Fruehling S, Swart R, Dolwick K M, Kremmer E, Longnecker R
Department of Microbiology-Immunology, Northwestern University Medical School, Chicago, Illinois 60611, USA.
J Virol. 1998 Oct;72(10):7796-806. doi: 10.1128/JVI.72.10.7796-7806.1998.
Latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV) is expressed on the plasma membrane of B lymphocytes latently infected with EBV and blocks B-cell receptor (BCR) signal transduction in EBV-immortalized B cells in vitro. The LMP2A amino-terminal domain that is essential for the LMP2A-mediated block on BCR signal transduction contains eight tyrosine residues. Association of Syk protein tyrosine kinase (PTK) with LMP2A occurs at the two tyrosines of the LMP2A immunoreceptor tyrosine-based activation motif, and it is hypothesized that Lyn PTK associates with the YEEA amino acid motif at LMP2A tyrosine 112 (Y112). To examine the specific association of Lyn PTK to LMP2A, a panel of LMP2A cDNA expression vectors containing LMP2A mutations were transfected into an EBV-negative B-cell line and analyzed for Lyn and LMP2A coimmunoprecipitation. Lyn associates with wild-type LMP2A and other LMP2A mutant constructs, but Lyn association is lost in the LMP2A construct containing a tyrosine (Y)-to-phenylalanine (F) mutation at LMP2A residue Y112 (LMP2AY112F). Next, the LMP2AY112F mutation was recombined into the EBV genome to generate stable lymphoblastoid cell lines (LCLs) transformed with the LMP2AY112F mutant virus. Analysis of BCR-mediated signal transduction in the LMP2AY112F LCLs revealed loss of the LMP2A-mediated block in BCR signal transduction. In addition, LMP2A was not tyrosine phosphorylated in LMP2AY112F LCLs. Together these data indicate the importance of the LMP2A Y112 residue in the ability of LMP2A to block BCR-mediated signal transduction and place the role of this residue and its interaction with Lyn PTK as essential to LMP2A phosphorylation, PTK loading, and down-modulation of PTKs involved in BCR-mediated signal transduction.
爱泼斯坦-巴尔病毒（EBV）的潜伏膜蛋白2A（LMP2A）在被EBV潜伏感染的B淋巴细胞质膜上表达，并在体外阻断EBV永生化B细胞中的B细胞受体（BCR）信号转导。LMP2A介导的对BCR信号转导的阻断所必需的LMP2A氨基末端结构域含有八个酪氨酸残基。Syk蛋白酪氨酸激酶（PTK）与LMP2A的结合发生在LMP2A基于免疫受体酪氨酸的激活基序的两个酪氨酸处，据推测Lyn PTK与LMP2A酪氨酸112（Y112）处的YEEA氨基酸基序结合。为了研究Lyn PTK与LMP2A的特异性结合，将一组含有LMP2A突变的LMP2A cDNA表达载体转染到EBV阴性B细胞系中，并分析Lyn和LMP2A的共免疫沉淀。Lyn与野生型LMP2A和其他LMP2A突变体构建体结合，但在LMP2A残基Y112（LMP2AY112F）处含有酪氨酸（Y）到苯丙氨酸（F）突变的LMP2A构建体中，Lyn的结合丧失。接下来，将LMP2AY112F突变重组到EBV基因组中，以产生用LMP2AY112F突变病毒转化的稳定淋巴母细胞系（LCL）。对LMP2AY112F LCL中BCR介导的信号转导分析显示，LMP2A介导的BCR信号转导阻断丧失。此外，LMP2A在LMP2AY112F LCL中未发生酪氨酸磷酸化。这些数据共同表明LMP2A Y112残基在LMP2A阻断BCR介导的信号转导能力中的重要性，并表明该残基的作用及其与Lyn PTK的相互作用对于LMP2A磷酸化、PTK加载以及参与BCR介导的信号转导的PTK的下调至关重要。
