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Activation of the EBV/C3d Receptor (CR2, CD21) on Human B Lymphocyte Surface Triggers Tyrosine Phosphorylation of the 95-kDa Nucleolin and Its Interaction with Phosphatidylinositol 3 Kinase

Monique Barel, Muriel Le Romancer and Raymond Frade
J Immunol March 1, 2001, 166 (5) 3167-3173; DOI: https://doi.org/10.4049/jimmunol.166.5.3167
Monique Barel
Immunochimie des Régulations Cellulaires et des Interactions Virales, Centre Institut National de la Santé et de la Recherche Médicale, Paris, France
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Muriel Le Romancer
Immunochimie des Régulations Cellulaires et des Interactions Virales, Centre Institut National de la Santé et de la Recherche Médicale, Paris, France
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Raymond Frade
Immunochimie des Régulations Cellulaires et des Interactions Virales, Centre Institut National de la Santé et de la Recherche Médicale, Paris, France
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Abstract

We previously demonstrated that CR2 activation on human B lymphocyte surface triggered tyrosine phosphorylation of a p95 component and its interaction with p85 subunit of phosphatidylinositol 3′ (PI 3) kinase. Despite identical molecular mass of 95 kDa, this tyrosine phosphorylated p95 molecule was not CD19, the proto-oncogene Vav, or the adaptator Gab1. To identify this tyrosine phosphorylated p95 component, we first purified it by affinity chromatography on anti-phosphotyrosine mAb covalently linked to Sepharose 4B, followed by polyacrylamide gel electrophoresis. Then, the isolated 95-kDa tyrosine phosphorylated band was submitted to amino acid analysis by mass spectrometry; the two different isolated peptides were characterized by amino acid sequences 100% identical with two different domains of nucleolin, localized between aa 411–420 and 611–624. Anti-nucleolin mAb was used to confirm the antigenic properties of this p95 component. Functional studies demonstrated that CR2 activation induced, within a brief span of 2 min, tyrosine phosphorylation of nucleolin and its interaction with Src homology 2 domains of the p85 subunit of PI 3 kinase and of 3BP2 and Grb2, but not with Src homology 2 domains of Fyn and Gap. These properties of nucleolin were identical with those of the p95 previously described and induced by CR2 activation. Furthermore, tyrosine phosphorylation of nucleolin was also induced in normal B lymphocytes by CR2 activation but neither by CD19 nor BCR activation. These data support that tyrosine phosphorylation of nucleolin and its interaction with PI 3 kinase p85 subunit constitute one of the earlier steps in the specific intracellular signaling pathway of CR2.

The receptor for C3d, CR2, 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), which interacts with the LYNVEA site expressed on 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 (1, 2, 3) (11, 12). CR2 also interacts with autoantibodies present in serum of polyarthritis rheumatoid patients (13) and with CD23 (14). Molecular cloning indicated that CR2 is constituted by a large extracellular domain of 954 aa composed of 15–16 short consensus repeats, a 24-aa transmembrane domain, and a 34-aa intracellular domain (15, 16). CR2 allows C3d and EBV to induce proliferation (17, 18) or transformation (8), respectively. One of the most early events in these two biological functions is the cross-linking of CR2 at 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 regulation of B lymphocyte proliferation was analyzed by determining its interaction with cellular components. At the cell surface, CR2 could interact with other Ags as IgM (22), CD19, and target of anti-proliferative Ab (TAPA-1)3 (23, 24, 25). These authors suggested that CR2 might play a role in B cell regulation only through its complex formation with CD19 and TAPA-1 (23, 24, 25). However, there is evidence 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 antioncoprotein (29), p68 calcium-binding protein (30), and nuclear p120 ribonucleoprotein (31), through distinct binding sites (32, 33). Third, a role of the CR2 intracytoplasmic tail, despite its short length of 34 aa, in early events associated to 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 pep34, a synthetic peptide whose sequence corresponded to the full intracytoplasmic tail of CR2, inhibited the specific proliferation induced through CR2. Fourth, analyzing the growth factor function of C3d (17), we demonstrated that in serum-free medium, C3d and pep16, a 16-aa 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-aa 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). In a previous work, we also suggested that CR2 activation in cell extracts triggered intracellular phosphorylation through a CD19 independent pathway (38).

More recently, we demonstrated (39) that CR2 activation on cell surface triggered phosphatidylinositol 3′ (PI 3) kinase activation through a pathway distinct to that triggered through CD19 activation. Indeed, in contrast to CD19 activation, CR2 activation did not trigger either interaction of PI 3 kinase p85 subunit with CD19 or the proto-oncogene Vav or coprecipitation of PI 3 kinase activity with CD19. However, CR2 activation induced interaction of PI 3 kinase p85 subunit through its Src homology (SH) 2 domains with a tyrosine phosphorylated p95 component. Despite identical molecular mass of 95 kDa, this tyrosine phosphorylated p95 was not CD19, Vav, or Gab1, an adaptator molecule. Therefore, the aim of this work was to identify this p95 cellular component whose tyrosine phosphorylation was induced by CR2 activation. Herein, we demonstrate that this p95 tyrosine phosphorylated component is nucleolin, which interacts with the p85 subunit of PI 3 kinase. In addition, tyrosine phosphorylation of nucleolin induced by activated CR2 occurs in B lymphoma cells as well as in normal B lymphocytes. This regulation in normal B lymphocytes is specific of activated CR2 as not induced by activated CD19 or surface Ig (sIg).

Materials and Methods

Cells

Raji cells are from a Burkitt B lymphoma cell line that express CR2. K562A cells express CR2, but not CD19, as derived from K562, wild-type cells transfected with the CR2 cDNA (38). Cells were grown in RPMI 1640 medium supplemented with 10% FCS, 2 mM l-glutamine, 1000 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in 5% CO2. Neomycin (200 μg/ml) were added for K562A cells. Normal B lymphocytes purified from tonsils of normal human donors were purified as previously described (35).

Abs and reagents

The following Abs and reagents were purchased from the indicated companies: anti-CR2 mAb: BL13 and anti-CD19 mAb (B4) from Immunotech (Westbrook, ME) and OKB-7 from Ortho Diagnostics (Raritan, NJ), goat anti-human IgM F(ab′)2 from Cappel (Durham, NC), and goat anti-mouse Ig (GAM) from Dako (Carpenteria, CA); polyclonal Ab anti-p85 subunit of PI 3 kinase, anti-phosphotyrosine residue (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); Grb2, Fyn, and GAP fusion proteins were obtained from PharMingen (Becton Dickinson, France). Anti-nucleolin mAb were generous gifts from Maria Lopez-Trascasa (Hospital La Paz, Madrid, Spain), Nancy Maizel (Yale University, CT), and Ning-Hsing Yeh (National Yang-Ming University, Taipei, Taiwan). Anti-Grb2 mAb was purchased from Transduction Laboratory (Lexington, KY).

Construction of recombinant SH2 domain of 3BP2

PCR samples containing 500 ng PSport-3BP2 kindly provided by Margaret Knowles (Marie Curie Research Institute, Oxted, U.K.) were heated for 5 min at 94°C, followed by 30 cycles of 1 min at 94°C, 1 min at 55°C, 1.30 min at 72°C, followed by a final extension of 7 min at 72°C. 5′ primer was 5′-CGGGATCCATTACCTTATACGTGATA-3′ (nt 3141–3156 with a BamHI site) and 3′ primer was 5′-TAGAATTCGCTGGCTGGGTTGTATGG-3′ (nt 3246–3261 with a EcoRI site). The PCR fragment obtained was purified after electrophoresis and excision of an agarose gel. Then, SH2 domain of 3BP2 was ligated into PGEX4T3 vector (Amersham, France). After sequencing the insert, fusion protein was produced in BL21 bacteria.

Cell activation

Cells (2 × 107) were washed with RPMI 1640 medium and incubated with the indicated mAb (5 μg/ml) in a final volume of 500 μl at 4°C for 30 min. Then, GAM (20 μg/ml) was added at 37°C for the indicated times to cross-link the first Abs only when BL13 or anti-CD19 was used and not with OKB7. Indeed, it was already well established that among the identified anti-CR2 mAb some, as OKB-7, induced CR2 activation without cross-linking by a secondary Ab, whereas some others, as BL13, needed to be cross-linked by a secondary Ab to activate CR2 (6). 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, phosphatase inhibitors as 50 mM NaF, 1 mM Na3VO4, 30 mM Na4P2O7, and protease inhibitors 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 × g for 15 min and were stored at −80°C. Protein content was normalized after a bicinchoninic acid assay.

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 LKB, Piscataway, NJ) or protein A/G-agarose beads (20 μl; Sigma, St. Louis, MO) for 2 h at 4°C. For each mAb used, an irrelevant mAb of the same isotype has been used in preliminary experiments to verify the specificity of immunoprecipitation assays (data not shown). Immunobeads were washed five times in lysis buffer. Then, proteins were eluted in sample buffer, divided in two or three samples depending on the experiments and submitted to 7% SDS-PAGE under reducing conditions. After SDS-PAGE, separated proteins were electrotransferred on Hybond-ECL nitrocellulose sheet (Amersham, Arlington Heights, IL). The membranes were incubated for 1 h in PBS 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 PBS/0.05% Tween 20, peroxidase-linked secondary Abs were added for 1 h at room temperature. Nitrocellulose sheets were then washed in PBS/0.05% Tween 20 and binding of second Abs was detected using the enhanced chemiluminescence kit (Amersham).

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.

Purification of p95 tyrosine phosphorylated protein

Raji cells (109) were activated by BL13, an anti-CR2 mAb, that was cross-linked with GAM at 37°C, for 2 min, as previously described (39). Then, total cellular components were solubilized in 1% Nonidet P-40 and immunoprecipitated on anti-PTyr mAb covalently linked to Sepharose 4B beads. After washing, proteins were eluted in sample buffer then submitted to 7.5% SDS-PAGE under reducing conditions and electrotransferred on polyvinylidene difluoride (PVDF) membranes (Amersham). Proteins present on PVDF membranes were stained with Ponceau Red. The membrane part that contained the 95-kDa protein detected in samples prepared from CR2-activated cells and absent in samples prepared from nonactivated cells was cut and submitted to high mass accuracy tryptic peptide mapping using matrix-assisted laser desorption/ionization mass spectrometry (Protana A/S; Odense, Denmark). Briefly, the extracted peptides were purified using micro column filled with Poros R2, 50-μm beads. The column was packed in a borosilicate purification needle. After packing, the microcolumn was equilibrated in 5% formic acid and the digest, dissolved in 5% formic acid, was loaded. Several column volumes of 5% formic acid were used for washing. The peptides were eluted with 1 μl of 5% formic acid/50% HPLC-grade methanol in to a nanoES needle. ESMS was performed on a prototype quadrupole time-of-flight mass spectrometer (Sciex, Thornhill, Toronto, Canada) equipped with a nanoES source from Protana A/S (Odense, Denmark). Matrix-assisted laser desorption/ionization mass spectrometry was performed on a Voyager-DE STR from PerSeptive Biosystems. Analysis of the ms/ms data was performed using BioMultiView version 1.4b (Sciex). Database searches were performed with PepSea Client 1.1.

Results

We previously clearly demonstrated that CR2 activation on cell surface triggered in vivo PI 3 kinase activation through a pathway distinct to that induced through CD19 activation and interaction of a p95-kDa tyrosine phosphorylated component with the p85 subunit of PI 3 kinase (39). To identify this p95-kDa component, the first step was to purify it and then to analyze its properties. In all the following experiments, except when indicated, the same experimental procedure was used according to our previous data, i.e., CR2 was activated on cell surface by two different anti-CR2 mAb, as OKB-7 and BL13, well known to trigger lymphocyte proliferation (6): OKB-7 induced B cell proliferation without needing cross-linking by a secondary Ab, whereas BL13 needed to be cross-linked with GAM. Cells incubated only with buffer or with the secondary GAM were not activated and used as control for OKB-7 and BL-13, respectively. Then, total cellular components were solubilized in 1% Nonidet P-40 and submitted to different immunoprecipitation experiments, which were analyzed by immunoblotting using specific Abs.

Identification of the p95 protein whose tyrosine phosphorylation was induced by CR2 activation

To identify the p95 tyrosine phosphorylated component, total solubilized proteins were immunoprecipitated on anti-phosphotyrosine (P-Tyr) mAb covalently linked to Sepharose 4B beads. After washing, eluted proteins were submitted to SDS-PAGE and electrotransferred on PVDF membranes that were stained with Ponceau Red. As shown in Fig. 1⇓A, a 95-kDa cellular protein was detected in samples prepared from CR2-activated cells (lane 2) and was absent in samples prepared from nonactivated cells (lane 1). In control, this p95-kDa component was tyrosine phosphorylated in CR2-activated cells (lane 4) and was not in nonactivated cells (lane 3). The PVDF membrane part, which contained this 95-kDa protein, was cut and submitted to sequencing by mass spectrometry method developed by Protana (as described in Materials and Methods). From this analysis (Fig. 1⇓B), two different peptides were chosen for the collision dissociation experiments (Protana) and characterized by the following sequences EVFEDAAEIR and GFGFVDFNSEEDAK. These two peptide sequences were 100% identical with two sequences of human nucleolin and were localized in two different RNA-binding domains (or RRM) of nucleolin, between aa 411–420 and 611–624, respectively. Subsequently, solubilized cellular components from CR2 activated or nonactivated Raji cells were immunoprecipitated on anti-nucleolin mAb. As shown in Fig. 2⇓, although nucleolin was present (B) in both CR2-activated (lanes 2–5) and nonactivated cells, incubated only with buffer (lane 1) or second Ab alone (lane 6), nucleolin was tyrosine phosphorylated (A) only after CR2 activation. The maximum of phosphorylation was reached at 2 min, i.e., a kinetics identical with that previously described for the tyrosine phosphorylated p95 (39). Identical results were obtained when CR2 was activated on Raji cell surface by OKB-7, another anti-CR2 mAb (data not shown). Furthermore, immunodepletion on anti-nucleolin mAb of cell extracts, prepared from CR2 activated cells, led to selective loss of the p95 tyrosine phosphorylated nucleolin (Fig. 2⇓, A and B, lanes 7–9), All these data demonstrated that the p95-kDa protein whose phosphorylation on tyrosine was induced by CR2 activation was nucleolin.

FIGURE 1.
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FIGURE 1.

Tyrosine-phosphorylated p95 protein is nucleolin. Total cellular components from 109 Raji cells nonactivated (lanes 1 and 3) or activated (lanes 2 and 4) by BL13, an anti-CR2 mAb, cross-linked with GAM at 37°C, for 2 min, were solubilized in 1% Nonidet P-40 and immunoprecipitated on anti-PTyr mAb covalently bound to Sepharose 4B beads. After washing, eluted proteins were submitted to 7.5% SDS-PAGE under reducing conditions and electrotransferred on PVDF membranes. Proteins present on PVDF membranes were either stained with Ponceau Red (A, lanes 1 and 2) or immunodetected with anti-Ptyr mAb (A, lanes 3 and 4). The part of the membrane that contained the 95-kDa protein detected in samples prepared from CR2-activated cells and absent in samples prepared from nonactivated cells was cut and submitted to high mass accuracy tryptic peptide mapping using matrix-assisted laser desorption/ionization mass spectrometry. Analysis of the ms/ms data was performed using BioMultiView, Version 1.4b. Database searches gave two different peptide sequences, which corresponded to different parts of human nucleolin (B).

FIGURE 2.
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FIGURE 2.

Activated CR2 triggers tyrosine phosphorylation of human nucleolin. Raji cells (2 × 107) were incubated with 5 μg/ml BL13 in a final volume of 500 μl at 4°C for 30 min (lanes 1–9), then without (lanes 1 and 7) or with 20 μg/ml GAM at 37°C for 2 min (lanes 2, 7, and 8), 4 min (lane 3), 6 min (lane 4), 8 min (lane 5) to cross-link the first Ab. Another control was made by incubating cells only with 20 μg/ml GAM at 37°C for 2 min (lane 6). One milligram of total solubilized proteins (lanes 1-8) or solubilized proteins preabsorbed four times on anti-nucleolin mAb (lane 9) were incubated for 2 h at 4°C with 5 μg anti-nucleolin mAb. Immune complexes were bound to 20-μl protein A/G-agarose beads for 2 h at 4°C. Immunobeads were washed five times in lysis buffer, then proteins were eluted in sample buffer, divided in two identical samples, and submitted to two 7% SDS-PAGE under reducing conditions. After SDS-PAGE and electrotransfer to nitrocellulose sheet, immunodetection was performed with 1 μg/ml anti-phosphotyrosine mAb (A) or anti-nucleolin mAb diluted 1/1000 (B).

CR2 activation triggers binding of tyrosine phosphorylated nucleolin on the p85 subunit of the PI 3 kinase

Our previous demonstration that CR2 activation on Raji cell surface induced the interaction of the p95 tyrosine phosphorylated protein with the p85 subunit of PI 3 kinase (39) led us to analyze the binding properties of nucleolin. Therefore (Fig. 3⇓), solubilized components, prepared from CR2-activated (lane 2) or nonactivated (lane 1) cells, were immunoprecipitated either on anti-p85 Ab or anti-nucleolin mAb and analyzed with different specific Abs. First, analysis with anti-nucleolin (A) or anti-phosphotyrosine (B) Abs of cellular components immunoprecipitated on anti-p85 subunit Abs demonstrated that after 2 min activation of CR2, nucleolin bound on the p85 subunit of PI 3 kinase (A). The demonstration that the tyrosine-phosphorylated p95 component bound on the p85 subunit (B) was nucleolin was completed by analyzing the solubilized components immunoprecipitated on anti-nucleolin mAb with anti-p85 subunit Ab (C) or anti-phosphotyrosine mAb (D). Results demonstrated that although nucleolin was always immunoprecipitated on anti-nucleolin, as already shown in Fig. 2⇑, the p85 subunit interacted with nucleolin (C) only when this latter was phosphorylated (D). In addition, as previously shown (39), the p85 subunit of P I3 kinase coprecipitated with nucleolin was not tyrosine phosphorylated (B and D).

FIGURE 3.
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FIGURE 3.

Activated CR2 triggers binding of tyrosine-phosphorylated nucleolin on the p85 subunit of the PI 3 kinase. Raji cells (2 × 107) were incubated with 5 μg/ml OKB7 in a final volume of 500 μl at 4°C for 30 min, then activated at 37°C for 2 min (lane 2) or not activated (lane 1). One milligram of solubilized proteins of each assay was incubated for 2 h at 4°C either with 5-μg rabbit anti-p85 subunit of PI 3 kinase (A and B) or with 5-μg anti-nucleolin mAb (C and D). Immune complexes were bound to 20-μl protein A/G-agarose beads for 2 h at 4°C. Immunobeads were washed five times in lysis buffer, then proteins were eluted in sample buffer, divided in two identical samples, and submitted to two 7% SDS-PAGE under reducing conditions. After SDS-PAGE and electrotransfer to nitrocellulose sheet, immunodetection was performed with 1 μg/ml anti-phosphotyrosine mAb (B and D), anti-nucleolin mAb diluted 1/1000 (A) or anti-p85 subunit Ab diluted 1/2000 (C).

Study of the interaction of tyrosine phosphorylated nucleolin with the p85 subunit induced by CR2 activation was completed by using different SH2 domains fused to GST proteins (Fig. 4⇓, I). Solubilized components prepared from CR2-activated (lane 2) or nonactivated (lane 1) Raji cells were incubated with SH2-containing proteins, as the p85 subunit of PI 3 kinase, 3BP2, Grb2, Fyn, or Gap, then bound components were analyzed by immunoblotting with anti-nucleolin mAb. Data demonstrated that nucleolin whose tyrosine phosphorylation was induced only when Raji cells were activated interacted with SH2 domains of p85 subunit (A), 3BP2 (B), or Grb2 (C) but not with SH2 domains of Fyn (D) or Gap (E) or on GST alone (data not shown). The specificity of the interaction of tyrosine-phosphorylated nucleolin with SH2 domains of p85 subunit, 3BP2 and Grb2 but not with SH2 domains of Fyn and Gap is in good agreement with our previous demonstration of the interaction of the tyrosine phosphorylated p95 with these same SH2 domains (39). In addition, taking into account that anti-Grb2 mAb was available (but not anti-3BP2 mAb), herein we also demonstrated that nucleolin coprecipitated with intracellular stores of Grb2 molecules (Fig. 4⇓, II).

FIGURE 4.
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FIGURE 4.

Binding of tyrosine-phosphorylated nucleolin on different SH2 domains. Raji cells (2 × 107) were incubated with 5 μg/ml OKB7 in a final volume of 500 μl at 4°C for 30 min, then activated at 37°C for 2 min (lane 2) or not activated (lane 1). One milligram of solubilized proteins of each assay was incubated for 2 h at 4°C: in I, with 5 μg GST-SH2 domain of p85 subunit of PI 3 kinase (A), 3BP2 (B), Grb2 (C), Fyn (D), or Gap (E); in II, with 5 μg anti-nucleolin mAb and immune complexes were bound to 20-μl protein A/G-agarose beads for 2 h at 4°C. Then, beads were washed five times in lysis buffer, then proteins were eluted in sample buffer and submitted to 7% SDS-PAGE under reducing conditions. After SDS-PAGE and electrotransfer to nitrocellulose sheet, immunodetection was performed in I, with anti-nucleolin mAb diluted 1/1000 and in II, either with anti-nucleolin mAb (diluted 1/1000) or anti-Grb2 mAb (diluted 1/5000).

Thus, CR2 activation on Raji cells induced interaction of tyrosine-phosphorylated nucleolin with SH2 domains of p85 subunit of PI 3 kinase and also with SH2 domains of 3BP2 and Grb2.

Tyrosine phosphorylation of nucleolin is induced by CR2 activation and not by CD19 or sIg activation

We previously demonstrated that the 95-kDa protein whose tyrosine phosphorylation was triggered by activated CR2 was not CD19 and that this phosphorylation was independent of CD19 activation (39). Thus, the following experiments were performed. First, CR2 was activated by OKB-7 on K562A cells, which are K562 CD19-negative and CR2-positive cells (as transfected with CR2 cDNA), in the same conditions used for Raji cells (Fig. 5⇓A). In these conditions, although expression of nucleolin was the same both in activated or nonactivated K562A cells (lanes 1 and 2), tyrosine phosphorylation of nucleolin was induced only after CR2 activation (lane 4) and not when K562A cells were not activated (lane 3). Second, because some molecules of CR2 were described associated to sIg or CD19 (22, 23, 24, 25), we also used normal B lymphocytes and analyzed whether cross-linking of sIg or CD19 on normal B lymphocytes surface could also trigger tyrosine phosphorylation of nucleolin. Using cross-linking conditions of CD19 or sIg widely described by others (22, 23, 24, 25), we demonstrated (Fig. 5⇓B) that nucleolin, expressed in normal B lymphocytes (lane 1), was tyrosine phosphorylated after CR2 activation (lane 5). In addition, in the same conditions, tyrosine phosphorylation of nucleolin was neither induced by sIg (lane 3) nor CD19 (lane 4) cross-linking. Nonactivated normal B lymphocytes were used in control (lane 2). All these data supported that tyrosine phosphorylation of nucleolin triggered by activated CR2 occurred in normal B lymphocytes as well as in lymphoma B cells. In addition, this regulation was specific of CR2 as not induced by activated CD19 or sIg.

FIGURE 5.
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FIGURE 5.

Tyrosine phosphorylation of nucleolin is induced by CR2 activation and not by CD19 or sIg activation. A, K562A, K562 cells transfected with CR2 cDNA, (2 × 107) were incubated with 5 μg/ml OKB7 in a final volume of 500 μl at 4°C for 30 min, then nonactivated (lanes 1 and 3) or activated at 37°C for 2 min (lane 2 or 4). One milligram of solubilized proteins of each assay was incubated for 2 h at 4°C with 5 μg anti-nucleolin mAb. Immune complexes were bound to 20-μl protein A/G-agarose beads for 2 h at 4°C. Immunobeads were washed five times in lysis buffer, then proteins were eluted in sample buffer, divided in two identical samples, and submitted to 7% SDS-PAGE under reducing conditions. After SDS-PAGE and electrotransfer to nitrocellulose sheet, immunodetection was performed either with 1 μg/ml anti-phosphotyrosine mAb (lanes 3 and 4) or anti-nucleolin mAb diluted 1/1000 (lanes 1 and 2). B, Normal human B lymphocytes (2 × 107) were incubated with 20 μg/ml F(ab′) 2 of anti-human IgM (lane 3), 4 μg/ml anti-CD19 mAb (lane 4), or 5 μg/ml OKB7 (lane 5), in a final volume of 500 μl at 4°C for 30 min then activated at 37°C for 2 min (lanes 3–5), as described in Material and Methods. In control, cells were incubated, in the same conditions, with GAM alone (lanes 1 and 2). One milligram of solubilized proteins of each assay was immunoprecipitated on anti-nucleolin mAb and processed as mentioned in A, until detection of components on nitrocellulose sheet: immunodetection was performed either with 1 μg/ml anti-phosphotyrosine mAb (lanes 2–5) or anti-nucleolin mAb diluted 1/1000 (lane 1).

Discussion

This is the first demonstration that the p95 component, whose tyrosine phosphorylation and interaction with the p85 subunit of PI 3 kinase was induced by CR2 activation, is nucleolin. The different results that support this demonstration are: 1) amino acid analysis by mass spectrometry of peptides isolated from the p95 tyrosine phosphorylated component purified by affinity chromatography on anti-phosphotyrosine mAb; 2) this p95 tyrosine-phosphorylated component was recognized by anti-nucleolin mAb; 3) after CR2 activation, nucleolin presented the same kinetics of tyrosine phosphorylation as the p95 component; 4) tyrosine-phosphorylated nucleolin interacted, as well as the p95 component, with the SH2 domains of the p85 subunit of PI 3 kinase and presented identical specificity for other SH2 domains (i.e., interactions with SH2 domains of 3BP2 and Grb2, but not with SH2 domains of Fyn and Gap).

In addition, although it was described by others that some CR2 molecules may interact with other cell surface Ags as IgM (22) and CD19 (23, 24, 25), suggesting identical pathways for sIg, CD19, and CR2, we previously clearly demonstrated that CR2 activation triggered tyrosine phosphorylation of a 95-kDa component that was not CD19 (39). Herein, we demonstrated that tyrosine phosphorylation of nucleolin was specifically induced by activated CR2. Indeed, this specific regulation was induced in: 1) K562A cells, which are CD19-negative cells; and 2) normal B lymphocytes, where activation of sIg or CD19 did not. Thus, nucleolin tyrosine phosphorylation triggered by activated CR2 was not restricted to EBV-transformed cells. The data presented herein strongly support the presence of distinct intracellular signaling pathways induced after activation of CR2 or sIg and CD19, where nucleolin could play a role.

Nucleolin is an abundant ubiquitous nonhistone nucleolar phosphoprotein of exponentially growing eukaryotic cells, which belong to a large family of RNA-binding protein (40). Nucleolin acts as a protein shuttling between nucleus and cytoplasm (41) and may be associated with the cell membrane (42). Therefore, nucleolin acts as a key molecule by transferring cytoplasmic signal between the cell surface, the cytoplasm, and the nucleus. Among its numerous functions (for review, see Ref. 40), nucleolin is involved in the regulation of ribosome biogenesis, one of the key element for control of cell growth (43). Nucleolin also participates in the B cell-specific transcription factor and switch region binding protein LR1 (44). In these events, phosphorylation of nucleolin has been shown to regulate its activity (40). Until now, nucleolin has been shown to be phosphorylated only on serine (45) and on threonine (46, 47) residues, despite the presence of a significant number of tyrosine residues. Phosphorylation of nucleolin on serine or on threonine residues occurs during interphase or mitosis, respectively (for review, see Ref. 40). Therefore, our data constitute the first report that demonstrates that nucleolin may be also phosphorylated on tyrosine, as detailed in the above paragraph. This tyrosine phosphorylation specifically occurred only after a brief span of 2 min CR2 activation on B lymphocyte surface and concerned only a subpopulation of nucleolin molecules. Indeed, quantification by densitometer analysis showed that among all nucleolin molecules immunoprecipitated on anti-nucleolin Abs, only a low amount (<5%) was tyrosine phosphorylated (data not shown). These data suggest that this tyrosine phosphorylation of nucleolin takes place in an early and brief important step involved in B lymphocyte proliferation induced by CR2 activation. In addition, this early and brief event may account more likely for the difficulty for others to observe this tyrosine phosphorylation.

Furthermore, our previous demonstration that CR2 activation led to activation of PI 3 kinase activity (39) and the herein identification of the p95 nucleolin suggest that interaction of tyrosine phosphorylated nucleolin with SH2 domains of the p85 subunit of PI 3 kinase more likely implies a similar mechanism to that already described by others (48) in activation of PI 3 kinase activity: i.e., Ptyr-containing protein by interacting with SH2 domain of p85 induces a conformational change in this regulatory subunit that activates the p110 catalytic subunit (48). One well-identified target of PI 3 kinase activation is the protein kinase Akt that plays a major role in protecting cells from apoptosis and in promoting cell proliferation (49, 50).

The mechanisms through which activated CR2 triggered interaction of tyrosine phosphorylated nucleolin with PI 3 kinase p85 subunit remain unknown. Previously, we ruled out a direct interaction between activated CR2 and p85 subunit of PI 3 kinase (39). Furthermore, direct interaction between CR2 and nucleolin could not be evidenced. Therefore, intermediate molecules, still unknown, should be involved in this activation pathway. Among these intermediate molecules, one may consider Grb2 and 3BP2 (51). Indeed, using GST fusion proteins, we found that CR2 activation on Raji cell surface induced specific interaction of the tyrosine-phosphorylated nucleolin with SH2 containing proteins as 3BP2 and Grb2, but not with Fyn or Gap. Relevance of intracellular interaction of Grb2 with tyrosine-phosphorylated nucleolin induced by activated CR2 was herein confirmed by coprecipitating Grb2 and nucleolin molecules. 3BP2 may be also considered because it is an intracellular molecule involved in signal transduction, through its SH2- and SH3-binding domains and pleckstrin homology domains, which allow protein anchoring to the cytoplasmic membrane (52). Further studies are also needed to relate this new signaling pathway to our previous demonstration that CR2 interacted through its C-terminal domain with p53 in human B lymphoma cells (29) and with a p68 calcium-binding protein in normal B lymphocytes (30). Thus, it will be also necessary to analyze whether CR2 activation triggers tyrosine phosphorylation of nucleolin also in murine B lymphocytes and its involvement in their immunomodulatory functions (53)

Although we clearly demonstrated that nucleolin could be phosphorylated after activation of CR2, the kinases that are involved in this phosphorylation remain unknown. The demonstration that Fyn, Lyn, and Lck were involved in CD19 and BCR signaling (54, 55) and our herein demonstration that tyrosine phosphorylation of nucleolin was not induced by CD19 or BCR activation, but only by CR2 activation, led us to suggest that other phosphotyrosine kinases may be involved in this latter pathway. Preliminary experiments suggested that pp60src, which was found expressed in Raji cells (as verified with a specific anti-pp60src mAb), could be involved. Indeed, herbimycin, a specific pp60src inhibitor, inhibited tyrosine phosphorylation of nucleolin and its subsequent binding on the p85 subunit of PI3-kinase, induced by CR2 activation (M. Barel, unpublished data). Further studies are needed to confirm this hypothesis.

In conclusion, our demonstration that CR2 activation triggers, within 2 min, tyrosine phosphorylation of nucleolin (which is well known to be involved in cell regulation) and interaction of the phosphorylated nucleolin with the SH2 domains of the p85 subunit of PI 3 kinase (which is known to play a role in cell proliferation) (56), supports that this regulation constitutes one of the earlier steps in human B lymphocyte proliferation specifically induced via CR2 activation (6), as not induced by CD19 or BCR activation.

Acknowledgments

We thank Carine De Ruyffelaere, Michelle Balbo, and Gérard Drevet for technical 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.

  • ↵2 Address correspondence and reprint requests to Dr. Raymond Frade, Institut National de la Santé et de la Recherche Médicale, Unité U.354, Center, Institut National de la Santé et de la Recherche Médicale, Hôpital Saint-Antoine, 184 rue du Faubourg Saint-Antoine, 75012, Paris, France. E-mail address: frade354{at}easynet.fr

  • 3 Abbreviations used in this paper: TAPA-1, target of anti-proliferative Ab; PI 3, phosphatidylinositol 3′; SH, Src homology; GAM, goat anti-mouse Ig; Ptyr, phosphotyrosine residue; PVDF, polyvinylidene difluoride; sIg, surface Ig; BCR, B cell receptor.

  • Received August 14, 2000.
  • Accepted December 29, 2000.
  • Copyright © 2001 by The American Association of Immunologists

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Activation of the EBV/C3d Receptor (CR2, CD21) on Human B Lymphocyte Surface Triggers Tyrosine Phosphorylation of the 95-kDa Nucleolin and Its Interaction with Phosphatidylinositol 3 Kinase
Monique Barel, Muriel Le Romancer, Raymond Frade
The Journal of Immunology March 1, 2001, 166 (5) 3167-3173; DOI: 10.4049/jimmunol.166.5.3167

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Activation of the EBV/C3d Receptor (CR2, CD21) on Human B Lymphocyte Surface Triggers Tyrosine Phosphorylation of the 95-kDa Nucleolin and Its Interaction with Phosphatidylinositol 3 Kinase
Monique Barel, Muriel Le Romancer, Raymond Frade
The Journal of Immunology March 1, 2001, 166 (5) 3167-3173; DOI: 10.4049/jimmunol.166.5.3167
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