Identification and Characterization of a Novel BASH N Terminus-associated Protein, BNAS2

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, PLCγ2, 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-κB, 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. 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, PLCγ2, 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-κB, 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. The membrane immunoglobulin (Ig) and Igα/Igβ heterodimers compose the B cell antigen receptor (BCR). 1The abbreviations used are: BCR, B cell antigen receptor; PLC, phospholipase C; MEK, mitogen-activated kinase; ERK, extracellular signal-regulated protein kinase; NF-AT, nuclear factor of activated T cells; NF-κB, nuclear factor-κB; SH2, Src homology 2; Btk, Bruton's tyrosine kinase; CD, clusters of differentiation; GST, glutathione S-transferase; PMA, phorbol 12-myristate 13-acetate; FITC, fluorescein isothiocyanate; BNAS, BASH N terminus-associated protein 2; MAP, mitogen-activated protein; ER, endoplasmic reticulum; RT, reverse transcriptase. BCR engagement evokes a signal that eventually causes various outcomes of B cells, such as activation, proliferation, differentiation, or apoptosis, depending on the developmental stage and circumstances of the cells. Cross-linking of BCR induces phosphorylation of tyrosine residues in the immunoreceptor tyrosine-based activation motifs (ITAM) of Igα and Igβ. When the ITAM of Igα is phosphorylated, the tyrosine kinase Syk is recruited and activated. The other tyrosine kinases belonging to the Src family (Lyn, Fyn, and Blk), as well as Btk, are also phosphorylated and activated through BCR engagement. These kinases phosphorylate and activate signaling intermediates like phospholipase C (PLC)γ, Vav, and phosphatidylinositol 3-kinase, and the following signaling cascades such as Ras-Raf-MEK-ERK pathway, and those leading to the activation of transcription factors, NF-AT, AP-1, or NF-κB (1Reth M. Wienands J. Annu. Rev. Immunol. 1997; 15: 453-479Crossref PubMed Scopus (377) Google Scholar, 2Kurosaki T. Nat. Rev. Immunol. 2002; 2: 354-363Crossref PubMed Scopus (177) Google Scholar, 3Campbell K.S. Curr. Opin. Immunol. 1999; 11: 256-264Crossref PubMed Scopus (184) Google Scholar). BASH (also known as BLNK or SLP-65) is a B cell-specific member of SLP-76 adapter protein family. The primary structure of BASH reveals a string of basic, acidic, prolinerich, and SH3 domains from N to C terminus. As a result of BCR cross-linking in B cells, BASH is rapidly tyrosine-phosphorylated by Syk and bound to the SH2 domains of PLCγ2, Btk, Vav, and Nck, or to the SH3 domain of Grb2 (4Fu C. Turck C.W. Kurosaki T. Chan A.C. Immunity. 1998; 9: 93-103Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar, 5Wienands B.J. Schweikert J. Wollscheid B. Jumaa H. Nielson P. Reth M. J. Exp. Med. 1998; 188: 791-795Crossref PubMed Scopus (236) Google Scholar, 6Goitsuka R. Fujimura Y. Mamada H. Umeda A. Morimura T. Uetsuka K. Doi K. Tsuji S. Kitamura D. J. Immunol. 1998; 161: 5804-5808PubMed Google Scholar, 7Ishiai M. Kurosaki M. Pappu R. Okawa K Ronko I. Fu C. Shibata M. Iwamatsu A. Chan A.C. Kurosaki T. 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Y. and Y. proteins that with the domain have not been have to such proteins by yeast two-hybrid chicken BASH N-terminal domain as a and with a from chicken B cell we the of a novel tetra-span transmembrane protein, which we have named BASH N terminus associated protein and the of of yeast and of as in the of from cells with and into and of of indicated that have been of the and of as a into a a The on and at on of the for and and of the was for A of chicken BASH amino amino acid was by with of and reverse and of as a and of The was with and and into an from was into a from the by the yeast was into the and T. Goitsuka R. Y. I. Reth M. Kitamura D. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). mouse BNAS2 was by with mouse as a and with the following and The and with and and of and and The into the and of a of to a with a The of from was into was with and and the and to the the chicken BASH from was into of was by with of and reverse and of as a and of The was with a of from and an of from the was into the and from was into was by with and and with as a The was with and and with an from T. Y. N. T. Y. S. 1993; Google Scholar). of from various cell and R. H. H. Fujimura Y. Y. A. C. Hayashi K. Kitamura D. Int. Immunol. 2000; 12: PubMed Scopus Google Scholar). The thus in of with of of the and and of The of for the and as for at by of for at at at of on and with The was also Cell cells and as M. Kurosaki M. Pappu R. Okawa K Ronko I. Fu C. Shibata M. Iwamatsu A. Chan A.C. Kurosaki T. Immunity. 1999; 10: 117-125Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). cells in of and and with These into cells. the cells For of was transfected into cells as For the of transfected into BASH-deficient cells by the cells or with mouse For the of or various dose of BNAS2 or to the of with with of of and of The transfected into cells by In the of the of or BNAS2 with of M. Y. H. K. K. Yamamoto T. H. T. J. Exp. Med. 1998; PubMed Scopus Google Scholar) and of the cells for with or with a of and phorbol 12-myristate and in the as Tsuji S. Y. Kurosaki T. Kitamura D. J. Immunol. 1998; Google Scholar). mouse BNAS2 protein was by with a to N-terminal residues of mouse BNAS2 with The serum was with the and and BASH was K. Nittono R. Okamoto N. Tsuji S. Hara Y. Goitsuka R. Kitamura D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2755-2760Crossref PubMed Scopus (124) Google Scholar). and and analysis as (11Tsuji S. Okamoto M. Yamada k. Okamoto N. Goitsuka R. Alnold R. Kiefer F. Kitamura D. J. Exp. Med. 2001; 194: 529-539Crossref PubMed Scopus (55) Google Scholar). For the and proteins expressed in and These proteins with a (11Tsuji S. Okamoto M. Yamada k. Okamoto N. Goitsuka R. Alnold R. Kiefer F. Kitamura D. J. Exp. Med. 2001; 194: 529-539Crossref PubMed Scopus (55) Google Scholar). of proteins bound to and cell the proteins bound to the by was For and and For the and and proteins expressed from in cells with and fluorescein of BASH N by BASH is known to with various signaling proteins through its and the SH2 domain as proteins to a conserved N-terminal domain domain is crucial for BASH in BCR signal we to proteins that to domain. a yeast two-hybrid the chicken BASH amino domain as a of we from of a from cells, which of was by of into the yeast the the of the and on of was and presumed to contain a by to as and also to chicken in a the protein deduced from as BNAS2. The primary structure of BNAS2 is shown in The chicken BNAS2 protein of amino and has an of The mouse and of BNAS2 identified in the for which been named as factor and to the chicken BNAS2 to have four transmembrane domains, as by analysis T P. 2000; PubMed Scopus Google Scholar). The that of a domain, in a central MARVEL proteins are in proteins, such as occludin and family proteins L. F. A. Sci. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The primary protein of BNAS2 some to member of the protein and to protein (also named as J. 1997; PubMed Google Scholar), for of which has been BNAS2 may be a transmembrane protein that with other proteins including of to the of RT-PCR was from various and cell As be in BNAS2 is expressed in and cell In a B cell BNAS2 is expressed in pre-B and mature B cell but not in plasma cell and that the of BNAS2 is but may be as B cells into cells. of BNAS2 to BASH in of BNAS2 and the N-terminal domain of BASH was in yeast two-hybrid as we BASH is of to BNAS2. The mouse BASH and mouse BNAS2 into cells, the in the cell lysate was with and was As shown in of BNAS2 was co-precipitated with As a mouse of the as the not BNAS2 the was the was also co-precipitated BASH protein was shown to to BNAS2. a BASH and BNAS2 proteins, we a that the protein, but not the protein, which was expressed in BASH-deficient cells of BCR cross-linking that BASH the and BNAS2 to a of expressed BNAS2 and BASH proteins by from a mouse B cell was with and the proteins by As shown in BASH was co-precipitated with the level of proteins was low in cell line. In addition, we that Btk and ERK, but not MEK, also co-precipitated with BNAS2 was that BNAS2 binds not to BASH but also some other signaling such as Btk and of BASH and BNAS2 in a the of BNAS2 in a mouse BNAS2 was transfected into cells. the cells and with by in cytoplasm was localized in a of ER with in the In some of at a was the of BNAS2 and BASH in cells, the BNAS2 and BASH transfected into cells, and the cells by As be in in the with from the the in which a domain amino been a but In was localized in a structure in the including nuclear and in which be a of protein or the cell not When and in cells, proteins co-localized in was localized the other in the cytoplasm of the of and The of was not by the of These that BASH binds to BNAS2 through its N-terminal domain in cells, and that of BASH is by its that of BNAS2 is of of Elk1, but NF-κB, by to a of BNAS2 in BCR we the of overexpression of BNAS2 on activation of transcription and is known to be phosphorylated and activated by ERK, used as for ERK or mouse BNAS2 was expressed in cells together with system, the cells with or with a of and As shown in chicken and mouse BNAS2 to enhancement of activation of In the activation of an gene was also upon overexpression of the BNAS2 proteins not In BNAS2 not the transcriptional activation of NF-κB These that BNAS2 plays a role in the BCR signaling leading to ERK activation, through with the BASH as well as Btk and we the and of a tetra-span transmembrane protein, which binds to an N-terminal domain of BASH, as well as ERK2 and BNAS2 was localized in an structure in the cytoplasm and nuclear membrane. BNAS2 overexpression resulted in an enhancement of BCR activation of Elk1, a transcription factor known to be phosphorylated and activated by The amino acid of BNAS2 is well conserved and in the transmembrane of protein as a transmembrane protein, which is with the intracellular of BNAS2 to ER and the nuclear membrane. of BNAS2 is from that of other tetra-span transmembrane proteins, such as CD9, CD37, CD53, CD63, CD81, CD82, and claudin, of which are known to be plasma membrane In addition, the four transmembrane in BNAS2 are involved in the MARVEL domain, a recently domain conserved in proteins of the and and occludin some of which are associated with membrane L. F. A. Sci. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). BNAS2 may a novel of tetra-span membrane that BNAS2 binds to the N-terminal domain amino of which not contain a tyrosine in yeast cells as well as in In the BNAS2 in the from and B cells binds to BASH tyrosine phosphorylation of proteins is to be necessary for The N-terminal domain of BASH has homology to known protein interacting and thus the of its to BNAS2 The of the conserved N-terminal domain of BASH has not been but have an essential role for domain in BCR signaling leading to the activation of a of indicated that BNAS2 binds to ERK2 in to BASH and may to ERK activation in response to BCR ligation BASH was shown to be not for ERK activation upon BCR ligation in B cell line M. Kurosaki M. Pappu R. Okawa K Ronko I. Fu C. Shibata M. Iwamatsu A. Chan A.C. Kurosaki T. Immunity. 1999; 10: 117-125Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). 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