Inui S, Kuwahara K, Mizutani J, Maeda K, Kawai T, Nakayasu H, Sakaguchi N
Department of Immunology, School of Life Science, Faculty of Medicine, Tottori University, Yonago, Japan.
J Immunol. 1995 Mar 15;154(6):2714-23.
We have shown previously that a 52-kDa phosphoprotein (p52) co-precipitated with Ig receptor (IgR)-associated MB-1 protein was inducibly phosphorylated by the stimulation with 12-O-tetradecanoyl-phorbol-13-acetate. By immunizing the 52-kDa protein co-precipitated with MB-1, we prepared a mAb (19-14), which can immunoprecipitate the p52 and the associated kinase molecule. Immune complex kinase assay with the 19-14 mAb showed that the p52 is associated with a novel kinase molecule and is involved in IgR-mediated signal transduction. Here we isolated a cDNA clone (alpha 4) from a murine bone marrow cDNA library in lambda gt-11 vector by the 19-14 mAb. The alpha 4 cDNA encodes a novel protein of 340 amino acids with multiple potential phosphorylation sites. The 1.4-kb alpha 4 mRNA is expressed in various cells including cell lines of B lineage, T lineage, monocytic, fibroblast, and normal organs such as liver, spleen, thymus, and brain. To study the molecule encoded by the alpha 4 cDNA, we produced a chimeric protein of the glutathione S transferase (GST)-alpha 4 in pGEX-3X expression vector. The 19-14 mAb binds to the GST-alpha 4 fusion protein. Rabbit anti-alpha 4 Ab prepared by immunizing the GST-alpha 4 fusion protein immunoprecipitated a 52-kDa phosphoprotein from WEHI-231 cells. The 52-kDa phosphoprotein immunoprecipitated by the anti-alpha 4 Ab is tightly associated with kinase activity that is similarly observed with the p52 protein reported previously. Moreover, the 52-kDa phosphoprotein immunoprecipitated with the anti-alpha 4 Ab is identical to the p52 by the protein comparison on non-equilibrium pH gradient gel electrophoresis/SDS-PAGE, isoelectric focusing/SDS-PAGE, and V8 protease mapping. The 52-kDa phosphoprotein is associated with functional components that are inducibly phosphorylated by anti-IgM stimulation. These results suggested that the alpha 4 gene-encoded molecule is functionally involved in the IgR-mediated signal transduction in B cells.
我们之前已经表明，一种与Ig受体（IgR）相关的MB-1蛋白共沉淀的52-kDa磷蛋白（p52）可被12-O-十四烷酰佛波醇-13-乙酸酯刺激诱导磷酸化。通过对与MB-1共沉淀的52-kDa蛋白进行免疫，我们制备了一种单克隆抗体（19-14），它可以免疫沉淀p52和相关的激酶分子。用19-14单克隆抗体进行的免疫复合物激酶分析表明，p52与一种新型激酶分子相关，并参与IgR介导的信号转导。在这里，我们通过19-14单克隆抗体从λgt-11载体中的小鼠骨髓cDNA文库中分离出一个cDNA克隆（α4）。α4 cDNA编码一种含有多个潜在磷酸化位点的340个氨基酸的新型蛋白质。1.4-kb的α4 mRNA在包括B细胞系、T细胞系、单核细胞、成纤维细胞的细胞系以及肝脏、脾脏、胸腺和大脑等正常器官的各种细胞中表达。为了研究由α4 cDNA编码的分子，我们在pGEX-3X表达载体中产生了谷胱甘肽S转移酶（GST）-α4的嵌合蛋白。19-14单克隆抗体与GST-α4融合蛋白结合。通过对GST-α4融合蛋白进行免疫制备的兔抗α4抗体从WEHI-231细胞中免疫沉淀出一种52-kDa磷蛋白。抗α4抗体免疫沉淀的52-kDa磷蛋白与激酶活性紧密相关，这与之前报道的p52蛋白情况类似。此外，通过非平衡pH梯度凝胶电泳/SDS-PAGE、等电聚焦/SDS-PAGE和V8蛋白酶图谱分析进行蛋白质比较，抗α4抗体免疫沉淀的52-kDa磷蛋白与p52相同。52-kDa磷蛋白与可被抗IgM刺激诱导磷酸化的功能成分相关。这些结果表明，α4基因编码的分子在功能上参与了B细胞中IgR介导的信号转导。
