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Establishment of Neural Cell Lines by Introducing cDNA for Immortalization ; Studies on their Surface Antigens and Growth/Differentiation Factors

通过引入 cDNA 进行永生化建立神经细胞系；

基本信息

批准号：
04807017
负责人：
SASAKI Hiroko
金额：
$ 1.22万
依托单位：
Sapporo Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

Primary cultured cells from newborn rat brain were immortalized by infecting retroviruses containing either SV40 large T antigen gene or adenovirus E1A gene. cDNAs encoding protein-tyrosine kinases (PTK) expressed in the immortalized cells were cloned by the polymerase chain reaction-based approach. A second PTK of the focal adhesion kinase (FAK) subfamily, which we call CAKbeta (cell adhesion kinase beta), was identified as one of the PTKs encoded by the cDNAs. The entire rat CAKbeta amino acid sequence was deduced from cDNA clones. CAKbeta is a large (115.7-kDa) PTK that contains N-terminal and C-terminal domains of 418 and 330 amino acid residues in addition to the central kinase domain of 261 amino acids. The amino acid sequence of the catalytic domain of CAKbeta is 60% identical with that of mouse FAK.The two proteins have a homology over their entire lengths except for the extreme N-terminal 88 residues and share 45% overall sequence identity. The CAKbeta gene is less evenly expressed in a variety of rat organs than the FAK gene. Anti-CAKbeta antibody affinity-purified from rabbit antiserum specifically immunoprecipitated a 113-kDa protein from rat brain, 3Y1 cells, and from COS-7 cells transfected with CAKbeta cDNA.Phosphorylation of CAKbeta was shown in immune-complex kinase assay. The tyrosine-phosphorylated state of CAKb in 3Y1 cells was not reduced on trypsinization, nor enhanced in response to plating the cells onto fibronectin. Subcellular localization of CAKbeta was studied by confocal laser scanning microscopy in the immunostained COS-7 cells transfected with epitope-tagged-CAKbeta cDNA.CAKbeta localized to sites of cell-to-cell contact at the middle to upper portions of the cells. The localization was different from that of FAK,which was found at the bottom of the cells. Thus, CAKbeta is a PTK with a possibility to participate in the signal transduction regulated by cell-to-cell contacts.

用含SV 40大T抗原基因或腺病毒E1 A基因的逆转录病毒感染原代培养的新生大鼠脑细胞，使其永生化。通过聚合酶链反应为基础的方法克隆编码蛋白酪氨酸激酶（PTK）在永生化细胞中表达的cDNA。粘着斑激酶（FAK）亚家族的第二个PTK，我们称之为CAKbeta（细胞粘附激酶β），被鉴定为由cDNA编码的PTK之一。从cDNA克隆中推导出整个大鼠CAK β氨基酸序列。CAKbeta是一种大的（115.7-kDa）PTK，除了包含261个氨基酸的中央激酶结构域外，还包含418个和330个氨基酸残基的N末端和C末端结构域。CAK β的催化结构域的氨基酸序列与小鼠FAK的催化结构域的氨基酸序列有60%的同源性，除N端88个残基外，两者在整个蛋白质长度上具有同源性，总的序列同源性为45%。CAKbeta基因在多种大鼠器官中的表达不如FAK基因均匀。从兔抗血清中亲和纯化的抗CAK β抗体特异性免疫沉淀来自大鼠脑、3 Y1细胞和来自转染CAK β cDNA的COS-7细胞的113-kDa蛋白。酪氨酸磷酸化状态的CAKb在3 Y1细胞中没有减少胰蛋白酶消化，也没有增强在响应平板细胞上纤连蛋白。用共聚焦激光扫描显微镜观察了转染表位标记的CAK β cDNA的COS-7细胞中CAK β的亚细胞定位，结果表明CAK β定位于细胞中上部细胞与细胞接触的部位。其定位与FAK不同，FAK定位于细胞底部。因此，CAKbeta是一种PTK，有可能参与细胞间接触调节的信号转导。