International Immunology Advance Access originally published online on February 15, 2006
International Immunology 2006 18(4):545-553; doi:10.1093/intimm/dxh395
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The BASH/BLNK/SLP-65-associated protein BNAS1 regulates antigen-receptor signal transmission in B cells
Division of Molecular Biology, Research Institute for Biological Sciences, Tokyo University of Science, 2669 Yamazaki, Noda, Chiba 278-0022, Japan
1 Present address: Department of Pharmacology, Matsumoto Dental University, 1780 Goubara, Hiro-oka, Shiojiri, Nagano 399-0781, Japan
Correspondence to: D. Kitamura; E-mail: kitamura{at}rs.noda.tus.ac.jp
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Abstract |
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BASH/BLNK/SLP-65 is an adaptor protein necessary for the B cell receptor (BCR) signal transduction. Here we report the identification through the yeast two-hybrid system of a novel 26-kDa protein, BASH N-terminus-associated protein 1 (BNAS1), which interacts with the conserved and functionally important N-terminal domain of BASH/BLNK/SLP-65. BNAS1 presumably contains four transmembrane domains and the leucine zipper (LZ) motif, and is expressed ubiquitously. The association of BNAS1 with BASH/BLNK/SLP-65 through its LZ motif in vertebrate cells was demonstrated by immunoprecipitation assay. Confocal microscopy revealed that exogenously expressed BNAS1 is localized to the endoplasmic reticulum (ER) and the nuclear envelope. BASH/BLNK/SLP-65 alone was present diffusely in the cytoplasm, but localized to the same position as BNAS1 when co-expressed with BNAS1. Their co-localization was dependent on the domains containing the LZ motif of both molecules. BCR-signaled transcriptional activation of Elk-1 was suppressed by over-expression of BNAS1 in DT40 chicken B cells, and conversely augmented in the BNAS1-deficient DT40 cells, which was restored by BNAS1 reconstitution. This augmentation of Elk-1 activation in the BNAS1-deficient cells was abolished selectively by Jun N-terminal kinase (JNK) inhibitor, suggesting that BNAS1 regulates Elk-1 activation through JNK. Taken together, these results suggest that BNAS1 interacts with BASH/BLNK/SLP-65 at the ER and/or the outer nuclear membrane and is involved in the regulation of the signal transmission via mitogen-activated protein kinases leading to Elk-1 activation.
Keywords: B cell receptor, Elk-1, leucine zipper, mitogen-activated protein kinase, signal transduction
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Introduction |
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The B cell receptor (BCR), composed of the membrane Ig and Ig
/Igß heterodimers, is essential for development, selection, maintenance and immune response of B cells (1). At a cellular level, BCR is known to signal a variety of events, such as proliferation, differentiation, apoptosis and induction/suppression of Ig gene recombination depending on the developmental stages of the B cells and their circumstances. BCR signaling must be properly regulated for the control of intensity and duration of the cellular responses. Incorrect regulation of the BCR signaling may cause autoimmunity, immunodeficiency or leukemogenesis. Therefore, it is important to understand a mechanism for the regulation of BCR signaling at a molecular level.
Upon BCR ligation, tyrosine kinases of Syk, Src and Bruton's tyrosine kinase (Btk) families are recruited to BCR and activated, which in turn phosphorylate various adaptor and effector molecules such as phospholipase C 
2 (PLC2), Vav, Grb2, followed by the activation of other effectors such as protein kinase C, phosphatidilinositol 3-kinase, Ras, Rac and mitogen-activated protein (MAP) kinases as well as transcription factors such as nuclear factor of activated T cells (NF-AT), AP-1 and nuclear factor-
B (NF-B) (2–4). BASH/BLNK/SLP-65 is a B cell-specific member of the SLP-76 family adaptor protein and plays a pivotal role in BCR as well as pre-BCR signal transduction (5–8). Studies using gene knockout mice have revealed that BASH/BLNK/SLP-65 is essential for BCR-mediated survival, activation and proliferation of B cells in vitro, and their efficient development in vivo (9–12). In humans, BASH/BLNK/SLP-65 is essential for developmental progress from pro-B to pre-B cell (13).
BASH/BLNK/SLP-65 is phosphorylated mainly by Syk on tyrosine residues scattering over its conserved N-terminal half and binds to Btk, PLC2, Vav and Nck through their Src homology 2 (SH2) domains, which is crucial for activation of PLC2 and Rac1 (5, 6, 14–16). Proline-based SH3-binding motifs scattering over the central part of BASH/BLNK/SLP-65 may be necessary for the interaction with Grb2 and the related adaptors. The C-terminal SH2 domain of BASH has been shown to interact with HPK1 (17). N-terminal basic domain (the first 
50 amino acids) is also conserved among species and has recently been shown to contain a leucine zipper (LZ) motif that is responsible for the plasma membrane association of BASH/BLNK/SLP-65 and pre-BCR signaling (18). These molecular interactions nucleated by BASH/BLNK/SLP-65 have been proposed to eventually lead to the activation of MAP kinases [extracellular signal-regulated kinase (ERK), JNK and p38], NF-AT and NF-B (5, 8, 19), although molecular pathway for this is not fully understood.
To further understand the mechanism that links BASH/BLNK/SLP-65 (called BASH hereafter for convenience) to the downstream pathways, we sought to identify the proteins that bind to BASH in an SH2/SH3-domain-independent manner. Using the N-terminal 62 amino acids of chicken BASH (cBASH) as a bait in a yeast two-hybrid system, we have recently identified a protein that we named BASH N-terminus-associated protein 2 (BNAS2). BNAS2 contains putative four transmembrane domains and the LZ motif, apparently localized to the endoplasmic reticulum (ER) and the nuclear envelope, and accelerates BCR-mediated Elk-1 activation in B cells (20, our unpublished result). In parallel to this screening, we performed another two-hybrid screening with a bait composed of the N-terminal 158 amino acids of cBASH, aiming to identify a broader range of binding partners. Here we report one of the clones isolated by this screening, the protein encoded by which has been named BNAS1. BNAS1 is likely to be a membrane protein localized to the ER and the nuclear envelope, contains the LZ motif that is necessary for recruiting BASH and may regulate antigen-receptor-signaled Elk-1 activation via MAP kinases.
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Methods |
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Yeast two-hybrid screening
The two-hybrid screening was performed using Hybrid Hunter (Invitrogen, San Diego, CA, USA) with a yeast strain L40 according to the manufacturers' manual. A bait construct pHybLex/Zeo-cBASH(1–158) contained a fragment encoding amino acid residues 1–158 of cBASH, which was made by PCR using primers, 5'-GGGAATTCGGATCCATGGACAAGCTGAACAAAC-3' and 5'-GGCTCGAGTCATGTGCTTGGAAGAGGTTTGTTG-3', and the cBASH cDNA as a template. Five hundred micrograms of the DT40 cDNA library were transformed into the L40 carrying the above bait construct. The screening, DNA isolation and sequence procedures were performed as previously described (20). The nucleotide sequence for the chicken BNAS1 (cBNAS1) mRNA has been deposited in the GenBank/European Bioinformatics Institute Data Bank database under GenBank accession number AB106886.
Plasmid constructions
pECFP-mBASH: A BamHI fragment from pAT7-mBASH (17) was inserted into pECFP-C1 (Clontech, Palo Alto, CA, USA). pCAT7-cBNAS1: The EcoRI–XhoI fragment from a positive clone (8B131-1) isolated by the two-hybrid screening was inserted into EcoRI–SalI sites of pCAT7-neo vector (21). pCAT7-mBNAS1: The mouse BNAS1 (mBNAS1) cDNA, made by PCR using primers, m8B131-1-1 (5'-GGGAATTCTATGGGCGCGCGCGCGTCCCAGGAGCCCCGG-3') and m8B131-1-A (5'-GGGTCGACTCACTTGTCCTTGGAGCGGTCAAGGTTCTC-3'), and mouse spleen cDNA as a template, was inserted into EcoRI–SalI sites of pCAT7-neo. pCAFLAG-mBNAS1: The mBNAS1 cDNA was inserted into EcoRI–SalI sites of pCAFLAG vector (20). pEYFP-mBNAS1: An EcoRI–SalI fragment from pCAT7-mBNAS1 was inserted into pEYFP-C1 (Clontech). pEYFP-mBNAS1
LZ: A fragment made by PCR using primers, 5'-GAAGATCTCGAGCTCAAGCTTC-3' and 5'-CCGCTCGAGCTTCTCCTCCACACTGG-3', and pEYFP-mBNAS1 as a template, was digested with BglII and XhoI, and ligated with a longer fragment of BglII/XhoI-digested pEYFP-mBNAS1. The plasmids pAT7-mBASH, pECFP-cBASH and pECFP-cBASH62 were described previously (20).
Reverse transcriptase–PCR
Isolation of poly A+ RNAs and synthesis of cDNA were described previously (22). The cDNA was amplified with the primer m8B131-1-1 and m8B131-1-A mentioned above and Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA, USA) in the following PCR condition: 94°C for 5min, and then 30 cycles of 94°C for 1 min, 68°C for 2 min, 72°C for 1 min. ß-Actin cDNA was amplified with the primers, 5'-TACAATGAGCTGCGTGTGGC-3' and 5'-ATAGCTCTTCTCCAGGGAGG-3'. Chicken cDNAs for BNAS1 and G3PDH were amplified with the following primers: BNAS1, 5'-ATGGCGGCGGGTCGGCTGCCGGCGGTGCTGCTG-3' and 5'-TCATTGGTCACTGCCTCTGTCCAGGTTCTCAAT-3'; G3PDH, 5'-ATTTGGCCGTATTGGCCGCC-3' and 5'-CATAAGACCCTCCACAATGCC-3'. The PCR products were electrophoresed with 3% agarose gel and stained with ethidium bromide.
Immunoprecipitation and Western blotting
All cell lines were cultured as described (20). For the transient expression, expression vectors (1 µg each), pCAFLAG-mBNAS1, pEYFP-mBNAS1, pEYFP-mBNAS1LZ or pEYFP-C1, and pAT7-mBASH, were co-transfected into 2 x 106 Cos-7 cells using TransIT-LT1 reagents (Mirus, Madison, WI, USA). After 2 days, the cells were harvested. Lysates from these or other cells were subjected to immunoprecipitation and Western blot analysis as described previously (17) using the following antibodies: mouse monoclonal anti-T7 and anti-FLAG (M2), and rabbit anti-green fluorescent protein (GFP) antibodies purchased from Novagen (Madison, WI, USA), Sigma-Aldrich (Irvine, CA, USA) and Molecular Probes (Eugene, OR, USA), respectively. Rabbit anti-BASH antibody was described previously (11).
Confocal microscopy
HeLa cells were transfected with pEYFP-mBNAS1 (0.5 µg) as above. Two days later, cells were fixed with 100% methanol and then blocked with 5% FCS in PBS followed by washing with PBS three times. Then cells were stained with mouse anti-laminA/C (1:50, JoL2, Chemicon, Temecula, CA, USA), mouse anti-Golgi zone (1:100, Chemicon) or mouse anti-calnexin antibody (1:50, Chemicon), and rhodamine (TRITC)-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch, West Grove, PA, USA) as a second antibody. After washing, fluorescence was observed using confocal laser-scanning microscope (Leica TCS SP). Excitation wavelengths for yellow fluorescent protein (YFP) and rhodamine were 488 and 568 nm, respectively. Emission signals were detected between 520 and 540 nm for YFP, and 585 and 610 nm for rhodamine. For the experiments in Fig. 5, various ECFP- and EYFP-tagged expression vectors (0.5 µg each) were transfected into HeLa cells and confocal laser-scanning microscopy was performed as described previously (20).
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Luciferase assay
Luciferase assay was performed as described previously (20), except that the control vector pRSV-ß-gal was replaced with the constitutive renilla luciferase expression vector phRL-TK (Promega, Madison, WI, USA). The firefly and renilla luciferase activities were measured using the Dual-Luciferase Assay System (Promega). AP-1-luc vector is composed of pGL2-promorter vector (Promega) and eight copies of AP-1p site (–161 to –143) of the mouse IL-2 gene promoter (a gift of K. Arai). Where indicated, MAP kinase/ERK kinase (MEK) inhibitor U0126 (Calbiochem, San Diego, CA, USA) at 10 µM, p38 inhibitor SB203580 (Calbiochem, 10 µM), JNK inhibitor SP600125 (Calbiochem, 30 µM) or an equal amount of solvent (dimethyl sulfoxide) alone were added 30 min before the stimulation with anti-IgM or phorbol 12-myristate 13-acetate and ionomycin.
Generation of BNAS1-deficient DT40 cells
Gene-targeting vector was constructed as follows: A cBNAS1 genomic fragment was obtained by PCR using the primers, cBNAS1-1 (5'-GGGCGGCCGCATGGCGGCGGGTCGGCTGCCGGCGGTGCTGCTG-3') and cBNAS1-B (5'-GGGGTACCGTATAATGGCTGAGTGTGTAATCTAGATCG-3'), and DT40 genomic DNA as a template, and cloned into pGEM-Teasy vector (Promega) to generate pGEM-cBNAS1-genome. The SacII–NdeI fragment from the pGEM-cBNAS1-genome was ligated with the SacII–AseI fragment of pEGFP-C1 vector containing pUC Ori. This construct was digested at the AseI site located in the exon 2 of BNAS1 gene, and ligated with the BamHI fragment of pLoxNeo (23) containing a neomycin-resistant cassette (neo) flanked with loxP sites, resulting in the generation of the targeting vector pcBNAS1-Neor. This construct was linearized with EcoRI and BamHI, and electroporated into DT40 cells stably expressing the Cre recombinase fused with estrogen receptor hormone-binding domains [DT40(M)] (21), and the cells were selected with 2 mg ml–1 G418 (Wako, Osaka, Japan). The drug-resistant clones were expanded and verified for the homologous recombination of the targeting vector by Southern blot analysis with XhoI-digested genomic DNA and NotI- and PstI-digested genomic fragment as a probe (Fig. 7). The targeted heterozygous clones (BNAS1+/neo) were then treated with 4-hydroxy-tamoxifen to remove the neo by the Cre recombinase. The resultant cells (BNAS1+/–) were transfected again with the same targeting vector as above, selected and verified in the same way to generate the homozygous mutant clone (BNAS1neo/–, Fig. 7). Disruption of both BNAS1 alleles was confirmed by reverse transcriptase (RT)–PCR analysis as mentioned above.
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Reconstruction of the BNAS1-deficient cells
The T7-tagged cBNAS1 fragment was generated by PCR with primers, 5'-GGGGTACCATGGCCAGCATGACCG-3' and 5'-ACGCGTCGACTCATTGGTCACTGCCTCTG-3', and pCAT7-cBNAS1 as a template. The PCR product was inserted into the EcoRV site of pExpress (23), and the SpeI fragment from the resultant plasmid was inserted into an NheI site of pLoxBsr (23) to generate pT7-cBNAS1-Bsr. The BNAS1-deficient DT40 cells (BNAS1neo/–) were transfected with the pT7-cBNAS1-Bsr vector, selected with 50 µg ml–1 blasticidin S (Funakoshi, Tokyo, Japan) and verified for the expression of BNAS1 by western blot analysis with anti-T7 antibody.
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Results |
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Identification of a novel BASH N-terminus-associated protein, BNAS1
To identify proteins interacting with the N-terminal domain of BASH, we screened the cDNA library from chicken B cell line DT40 by the yeast two-hybrid system with the cBASH 1–158 amino acids region [BASH(1–158)] as a bait. After three rounds of screening, we finally isolated six independent clones from 7.5 x 106 colony-forming units of a cDNA library. Binding of these clones with the bait was confirmed by the back transformation of each of these clones into the yeast reporter strain expressing the bait construct. Four out of the six clones turned out to bind with another bait consisting of 1–62 amino acids of BASH [BASH(1–62)] through another set of back transformation experiment. After sequencing these clones, we focused on one of these clones containing a novel sequence. By comparing with nucleotide and amino acid sequences in the database, we presumed the coding region of this clone encoding a 237-amino acid protein with a calculated molecular mass of approximately 26 kDa, which we named BNAS1 (GenBank accession number: AB106886). We found in a database the possible human and mouse orthologs of the cBNAS1, which are highly conserved (62.2% and 61.7% identical to cBNAS1, respectively; Fig. 1). We also cloned a cDNA encoding the mBNAS1 by PCR. Except for the presumable orthologs of other species, no proteins having any similarity to BNAS1 could be found in the database. The BNAS1 protein is predicted to have four transmembrane domains by InterProScan analysis. We also found a putative LZ motif at the C-terminal region of the protein (Fig. 1). The human ortholog of BNAS1 has been reported as BRI3BP, which was identified by yeast two-hybrid system as a protein binding with BRI3, a lysosomal protein involved in tumor necrosis factor-induced cell death (24). BRI3BP gene was shown to be expressed in several tissues, but neither confirmation of the BRI3 binding nor functional characterization of BRI3BP protein has been reported so far (25).
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Expression profile of BNAS1 mRNA was assessed by RT–PCR using a variety of cell lines. As shown in Fig. 2, BNAS1 mRNA was detected in any examined cell lines of lymphoid and other hematopoietic as well as non-hematopoietic origin. Thus, BNAS1 appears to be expressed ubiquitously in various types of cells.
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Interaction between BNAS1 and BASH in vitro
We next examined whether BNAS1 interacts with a full-length BASH protein in mammalian cells. The expression vectors encoding T7-tagged mouse BASH (T7-mBASH) and FLAG-tagged mouse BNAS1 (FLAG-mBNAS1) were co-transfected into Cos-7 cells. When T7-mBASH was immunoprecipitated from the cell lysate with anti-T7 antibody, mBNAS1 was co-precipitated (Fig. 3A). Conversely, when FLAG-mBNAS1 was immunoprecipitated with anti-FLAG antibody, mBASH was co-precipitated (Fig. 3B). To examine the association of constitutively expressed endogenous proteins in the absence of available antibody against BNAS1, we used BNAS1-deficient DT40 (BNAS1–) cells and the BNAS1– cells stably reconstituted with T7-tagged cBNAS1 protein (see below). As shown in Fig. 3(C), the endogenous cBASH was co-precipitated with the T7-cBNAS1 by anti-T7 antibody in the latter cells, but not in the former, irrespective of BCR cross-linking by anti-IgM antibody. As shown in Fig. 3(D), mBNAS1 carrying a deletion (amino acids 226–243) in its LZ motif (mBNAS1
LZ) failed to co-precipitate with mBASH, indicating that BNAS1 interacts with BASH through its LZ motif.
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Subcellular localization of BNAS1
We next examined the subcellular localization of BNAS1 with confocal laser-scanning fluorescence microscopy. HeLa cells were transiently transfected with the expression plasmid encoding the YFP-tagged mouse BNAS1 protein (YFP–mBNAS1), and the cells were stained with mAbs specific to calnexin or laminA/C (Fig. 4). YFP–mBNAS1 was clearly localized to a mesh-like network in the cytoplasm, a typical pattern of the ER, and the nuclear envelope, where calnexin was co-localized (A–C). The nuclear envelope localization of mBNAS1 was further supported by its nuclear rim-like structure lined with laminA/C, a protein localized to the inner surface of the inner nuclear membrane (D–F). BNAS1 localization was not overlapped with the staining of the Golgi apparatus (data not shown). These results indicate that BNAS1 is specifically localized to the ER and the continuous outer nuclear membrane.
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Co-localization of BNAS1 and BASH in a cell
To ascertain the binding of BNAS1 and BASH in cells, we transfected HeLa cells with the expression vectors encoding YFP–mBNAS1, cyan fluorescent protein (CFP)–mBASH or CFP–cBASH fusion proteins, and examined by confocal laser-scanning fluorescence microscopy. The CFP-tagged mBASH and cBASH proteins were present diffusely in the cytoplasm, and occasionally in the nucleus as well (Fig. 5A and B; data not shown), whereas the YFP-tagged mBNAS1 protein appeared to be localized to the ER and the nuclear membrane (Fig. 5C). When the CFP–mBASH (Fig. 5D–F) or CFP–cBASH (Fig. 5G–I) and YFP–mBNAS1 were co-expressed in the cells, both proteins were co-localized to the region where YFP–mBNAS1 alone was localized. This co-localization was abolished when the N-terminal 62-amino acid domain, which contains a putative LZ motif (18), was deleted from cBASH (CFP–cBASH
62; Fig. 5J–L), and the CFP–cBASH62 was diffused in the cytoplasm and the nucleus except for some unidentified compartments. In addition, CFP–mBASH was not co-localized with YFP–mBNAS1LZ carrying a deletion in the LZ motif (Fig. 5M–O). These data confirm the physical interaction between BASH and BNAS1 in living cells, and suggest that the LZ motifs in both proteins mediate this interaction.
BNAS1 negatively regulates antigen-receptor-mediated Elk-1 activation in B cells
To elucidate the function of BNAS1 in the BCR signal transduction, we first assessed the effect of over-expression of cBNAS1 in chicken B cell line DT40 by the luciferase assay. We found that BCR ligation-induced transcriptional activation of Elk-1, a transcription factor known to be activated by three major MAP kinases (ERK, JNK, p38), was repressed by the over-expression of BNAS1 in a dose-dependent manner (Fig. 6A).
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To further investigate the function of BNAS1, we generated BNAS1-deficient DT40 cells through gene targeting (Fig. 7). The neomycin-resistant gene cassette (neo) flanked with loxP sequences was first inserted into the exon 2, the neo was then deleted via Cre-mediated recombination and finally the BNAS1 gene on the second allele was disrupted in the same way as the first allele (Fig. 7A and B). In the resultant DT40 clone (BNAS1neo/–), no intact BNAS1 mRNA, but only the truncated one, was detected by RT–PCR (Fig. 7C and D). The truncated RNA turned out to be an alternative splicing product that caused out-of-frame join between exons 1 and 3, which was confirmed by sequencing of the PCR product (data not shown). Thus, we call this BNAS1neo/– clone as BNAS1– hereafter. We further generated a stable transfectant of the BNAS1– cells with the cBNAS1 expression vector (cBNAS1/BNAS1–) to reconstitute the BNAS1 expression. Expression levels of BCR on the cell surface and growth rates of the DT40 cell clones used in the following study were essentially the same (data not shown).
We assessed the impact of the BNAS1 deficiency on BCR-mediated MAP kinase signaling pathway by Elk-1 activation-mediated luciferase assay. Transcriptional activity of Elk-1 after BCR cross-linking was significantly augmented in BNAS1– cells compared with the parental DT40 cells, and this augmentation was restored in the cBNAS1-reconstituted BNAS1– (cBNAS1/BNAS1–) cells (Fig. 6B, D and E). Elk-1 is known to induce the expression of c-fos, which is dimerized with c-Jun to form a transcription factor AP-1. BCR-signaled AP-1 activation was also augmented in the BNAS1– cells, albeit to a lesser extent, and restored in the cBNAS1/BNAS1– cells as examined by luciferase assay (Fig. 6C). The BCR-induced Elk-1 activation was completely blocked by MEK inhibitor U0126 (U, Fig. 6D). Therefore, MEK, and perhaps downstream ERK as well, is essential for the Elk-1 activation. Unexpectedly it was augmented by p38 inhibitor SB203580 (SB, Fig. 6D and E; treatment with 10, 25 and 50 µM of SB resulted in almost the same extent of augmentation, data not shown), revealing an inhibitory role for p38 in the BCR-induced Elk-1 activation. However, this does not appear to be responsible for the BNAS1-mediated regulation, because the augmentation of the BCR-induced Elk-1 activation due to the lack of BNAS1 was maintained when p38 kinase was inhibited (Fig. 6D and E). By contrast, JNK inhibitor SP600125 diminished the BCR-induced Elk-1 activation to the similar level among the parental DT40, BNAS1– and cBNAS1/BNAS1– cells, and augmentation was no longer observed in the BNAS1– cells (Fig. 6E). This suggests that JNK functions to enhance the ERK-dependent Elk-1 activation and that this function is regulated by a mechanism where BNAS1 is involved.
In contrast to the Elk-1 response, BCR-induced NF-B and NF-AT activations, as well as apoptosis of the cells, were not significantly affected by the BNAS1 deficiency (data not shown). In addition, BCR ligation-mediated increase of intracellular calcium was almost identical among the parental, BNAS1– and cBNAS1/BNAS1– DT40 cells (data not shown). Taken together, these data suggest that BNAS1 is not involved in the initial phase of BCR signal transduction, but negatively regulates the selective downstream pathway leading to Elk-1 activation perhaps through JNK. However, BCR-mediated active-state-specific phosphorylation of JNK, as well as ERK and p38, did not significantly differ among the parental, BNAS1–, and cBNAS1/BNAS1– DT40 cells (data not shown). Therefore, augmented Elk-1 transcriptional activity in BNAS1– cells is not likely due to the enhanced signaling cascade upstream of JNK. A possible mechanism for the BNAS1-mediated regulation of BCR-induced Elk-1 activation is discussed below.
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Discussion |
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Adaptor proteins bind to multiple signaling intermediates simultaneously or successively in a temporally regulated manner. Thus, identification of their binding partners is essential to clarify how the adaptors regulate the receptor signaling. BASH has multiple common protein-binding sites, such as phosphotyrosine- or proline-based sequences and an SH2 domain, and proteins bound to these domains have been identified. BASH also has a conserved N-terminal basic domain that shares little homology among the SLP-76 family proteins but contains a LZ motif required for the function of BASH (18, 26). We report here the identification of BNAS1 bound to the basic domain of BASH, in addition to the previously reported BNAS2 (20). BNAS1 and BNAS2 share no homology, but unexpectedly both possess four putative transmembrane domains as well as a putative LZ motif. Significance of the LZ motif located in the N-terminal part of BNAS2 has not been assessed so far, but the LZ motif in BNAS1 is suggested to bind to the LZ motif in BASH as documented in the present study. Both BNAS1 (this study) and BNAS2 (20, our unpublished results) are localized to the ER and the nuclear envelope, and may function to recruit BASH and its associated proteins to these sites and to regulate the action of such signaling complex. As BASH is recruited also to the plasma membrane through its LZ motif (18), another LZ motif-containing protein localized at plasma membrane might exist.
Elk-1 activation-driven luciferase assays with DT40 cells have revealed that BASH is necessary for BCR-signaled activation of Elk-1 (our unpublished data), in accord with the previous data showing severely attenuated ERK activation in BLNK(BASH)-deficient DT40 cells (8). Therefore, BASH is required for initial activation of ERK, perhaps through activation of PLC2 (8). On the other hand, BNAS1 negatively regulates the BCR-signaled Elk-1 activation (Fig. 6). It is currently unclear how the binding of BASH to BNAS1 is related to the positive and the negative signaling roles for BASH and BNAS1, respectively, in Elk-1 activation. It is possible that the BASH binding to BNAS1 inhibits negative regulatory function of BNAS1, and thus promotes Elk-1 activation. On the contrary, it is equally possible that BASH binding to BNAS1 promotes the negative regulatory function of BNAS1. Regarding to the latter possibility, BASH has been demonstrated to play ambivalent roles in pre-BCR-dependent proliferation of pre-B cells: it suppresses proliferation of pre-B cells in vitro in the culture with IL-7, while it is necessary for cell-cycle progression of large pre-B cells in vivo (26, 27).
The luciferase assays using MAP kinase inhibitors revealed that MEK, and perhaps downstream ERK as well, is essential for this Elk-1 activation, and JNK is dispensable but greatly enhances it, whereas p38 suppresses it. The augmentation of Elk-1 activation by the BNAS1 deficiency was maintained when p38 was inhibited, but lost when JNK was inhibited. Therefore, it is presumable that BNAS1 regulates the Elk-1 activation through inhibiting the JNK function to enhance the Elk-1 activation, but not through mediating p38 inhibitory function. However, bulk activation of JNK, as well as ERK and p38, after BCR cross-linking determined by western blot analysis with the phospho-specific antibodies appeared to be unaffected in the absence of BNAS1 (data not shown). Therefore, it is unlikely that BNAS1 regulates the BCR signaling pathway at a level upstream of these MAP kinases. In any case, assuming that only a fraction of activated MAP kinases are actually involved in the Elk-1 activation in the nucleus, it would be difficult to demonstrate biochemically a quantitative difference of the active MAP kinases that might be raised by BNAS1 deficiency. Considering its intracellular localization at the ER/nuclear membrane, we propose that BNAS1 may be involved in a machinery that regulates either local activity or nuclear entry of MAP kinases. It remains to be elucidated whether BASH binding is necessary for such function of BNAS1 and how JNK function is selectively suppressed by the BNAS1-involved mechanism. In contrast to BNAS1, over-expressed BNAS2 accelerated BCR-mediated Elk-1 activation in DT40 cells in a dose-dependent manner (20). Thus, both BNAS1 and BNAS2 might be involved in the same machinery that regulates activity of MAP kinases at the ER/nuclear membrane.
Elk-1 is a member of the ternary complex factor subgroup of the Ets family transcription factor, and was shown to promote cell growth and to inhibit apoptosis in some cell lines (28). Although we have not assessed whether the endogenous Elk-1 activation is affected by the absence of BNAS1, unchanged growth rate or BCR-induced apoptosis in the BNAS1-deficient DT40 cells (data not shown) suggests that BNAS1 is not critical for these processes. It is possible that the BCR-stimulated Elk-1 has other functions in B cells. Alternatively, such effects on cell proliferation by the absence of BNAS1 in DT40 cells might be masked by de-regulated expression of c-myc by viral integration (29) and other possible abnormalities in this lymphoma cell line. Further studies with non-transformed cells may unmask the physiological role for BNAS1.
In summary, we have identified BNAS1 as a novel binding partner of BASH, which likely interacts with BASH through LZ motifs of both molecules. BNAS1 appears to localize at the ER and the outer nuclear membrane and to regulate BCR-signaled activation of Elk-1 through MAP kinases. These results imply a new focus in the study of BCR-signal transduction, namely, the processing of the receptor-mediated signaling at the ER/nuclear envelope.
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Acknowledgements |
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We thank Ryo Goitsuka for chicken cDNA and genomic library, Hiroshi Arakawa for pLoxNeo, pLoxBsr and pExpress vectors and Yasushi Hara for helping with the confocal laser-scanning microscopy. This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology in Japan and the Japan Society for the Promotion of Science.
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Abbreviations |
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| BCR | B cell receptor |
| BNAS | BASH N-terminus-associated protein |
| Btk | Bruton's tyrosine kinase |
| cBASH | chicken BASH |
| cBNAS1 | chicken BNAS1 |
| CFP | cyan fluorescent protein |
| ER | endoplasmic reticulum |
| ERK | extracellular signal-regulated kinase |
| GFP | green fluorescent protein |
| LZ | leucine zipper |
| MAP | mitogen-activated protein |
| mBNAS1 | mouse BNAS1 |
| MEK | MAP kinase/ERK kinase |
| NF-AT | nuclear factor of activated T cells |
| NF-B | nuclear factor-B |
| PLC | phospholipase C |
| RT | reverse transcriptase |
| SH2 | Src homology 2 |
| YFP | yellow fluorescent protein |
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Notes |
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* These authors contributed equally to this work.
Transmitting editor: H. Karasuyama
Received 28 November 2005, accepted 6 January 2006.
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