Skip to main content
NCBI home page
Genes & Development logoLink to Genes & Development
. 2000 Jan 1;14(1):11–16.

Role of the rasGAP-associated docking protein p62dok in negative regulation of B cell receptor-mediated signaling

Yuji Yamanashi 1,2, Toshiki Tamura 3, Toshihide Kanamori 1, Hidehiro Yamane 3, Hideo Nariuchi 3, Tadashi Yamamoto 1, David Baltimore 4
PMCID: PMC316343  PMID: 10640270

Abstract

Antigenic stimulation of the B-cell receptor (BCR) is a central event in the immune response. In contrast, antigen bound to IgG negatively regulates signals from the BCR by cross-linking it to the inhibitory receptor FcγRIIB. Here we show that upon cross-linking of BCR or BCR with FcγRIIB, the rasGAP-associated protein p62dok is prominently tyrosine phosphorylated in a Lyn-dependent manner. Inactivation of the dok gene by homologous recombination has shown that upon BCR cross-linking, p62dok suppresses MAP kinase and is indispensable for FcγRIIB-mediated negative regulation of cell proliferation. We propose that p62dok, a downstream target of many PTKs, plays a negative role in various signaling situations.

Keywords: Dok, Lyn, B cell receptor, FcγRIIB, tyrosine kinase, MAP kinase

The p120 rasGAP-associated p62 protein was originally defined as a tyrosine-phosphorylated 62-kD protein coimmunoprecipitated with p120 rasGAP, a negative regulator of p21ras (Ellis et al. 1990; White and Yenush 1998). A p62 is tyrosine phosphorylated downstream of a wide range of protein-tyrosine kinases (PTKs), such as receptors for platelet-derived growth factor (PDGF), colony stimulating factor-1 (CSF-1), insulin, insulin-like growth factor (IGF), Kit ligand, and vascular endothelial growth factor (VEGF) or oncoproteins like Bcr–Abl, a causative factor of chronic myelogenous leukemia(CML)(Carpino et al. 1997; Yamanashi and Baltimore 1997; White and Yenush 1998; Noguchi et al. 1999). A p62 is also tyrosine phosphorylated downstream of receptors coupled with cytoplasmic PTKs. We and others have independently purified a p62 from v-Abl- or Bcr–Abl-transformed hematopoietic cells and identified it as a docking protein (Dok) (Carpino et al. 1997; Yamanashi and Baltimore 1997). p62dok has overall structural similarity to insulin receptor substrate-1 (IRS-1), having both pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains in its amino-terminal portion, followed by many binding sites for SH2 domains (Carpino et al. 1997; Yamanashi and Baltimore 1997; White and Yenush 1998). IRS-1 binds to activated and autophosphorylated insulin receptor through its amino-terminal PH and PTB domains and is subsequently tyrosine phosphorylated, allowing its carboxy-terminal region to act as docking sites for SH2-containing signaling molecules such as PI-3 kinase (White and Yenush 1998). p62dok appears to recruit a set of SH2-containing proteins different from those recruited by IRS-1 (Carpino et al. 1997; Yamanashi and Baltimore 1997; Noguchi et al. 1999); when tyrosine phosphorylated, it binds to rasGAP, Nck, and probably other SH2-containing proteins. Although it was recently found that ectopic overexpression of p62dok in a cell line up-regulates motility of the cells (Noguchi et al. 1999), the physiological function of p62dok has yet to be revealed. We have therefore generated a null mutation in the mouse dok gene and have studied the physiological role of p62dok in primary B cells.

Results and Discussion

To address the role of p62dok, we have generated a null mutation in the dok gene, in which the entire coding sequence of the gene was deleted by homologous recombination (Fig. 1A). dok−/− mice did not express the dok protein (Fig. 1B,C). Such mice were fertile and have lived at least 1 year with normal populations of hematopoietic cells. In the equivalent cells of normal mice, however, the dok gene is expressed at relatively high level (data not shown). It might be that dok-related gene(s) such as dok-2/frip/dok-R can compensate for the inactivated dok gene (Di Cristofano et al. 1998; Jones and Dumont 1998; Nelms et al. 1998; Lock et al. 1999). p56dok-2 is closely related to p62dok and also binds to rasGAP and Nck when tyrosine phosphorylated. However, dok-2 gene expression is not normally detectable in B cells (Nelms et al. 1998).

Figure 1.

Figure 1

Figure 1

Figure 1

Open in a new tab

Generation of dok-null-mutant mice. (A) Restriction map of wild-type allele, targeting vector, and null allele of the dok gene. Positions of exons covering the entire coding region and of the initiation and stop codons are indicated by shaded boxes and arrows, respectively. The dok probe used for Southern blotting, as well as for thymidine kinase (TK) and neomycin-resistant (neo) genes are indicated. (Stu) StuI; (Hd) HindIII; (Bam) BamHI. (B) Southern blot analysis of genomic DNA isolated from dok+/+, dok+/−, and dok−/− mice. BamHI-digested genomic DNA was hybridized with the dok probe. Wild-type (5.2-kb) and recombinant (4.2-kb) bands are indicated. (C) Absence of Dok protein in dok−/− mice. NP-40-soluble proteins (50 μg) from dok+/+ and dok−/− spleens are subjected to immunoblot analysis with mAb to p62dok. Immunoglobulin heavy chain (IgH) in the lysates reactive to the secondary antibody are indicated.

In B lymphoma cell lines, a rasGAP-associated 62-kD protein is tyrosine phosphorylated upon either BCR cross-linking or BCR/FcγRIIB cocross-linking (Gold et al. 1993; Vuica et al. 1997). Thus we examined tyrosine phosphorylation of p62dok in primary B cells after these treatments. When mouse splenic B cells were stimulated with the F(ab′)2 form of rabbit IgG to mouse IgM, which crosslinks only BCR (membrane IgM), p62dok was tyrosine phosphorylated within 2 min after cross-linking (Fig. 2A). To our surprise, p62dok was more strongly phosphorylated when B cells were stimulated with intact rabbit IgG, which cocross-links BCR and FcγRIIB. In both cases, p62dok was the most prominently phosphorylated band (Fig. 2B). Tyrosine phosphorylation of p62dok after these treatments was hardly detected in B cells from the lyn−/− mice at least during the early response (Fig. 2C) (Nishizumi et al. 1995, 1998). This suggests that p62dok has a role downstream of the Src-like PTK Lyn in both BCR- and FcγRIIB-mediated signaling. There are several apparent target sites for Src-like PTK in p62dok (Carpino et al. 1997; Yamanashi and Baltimore 1997) and coexpression of it with Lyn in 293T cells induced hyperphosphorylation of p62dok (data not shown), suggesting that Lyn may phosphorylate p62dok. B cells from lyn−/− mice exhibit hyperproliferation to BCR or BCR/FcγRIIB stimulation, along with enhanced MAP kinase activation and Ca2+ influx (Wang et al. 1996; Chan et al. 1997a,b; Nishizumi et al. 1998). This hyperresponsiveness is thought to be a cause of the autoimmune disease that develops in lyn−/− mice (Hibbs et al. 1995; Nishizumi et al. 1995). Thus, Lyn is a negative regulator of cell proliferation, MAP kinase, and Ca2+ influx, suggesting that p62dok acts at least partially as a novel signaling molecule in pathways downstream of Lyn.

Figure 2.

Figure 2

Figure 2

Figure 2

Figure 2

Open in a new tab

Lyn-dependent tyrosine phosphorylation of p62dok and its role in B-cell proliferation upon BCR or BCR/FcγRIIB stimulation. (A,B) Tyrosine phosphorylation of p62dok. Wild-type or dok−/− B cells were stimulated with the intact (Ig) or F(ab′)2 form of antibody (30 μg/ml) to IgM for the indicated time. Immunoprecipitated p62dok (from 6 × 106 cells) (IP:Dok) or total lysate (from 2 × 105 cells) of the B cells was subjected to immunoblotting with the mAb to p62dok (IB:Dok) or with RC20–HRP to detect phosphotyrosine (IB:PY). Positions of p62dok and standard protein markers (in kD) are indicated. (C) Lyn-dependent phosphorylation of p62dok. lyn−/− or wild-type (C57BL/6) B cells were stimulated with the intact (Ig-) or F(ab′)2 (F-) form of antibody (30 μg/ml) to IgM for the indicated time. Immunoprecipitated p62dok (from 3 × 106 cells) (IP:Dok) or total lysate (from 3 × 105 cells) of the B cells was subjected to immunoblotting with the mAb to p62dok (IB:Dok) or with RC20–HRP (IB:PY). The position of p62dok is indicated. (D) Effect of dok gene inactivation in B-cell proliferation. dok−/− (●) or wild-type (○) B cells were stimulated with the intact (Ig) or F(ab′)2 form of antibody (30 μg/ml) to IgM for 2 days in the presence (+) or absence (−) of pretreatment of mAb 2.4G2 to FcγRIIB. [3H]TdR incorporation for the last 8 hr of culture was measured (counts/3 min).

To address the function of p62dok, we examined the proliferative response of splenic B cells from dok−/− mice (Fig. 2D). Stimulation with F(ab′)2 to IgM induced proliferation of B cells irrespective of dok gene inactivation. However, stimulation with intact antibodies to IgM induced proliferation of B cells only from the dok−/− mice. In addition, pretreatment with mAb 2.4G2, which blocks FcγRIIB, allowed intact antibody to induce proliferation of wild-type B cells. These results indicate that the pathway of negative regulation of BCR-mediated proliferation by FcγRIIB is dysfunctional in dok−/− B cells at least in these experimental conditions.

Because tyrosine phosphorylation of proteins upon BCR stimulation is a critical event for subsequent cell proliferation (DeFranco 1997; Kurosaki 1997; Reth and Wienands 1997), p62dok might be expected to alter the phosphorylation pattern when FcγRIIB is cross-linked to BCR. However, except for phosphorylation of p62dok itself, overall tyrosine phosphorylation of proteins after BCR or BCR/FcγRIIB stimulation was not substantially altered by inactivation of the dok gene (Fig. 2Bb). Thus, the inhibitory effect of p62dok occurs without affecting the phosphorylation of major cellular substrates for tyrosine kinases.

Upon BCR stimulation, MAP kinase activation and Ca2+ influx are induced and thought to be important events (DeFranco 1997; Kurosaki 1997; Reth and Wienands 1997). There are three reports on Dok family function in MAP kinase regulation. When p62dok was constitutively overexpressed in a cell line, CHO-IR, which also constitutively overexpresses insulin receptor, there was a marked increase of membrane association of rasGAP upon insulin stimulation (Noguchi et al. 1999). However, the overexpression of p62dok did not significantly affect MAP kinase activation upon insulin stimulation of the CHO-IR cells. On the contrary, when p56dok-2 was transiently overexpressed in a cell line, 32D, which also transiently overexpresses IL-2 receptor, MAP kinase activation was suppressed upon IL-2 stimulation of the cells (Nelms et al. 1998). Overexpression of p56dok-2 also negatively regulated EGF receptor-mediated activation of MAP kinase in COS1 cells (Jones and Dumont 1999). Therefore, we first examined if the dok gene inactivation in primary B cells affected MAP kinase activity upon BCR cross-linking or cocross-linking of BCR with FcγRIIB. Active Erk1 or Erk2 MAP kinases can be detected by their phosphorylation on both threonine and tyrosine residues in the TEY motif. We used a mAb to the active/phosphorylated form of Erk (P-Erk) to monitor its activation. Because Erk1 activation was very weak upon BCR stimulation (data not shown), we focused on Erk2. As reported previously, Erk2 activation was suppressed by costimulation of FcγRIIB with intact IgG (Fig. 3A; Chan et al. 1997a). However, B cells prepared from dok−/− spleen showed higher Erk2 activation than normal cells upon BCR/FcγRIIB cocross-linking. In addition, Erk2 was also activated to a higher extent in the dok-null B cells than in normal cells even when BCR alone was cross-linked. These results indicate that p62dok is a negative regulator of MAP kinase upon cross-linking of BCR or BCR with FcγRIIB.

Figure 3.

Figure 3

Figure 3

Figure 3

Figure 3

Figure 3

Open in a new tab

Role of p62dok in negative regulation of MAP kinase and [Ca2+]i by FcγRIIB. (A) p62dok-dependent negative regulation of Erk2. dok−/− or wild-type B cells were stimulated with the intact (Ig) or F(ab′)2 form of antibody (30 μg/ml) to IgM for the indicated time. Immunoprecipitated Erk2 (from 4 × 106 cells) was subjected to immunoblotting with mAb to P-Erk2 and reprobed with antibody to Erk2. Positions of P-Erk2 (oval) and Erk2 (rectangle) are indicated (top panels). At each time point, the phosphorylation level of Erk2 was quantitated and normalized to the amount of immunoprecipitated Erk2 (bottom panel). The normalized phosphorylation level of Erk2 of dok−/− B cells at 5 min after BCR stimulation was defined as 10 in arbitrary units. (B) Association of rasGAP to p62dok. C57BL/6 B cells were stimulated with the intact (Ig-2) or F(ab′)2 form (F-2) of antibody (30 μg/ml) to IgM for 2 min or unstimulated (0). Proteins in anti-p62dok immunoprecipitates (from 1.5 × 107 cells) (Dok-IP) or total lysates (from 4 × 105 cells) (lysate) were subjected to immunoblotting with the mAb to rasGAP. The position of rasGAP is indicated. (C) Tyrosine phosphorylation of FcγRIIB in dok−/− B cells. dok−/− or wild-type B cells were stimulated with the intact (Ig) or F(ab′)2 form of antibody (30 μg/ml) to IgM for the indicated time. Immunoprecipitated FcγRIIB (from 3 × 106 cells) was subjected to immunoblotting with RC20–HRP to detect tyrosine phosphorylation. (D) Association of SHIP with FcγRIIB in dok−/− B cells. B cells were stimulated with intact antibody to IgM for 2 min (2) or unstimulated (0). Proteins in anti-FcγRIIB immunoprecipitates (from 1 × 107 cells) (FcγRIIB-IP) or total lysates (from 2 × 105 cells) (lysate) were subjected to immunoblotting with antibody to SHIP or FcγRIIB. The FcγRIIB–IP blotting with antibody to FcγRIIB was visualized separately from the total lysate blotting. Positions of SHIP and FcγRIIB are indicated. (E) Negative regulation of [Ca2+]i in dok−/− B cells. dok−/− or wild-type B cells, which were loaded with fura-2, were stimulated with the intact (dotted line) or F(ab′)2 (solid line) form of antibody (30 μg/ml) to IgM at t = 0 min. [Ca2+]i was calculated based on the fluorescence of fura-2 bound to Ca2+.

Because p120 rasGAP was associated with p62dok upon either BCR or BCR/FcγRIIB stimulation (Fig. 3B), p21ras, an upstream positive regulator of MAP kinase, may be suppressed by rasGAP. However, it was reported recently that overexpression of a mutant p56dok-2 that could not bind to either SH2 of rasGAP in vitro could still attenuate MAP kinase (Jones and Dumont 1999). The molecular basis of Dok family suppression of MAP kinase awaits further analysis. On the other hand, even in dok−/− B cells, Erk2 activation following BCR stimulation was still suppressed by cocross-linking it with FcγRIIB (Fig. 3A), suggesting that there is another molecule also suppressing MAP kinase downstream of FcγRIIB. SHIP might be the other negative regulator of MAP kinase, although that is yet controversial. (Liu et al. 1998, 1999; Okada et al. 1998).

Tyrosine phosphorylation of the ITIM (Immunoreceptor Tyrosine-based Inhibitory Motif) of FcγRIIB and subsequent association of SHIP to the motif are critical events for FcγRIIB-mediated inhibition of Ca2+ influx and cell proliferation (Ono et al. 1997; Ravetch 1997; Bolland et al. 1998; Coggeshall 1998; Liu et al. 1998; Scharenberg et al. 1998). Thus, p62dok might be involved in the FcγRIIB/SHIP pathway. However, we found that p62dok is dispensable for FcγRIIB phosphorylation and its subsequent binding to SHIP upon BCR/FcγRIIB stimulation (Fig. 3C,D). Also, increase of intracytoplasmic Ca2+ concentration ([Ca2+]i) following BCR cross-linking was suppressed to substantially the same extent by FcγRIIB engagement irrespective of the presence of p62dok (Fig. 3E). As reported previously for wild-type B cells (Chan et al. 1997b; Nishizumi et al. 1998), intracellular released Ca2+, as judged by the relative [Ca2+]i in the presence of EGTA in the medium returned to a basal level within 5 min after BCR or BCR/FcγRIIB stimulation in both dok−/− and wild-type B cells (data not shown). Thus, p62dok does not appear to be either a regulator or an effector of SHIP in the negative regulation of Ca2+ influx upon BCR/FcγRIIB cocross-linking.

FcγRIIB null mice show a relative enhancement of antibody production following both thymus-dependent (TD) and -independent (TI) antigen stimulation (Takai et al. 1996). However, Ship−/− lymphocytes reconstituted in Rag-1−/− mouse showed normal antibody production to vesicular stomatitis virus stimulation (Bolland et al. 1998). Therefore, SHIP is unlikely to be fully responsible for FcγRIIB-mediated negative signaling. The same was true of p62dok; dok-null mice had normal levels of IgG1 production to a TD antigen (Fig. 4A). In addition, dok-null mice showed normal levels of IgM and IgG1 production to TI antigens (data not shown). Unexpectedly, the level of IgM production to the TD antigen in the dok−/− mice was lower than in wild-type controls (Fig. 4A). Consistently, total IgM levels in unstimulated dok−/− mice were also lower than in wild-type controls (Fig. 4B). Apart from this phenotype, however, p62dok is unlikely to be fully responsible for FcγRIIB-mediated negative signaling. Either SHIP or p62dok might be sufficient, possibly in cooperation with other molecule(s), in balancing immunoglobulin production.

Figure 4.

Figure 4

Open in a new tab

Low level of IgM production in dok−/− mice. (A) IgM or IgG1 response to TD antigen. Relative DNP-specific IgM or IgG1 levels in sera from wild-type(○) or dok−/− (●) mice immunized with DNP–hemocyanine were determined by ELISA, in which the mean value of immunoglobulin levels in sera from wild-type mice was defined as 1. Sera were prepared at day 7 or day 14 postimmunization for IgM and IgG1, respectively. (B) IgM or IgG1 concentrations (mg/ml) of preimmunization sera from wild-type (○) or dok−/− (●) mice were determined by ELISA.

Because p62dok is phosphorylated downstream of multiple growth factor receptors, and its tyrosine phosphorylation correlates with the transforming activity of Bcr-Abl (Carpino et al. 1997; Yamanashi and Baltimore 1997; Bhat et al. 1998; White and Yenush 1998), we had expected that p62dok would be a positive regulator of cell proliferation. Its relative, IRS-1, has a positive role in insulin receptor signaling. However, our data show that p62dok, at least in B cells, is a negative regulator of MAP kinase and cell proliferation. It appears that p62dok could be important for negatively regulating the strength of signals downstream of many PTKs. In this regard, it is important that overexpression of p56dok-2 negatively regulates MAP kinase activity in a cell lines (Nelms et al. 1998; Jones and Dumont 1999). Because p62dok and p56dok-2 are two major tyrosine-phosphorylated proteins in bcr-abl-positive primary CML cells (Carpino et al. 1997; Nelms et al. 1998), they may have a role in the establishment of the chronic phase of human CML by negatively regulating signal(s) from Bcr–Abl.

Materials and methods

Generation of dok-null-mutant mice

Murine dok genomic DNA fragments were obtained from a 129/SVJ mouse genomic library by screening with a fragment (1–448) of the murine dok cDNA (GenBank accession no. U78818). To construct the targeting vector, a 2.6-kb HindIII–HindIII 5′ fragment and a 6.8-kb StuI–StuI 3′ fragment were placed into the XhoI and PmeI sites of pPNT plasmid (Tybulewicz et al. 1991), respectively. This vector was linearized by digestion with PacI and electroporated into J1 ES cells, which were selected with G418 and Gancyclovir. Southern blot analysis identified 5 recombinant clones with a single targeted allele among 326 clones. Targeted cells were injected into fertilized blastocysts from C57BL/6 female mice. Chimeric male mice were crossed with C57BL/6 females for germ-line transmission. Homozygotes were generated following heterozygous matings. The absence of p62dok in homozygotes was confirmed by Western blotting of solubilized proteins (prepared from spleens of 8- to 11-week-old homozygous mice). All dok−/− and control mice used were 8–11 weeks old with no splenomegaly, on a 129/C57BL/6 background or otherwise noted. All mice were maintained under specific pathogen-free conditions.

Antibodies

MAb A-3 to mouse p62dok (Santa Cruz Biotechnology) was raised against its amino-terminal portion (1–276) (Yamanashi and Baltimore 1997). We confirmed that it reacts with p62dok but not p56dok-2 by immunoblotting (data not shown). Rabbit IgG to mouse IgM was affinity purified with μ-chain, and the F(ab′)2 form of it was prepared as described (Kato et al. 1994). mAb 2.4G2 to FcγRIIB was purified with protein A–Sepharose from ascites. mAb HO-13-4 to Thy 1.2 was used in the form of ascites. Recombinant variable region of the PY20-based mAb RC20 to phosphotyrosine, conjugated with HRP (RC20–HRP), was from Transduction Laboratory. Polyclonal antibodies to Erk2 (C14), and to SHIP (N-1) were from Santa Cruz Biotechnology. Anti-mβ1 rabbit antiserum used for FcγRIIB immunoblotting was a kind gift from J.V. Ravetch (Rockefeller University, New York, NY). mAbs to phospho-Erk (E10) and p120 rasGAP (B4F8) were from New England Biolabs and Santa Cruz Biotechnology, respectively.

Preparation of resting B cells from spleen

Splenic B cells were prepared as described previously (Kato et al. 1994). Notably, all B cells used were purified with Percoll density gradient centrifugation, which enriched small resting B cells, and the purified cells contained >95% surface Immunoglobulin-positive B cells. The same expression level of the surface FcγRIIB was confirmed on dok−/− B cells as on wild-type controls by FACScan analysis (data not shown). With lyn−/− and C57BL/6 B cells, they were prepared from 7-week-old mice, that did not show any splenomegaly. The lyn−/− mice had been generated as described and backcrossed at least six generations to C57BL/6 mice (Nishizumi et al. 1995, 1998).

Immunoprecipitation and immunoblotting

B cells (1 × 107) were suspended in 200 μl of minimal essential medium (MEM) and incubated with intact or F(ab′)2 form of rabbit IgG to mouse IgM for 0, 2, 5, or 30 min at 37°C. Cells were then solubilized by adding 1 ml of 1.2× TNN buffer (1× = 50 mm Tris at pH 8.0, 150 mm NaCl, 1% NP-40, 0.2 mm sodium orthovanadate, 20 μg/ml aprotinin). The lysate was centrifuged and the supernatant was precleared by treatment with excess protein G–Sepharose. The cleared lysate was incubated with 1–2 μg of antibody to p62dok, Erk2, or FcγRIIB for 1.5–3 hr, and the immune complexes were precipitated with protein G–Sepharose and washed five times with 1× TNN buffer. Proteins in the immunoprecipitates or the cleared lysates were resolved by SDS-PAGE and transferred to PVDF membrane (Bio-Rad). Immunoblotting was performed by standard methods and visualized by chemiluminescence (NEN) or by alkaline phosphatase-based coloring (Promega). The identity of tyrosine-phosphorylated p62dok in the cleared lysates (Fig. 2B) was verified by immunodepletion assay (data not shown). Quantitation of P-Erk and Erk2 immunoblotting (Fig. 3A) was performed with NIH Image software (v. 1.61).

B-cell proliferation assay

B cells (1 × 105) in 100 μl of RPMI-1640 medium, supplemented with 10% FCS, 50 μm β-mercaptoethanol, and 100 μg/ml kanamycin, were cultured in a 96-well plate in the presence of the intact or F(ab′)2 form of rabbit IgG to mouse IgM with or without pretreatment with 10 μg/ml 2.4G2 mAb to FcγRIIB, which was added to the culture at 30 min before anti-IgM stimulation. Proliferation was evaluated by pulsing with 0.25 μCi [3H]thymidine ([3H]TdR, ICN Biochemicals) for the last 8 hr of the culture. [3H]TdR incorporation was measured in a Matrix 96 system (Packard) and represented as the mean value of triplicate assays as described (Kato et al. 1994). Standard deviation was less than 5% of each readout.

[Ca2+]i assay

B cells (1 × 107 cells/ml) in MEM containing 0.025% BSA were incubated with 4 μm fura-2AM at 37°C for 45 min. After washing three times, 2 × 106 fura-2-loaded B cells were suspended in 0.5 ml of the same medium and stimulated with the intact or F(ab′)2 form of rabbit IgG to mouse IgM with stirring. Emission at 500 nm was monitored under excitation of the sample at two different wavelengths (340 and 380 nm) with a CAF-110 fluorescence spectrophotometer (Japan Spectroscopic Co.). [Ca2+]i was determined as described previously (Tamura and Nariuchi 1992).

Immunization and ELISA

Each 8- to 9-week-old mouse was immunized intraperitoneally with 10 μg of dinitrophenyl (DNP)–hemocyanin in incomplete Freund's adjuvant (TD), 2 μg of DNP–Ficoll in PBS (TI), or 10 μg of trinitrophenyl (TNP)–LPS in saline (TI) at day 0. Sera were prepared at day 7 or day 14 postimmunization or preimmunization. Amounts of IgM or IgG1 in preimmunization sera were determined by ELISA with isotype-specific antibody as described previously (Nishizumi et al. 1995, 1998). For postimmunization sera, relative amounts of IgM or IgG1 reacting with DNP-conjugated BSA were determined by ELISA. At least seven of nine dok−/− mice mounted IgM response to TD antigen (data not shown).

Acknowledgments

We thank M. Scott, J. Harrison, A. Koleske, E. Brown, and P. Svec for help in generating dok+/− mice, and members of the animal facilities of both Massachusetts Institute of Technology and Institute of Medical Science (University of Tokyo) for animal care. We also thank S. Tronick and J. Ravetch for antibodies and K. Horikawa, H. Nishizumi, H. Yoshida, B. Chen, G. Cohen, C. Lois, C. Roman, B. Horwitz and other members of the Baltimore, Yamamoto, and Nariuch laboratories for helpful discussions during the course of this work. Y.Y. thanks S. Nishikawa and K. Toyoshima for discussion and encouragement. This work was supported by a U.S. Public Health Service research grant (D.B.) and by grants from the Ministry of Education, Sciences, Sports, and Culture of Japan (Y.Y., H.N., and T.Y.).

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC section 1734 solely to indicate this fact.

Footnotes

E-MAIL yamanash@ims.u-tokyo.ac.jp; FAX 81-3-5449-5413.

References

  1. Bhat A, Johnson KJ, Oda T, Corbin AS, Druker BJ. Interactions of p62dok with p210bcr-abl and Bcr-Abl-associated proteins. J Biol Chem. 1998;273:32360–32368. doi: 10.1074/jbc.273.48.32360. [DOI] [PubMed] [Google Scholar]
  2. Bolland S, Pearse RN, Kurosaki T, Ravetch JV. SHIP modulates immune receptor responses by regulating membrane association of Btk. Immunity. 1998;8:509–516. doi: 10.1016/s1074-7613(00)80555-5. [DOI] [PubMed] [Google Scholar]
  3. Carpino N, Wisniewski D, Strife A, Marshak D, Kobayashi R, Stillman B, Clarkson B. p62dok: A constitutively tyrosine-phosphorylated, GAP-associated protein in chronic myelogenous leukemia progenitor cells. Cell. 1997;88:197–204. doi: 10.1016/s0092-8674(00)81840-1. [DOI] [PubMed] [Google Scholar]
  4. Chan VW, Meng F, Soriano P, DeFranco AL, Lowell CA. Characterization of the B lymphocyte populations in Lyn-deficient mice and the role of Lyn in signal initiation and down-regulation. Immunity. 1997a;7:69–81. doi: 10.1016/s1074-7613(00)80511-7. [DOI] [PubMed] [Google Scholar]
  5. Chan VW, Lowell CA, DeFranco AL. Defective negative regulation of antigen receptor signaling in Lyn-deficient B lymphocytes. Curr Biol. 1997b;8:545–553. doi: 10.1016/s0960-9822(98)70223-4. [DOI] [PubMed] [Google Scholar]
  6. Coggeshall KM. Inhibitory signaling by B cell FcγRIIb. Curr Opin Immunol. 1998;10:306–312. doi: 10.1016/s0952-7915(98)80169-6. [DOI] [PubMed] [Google Scholar]
  7. DeFranco AL. The complexity of signaling pathways activated by the BCR. Curr Opin Immunol. 1997;9:296–308. doi: 10.1016/s0952-7915(97)80074-x. [DOI] [PubMed] [Google Scholar]
  8. Di Cristofano A, Carpino N, Dunant N, Friedland G, Kobayashi R, Strife A, Wisniewski D, Clarkson B, Pandolfi PP, Resh MD. Molecular cloning and characterization of p56dok-2 defines a new family of rasGAP-binding proteins. J Biol Chem. 1998;9:4827–4830. doi: 10.1074/jbc.273.9.4827. [DOI] [PubMed] [Google Scholar]
  9. Ellis C, Moran M, McCormick F, Pawson T. Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases. Nature. 1990;343:377–381. doi: 10.1038/343377a0. [DOI] [PubMed] [Google Scholar]
  10. Gold MR, Crowley MT, Martin GA, McCormick F, DeFranco AL. Targets of B lymphocyte antigen receptor signal transduction include the p21ras GTPase-activating protein (GAP) and two GAP-associated proteins. J Immunol. 1993;150:377–386. [PubMed] [Google Scholar]
  11. Hibbs ML, Tarlinton DM, Armes J, Grail D, Hodgson G, Maglitto R, Stacker SA, Dunn AR. Multiple defects in the immune system of Lyn-deficient mice, culminating in autoimmune disease. Cell. 1995;83:301–311. doi: 10.1016/0092-8674(95)90171-x. [DOI] [PubMed] [Google Scholar]
  12. Jones N, Dumont DJ. The Tek/Tie2 receptor signals through a novel Dok-related docking protein, Dok-R. Oncogene. 1998;17:1097–1108. doi: 10.1038/sj.onc.1202115. [DOI] [PubMed] [Google Scholar]
  13. ————— Recruitment of Dok-R to the EGF receptor through its PTB domain is required for attenuation of Erk MAP kinase activation. Curr Biol. 1999;9:1056–1060. doi: 10.1016/s0960-9822(99)80458-8. [DOI] [PubMed] [Google Scholar]
  14. Kato T, Kokuho T, Tamura T, Nariuchi H. Mechanisms of T cell contact-dependent B cell activation. J Immunol. 1994;152:2130–2138. [PubMed] [Google Scholar]
  15. Kurosaki T. Molecular mechanisms in B cell antigen receptor signaling. Curr Opin Immunol. 1997;9:309–318. doi: 10.1016/s0952-7915(97)80075-1. [DOI] [PubMed] [Google Scholar]
  16. Liu Q, Oliveira-Dos-Santos AJ, Mariathasan S, Bouchard D, Jones J, Sarao R, Kozieradzki I, Ohashi PS, Penninger JM, Dumont DJ. The inositol polyphosphate 5-phosphatase Ship is a crucial negative regulator of B cell antigen receptor signaling. J Exp Med. 1998;188:1333–1342. doi: 10.1084/jem.188.7.1333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Liu Q, Sasaki T, Kozieradzki I, Wakeham A, Itie A, Dumont DJ, Penninger JM. SHIP is a negative regulator of growth factor receptor-mediated PKB/AKT activation and myeloid cell survival. Genes & Dev. 1999;13:786–791. doi: 10.1101/gad.13.7.786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lock P, Casagrands F, Dunn A R. Independent SH2-binding sites mediate interaction of Dok-related protein with RasGTPase-activating protein and Nck. J Biol Chem. 1999;274:22775–22784. doi: 10.1074/jbc.274.32.22775. [DOI] [PubMed] [Google Scholar]
  19. Nelms K, Snow AL, Hu-Li J, Paul WE. FRIP, a hematopoietic cell-specific rasGAP-interacting protein phosphorylated in response to cytokine stimulation. Immunity. 1998;9:13–24. doi: 10.1016/s1074-7613(00)80584-1. [DOI] [PubMed] [Google Scholar]
  20. Nishizumi H, Taniuchi I, Yamanashi Y, Kitamura D, Ilic D, Mori S, Watanabe T, Yamamoto T. Impaired proliferation of peripheral B cells and indication of autoimmune disease in lyn-deficient mice. Immunity. 1995;3:549–560. doi: 10.1016/1074-7613(95)90126-4. [DOI] [PubMed] [Google Scholar]
  21. Nishizumi H, Horikawa K, Rascan IM, Yamamoto T. A double-edged kinase Lyn: A positive and negative regulator for antigen receptor-mediated signals. J Exp Med. 1998;187:1343–1348. doi: 10.1084/jem.187.8.1343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Noguchi T, Matozaki T, Inagaki K, Tsuda M, Fukunaga K, Kitamura Y, Kitamura T, Shii K, Yamanashi Y, Kasuga M. Tyrosine phosphorylation of p62dok induced by cell adhesion and insulin: possible role in cell migration. EMBO J. 1999;18:1748–1760. doi: 10.1093/emboj/18.7.1748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Okada H, Bolland S, Hashimoto A, Kurosaki M, Kabuyama Y, Iino M, Ravetch FV, Kurosaki T. Role of the inositol phosphatase SHIP in B cell receptor-induced Ca2+ oscillatory response. J Immunol. 1998;161:5129–5132. [PubMed] [Google Scholar]
  24. Ono M, Okada H, Bolland S, Yanagi S, Kurosaki T, Ravetch J V. Deletion of SHIP or SHP-1 reveals two distinct pathways for inhibitory signaling. Cell. 1997;90:293–301. doi: 10.1016/s0092-8674(00)80337-2. [DOI] [PubMed] [Google Scholar]
  25. Ravetch JV. Fc Receptors. Curr Opin Immunol. 1997;9:121–125. doi: 10.1016/s0952-7915(97)80168-9. [DOI] [PubMed] [Google Scholar]
  26. Reth M, Wienands J. Initiation and processing of signals from the B cell antigen receptor. Annu Rev Immunol. 1997;15:453–479. doi: 10.1146/annurev.immunol.15.1.453. [DOI] [PubMed] [Google Scholar]
  27. Scharenberg AM, El-Hillal O, Fruman DA, Beitz LO, Li Z, Lin S, Gout I, Cantley LC, Rawlings DJ, Kinet J-P. Phosphatidylinositol-3,4,5-triphosphate (PtdIns-3,4,5-P3)/Tec kinase-dependent calcium signaling pathway: A target for SHIP-mediated inhibitory signals. EMBO J. 1998;17:1961–1972. doi: 10.1093/emboj/17.7.1961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Takai T, Ono M, Hikida M, Ohmori H, Ravetch JV. Augmented humoral and anaphylactic responses in FcγRII-deficient mice. Nature. 1996;370:346–349. doi: 10.1038/379346a0. [DOI] [PubMed] [Google Scholar]
  29. Tamura T, Nariuchi H. T cell activation through TCR/-CD3 complex; IL-2 production of T cell clones stimulated with anti-CD3 without cross-linkage. J Immunol. 1992;148:2370–2377. [PubMed] [Google Scholar]
  30. Tybulewicz VL, Crawford CE, Jackson PK, Bronson RT, Mulligan RC. Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene. Cell. 1991;65:1165–1175. doi: 10.1016/0092-8674(91)90011-m. [DOI] [PubMed] [Google Scholar]
  31. Vuica M, Desiderio S, Schneck JP. Differential effects of B cell receptor and B cell receptor-FcγRIIB1 engagement on docking of Csk to GTPase-activating protein (GAP)-associated p62. J Exp Med. 1997;186:259–267. doi: 10.1084/jem.186.2.259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wang J, Koizumi T, Watanabe T. Altered antigen receptor signaling and impaired Fas-mediated apoptosis of B cells in lyn-deficient mice. J Exp Med. 1996;184:831–838. doi: 10.1084/jem.184.3.831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. White MF, Yenush L. The IRS-signaling system—A network of docking proteins that mediated insulin and cytokine action. In: Pawson AJ, editor. Protein modules in signal transduction. Berlin, Germany: Springer-Verlag; 1998. pp. 179–208. [DOI] [PubMed] [Google Scholar]
  34. Yamanashi Y, Baltimore D. Identification of the Abl- and rasGAP-associated 62 kDa protein as a docking protein, Dok. Cell. 1997;88:205–211. doi: 10.1016/s0092-8674(00)81841-3. [DOI] [PubMed] [Google Scholar]

Articles from Genes & Development are provided here courtesy of Cold Spring Harbor Laboratory Press

RESOURCES