortmannin and LY294002. CR2 activation did not trigger tyrosine phosphorylation of PI 3-kinase p85 subunit, but induced direct interaction of tyrosine phosphorylated p95 with the Src homology 2 domain of p85 subunit, as shown using glutathione-S-transferase fusion proteins. Despite identical molecular masses, immunoblotting analysis demonstrated that tyrosine-phosphorylated p95 that interacted in vivo and in vitro with p85 was neither CD19, the 95-kDa proto-oncogene vav, nor Gab1 (a 95-kDa adaptor molecule). Furthermore, p95 tyrosine phosphoprotein also expressed in K562A cells (CR2+ CD19- cells) interacted with Src homology 2 domain of PI 3-kinase p85 subunit after CR2 activation. Activated CR2 did not interact directly with p85 subunit or tyrosine-phosphorylated p95. This suggests the presence of an intermediate molecule between activated CR2 and tyrosine-phosphorylated p95, which may be 3BP2. In addition, in contrast to CD19 activation, CR2 activation did not trigger interaction of CD19 or Vav with PI 3-kinase p85 subunit or coprecipitation of PI 3-kinase activity with CD19. Together, these data clearly demonstrated that CR2 activation triggered in vivo PI 3-kinase activation through a pathway distinct from that triggered through CD19 activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CR2, the receptor for C3d, the 33-kDa fragment of the third component of complement (1, 2, 3), is a 140-kDa membrane glycoprotein isolated from human B lymphoma Raji cell surface (4) that interacts with the LYNVEA site expressed on the C3d fragment (5, 6). CR2 is also the EBV receptor (EBV/C3dR, CD21) (7, 8). CR2 binds to its two extracellular ligands, the C3d and the EBV capsid glycoprotein gp350/220 (9), through two distinct binding sites (10) localized on the first three short consensus repeats (SCR1 to SCR3) (11, 12). CR2 also interacts with autoantibodies present in serum of polyarthrite rheumatoid patients (13) and with CD23 (14). Molecular cloning indicated that CR2 is constituted by a large extracellular domain of 954 amino acids composed of 15 or 16 short consensus repeats, a 24-amino acid transmembrane domain, and a 34-amino acid intracellular domain (15, 16). CR2 allows C3d and EBV to induce proliferation (17, 18) or transformation (8), respectively. One of the earliest events in these two biological functions is the cross-linking of CR2 at the cell surface by specific extracellular multivalent ligands, as F(ab')2 of polyclonal anti-CR2 Ab (18), OKB7 an anti-CR2 mAb (19), particle-bound C3d (20), EBV envelope (21), or gp350, the EBV capsid protein (9).

The role of CR2 in the regulation of B lymphocyte proliferation was analyzed by determining its interaction with cellular components. At the cell surface, CR2 could interact with other Ags such as IgM (22), CD19, and TAPA-13 (23, 24, 25). These authors suggested that CR2 may play a role in B cell regulation only through its complex formation with CD19 and TAPA-1 (23, 24, 25). However, evidence exists that CR2 and its intracytoplasmic tail may have specific interactions with other intracellular components and may act in cell regulation independently from CD19 and TAPA-1. First, CR2 interacted with kinases as it was phosphorylated during B cell activation (26, 27, 28). Second, CR2 could also interact, depending on the normal or transformed state of human B lymphocytes, with three other distinct intracellular proteins, i.e., p53 anti-oncoprotein (29), p68 calcium-binding protein (30), and nuclear p120 ribonucleoprotein (p120RNP) (31), through distinct binding sites (32, 33). Third, a role of the CR2 intracytoplasmic tail, despite its short length of 34 amino acids, in early events associated with CR2 activation was suggested: 1) Carel et al. (34) showed that cells transfected with CR2 deleted of its C-terminal domain did not allow cell transformation by EBV, while the virus bound on cell surface; and 2) we showed (35) that increasing the intracellular concentration of pep34, a synthetic peptide whose sequence corresponded to the full intracytoplasmic tail of CR2, inhibited the specific proliferation induced through CR2 (35). Fourth, analyzing the growth factor function of C3d (17), we demonstrated that in serum-free medium, C3d and pep16, a 16-amino acid synthetic peptide carrying the LYNVEA binding site of C3d to CR2 (5), stimulated in vitro proliferation of human B normal or lymphoma cells (36). Servis and Lambris (37) found identical results using a 28-amino acid synthetic peptide, derived from C3d. CR2 activation by C3d and pep16 also enhanced in vitro and in vivo tyrosine phosphorylation of pp105, an intracellular 105-kDa component in Raji cells (36). Activated CR2 triggered in vitro regulation of pp105 phosphorylation through two distinct pathways: one required the presence of nonactivated CD19, and the other was CD19 independent; both pathways were TAPA-1 independent (38).

Despite these data, intracellular signal transduction associated with or triggered by CR2 activation in human B lymphocytes remained poorly documented. Indeed, while it was postulated that some CR2 molecules could form a complex with CD19 (23), and it was demonstrated that CD19 activation triggered CD19 phosphorylation and its binding to phosphatidylinositol 3-kinase (PI 3-kinase) (39), no data were available to demonstrate that CR2 activation could trigger PI 3-kinase activity through a mechanism identical with CD19 activation. PI 3-kinase activation has been associated with growth factor receptors (40). PI 3-kinase consists of a p110 catalytic subunit associated with a p85 regulatory polypeptide (41), this latter containing one SH3 and two SH2 domains (42).

We herein demonstrate that CR2 activation on the B lymphocyte surface triggers PI 3-kinase activity and interaction of the nontyrosine-phosphorylated p85 subunit of PI 3-kinase with a tyrosine-phosphorylated p95 component that is neither CD19, Vav, nor Gab1. The p95 protein directly interacts with SH2 domains of p85. These data also demonstrate that CR2 activation on lymphocyte surface triggers PI 3-kinase activity through an intracellular pathway that is fully distinct from that triggered via CD19 activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells

Raji or Daudi cells are Burkitt B lymphoma cell lines that express CR2 and CD19. K562W (wild-type) cells are an erythroleukemia cell line that originally did not express CR2 and CD19. K562A cells expressed CR2, but not CD19, as derived from K562W transfected with the CR2 cDNA (38). Cells were grown in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 1000 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in 5% CO2. Stable cDNA-transfected cells were selected using G418 (1 mg/ml; Life Technologies/BRL, Gaithersburg, MD).

Abs and reagents

The following Abs and reagents were purchased from the indicated companies: anti-CR2 mAb (BL13), anti-CD19 mAb (B4), and anti-CD40 (mAb89) from Immunotech (Westbrook, ME); anti-CD19 mAb (HD37) and goat anti-mouse Ig (GAM) from Dako (Carpenteria, CA); rabbit anti-CD19 Ab from Transduction Laboratory (Lexington, KY); polyclonal anti-p85 subunit of PI 3-kinase, anti-Gab1 Ab, anti-PTyr mAb (4G10), and GST fusion proteins of the N-terminal SH2 domain of p85 (SH2-Nt-p85) from Upstate Biotechnology (Lake Placid, NY); and the GST fusion protein of the SH3 domain of p85 from PharMingen (San Diego, CA; GST-SH3-p85).

Cell activation

Raji, Daudi, K562A or K562W cells (2 x 107) were washed with RPMI and incubated with the indicated mAb (1 µg/ml) in a final volume of 500 µl at 4°C for 20 min, then with GAM (20 µg/ml) at 37°C for the indicated times to cross-link the first Abs. Cells were collected by centrifugation and lysed for 30 min at 4°C in buffer containing 50 mM Tris (pH 7.6), 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 50 mM NaF, 1 mM Na3VO4, 30 mM Na4P2O7, and protease inhibitors, such as 1 mM PMSF, 60 mM EACA, 12.5 mM benzamidine, and 12.5 mM iodoacetamide. Solubilized proteins were collected by centrifugation at 15,000 x g for 15 min and were stored at -80°C.

Immunoprecipitation and immunoblotting

One percent Nonidet P-40 solubilized proteins were incubated for 2 h at 4°C with indicated Abs as recommended by the manufacturer. Immune complexes were bound to protein A-Sepharose (15 mg; Pharmacia, Piscataway, NJ) or protein G-agarose beads (10 µl; Sigma, St. Louis, MO) for 2 h at 4°C. Immunobeads were washed five times in lysis buffer, then proteins were eluted in sample buffer and submitted to 7.5% SDS-PAGE under reducing conditions. After SDS-PAGE, separated proteins were electrotransferred on a Hybond-ECL nitrocellulose sheet (Amersham, Arlington Heights, IL). The membranes were incubated for 1 h in Tris-buffered saline containing 3% powdered milk and then washed. Specific Abs to appropriate molecules were added and incubated for 2 h at room temperature. After washing in Tris-buffered saline/0.05% Tween-20, peroxidase-linked secondary Abs were added for 1 h at room temperature. Nitrocellulose sheets were then washed in Tris-buffered saline/0.05% Tween-20, and binding of second Abs was detected using the enhanced chemiluminescence (ECL) kit (Amersham). For sequential analysis of the same nitrocellulose sheet with distinct Abs, stripping was performed according to the manufacturer’s instructions.

In vitro binding assay

GST fusion proteins (5 µg) were bound to glutathione-Sepharose 4B beads (Pharmacia-LKB) for 1 h at 4°C and then incubated for 2 h at 4°C with 1% Nonidet P-40 solubilized proteins prepared from nonactivated or activated cells as described above. After this incubation, beads were washed five times with lysis buffer. Bound proteins were eluted, submitted to 7.5% SDS-PAGE, then analyzed by immunoblotting using the indicated Abs.

PI 3-kinase assays

PI 3-kinase activity was essentially measured as previously described (43) with minor modifications. Briefly, CR2- or CD19-activated Raji or Daudi cells were lysed in 20 mM Tris (pH 8), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, and 10% glycerol (with protease and phosphatase inhibitors as described above), and solubilized proteins were submitted to immunoprecipitation using indicated Abs. Beads were washed twice with lysis buffer and three times with 10 mM Tris-HCl (pH 7.4). Then, 10 µl of 1 mg/ml sonicated phosphatidylinositide (Avanti Polar Lipids) in 20 mM HEPES (pH 7.4) was added, and samples were incubated for 20 min on ice. After this preincubation, the phosphorylation reaction was started by addition of 20 µCi of [{gamma}-32P]ATP in 20 mM HEPES (pH 7.4), 5 mM MgCl2, 200 µM adenosine, and 50 µM ATP. The mixture was incubated for 15 min at 25°C and was stopped by addition of 100 µl 1 M HCl. Phospholipids were then immediately extracted with 200 µl of CHCl3/MeOH (1/1), and the organic phase was washed once with 80 µl of MeOH/HCl (1/1). Phosphorylated products were submitted to chromatography on silica gel 60 plates (Merck, Rahway, NJ) impregnated with 1% potassium oxalate in a CHCl3/MeOH/4 M NH4OH (9/7/2) developing solvent for 1 h. Phosphorylated products were visualized by autoradiography. In some experiments cells were preincubated with 100 nM wortmannin (Sigma) or 20 µM LY294002 (Biomol, Plymouth Meeting, PA), two PI-3 kinase specific inhibitors, at 37°C for 20 min.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CR2 activation triggers in vivo PI 3-kinase activation

CR2 was activated on Raji or Daudi cells by BL13, an anti-CR2 mAb that was cross-linked with GAM at 37°C, at the indicated times. Then, total cellular components were solubilized as indicated and immunoprecipitated on anti-PTyr mAb. The presence of PI 3-kinase activity or p85, the noncatalytic subunit of PI 3-kinase, was analyzed among these tyrosine-phosphorylated components, using specific assays. As shown in Fig. 1Go, PI 3-kinase activity was detected among solubilized components immunoprecipitated on anti-PTyr mAb only from Raji cells activated through CR2, but not in nonactivated Raji cells (lane 1). Maximum PI 3-kinase activity was reached within 2 min (lane 2) compared with 4 (lane 3) and 6 (lane 4) min. The specificity of PI 3-kinase activity was controlled in the presence of 100 nM wortmannin (lane 5) or 20 µM LY294002 (data not shown), two PI 3-kinase-specific inhibitors. Furthermore, components immunoprecipitated on anti-PTyr mAb were analyzed by immunoblotting using polyclonal anti-p85 Ab. As shown in Fig. 2Go, the p85 subunit of PI 3-kinase coprecipitated with tyrosine-phosphorylated proteins from samples of Raji cells was only activated through CR2 (lanes 3–5) and not of unstimulated cells (lane 2). The slight difference observed in gel migration of p85 subunit immunoprecipitated from Raji cells or present in total solubilized proteins (TSP) was due to the presence of a higher protein concentration in TSP. Identical results were obtained using Daudi cells.



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  		FIGURE 1. CR2 activation triggers PI 3-kinase activity. Raji cells (2 x 107) were incubated for 20 min at 4°C with buffer (lane 1) or BL13, an anti-CR2 mAb (lanes 2–5), then activated by cross-linking with GAM at 37°C for 2 min (lanes 1 and 2), 4 min (lanes 3 and 5), or 6 min (lane 4). Triton X-100 (1%)-solubilized components were immunoprecipitated on anti-PTyr mAb, then tested for PI 3-kinase activity in the absence (lanes 1–4) or the presence (lane 5) of 100 NM wortmannin. Identical results were obtained using Daudi cells in the same conditions. This figure is representative of the three experiments made with Raji cells and two others made with Daudi cells.

 


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  		FIGURE 2. CR2 activation triggers interaction of the p85 subunit of PI 3-kinase with tyrosine-phosphorylated proteins. Raji cells (2 x 107) were incubated for 20 min at 4°C with buffer (lane 2) or BL13 (lanes 3–5), then activated by cross-linking with GAM at 37°C for 2 min (lanes 2 and 3), 4 min (lane 4), and 6 min (lane 5). Nonidet P-40 (1%)-solubilized components immunoprecipitated on anti-PTyr mAb (lanes 2–5) or TSP (lane 1) were analyzed by immunoblotting with polyclonal anti-p85. Identical results were obtained using Daudi cells in the same conditions. This figure is representative of the two experiments performed with Raji cells and one other performed with Daudi cells.

 
These data demonstrated that CR2 activation on B cell surface triggered in vivo PI 3-kinase activity and interaction of PI 3-kinase p85 subunit with tyrosine-phosphorylated proteins.

CR2 activation induces interaction of a phosphorylated p95 component with the SH2 domain of the p85 subunit of PI 3-kinase

Coprecipitation of PI 3-kinase p85 subunit with tyrosine-phosphorylated proteins after CR2 activation led us to analyze whether the p85 subunit of PI 3-kinase in vivo interacted with tyrosine-phosphorylated proteins or was phosphorylated, two putative mechanisms of PI 3-kinase regulation (44, 45, 46). For this purpose, Raji or Daudi cells were activated by BL13, an anti-CR2 mAb, cross-linked with GAM as described above. Then, solubilized proteins were immunoprecipitated on polyclonal anti-p85 Ab and analyzed by immunoblotting using anti-PTyr mAb (Fig. 3GoA); the data demonstrated the presence of tyrosine-phosphorylated proteins, with apparent molecular masses of 95 and 120/130 kDa. A minimum increase in tyrosine phosphorylation of p120/130 was observed at 2 min compared with that in unstimulated cells and decreased at 4 min. In parallel, a stronger tyrosine phosphorylation of a p95 component was reached at 2 min and decreased after 4 min. In addition, anti-PTyr did not reveal any phosphorylated component at the level of 85 kDa molecular mass despite the presence of the same amount of the p85 subunit in all samples (Fig. 3GoB). Thus, these data demonstrated that CR2 activation did not trigger tyrosine phosphorylation of the p85 subunit of PI 3-kinase. Furthermore, CR2 activation triggered maximum tyrosine phosphorylation of the p95 component within 2 min, compared with 4 (lane 3) and 6 (lane 4) min, i.e., kinetics identical with the PI 3-kinase activation induced by CR2 activation (as shown in Fig. 1Go). In controls, using a polyclonal anti-PI 3-kinase p110 subunit Ab, we found that the p95 or 120/130-kDa components did not share an antigenic relationship with the 110-kDa chain of PI 3-kinase (data not shown).



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  		FIGURE 3. Tyrosine phosphoproteins interacting in vivo with the p85 subunit of PI 3-kinase. Raji cells (2 x 107) were incubated for 20 min at 4°C with buffer (lane 1) or BL13 (lanes 2–4), then activated by cross-linking with GAM at 37°C for 2 min (lanes 1 and 2), 4 min (lane 3), and 6 min (lane 4). Nonidet P-40 (1%)-solubilized components immunoprecipitated on polyclonal anti-p85 Ab were analyzed by immunoblotting with anti-PTyr mAb (A). The same sheet was stripped and immunoblotted with polyclonal anti-p85 Ab (B). Identical results were obtained using Daudi cells in the same conditions. This figure is representative of the five experiments performed with Raji cells and three others performed with Daudi cells.

 
To further analyze the interaction of the p85 subunit with tyrosine-phosphorylated p95 and to determine the specific p85 binding site involved in this interaction, we determined the ability of tyrosine-phosphorylated p95 to interact in vitro with GST fusion proteins containing the SH2-N-terminal (GST-SH2-Nt-p85) or SH3 (GST-SH3) domains of p85. As shown in Fig. 4Go, tyrosine-phosphorylated p95 bound to GST-SH2-Nt-p85 (lane 6) only after CR2 activation and not in nonactivated cells (lane 5). The specificity of p95 binding on the SH2 domain was also supported by the demonstration that p95 did not interact with the SH3 domain of p85 (lanes 3 and 4) or with GST alone (lanes 1 and 2). Identical results were obtained using Daudi cells.



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  		FIGURE 4. p95 interacts with the SH2 domain of the p85 subunit of PI 3-kinase. Raji cells (2 x 107) were incubated for 20 min at 4°C with buffer (lanes 1, 3, and 5) or BL13 (lanes 2, 4, and 6), then activated by cross-linking with GAM at 37°C for 4 min. Nonidet P-40 (1%)-solubilized components bound on GST (lanes 1 and 2), GST-SH3-p85 (lanes 3 and 4), and GST-SH2-Nt-p85 (lanes 5 and 6) were analyzed by immunoblotting with anti-PTyr mAb. Identical results were obtained using Daudi cells in the same conditions. This figure is representative of the two experiments performed with Raji cells and two others performed with Daudi cells.

 
Thus, CR2 activation on the B cell surface induced direct interaction of a tyrosine-phosphorylated p95 component with the SH2 domain of the nonphosphorylated p85 subunit of PI 3-kinase.

Tyrosine-phosphorylated p95 interacting with PI 3-kinase is not CD19

Tuveson et al. (39) have shown that activation of CD19 on B lymphocyte surface induced phosphorylation of CD19, a 95-kDa membrane Ag, and its interaction with p85, the subunit of PI 3-kinase. Therefore, we analyzed whether the tyrosine-phosphorylated p95 whose activation was induced by activated CR2 was CD19. For this purpose, Raji or Daudi cells were activated for 4 min by BL13, an anti-CR2 mAb, or by B4, an anti-CD19 mAb, cross-linked by GAM at 37°C. Then, total solubilized cellular proteins were immunoprecipitated on polyclonal anti-p85 Ab and analyzed by immunoblotting using anti-PTyr mAb. As shown in Fig. 5Go, activation of CR2 or CD19 at the Raji cell surface induced in both cases tyrosine phosphorylation of a p95 component that interacted with p85 subunit (Fig. 5GoA). However, when the same nitrocellulose sheet was stripped and immunoblotted with polyclonal anti-CD19 (Fig. 5GoB), data demonstrated that 1) after CR2 activation, CD19 was not present among phosphorylated p95 components that interacted with p85 subunit (lane 3) despite its presence among total solubilized components (lane 4); and 2) after CD19 activation, CD19 was present among p95 components that interacted with p85 subunit (lane 2). In addition, solubilized cellular proteins prepared from Raji cells activated through CD19 or CR2 were incubated with GST-SH2-Nt-p85, as described above (see Fig. 4Go). Then, proteins bound on this fusion protein were analyzed by immunoblotting using polyclonal anti-CD19 Ab. As shown in Fig. 6Go, no CD19 bound to the SH2 domain of p85 when Raji cells were activated through CR2 (lane 2). In control, CD19 bound on SH2 domain of p85 when Raji cells were activated through CD19 (lane 1). These results obtained in vitro are in good agreement with those observed in vivo. For both types of experiments, identical results were obtained using Daudi cells. Together, these data demonstrated that tyrosine-phosphorylated p95 was not CD19.



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  		FIGURE 5. CR2 activation triggers in vivo interaction of the p85 subunit of PI 3-kinase with a p95 component, which is not CD19. Raji cells (2 x 107) were incubated for 20 min at 4°C with buffer (lane 1); B4, an anti-CD19 mAb (lane 2); or BL13 (lane 3), then activated by cross-linking with GAM at 37°C for 4 min (lanes 1–3). Nonidet P-40 (1%) TSP were either immunoprecipitated on polyclonal anti-p85 Ab (lanes 1–3) or run directly in gel (lane 4), then analyzed by immunoblotting with anti-PTyr Ab (A). The same sheet was stripped and immunoblotted with polyclonal anti-CD19 Ab (B). Identical results were obtained using Daudi cells in the same conditions. This figure is representative of the four experiments performed with Raji cells and two others performed with Daudi cells.

 


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  		FIGURE 6. CD19 interacts with the SH2 domain of p85 in cells activated through CD19 but not through CR2. Raji cells (2 x 107) were incubated with B4 (lane 1), BL13 (lane 2), or mAb89, an anti-CD40 mAb (lane 3), then activated by cross-linking with GAM at 37°C for 4 min. Nonidet P-40 (1%) TSP incubated with GST-SH2-Nt-p85 bound to glutathione-Sepharose 4B beads (lanes 1–3) or run directly (lane 4) were analyzed by immunoblotting with polyclonal anti-CD19 Ab. Identical results were obtained using Daudi cells in the same conditions. This figure is representative of the four experiments performed with Raji cells and two others performed with Daudi cells.

 
Furthermore, the same experiments were performed using K562A cells, which were established by stable transfection with CR2 cDNA of the K562W (wild type), a cell line that originally did not express CR2 and CD19 (38). First, we verified that CR2 activation at the K562A cell surface triggered PI 3-kinase activity (data not shown). Second, as shown in Fig. 7Go, CR2 activation on the K562A cell surface induced interaction of the p85 subunit of PI 3-kinase with the tyrosine-phosphorylated p95 despite the absence of CD19. In controls, anti-CR2 activation of K562W cells did not trigger any significant amount of p95 tyrosine phosphorylation. Analysis demonstrated that the tyrosine-phosphorylated p95 component present in K562A cells was also able to bind to the SH2 domain of p85 (data not shown).



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  		FIGURE 7. CR2 activation triggers in vivo interaction of the p85 subunit of PI 3-kinase with a p95 component in K562A cells. K562A (left panel) or K562W (right panel) cells (2 x 107) were incubated for 20 min at 4°C with buffer (lanes 1 and 6) or BL13 (lanes 2–5 and 7), then activated by cross-linking with GAM at 37°C for 1 min (lane 2), 3 min (lanes 1, 3, 6, and 7), 6 min (lane 4), and 12 min (lane 5). Nonidet P-40 (1%)-solubilized components immunoprecipitated on polyclonal anti-p85 Ab were analyzed by immunoblotting with anti-PTyr mAb (A). The same sheet was stripped and immunoblotted with polyclonal anti-p85 Ab (B). This figure is representative of the three experiments performed with K562A cells and one performed with K562W cells.

 
These data clearly demonstrated that the p95 component whose tyrosine phosphorylation was induced by CR2 activation and which interacted with the SH2 domain of the p85 subunit of PI 3-kinase was not CD19.

Difference in PI 3-kinase activation pathways between CR2 and CD19 activation

These data led us to analyze whether PI 3-kinase activity was associated in vivo with CD19 on Raji or Daudi cells after CR2 activation. For this purpose, Raji cells were activated by anti-CR2 or anti-CD19 Ab as described above. Then, total solubilized proteins were immunoprecipitated on anti-CR2, anti-CD19, or anti-PTyr Ab and tested for PI 3-kinase activity. As shown in Fig. 8Go (left panel), CR2 activation did not trigger significant interaction of PI 3-kinase activity with CD19 (lane 5) compared with unstimulated cells (lanes 1 and 4), while PI 3-kinase activity interacted with tyrosine-phosphorylated components immobilized on anti-PTyr mAb (lane 2). In controls, CD19 activation triggered interaction of PI 3-kinase activity with CD19 (lane 6) as well as with tyrosine-phosphorylated components immobilized on anti-PTyr mAb (lane 3). Furthermore, we analyzed the interaction of PI 3-kinase activity with CR2 after CR2 or CD19 activation on the cell surface. As shown in Fig. 8Go (right panel), CR2 or CD19 activation did not induce coprecipitation of PI 3-kinase activity with CR2 immobilized on anti-CR2 mAb.



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  		FIGURE 8. CR2 activation does not trigger in vivo interaction of PI 3-kinase activity with CD19. Raji cells (2 x 107) were incubated for 20 min at 4°C with buffer (lanes 1, 4, and 7), BL13 (lanes 2, 5, and 8), or B4 (lanes 3, 6, and 9), then activated by cross-linking with GAM at 37°C for 4 min (lanes 1–9). Nonidet P-40 (1%)-solubilized components immunoprecipitated on anti-PTyr (lanes 1–3), anti-CD19 (lanes 4–6), or anti-CR2 (lanes 7–9) mAbs were tested for PI 3-kinase activity. Identical results were obtained using Daudi cells in the same conditions. This figure is representative of the three experiments performed with Raji cells and two others performed with Daudi cells.

 
As CD19 activation also triggered interaction of PI 3-kinase p85 subunit with the tyrosine-phosphorylated 95-kDa proto-oncogene vav (47), we analyzed whether tyrosine-phosphorylated p95 induced by CR2 activation was antigenically related to Vav. As shown in Fig. 9Go, after CR2 activation, the p95 Ag that coprecipitated with the p85 subunit of PI 3-kinase was not recognized by polyclonal anti-Vav (lane 2) despite the presence of Vav among solubilized components (lane 3). Identical data were obtained for all of the above experiments with Daudi cells.



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  		FIGURE 9. p95 is not Vav. Raji cells (2 x 107) were incubated for 20 min at 4°C with buffer (lane 1) or BL13 (lane 2), then activated by cross-linking with GAM at 37°C for 4 min (lanes 1 and 2). Nonidet P-40 (1%)-solubilized components immunoprecipitated on polyclonal anti-p85 (lanes 1 and 2) or run directly in gel (lane 3) were analyzed by immunoblotting with polyclonal anti-Vav Ab. This figure is representative of the two experiments performed with Raji cells and one performed with Daudi cells.

 
Together, these data clearly demonstrated that CR2 activation on the B cell surface triggered in vivo PI 3-kinase activation through a pathway distinct from that triggered through CD19 activation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We analyzed the intracellular events associated with CR2 activation on the human B lymphocyte surface. We herein present the first demonstration that CR2 activation on the Raji or Daudi cell surface increases PI 3-kinase activity and induces tyrosine phosphorylation of a p95 component that interacts with SH2 domains of the p85 subunit of PI 3-kinase. Despite an identical molecular mass, this phosphorylated p95 component is neither CD19, a tyrosine-phosphorylated 95-kDa component that interacts with p85 subunit after CD19 activation (36, 47), nor Vav (47). In addition, these data clearly demonstrate that CR2 activation triggers PI 3-kinase activation through a pathway distinct from that triggered by CD19 activation.

Indeed, CR2 activation triggered with identical kinetics the increase in PI 3-kinase activity and the interaction of the p85 subunit of PI 3-kinase with a tyrosine-phosphorylated p95 component. The specificity of PI 3-kinase activity increased by CR2 activation was controlled as inhibited by wortmannin and LY294002, two specific inhibitors. It is well known that activation of PI 3-kinase, which is a major event occurring during cell proliferation induced through cell surface receptors, may be mainly regulated either by phosphorylation of its p85 subunit or by its association through its SH2 domains with phosphorylated proteins (44, 45, 46). We herein demonstrated that CR2 activation specifically triggered PI 3-kinase activity through interaction of the SH2 domain of its p85 subunit with the tyrosine-phosphorylated p95 protein. Indeed, 1) p85 was not recognized by anti-PTyr Abs, ruling out a putative phosphorylation of this molecule during CR2 activation; 2) the p85 subunit, despite the absence of tyrosine phosphorylation, coprecipitated with tyrosine-phosphorylated components on anti-PTyr Abs; 3) tyrosine-phosphorylated p95 coprecipitated with p85 subunit despite the fact that p95 was not recognized by anti-p85 Abs; 4) tyrosine-phosphorylated p95 bound to SH2, but not to SH3, domains of p85; and 5) all the events described above occurred only on cells activated through CR2, not on nonactivated cells.

Furthermore, tyrosine-phosphorylated p95, whose interaction with p85 subunit was triggered by CR2 activation on Raji or Daudi cell surface was not CD19, as this tyrosine phosphorylated p95 was: 1) not recognized by anti-CD19 Abs, as compared to positive controls. In addition, after CR2 activation, CD19 did not bind to the SH2 domain of p85 subunit, whereas in control CD19, activation induced CD19 interaction with p85 SH2 domains; 2) also present in the CR2-positive K562A cells which did not express CD19: CR2 activation on the K562A cell surface triggered identical binding of this tyrosine-phosphorylated p95 on SH2 domains of the p85 subunit as well as the p95 component identified in Raji or Daudi cells. In addition, despite the similarity of molecular mass and phosphorylation properties, we did not find any antigenic relationship between the tyrosine-phosphorylated p95 and: 1) the 95-kDa proto-oncogene vav, which interacted with PI 3-kinase p85 subunit after CD19 activation (47) (polyclonal anti-Vav did not react with p95); and 2) Gab1, an intermediate signaling molecule that interacted directly with PI 3-kinase p85 subunit after nerve growth factor receptor activation (48) (polyclonal anti-Gab1 did not recognize p95; data not shown). Although the tyrosine-phosphorylated p95 component that interacted with the p85 subunit after CR2 activation remains unidentified, tyrosine-phosphorylated components characterized by identical apparent molecular masses and that also interact with p85 SH2 domains after cell activation by IL-3 have been recently described (49, 50).

While the mechanisms through which activated CR2 triggers interaction of tyrosine-phosphorylated p95 with PI 3-kinase p85 subunit remain unknown, a direct interaction between activated CR2 and the p85 subunit of PI 3-kinase was ruled out. Indeed, CR2 activation did not allow coprecipitation of CR2 immobilized on anti-CR2 mAb with PI 3-kinase activity or p85 subunit. This is in good agreement with the analysis of CR2 amino acid sequence, which demonstrated that CR2 did not carry the consensus sequence YXXM that interacted with the SH2 domain of p85 (51). Furthermore, CR2 activation induced interaction of tyrosine-phosphorylated p95 with p85, without CR2 tyrosine phosphorylation or interaction of CR2 or CD19 with the p85 subunit of PI 3-kinase. These results strongly suggest that CR2 can activate PI 3-kinase via a mechanism in which an intermediate molecule is involved. Similar indirect mechanisms have been proposed for other receptors: 1) binding of PI 3-kinase to erythropoietin receptor was not required for erythropoietin-induced PI 3-kinase activation, while erythropoietin receptor also devoid of the sequence YXXM still activated PI 3-kinase (52); and 2) nerve growth factor induced the activation of PI 3-kinase, while this enzyme did not bind to the nerve growth factor receptor (53).

Among the components that may act as an intermediate molecule between activated CR2 and tyrosine-phosphorylated p95, one may consider 3BP2 (54). Indeed, in preliminary studies using GST fusion polypeptides, we found that CR2 activation on Raji or Daudi cell surface induced specific interaction of the tyrosine-phosphorylated p95 with SH2-containing proteins, such as 3BP2 and Grb2, but not with Fyn or Gap (S. Bouillie et al., unpublished observations). 3BP2 appears to be an intracellular molecule involved in signal transduction and/or a potential regulator of the tyrosine kinase Abl. 3BP2 carries SH2 and SH3 binding domains (this latter allowing its interaction with Abl) and pleckstrin homology domains (which allow protein anchoring to the cytoplasmic membrane) (55).

In addition, the data presented herein represent the first clear evidence that a strong difference exists between the intracellular events associated with CR2 activation and those associated with CD19 activation. Indeed, in contrast to CD19 activation, CR2 activation of PI 3-kinase activity did not induce 1) in vivo direct interaction of the p85 subunit of PI 3-kinase with CD19 or association of PI 3-kinase activity with CD19; 2) in vitro CD19 interaction with the SH2 domain of the PI 3-kinase p85 subunit; or 3) interaction of Vav with the PI 3-kinase p85 subunit. In addition, CR2 activation triggered PI 3-kinase activity and interaction of tyrosine-phosphorylated p95 with p85 subunit even in the absence of CD19, as shown in K562A cells.

Furthermore, the demonstration that CR2 activation triggered both short-lived PI 3-kinase activation and cell proliferation (9, 18, 19, 20, 21, 36) raised the question of their possible relationship. Preliminary data, obtained by measuring B lymphocyte proliferation in the presence of wortmannin, demonstrated that this specific PI 3-kinase inhibitor also inhibited Raji cell proliferation induced through activated CR2 (S. Bouillie et al., unpublished observations). In addition, the short-lived activation of PI 3-kinase seems to have a biological significance in lymphocyte activation, as suggested for other cell activators (56, 57).

In conclusion, this suggests that the short-lived PI 3-kinase activation triggered by activated CR2, independently of CD19, may represent one of the first events involved in B cell activation pathways.


   Acknowledgments
 
We thank Michelle Balbo and Gérard Drevet for technical assistance and Christel Leger for secretarial assistance.


   Footnotes
 
1 This work was supported by Institut National de la Santé et de la Recherche Médicale, Association pour la Recherche contre le Cancer and Comité de Paris de La Ligue Nationale Française contre le Cancer. Back

2 Address correspondence and reprint requests to Dr. Raymond Frade, Immunochimie des Régulations Cellulaires et des Interactions Virales, Institut National de la Santé et de la Recherche Médicale Unit 354, Centre INSERM, Hôpital Saint-Antoine, 75012 Paris, France. E-mail address: Back

3 Abbreviations used in this paper: TAPA-1, target of antiproliferative Ab; PI 3-kinase, phosphatidylinositol 3-kinase; SH2/SH3, Src homology 2/3; GAM, goat anti-mouse Ig; GST, glutathione-S-transferase; PTyr, phosphotyrosine residue; TSP, total solubilized proteins. Back

Received for publication June 1, 1998. Accepted for publication September 8, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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