A B cell-specific adaptor protein, BASH (also known as BLNK or SLP-65), is crucial for B cell receptor (BCR) signaling. BASH binds to various signaling intermediates, such as Btk, PLC␥2, Vav, and Grb2, through its well defined motifs. Although functional significance of such interactions has been documented, BASH-mediated signal transduction mechanism is not fully understood. Using the yeast two-hybrid system, we have identified a novel protein that binds to a conserved N-terminal domain of BASH, which we named BNAS2 (BASH N terminus associated protein 2). From its deduced amino acid sequence, BNAS2 is presumed to contain four transmembrane domains, which are included in a central MARVEL domain, and to localize to endoplasmic reticulum. BNAS2 was co-precipitated with BASH as well as Btk and ERK2 from a lysate of mouse B cell line. In the transfected cells, the exogenous BNAS2 was localized in a mesh-like structure in the cytoplasm resembling that of endoplasmic reticulum (ER) and nuclear membrane. BASH was co-localized with BNAS2 in a manner dependent on its N-terminal domain. RT-PCR analysis indicated that BNAS2 mRNA is expressed ubiquitously except for plasma cells. In chicken B cell line DT40, overexpression of BNAS2 resulted in an enhancement of BCR ligation-mediated transcriptional activation of Elk1, but not of NF-B, in a manner dependent on the dose of BNAS2. Thus BNAS2 may serve as a scaffold for signaling proteins such as BASH, Btk, and ERK at the ER and nuclear membrane and may facilitate ERK activation by signaling from cell-surface receptors.
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.
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