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

用含有SV40大T抗原基因或腺病毒E1A基因的逆转录病毒感染新生大鼠脑原代培养细胞，使其永生化。通过基于聚合酶链反应的方法克隆了永生化细胞中表达的编码蛋白酪氨酸激酶（PTK）的cdna。局灶黏附激酶（FAK）亚家族的第二个PTK，我们称之为cakβ（细胞黏附激酶β），被鉴定为由cdna编码的PTK之一。从cDNA克隆中推导出大鼠cak - β氨基酸的完整序列。CAKbeta是一个大的（115.7 kda） PTK，包含418和330个氨基酸残基的n端和c端结构域，以及261个氨基酸的中央激酶结构域。cakβ催化结构域的氨基酸序列与小鼠FAK的相同度为60%。这两个蛋白除了n端末端的88个残基外，在整个长度上具有同源性，并且具有45%的序列一致性。与FAK基因相比，cak β基因在大鼠各种器官中的表达不均匀。抗CAKbeta抗体亲和纯化自兔抗血清，特异免疫沉淀来自大鼠脑、3Y1细胞和转染CAKbeta cDNA的COS-7细胞的113-kDa蛋白。免疫复合物激酶试验显示cakβ的磷酸化。在3Y1细胞中，酪氨酸磷酸化的CAKb状态在胰蛋白酶化过程中没有降低，也没有在将细胞镀在纤维连接蛋白上时增强。在转染表位标记CAKbeta cDNA的免疫染色COS-7细胞中，用共聚焦激光扫描显微镜研究了CAKbeta的亚细胞定位。CAKbeta定位于细胞间接触的位置，在细胞的中间到上部。这种定位与FAK不同，FAK位于细胞底部。因此，CAKbeta是一种可能参与细胞间接触调节的信号转导的PTK。

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.