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Research Article

Tyrosine phosphorylation of 3BP2 is indispensable for the interaction with VAV3 in chicken DT40 cells Kazuyasu Chiharaa,d, Yukihiro Kimuraa,b, Chisato Honjoha,c, Shota Yamauchia,d, Kenji Takeuchia,d, Kiyonao Sadaa,d,n a

Division of Genome Science and Microbiology, Department of Pathological Sciences, Faculty of Medical Sciences, Fukui 910-1193, Japan b Division of Otorhinolaryngology Head and Neck Surgery, Department of Sensory and Locomotor Medicine, Faculty of Medical Sciences, Fukui 910-1193, Japan c Third Department of Internal Medicine, Faculty of Medical Sciences, Fukui 910-1193, Japan d Organization for Life Science Advancement Programs, University of Fukui, Fukui 910-1193, Japan

article information

abstract

Article Chronology:

Adaptor protein c-Abl SH3 domain-binding protein-2 (3BP2) is known to play regulatory roles in

Received 26 September 2013

immunoreceptor-mediated signal transduction. We have previously demonstrated that Tyr174,

Received in revised form

Tyr183 and Tyr446 in mouse 3BP2 are predominantly phosphorylated by Syk, and the phosphor-

25 December 2013

ylation of Tyr183 and the Src homology 2 (SH2) domain of mouse 3BP2 are critical for B cell

Accepted 28 December 2013

receptor (BCR)-induced activation of nuclear factor of activated T cells (NFAT) in human B cells.

Available online 6 January 2014

In this report, we have shown that Syk, but not Abl family protein-tyrosine kinases, is critical for

Keywords: Adaptor proteins 3BP2 Syk Abl Vav3 B cell receptor

BCR-mediated tyrosine phosphorylation of 3BP2 in chicken DT40 cells. Mutational analysis showed that Tyr174, Tyr183 and Tyr426 of chicken 3BP2 are the major phosphorylation sites by Syk and the SH2 domain of 3BP2 is critical for tyrosine phosphorylation. In addition, phosphorylation of Tyr426 is required for the inducible interaction with the SH2 domain of Vav3. Moreover, the expression of the mutant form of 3BP2 in which Tyr426 was substituted to Phe resulted in the reduction in BCR-mediated Rac1 activation, when compared with the case of wild-type. Altogether, these data suggest that 3BP2 is involved in the activation of Rac1 through the regulation of Vav3 by Syk-dependent phosphorylation of Tyr426 following BCR stimulation. & 2014 Elsevier Inc. All rights reserved.

Introduction Cross-linking of BCR triggers a number of biochemical events. Tyrosine phosphorylation of receptor subunits within the immunoreceptor tyrosine-based activation motif provides the docking site for the SH2 domain of nonreceptor type protein-tyrosine

kinase Syk to recruit Syk to the plasma membrane and to activate Syk. Activated Syk phosphorylates various downstream molecules, which in turn induce the signaling cascades to regulate B cell responses. Studies by using Syk-deficient DT40 cells have demonstrated that Syk is essential for BCR-mediated tyrosine phosphorylation of cellular proteins including phospholipase C

Abbreviations: 3BP2, c-Abl SH3 domain-binding protein-2; SH2, Src homology 2; BCR, B cell receptor; NFAT, nuclear factor of activated T cells; PLC, phospholipase C; SH3, Src homology 3; mAb, monoclonal antibody; BLNK, B cell linker protein n Corresponding author at: Division of Genome Science and Microbiology, Department of Pathological Sciences, School of Medicine, Faculty of Medical Sciences, University of Fukui, 23-3 Matsuoka-Shimoaizuki, Eiheiji, Fukui 910-1193, Japan. E-mail address: [email protected] (K. Sada).

0014-4827/$ - see front matter & 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yexcr.2013.12.026

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(PLC)-γ2, which are known to form signaling complex with the aid by adaptor proteins [1,2]. Adaptor proteins have tyrosine phosphorylation sites, multiple motif, and domains those allow targeting, assembling and regulating the other signaling molecules to orchestrate the immunoreceptor-mediated intracellular signaling. Adaptor protein 3BP2 was originally identified as a c-Abl–Src homology 3 (SH3) domain-binding protein of unknown function [3]. The predominant expression of 3BP2 mRNA was found in B cells, and the analysis of 3BP2-deficient mice demonstrated that 3BP2 is required for B cell proliferation and cell cycle progression [4,5]. It was demonstrated that 3BP2 interacts with Vav family proteins to regulate BCR-mediated Rac1 and NFAT activation [6]. 3BP2 was also identified as a Syk kinase-interacting protein by yeast twohybrid screening [7]. In the previous study, we demonstrated that Syk could phosphorylate Tyr174, Tyr183 and Tyr446 in mouse 3BP2 in COS cells [8]. Among these phosphorylation sites, Tyr183 is necessary for the direct interaction with PLC-γ2 and Vav1 in B cells. Phosphorylation of Tyr183 and the SH2 domain of 3BP2 play critical roles on the BCR-mediated NFAT activation in B cells. These findings demonstrated that adaptor protein 3BP2 connects Syk to the downstream effectors to positively regulate BCR signaling. Phosphorylation of Tyr174 and Tyr446 in 3BP2 is not required for BCR-mediated activation of NFAT in mammalian B cells [9]. Recently, the sequence of mRNA encoding for chicken 3BP2 was identified [10]. The analysis by Basic Local Alignment Search Tool (BLAST) algorithm [11] revealed that the amino acid sequence of chicken 3BP2 has 58% identity with mouse 3BP2, whereas it is almost identical between human and mouse (86%). Although the tyrosine residues corresponding to Tyr174, Tyr183 and Tyr446 of mouse 3BP2 were all conserved in chicken 3BP2, the amino acid sequence around the tyrosine phosphorylation sites are not completely identical. These findings led us to consider whether chicken 3BP2 is tyrosine phosphorylated particularly by Syk in response to BCR stimulation. In addition, the physiological roles of tyrosine phosphorylation of chicken 3BP2 in BCR signaling are remained to be determined. In this report, we have characterized BCR-mediated tyrosine phosphorylation of chicken 3BP2. DT40 cells have been utilized because of the merit of genetical analysis by the deletion of BCR signaling molecules. In addition, we addressed the requirement of phosphotyrosyl residues for the interaction with the other signaling molecules. Present study demonstrated that BCR stimulation induces Syk-dependent tyrosine phosphorylation of chicken 3BP2, and phosphorylation of Tyr426 in 3BP2 is required for the association with Vav3 to regulate the activation of Rac1.

Materials and methods Antibodies and cDNAs Anti-chicken IgM monoclonal antibody (mAb) (clone M4) was purchased from Southern Biotech (Birmingham, AL). Antiphosphotyrosine (4G10) and anti-glyceraldehyde-3-phosphate dehydrogenase mAbs were obtained from Millipore (Bedford, MA). Anti-HA mAb was from Covance (Princeton, NJ). Antichicken Vav3 rabbit polyclonal antibody was kindly provided by Dr. Tomohiro Kurosaki (Osaka University, Japan) [12]. Anti-FLAG

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mAb (clone M2) was from Sigma (St. Louis, MO). Anti-GST mAb was from Nacalai (Kyoto, Japan). Anti-Rac1 mAb was from BD Biosciences (San Jose, CA). The chicken 3BP2 and Vav3 cDNAs were obtained as follows. Total RNA from chicken DT40 cells was purified by using RNeasy mini kit (Qiagen, Valencia, CA) and the first strand cDNA was generated by superscript III (Life technologies, Carlsbad, CA) with random hexamer according to manufacture0 s instruction. The full-length cDNA encoding each protein were amplified by PCR using following primer sets: and 50 –AAG50 –AAGGATCCATGGCCTCAGAGGAGCAGGTCTG-30 0 0 GATCCTCACCTTGGTTTAGAGTAACCAT-3 for chicken 3BP2; 5 –GAATTCATGGAGCCGTGGAAGCAGTGCGG-30 and 50 –GAATTCTCATTCATC TTCTTCAACATAAG-30 for chicken Vav3. The PCR products were subcloned into pGEM-T easy vector (Promega, Madison, WI), and DNA sequences were verified. The cDNA fragment was inserted into pcDNA3.1(þ)-HA expression vector (3BP2) or pcDNA3.1(þ)-FLAG expression vector (Vav3) (kindly provided from Dr. Keiji Tanaka, Tokyo Metropolitan Institutes of Medical Science, Japan) to add epitope tag at the N-terminus of each protein. The cDNA fragment encoding FLAG-tagged Vav3 was transferred into pApuro vector (kindly provided from Dr. Tomohiro Kurosaki). We generated point mutations of Tyr174 to Phe (Y174F) in chicken 3BP2 with 2 primers 50 -CTCATCATTCAGCAGATAATGAAGATTTTGACCAGGAGGAAG-30 and 50 -CTTCCTCCTGGTCAAAATCTTCATTATCTGCTGAATGATGAG-30 , Tyr183 to Phe (Y183F) with 50 -GGAAGATGATGAGTCCTTCTTGCAACCGGATACTT-30 and 50 -AAGTATCCGGTTGCAAGAAGGACTCATCATCTTCC-30 , Tyr426 to Phe (Y426F) with 50 -AAACGATGATTCTGATGACGACTTTGAAAAAGTTGAGCTGC-30 and 50 -GCAGCTCAACTTTTTCAAAGTC 466 0 GTCATCAGAATCATCGTTT-3 , Arg to Lys (RK) with 50 -CAGAATGGAT TATACTGCATTAAGAACTCATCTACTAAGGCTGG-30 and 50 -CCAGCCTTAGTAGATGAGTTCTTAATGCAGTATAATCCATTCTG-30 by site-directed mutagenesis kit (Agilent technologies, Santa Clara, CA). The resulted point mutations were confirmed by the DNA sequencing. To generate the double mutant of Tyr183 and Tyr426 of chicken 3BP2 (DM), pcDNA3.1(þ)-HA chicken 3BP2 Y183F was digested with HindIII. The cDNA fragment contained Y183F mutation was inserted into the backbone of pcDNA3.1(þ)-HA chicken 3BP2 Y426F digested with HindIII.

Cell culture and transfection DT40 cells were maintained in RPMI 1640 medium supplemented with 100 units/ml of penicillin, 10% (v/v) heat-inactivated fetal calf serum and 1% chicken serum at 39.5 1C. Lyn-deficient (Lyn  ) and Syk-deficient (Syk  ) DT40 cells were from Dr. Tomohiro Kurosaki [1]. Abl-deficient (Abl  ) and Arg-deficient (Arg  ) DT40 cells were from Dr. Ken-ichi Yamamoto and Dr. Hiroko Shimizu (Kanazawa University, Japan) [13,14]. To express HA-tagged 3BP2 and/or FLAG-tagged Vav3 in DT40 cell lines, expression vectors were stably transfected into these cells. In brief, 30 μg of linealized expression constructs were transfected into 5  106 cells by electroporation (25 μF, 550 V) using GenePulser Xcell (Bio-Rad, Hercules, CA) as described [1]. Stably transfected cell lines were selected with 2 mg/ml active G418 (Nacalai) and/or 0.5 mg/ml puromycin (Nacalai). Cell lines were screened by the level of protein expression by the immunoblotting with anti-HA or antiFLAG mAb. Transient transfection of 2  105 COS-7 cells was performed by X-tremeGene9 DNA transfection reagent (Roche, Indianapolis, IN) according to the manufacturer0 s instruction.

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Cell activation, immunoprecipitation and immunoblotting DT40 cell lines were stimulated with 4 μg/ml anti-chicken IgM mAb for 5 min followed by washing twice with ice-cold PBS. To inhibit the kinase activity of Syk, R406 (Selleck Chemiclas, Houston, TX) was incubated with cells for 5 min prior to stimulation. For immunoprecipitation, cells were washed twice with icecold PBS, and solubilized in the lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 10 mM EDTA, 100 mM NaF, 1 mM Na3VO4, 1% Triton X-100, 1 mM PMSF and 2 μg/ml aprotinin). Precleared cell lysates were incubated with anti-HA mAb prebound to the beads (antiHA Affinity Matrix, Roche). After rotation for 2 h at 4 1C, the beads were washed 4 times with the lysis buffer. Immunoprecipitated proteins were eluted by heat treatment for 5 min at 100 1C with 2  sampling buffer, separated by SDS-PAGE and analyzed by the immunoblotting. Immunoblotting experiments were performed as described [15,16]. In brief, proteins were separated by SDS-PAGE and electronically transferred to polyvinylidene difluoride (PVDF) membrane (Millipore). Membranes were blocked with TBST (25 mM Tris, pH 8.0, 150 mM NaCl and 0.1% Tween 20) containing 5% milk for 1 h. The blots were reacted with the indicated primary antibodies for 1 h. After the extensive washing, the blots were reacted with horseradish peroxidase-conjugated secondary antibodies for 30 min. Proteins were visualized by the Enhanced Chemiluminescence (ECL) reagent (Western Lightning, Perkin Elmer Life Sciences, Boston, MA). All immunoblotting procedures were performed at room temperature.

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transferred to PVDF membrane. After blocking with 5% milk in TBST for 1 h at 4 1C, the membranes were incubated with 2.5 μg/ml of GST-Vav3-SH2 for 1 h at 4 1C. After the extensive washing, membranes were reacted with anti-GST mAb, followed by horseradish peroxidase-conjugated secondary antibody. Proteins were visualized by the ECL detection [9].

Rac1 activity assay The analysis of Rac1 activity in DT40 cells were performed as described [17]. In brief, DT40 cells stably expressing different kinds of 3BP2 were either unstimulated or stimulated with antichicken IgM mAb. Cells were then solubilized with Mg2þ binding buffer (1% NP-40, 50 mM Tris, pH7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, 1 mM PMSF and 2 μg/ml aprotinin). Precleared cell lysates were reacted with 20 μg of GST-human Pak1PBD (p21-binding domain in serine/threonine kinase Pak1) [17] prebound to glutathione Sepharose 4B beads for 90 min at 4 1C. The beads were washed 4 times with Mg2þ binding buffer. The binding proteins were eluted by heat treatment for 5 min at 100 1C with 2  sampling buffer, separated by SDS-PAGE and analyzed by immunoblotting with anti-Rac1 mAb.

Results Syk, but not Abl family of protein-tyrosine kinases, phosphorylates chicken 3BP2 after BCR stimulation

Pull-down assay The cDNA fragment encoding SH2 domain of chicken Vav3 (Asp665 to Glu767) was amplified by PCR using two primers, 50 -GGATCCGACTACTCGTCACAACTGTGGTTTGCAGGA-30 and 50 -CTC GAGTCATTCTTTGTACGGAAATTGAAGTGTAGTATC-30 , from pcDNA 3.1(þ)-FLAG-Vav3 as a template. Resulted PCR fragment was subcloned into pGEX4T.3 vector (GE Healthcare, Buckinghamshire, UK). GST-fusion proteins were expressed in bacteria and purified by glutathione Sepharose 4B beads (GE Healthcare). Preparation of GST-fusion proteins was confirmed by SDS-PAGE and Coomassie brilliant blue staining. DT40 cells stably expressing different kinds of 3BP2 were either unstimulated or stimulated with anti-chicken IgM mAb. COS-7 cells transiently expressing different kinds of 3BP2 were either unstimultated or stimulated with 100 μM pervanadate for 5 min. Cells were then washed twice with ice-cold PBS, and solubilized with the lysis buffer. Precleared cell lysates were reacted with 20 μg of GST fusion proteins prebound to glutathione Sepharose 4B beads for 90 min at 4 1C. The beads were washed 4 times with the lysis buffer. Proteins interacting with GST fusion proteins were eluted by heat treatment for 5 min at 100 1C with 2  sampling buffer, separated by SDS-PAGE and analyzed by the immunoblotting.

Far western analysis COS-7 cells expressing HA-3BP2 wild-type or HA-3BP2 Y426F were either unstimulated or stimulated with 100 μM pervanadate for 5 min. Cells were then washed with ice-cold PBS twice, and solubilized with the lysis buffer. Anti-HA immunoprecipitates from precleared lysate were separated by SDS-PAGE and

Previously, we reported that Syk is essential for BCR-mediated tyrosine phosphorylation of mouse 3BP2, and tyrosine phosphorylation of 3BP2 plays important roles on the association with PLCγ2 and Vav1 [9]. In addition to Syk, recently we found that 3BP2 is also tyrosine phosphorylated when it was coexpressed with Abl in COS-7 cells (data not shown). Therefore, we performed additional experiments to examine the possible involvement of Abl-family kinases for BCR-mediated tyrosine phosphorylation of 3BP2 using various DT40 cell lines each lacks different protein-tyrosine kinase by gene targeting. Each cell line stably expresses HAtagged chicken 3BP2. As shown in Fig. 1A, BCR-mediated tyrosine phosphorylation of chicken 3BP2 was reduced in Lyn-deficient cells, and was completely abrogated in Syk-deficient cells. In contrast, the deficiency of Abl or Arg caused no significant changes on the level of BCR-mediated tyrosine phosphorylation. This result conclusively demonstrated that Syk, but not Abl-family kinases, is critical for BCR-mediated tyrosine phosphorylation of chicken 3BP2. Recently, novel Syk inhibitors were highlighted in the clinical trial for the autoimmune diseases, and blockade of Syk function could be useful for the treatment of rheumatoid arthritis, idiopathic thrombocytopenic purpura, and systemic lupus erythematosus [18–20]. Therefore, next we examined the effect of Syk inhibitor in BCR signaling. As shown in Fig. 1B, BCR-mediated tyrosine phosphorylation of cellular proteins was inhibited by the pretreatment of cells with R406 in a dose response manner. Immunoprecipitation experiment revealed that the level of tyrosine phosphorylation of chicken 3BP2 was also dramatically reduced by the pretreatment of cells with R406 (Fig. 1C). Taken together, these results demonstrated that Syk is indispensable

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Fig. 1 – Syk, but not Abl family of protein-tyrosine kinases, is indispensable for tyrosine phosphorylation of chicken 3BP2 following BCR stimulation. (A) Analysis of tyrosine phosphorylation of HA-chicken 3BP2 in DT40 wild-type (WT), Lyn-deficient (Lyn  ), Syk-deficient (Syk  ), Abl-deficient (Abl  ), and Arg-deficient cells (Arg  ). Cells were stimulated without () or with (þ) anti-chicken IgM mAb for 5 min. Anti-HA immunoprecipitates were separated by SDS-PAGE and analyzed by immunoblotting with anti-phosphotyrosine mAb (pTyr) (top panel). The membrane was stripped and reprobed with anti-HA mAb (bottom panel). (B and C) The effect of Syk inhibitor on BCR-mediated tyrosine phosphorylation of cellular proteins. DT40 cells were preincubated with the indicated concentration of R406 for 5 min, and then stimulated with anti-IgM mAb for 5 min. Detergent-soluble lysates (B) or anti-HA immunoprecipitates (IP) (C) were separated by SDS-PAGE and analyzed by immunoblotting with antiphosphotyrosine mAb (pTyr) (top panel). The membrane was stripped and reprobed with anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mAb (B) or anti-HA mAb (C) (bottom panel). Molecular size markers are indicated at the left in kDa. The results were representative of 3 independent experiments.

for tyrosine phosphorylation of chicken 3BP2 in BCR-stimulated DT40 cells.

Identification of Vav3 as a tyrosine phosphorylated binding partner of chicken 3BP2 in DT40 cells To characterize tyrosine phosphorylation of 3BP2 in B cells, we utilized chicken DT40 wild-type cells stably expressing HA-tagged chicken 3BP2. Immunoprecipitation experiment demonstrated that engagement of BCR induced tyrosine phosphorylation of 3BP2 (Fig. 2A, top panel, arrowhead) with some additional tyrosine phosphorylated proteins associated with 3BP2. Benefit of this experiment using chicken 3BP2 is the efficiency of immunoprecipitation, because we have been faced to the difficulty of the immunoprecipitation of HA-tagged mouse 3BP2 due

to the limited amount of polyclonal antibodies or lower yield of immunoprecipitation by using anti-HA mAb (Fig. 2A, middle panel, data not shown). Among those 3BP2-interacting proteins, protein whose molecular weight was around 100 kDa was highly tyrosine phosphorylated in response to the stimulation with anti-IgM (top panel, arrow). The candidate of this molecule is Vav family protein which is known to be tyrosine phosphorylated in response to BCR stimulation. In fact, Vav1, Vav2, and Vav3 were reported to form complex with 3BP2 in human B cell line through multiple mechanisms [6]. In addition, we recently showed that mouse 3BP2 forms inducible complex with Vav1 in response to BCR stimulation through the inducible interaction of phospho-Tyr183 in mouse 3BP2 and the SH2 domain of Vav1 [9]. Because Vav3 is the predominant member of Vav family proteins expressed in DT40 cells [12,21], we next examined whether

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Fig. 2 – Identification of Vav3 as a 3BP2-interacting protein. (A) Identification of 3BP2-interacting proteins in DT40 cells. DT40 cells expressing HA-tagged 3BP2 were stimulated without () or with (þ) anti-IgM mAb for 5 min. Cell lysates were subjected to the immunoprecipitation with anti-HA mAb. Detergent-soluble lysates (DSL) and anti-HA immunoprecipitates (IP) were separated by SDS-PAGE and analyzed by immunoblotting with anti-phosphotyrosine mAb (pTyr), anti-HA mAb, and anti-Vav3 polyclonal antibody. (B) The interaction of Vav3 with 3BP2. DT40 cells expressing FLAG-tagged Vav3 (FLAG-Vav3), HA-tagged 3BP2 (HA-3BP2) or both of them (FLAG-Vav3þHA-3BP2) were stimulated without ( ) or with (þ) anti-IgM mAb for 5 min. Cells were then solubilized with the lysis buffer, and subjected to immunoprecipitation with anti-HA mAb. Anti-HA immunoprecipitates were separated by SDS-PAGE and analyzed by immunoblotting with anti-FLAG and anti-HA mAbs. Molecular size markers are indicated at the left in kDa. The results were representative of 3 independent experiments.

Vav3 could interact with chicken 3BP2 in response to BCR stimulation. Immunoblotting experiment revealed that BCR stimulation causes inducible complex formation of HA-tagged 3BP2 with endogenous Vav3 (Fig. 2A, lower panel). To further evaluate the association of Vav3 with 3BP2 by using the different antibodies, we generated DT40 cell lines stably expressing HA-tagged 3BP2 and/or FLAG-tagged Vav3. As shown in Fig. 2B, BCR stimulation strongly induced the complex formation of HA-tagged 3BP2 with FLAGtagged Vav3 as in the case of endogenous Vav3. These results demonstrated that Vav3 is one of the tyrosine phosphorylated inducible binding partners of 3BP2 in B cells.

Characterization of tyrosine phosphorylation of chicken 3BP2 in DT40 cells We previously demonstrated that Tyr174, Tyr183 and Tyr446 in mouse 3BP2 were predominantly phosphorylated by Syk in COS-7 cells [8]. Expression of 3BP2 with the mutation of each tyrosine residue resulted in the reduced tyrosine phosphorylation of 3BP2 after BCR stimulation [9]. Because the amino acid sequences of chicken 3BP2 corresponding to the tyrosine phosphorylation sites of mouse protein are not completely identical (Fig. 3A), it is necessary to determine which tyrosine residues in chicken 3BP2 are phosphorylated in response to BCR stimulation. To address

this issue, we made various mutant forms of chicken 3BP2 whose tyrosine phosphorylation sites were substituted for phenylalanine. Unlike mouse 3BP2, Tyr174 of chicken 3BP2 is not located within the Asp–Tyr–Glu sequence which is preferred to be phosphorylated by Syk as a substrate [8,22]. However, BCRmediated tyrosine phosphorylation of 3BP2 was partially reduced by the point mutation of Tyr174 to Phe (Y174F), suggesting that Tyr174 is one of tyrosine phosphorylation sites in BCR-activated DT40 cells (Fig. 3B). In addition, the point mutation of Tyr183 to Phe or Tyr426 to Phe (Y183F and Y426F, respectively) resulted in the suppression of BCR-mediated tyrosine phosphorylation of chicken 3BP2. Furthermore, tyrosine phosphorylation was dramatically reduced to almost undetectable level by the double mutation of both Tyr183 and Tyr426 to Phe (DM) (Fig. 3C). Taken together, Tyr174, Tyr183 and Tyr426 were the major phosphorylation sites of chicken 3BP2 in BCR-stimulated DT40 cells. Our previous study demonstrated that the SH2 domain is required for the BCR-mediated tyrosine phosphorylation of mouse 3BP2 [9]. Similar to this report, substitution of Arg466 to Lys (RK) in the SH2 domain of chicken 3BP2 resulted in the abrogation of BCR-mediated tyrosine phosphorylation, presumably due to the loss of the interaction with Syk (Fig. 3C) [6,7]. This result clearly showed that the requirement of the SH2 domain for tyrosine phosphorylation of 3BP2 by Syk is inherited beyond species.

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Phosphorylation of Tyr426 of chicken 3BP2 is required for the direct binding to Vav3 To examine whether BCR-mediated tyrosine phosphorylation of 3BP2 is required for the binding to Vav3, immunoprecipitation experiments were carried out. As shown in Fig. 4A, the point mutation of Tyr183 caused reduction in the binding of chicken 3BP2 with endogenous Vav3. This binding was completely abrogated by the double mutation of Tyr183 and Tyr426, or the point mutation of Arg466. To further analyze molecular mechanisms underlying the interaction of chicken 3BP2 with Vav3, pull-down experiments were carried out. As shown in Fig. 4B, incubation of GST-Vav3-SH2 domain with detergent-soluble lysates resulted in the binding to HA-tagged chicken 3BP2 wild-type in BCR stimulation-dependent manner. In contrast, the interaction was dramatically diminished by the point mutation of Tyr426 or the double mutation of Tyr183 and Tyr426. It is important to note that the point mutation of Arg466 in the SH2 domain caused dramatic reduction in the BCR-mediated binding to Vav3. Similar studies were examined by using COS-7 cells transiently expressing HA-tagged 3BP2. As shown in Fig. 4C,

Fig. 3 – Analysis of BCR-mediated tyrosine phosphorylation of chicken 3BP2. (A) The structure of chicken 3BP2 used in this study and comparison of the amino acid sequence around possible tyrosine phosphorylation sites among chicken, mouse and human 3BP2. The HA tag (HA), N-terminal pleckstrin homology domain (PH), 3 proline-rich regions (Pro-rich), and SH2 domain (SH2) and a critical residue for the function of SH2 domain (Arg466) were represented. The sequences around the tyrosine phosphorylation sites in various species are shown at the bottom. (B and C) Analysis of BCR-mediated tyrosine phosphorylation of chicken 3BP2. DT40 cells stably expressing HA-3BP2 wild-type (WT) and HA-3BP2-Y174F (Y174F) (B), HA3BP2-Y183F (Y183F), HA-3BP2-Y426F (Y426F), HA-3BP2-Y183F/ Y426F (DM) and HA-3BP2-R466K (RK) (C) were stimulated without () or with (þ) anti-IgM mAb for 5 min. Anti-HA immunoprecipitates were separated by SDS-PAGE and analyzed by immunoblotting with anti-phosphotyrosine mAb (pTyr) (top panel). The membrane was stripped and reprobed with anti-HA mAb (bottom panel). Molecular size markers are indicated at the left in kDa. The results were representative of 3 independent experiments.

Fig. 4 – Analysis of the mechanism of interaction of 3BP2 with Vav3. (A) Interaction of Vav3 with 3BP2 expressed in DT40 cells. DT40 cells expressing different kinds of HA-chicken 3BP2 were stimulated without ( ) or with (þ) anti-IgM mAb for 5 min. AntiHA immunoprecipitates were separated by SDS-PAGE and analyzed by immunoblotting with anti-Vav3 polyclonal antibody (top panel). The membrane was stripped and reprobed with antiHA mAb (bottom panel). (B and C) Pull-down assay. (B) Interaction of the SH2 domain of Vav3 with 3BP2 expressed in DT40 cells. DT40 cells expressing different kinds of HA-chicken 3BP2 were stimulated without () or with (þ) anti-IgM mAb for 5 min. (C) Interaction of the SH2 domain of Vav3 with 3BP2 expressed in COS-7 cells. COS-7 cells expressing different kinds of HA-3BP2 were stimulated without () or with (þ) pervanadate (PV) for 5 min. Detergent-soluble lysates were reacted with GST-Vav3-SH2 prebound to glutathione Sepharose 4B beads. The interacted proteins (pull-down) and the source of precipitation (input) were separated by SDS-PAGE and analyzed by immunoblotting with anti-HA mAb. The preparation of GST-Vav3-SH2 was confirmed by Coomassie brilliant blue (CBB) staining. (D) Direct interaction of tyrosine-phosphorylated 3BP2 with the SH2 domain of Vav3. Detergent-soluble lysates were obtained from COS-7 cells expressing HA-chicken 3BP2 wild-type (WT) or HA-chicken 3BP2 Y426F (Y426F) stimulated without () or with (þ) pervanadate (PV) for 5 min. Anti-HA immunoprecipitates were separated by SDS-PAGE and subjected to far western analysis with GST-Vav3SH2 as a probe (top panel). The membrane was stripped and then reprobed with anti-phosphotyrosine (pTyr) mAb (middle panel). The amount of immunoprecipitated HA-chicken 3BP2 were analyzed by immunoblotting with anti-HA mAb (bottom panel). Molecular size markers are indicated at the left in kDa. The results were representative of 3 independent experiments.

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the treatment of cells with pervanadate enhanced the interaction of HA-tagged chicken 3BP2 wild-type with GST-Vav3-SH2, whereas the interaction was completely abrogated by the point mutation of Tyr426 or the double mutation of Tyr183 and Tyr426. In contrast to the case of BCR stimulation, the point mutation of Arg466 caused no effect on the binding to the SH2 domain of Vav3 in this experimental condition. These results highlighted that phosphorylation of Tyr426 in BCR signaling is necessary for the interaction of 3BP2 with Vav3. 3BP2-RK has a capacity to interact with Vav3 when it is tyrosine phosphorylated. Taken together, these results demonstrated that BCR-mediated tyrosine phosphorylation of chicken 3BP2, specifically the phosphorylation of Tyr426, plays critical roles on the interaction with the SH2 domain of Vav3. Next, we examined whether chicken 3BP2 directly interacts with the SH2 domain of Vav3. As shown in Fig. 4D, far western analysis demonstrated that GST-Vav3-SH2 domain directly bound to HA-tagged chicken 3BP2 wild-type, but not 3BP2-Y426F immunoprecipitated from pervanadate-treated COS-7 cells, suggesting that phosphorylated Tyr426 in chicken 3BP2 is directly recognized by the SH2 domain of Vav3.

Phosphorylation of Tyr426 of chicken 3BP2 positively regulates BCR-mediated activation of Rac1 It was reported that Vav3 plays important roles on BCR-mediated activation of Rac1 in DT40 cells [12,23]. Therefore, we finally examined whether the interaction of Vav3 with phospho-Tyr426 in chicken 3BP2 is involved in the regulation of BCR-mediated activation of Rac1. As shown in Fig. 5, Rac1 activity in DT40 cells expressing HA-tagged chicken 3BP2 wild-type was increased after BCR stimulation, whereas the expression of HA-tagged chicken 3BP2-Y426F resulted in the dramatic suppression in Rac1 activation after BCR stimulation, suggesting that tyrosine phosphorylation of Tyr426 in chicken 3BP2 positively regulates the BCRmediated activation of Rac1.

Fig. 5 – Analysis of BCR-mediated activation of Rac1. DT40 cells expressing HA-chicken 3BP2 wild-type (WT) or HA-chicken 3BP2 Y426F (Y426F) were stimulated with anti-IgM mAb for the indicated periods of time. Cells were solubilized in Mg2þ binding buffer and detergent-soluble lysates were reacted with GST-Pak1-PBD prebound to glutathione Sepharose 4B beads. The interacted proteins (pull-down) and the source of precipitation (input) were separated by SDS-PAGE and analyzed by immunoblotting with anti-Rac1 mAb (top and middle panels). The preparation of GST-Pak1-PBD was confirmed by Coomassie brilliant blue (CBB) staining (bottom panel). Molecular size markers are indicated at the left in kDa. The results were representative of 3 independent experiments.

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Discussion 3BP2 was originally isolated as a protein-tyrosine kinase Abl binding protein [3]. c-Abl could phosphorylate 3BP2 and coexpression of 3BP2 increased phosphorylation of the kinase domain of c-Abl in COS cells (data not shown). This suggests that 3BP2 is a substrate of c-Abl and 3BP2 regulates the kinase activity of upstream kinase c-Abl. Our genetical and pharmacological experiments clearly demonstrated that Syk, but not Abl-family of kinase, is critical for BCR-mediated tyrosine phosphorylation of 3BP2 in B cells (Fig. 1). Considering the structural similarity between c-Abl and Src-family kinases, it is possible that phosphorylated 3BP2 acts as a ligand of c-Abl to induce the partially open structure, as predicted by our initial study of 3BP2 and Lyn kinase [8]. In fact, the interaction of 3BP2 enhances the kinase activity of c-Abl in osteoblast [24], supporting our hypothesis that 3BP2 is the ligand of protein-tyrosine kinase to upregulate the kinase activity. 3BP2 is one of the adaptor molecules to connect proximal protein-tyrosine kinases to Vav family proteins. There are 3 possible mechanisms of 3BP2–Vav interactions. Foucault et al. demonstrated that 3BP2 constitutively interacts with Vav proteins, and BCR-stimulation further enhances the interaction of 3BP2 with Vav1 [6]. Deletion mapping analysis using transient expression system in COS-1 cells showed that one of the proline-rich regions of mouse 3BP2 (amino acids 201–240) is required to associate with Vav1 and Vav2 [6]. We could also detect some binding of 3BP2 with Vav3 in resting condition (Fig. 2). Thus, the constitutive binding with Vav3 through its SH3 domain might influence the function of 3BP2 in BCR-stimulated B cells. Secondary, we have showed that BCR stimulation induces Syk-dependent tyrosine phosphorylation of 3BP2, and phosphorylation of Tyr183 (mouse 3BP2) or Tyr426 (chicken 3BP2) are required for the inducible interaction of 3BP2 with the SH2 domain of Vav1 and Vav3 in response to BCR stimulation, respectively (Fig. 4) [8]. Tyr183 in mouse 3BP2 is located in the part of consensus binding motif (Tyr– Leu–Glu–Pro) of the SH2 domain of Vav1 [25]. However, this motif is not completely conserved in chicken 3BP2 (Tyr–Leu–Gln–Pro, Fig. 3A). This may be the reason why phosphorylation of Tyr183 in chicken 3BP2 is not critical for the binding with Vav3 (Fig. 4). In addition, there is a possibility that the SH2 domain of chicken Vav3 has different binding specificity when it is compared with that of Vav1 or Vav2. Tertiary, it is also possible that tyrosine phosphorylated Vav family proteins directly interact with the SH2 domain of 3BP2. Indeed, Vav1 was co-immunoprecipitated with the SH2 domain of 3BP2 in B cells [6]. Vav proteins are rapidly tyrosine phosphorylated in response to BCR stimulation [26], and contain at least 3 conserved tyrosine residues at N-terminus [27]. Of these, Tyr174 (corresponding to Tyr173 of Vav3) is thought to be a key regulatory site for the structural changes and regulation of guanine nucleotide exchange activity towards Rho family GTPases [28,29]. Based on these observations, we examined the roles of Tyr173 of Vav3 on the binding to chicken 3BP2. In pervanadate-treated COS-7 cell, Vav3 was capable to form complex with 3BP2, whereas the phosphorylation of Tyr173 had almost no effect on the binding to 3BP2 (supplemental Fig. 1). Although the experimental condition is largely nonphysiological when compared with anti-IgM stimulation, this result suggested that the binding with the SH2 domain of 3BP2 might not be crucial for the guanine nucleotide exchange activity of Vav3.

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To understand molecular mechanisms underlying the SH2 domain-mediated tyrosine phosphorylation of 3BP2, we explored the interacting molecules with the SH2 domain of 3BP2 in BCRactivated DT40 cells. Pull-down experiment showed that some tyrosine-phosphorylated proteins forms protein complex with the SH2 domain of chicken 3BP2 (supplemental Fig. 2). Proteomics analysis further demonstrated that one of the 3BP2-interacting protein nucleolin [30] was detected in the band whose molecular weight was around 100 kDa. Nucleolin is known to be tyrosine phosphorylated in response to the activation of complement component receptor 2, and forms protein complex with the SH2 domain of 3BP2 in human B cell line [30]. However, we could not detect any tyrosine phosphorylation of chicken nucleolin even in pervanadate-treated COS-7 cells (supplemental Fig. 3) suggesting that chicken nucleolin was indirectly bound to 3BP2. Therefore, it seems likely that the binding of chicken nucleolin is independent on protein-tyrosine phosphorylation induced by BCR stimulation. It was shown that Rac1 is required for the activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase in BCR-stimulated DT40 B cells [31]. BCR-mediated activation of Rac1 is remarkably diminished in Vav3-deficient DT40 cells [12], indicating that the regulation of Vav3 is critical for Rac1 activation in BCR-activated DT40 cells. Regarding the activation of Vav family proteins, the translocation of Vav family proteins to plasma membrane is thought to play important roles on the guanine nucleotide exchange activity [27,32]. In fact, it was shown that adaptor protein B cell linker protein (BLNK) associates with the SH2 domain of Vav proteins in response to BCR stimulation [33] and regulates the translocation of Vav3 into membrane raft following BCR stimulation, which is critical for the optimal activation of Rac1 [23]. Previously, we found that the SH2 domain of 3BP2 directly interacts with BLNK in response to BCR stimulation [9]. Therefore, BCR stimulation could induce translocation of 3BP2 into plasma membrane by interacting with tyrosinephosphorylated BLNK. The translocation of 3BP2 could facilitate that of Vav3 by providing phosphorylated Tyr426 as the docking site. This might be one possible reason that BCR-mediated activation of Rac1 was reduced in DT40 cells expressing 3BP2Y426F, which lacks the interaction site with Vav3 (Fig. 5).

Conclusions Our present studies clearly demonstrated that 3BP2 is the substrate of Syk to connect Syk to Vav family proteins in BCR signaling pathways. 3BP2 is tyrosine phosphorylated in response to BCR stimulation by Syk, but not Abl family protein-tyrosine kinases. Tyr183 and Tyr426 in chicken 3BP2 are identified as major phosphorylation site by Syk. Among them, phosphorylation of Tyr426 plays important roles for the interaction with the SH2 domain of Vav3 and BCR-mediated activation of Rac1. These observations could provide further insights into the roles of Vav family proteins on BCR-stimulated B cell function.

Acknowledgments We are grateful to Dr. Tomohiro Kurosaki, Dr. Keiji Tanaka, Dr. Ken-ichi Yamamoto, and Dr. Hiroko Shimizu for providing the reagents. We also thank Ms. Satomi Yamamoto, Ms. Kuniyo Miyagoshi, and Life

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Science Research Laboratory, University of Fukui, for the helpful assistance. This study was supported in part by the research grants from the Research and Education Program for Life Science, University of Fukui (to K.C.), the research grants from the University of Fukui (to K.C.), and the Grant-in-Aids from the Japan Society for the Promotion of Science and the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Appendix A.

Supplementary materials

Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.yexcr.2013.12.026.

references [1] M. Takata, H. Sabe, A. Hata, T. Inazu, Y. Homma, T. Nukada, H. Yamamura, T. Kurosaki, Tyrosine kinases Lyn and Syk regulate B cell receptor-coupled Ca2þ mobilization through distinct pathways, EMBO J. 13 (1994) 1341–1349. [2] T. Kurosaki, S.A. Johnson, L. Pao, K. Sada, H. Yamamura, J.C. Cambier, Role of the Syk autophosphorylation site and SH2 domains in B cell antigen receptor signaling, J. Exp. Med. 182 (1995) 1815–1823. [3] R. Ren, B.J. Mayer, P. Cicchetti, D. Baltimore, Identification of a ten-amino acid proline-rich SH3 binding site, Science 259 (1993) 1157–1161. [4] M.A. de la Fuente, L. Kumar, B. Lu, R.S. Geha, 3BP2 deficiency impairs the response of B cells, but not T cells, to antigen receptor ligation, Mol. Cell Biol. 26 (2006) 5214–5225. [5] G. Chen, I.D. Dimitriou, J. La Rose, S. Ilangumaran, W.C. Yeh, G. Doody, M. Turner, J. Gommerman, R. Rottapel, The 3BP2 adaptor protein is required for optimal B-cell activation and thymus-independent type 2 humoral response, Mol. Cell Biol. 27 (2007) 3109–3122. [6] I. Foucault, S. Le Bras, C. Charvet, C. Moon, A. Altman, M. Deckert, The adaptor protein 3BP2 associates with VAV guanine nucleotide exchange factors to regulate NFAT activation by the B-cell antigen receptor, Blood 105 (2005) 1106–1113. [7] M. Deckert, S. Tartare-Deckert, J. Hernandez, R. Rottapel, A. Altman, Adaptor function for the Syk kinases-interacting protein 3BP2 in IL-2 gene activation, Immunity 9 (1998) 595– 605. [8] K. Maeno, K. Sada, S. Kyo, S.M. Miah, K. Kawauchi-Kamata, X. Qu, Y. Shi, H. Yamamura, Adaptor protein 3BP2 is a potential ligand of Src homology 2 and 3 domains of Lyn protein-tyrosine kinase, J. Biol. Chem. 278 (2003) 24912–24920. [9] U. Shukla, T. Hatani, K. Nakashima, K. Ogi, K. Sada, Tyrosine phosphorylation of 3BP2 regulates B cell receptor-mediated activation of NFAT, J. Biol. Chem. 284 (2009) 33719–33728. [10] R.B. Caldwell, A.M. Kierzek, H. Arakawa, Y. Bezzubov, J. Zaim, P. Fiedler, S. Kutter, A. Blagodatski, D. Kostovska, M. Koter, J. Plachy, P. Carninci, Y. Hayashizaki, J.M. Buerstedde, Full-length cDNAs from chicken bursal lymphocytes to facilitate gene function analysis, Genome Biol. (2005), http://dxdoi.org/10.1186/gb2004-6-1-r6 (R6). [11] S.F. Altschul, W. Gish, W. Miller, E.W. Myers, D.J. Lipman, Basic local alignment search tool, J. Mol. Biol. 215 (1990) 403–410. [12] K. Inabe, M. Ishiai, A.M. Scharenberg, N. Freshney, J. Downward, T. Kurosaki, Vav3 modulates B cell receptor responses by regulating phosphoinositide 3-kinase activation, J. Exp. Med. 195 (2002) 189–200. [13] N. Takao, R. Mori, H. Kato, A. Shinohara, K. Yamamoto, c-Abl tyrosine kinase is not essential for ataxia telangiectasia mutated

EX P ER I ME NTAL C E LL RE S E ARCH

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

functions in chromosomal maintenance, J. Biol. Chem. 275 (2000) 725–728. Y. Li, H. Shimizu, S.L. Xiang, Y. Maru, N. Takao, K. Yamamoto, Arg tyrosine kinase is involved in homologous recombinational DNA repair, Biochem. Biophys. Res. Commun. 299 (2002) 697–702. K. Ogi, K. Nakashima, K. Chihara, K. Takeuchi, T. Horiguchi, S. Fujieda, K. Sada, Enhancement of B-cell receptor signaling by a point mutation of adaptor protein 3BP2 identified in human inherited disease cherubism, Genes Cells 16 (2011) 951–960. K. Chihara, K. Nakashima, K. Takeuchi, K. Sada, Association of 3BP2 with SHP-1 regulates SHP-1-mediated production of TNFalpha in RBL-2H3 cells, Genes Cells 16 (2011) 1133–1145. S.M. Miah, T. Hatani, X. Qu, H. Yamamura, K. Sada, Point mutations of 3BP2 identified in human-inherited disease cherubism result in the loss of function, Genes Cells 9 (2004) 993– 1004. A. Podolanczuk, A.H. Lazarus, A.R. Crow, E. Grossbard, J.B. Bussel, Of mice and men: an open-label pilot study for treatment of immune thrombocytopenic purpura by an inhibitor of Syk, Blood 113 (2009) 3154–3160. M.E. Weinblatt, A. Kavanaugh, M.C. Genovese, T.K. Musser, E.B. Grossbard, D.B. Magilavy, An oral spleen tyrosine kinase (Syk) inhibitor for rheumatoid arthritis, N. Engl. J. Med. 363 (2010) 1303–1312. S. Iwata, K. Yamaoka, H. Niiro, K. Nakano, S.P. Wang, K. Akashi, Y. Tanaka, Amplification of Toll-like receptor-mediated signaling through spleen tyrosine kinase in human B-cell activation, J. Allergy Clin. Immunol. 129 (2012) 1594–1601. M. Weber, B. Treanor, D. Depoil, H. Shinohara, N.E. Harwood, M. Hikida, T. Kurosaki, F.D. Batista, Phospholipase C-gamma2 and Vav cooperate within signaling microclusters to propagate B cell spreading in response to membrane-bound antigen, J. Exp. Med. 205 (2008) 853–868. R. Schmitz, G. Baumann, H. Gram, Catalytic specificity of phosphotyrosine kinases Blk, Lyn, c-Src and Syk as assessed by phage display, J. Mol. Biol. 260 (1996) 664–677. S. Johmura, M. Oh-hora, K. Inabe, Y. Nishikawa, K. Hayashi, E. Vigorito, D. Kitamura, M. Turner, K. Shingu, M. Hikida, T. Kurosaki, Regulation of Vav localization in membrane rafts by adaptor molecules Grb2 and BLNK, Immunity 18 (2003) 777–787. N. Levaot, P.D. Simoncic, I.D. Dimitriou, A. Scotter, J. La Rose, A.H. Ng, T.L. Willett, C.J. Wang, S. Janmohamed, M. Grynpas, E.

3 22 (2 014 ) 99 – 107

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

107

Reichenberger, R. Rottapel, 3BP2-deficient mice are osteoporotic with impaired osteoblast and osteoclast functions, J. Clin. Invest. 121 (2011) 3244–3257. Z. Songyang, S.E. Shoelson, J. McGlade, P. Olivier, T. Pawson, X.R. Bustelo, M. Barbacid, H. Sabe, H. Hanafusa, T. Yi, Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav, Mol. Cell Biol. 14 (1994) 2777–2785. X.R. Bustelo, M. Barbacid, Tyrosine phosphorylation of the vav proto-oncogene product in activated B cells, Science 256 (1992) 1196–1199. S. Katzav, Vav1: a hematopoietic signal transduction molecule involved in human malignancies, Int. J. Biochem. Cell Biol. 41 (2009) 1245–1248. B. Aghazadeh, W.E. Lowry, X.Y. Huang, M.K. Rosen, Structural basis for relief of autoinhibition of the Dbl homology domain of proto-oncogene Vav by tyrosine phosphorylation, Cell 102 (2000) 625–633. A.V. Miletic, K. Sakata-Sogawa, M. Hiroshima, M.J. Hamann, T.S. Gomez, N. Ota, T. Kloeppel, O. Kanagawa, M. Tokunaga, D.D. Billadeau, W. Swat, Vav1 acidic region tyrosine 174 is required for the formation of T cell receptor-induced microclusters and is essential in T cell development and activation, J. Biol. Chem. 281 (2006) 38257–38265. M. Barel, M. Le Romancer, R. Frade, 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, J. Immunol. 166 (2001) 3167–3173. A. Hashimoto, H. Okada, A. Jiang, M. Kurosaki, S. Greenberg, E.A. Clark, T. Kurosaki, Involvement of guanosine triphosphatases and phospholipase C-gamma2 in extracellular signal-regulated kinase, c-Jun NH2-terminal kinase, and p38 mitogen-activated protein kinase activation by the B cell antigen receptor, J. Exp. Med. 188 (1998) 1287–1295. C. Charvet, A.J. Canonigo, D.D. Billadeau, A. Altman, Membrane localization and function of Vav3 in T cells depend on its association with the adaptor SLP-76, J. Biol. Chem. 280 (2005) 15289–15299. C. Fu, C.W. Turck, T. Kurosaki, A.C. Chan, BLNK: a central linker protein in B cell activation, Immunity 9 (1998) 93–103.