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Establishment of cell lines which specifically express a subtype of protein kinases and protein phosphatases and its application

特异表达蛋白激酶和蛋白磷酸酶亚型细胞系的建立及其应用

基本信息

批准号：
03557011
负责人：
TANAKA Chikako
金额：
$ 11.58万
依托单位：
Kobe University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

项目摘要

Protein Kinases and protein phosphatases have been thought to play pivotal roles in cellular signal transduction. Recent advances in molecular biological techniques have revealed that many of these enzymes have several subtypes coded by distinct genes. In this study, we aimed to establish cell lines which specifically express a subtype of these enzymes to clarify the physiological role of these subtypes. Obtained results are as follows: 1) Two subtypes of the catalytic subunit of calcineurin (alpha and beta) were expressed in E. coli and specific antibodies were prepared by injecting them to rabbit. Distribution of these two subtypes in the rat brain were studied immunohistochemically using these specific antibodies. Effects of FK506/FKBP complex on the activity of calcineurin containing these subtypes were also studied using immunoprecipitated samples by these antibodies. 2) We cloned a cDNA which encodes a novel testis-specific subtype of the regulatory subunit of calcineurin, providing the evidence that there are at least two distinct genes coding the regulatory subunit of calcineurin. 3) We cloned two subtypes of the catalytic subunit of protein kinase A (alpha and beta), and their distribution in various rat tissues and various cell lines were studied. 4) Distribution of various subtypes of protein kinase C in nigro-striatal pathway was studied. 5) Change in the expression of protein kinase C subtypes during development of nervous tissue was studied and it is suggested that ontogeny of each subtype is different. 6) GABAB receptor and protein kinase C were expressed in Xenopus oocyte and it is shown that the function of GABA_B receptor is regulated by protein kinase C.

蛋白激酶和蛋白磷酸酶被认为在细胞信号转导中发挥着关键作用。分子生物学技术的最新进展表明，许多酶具有由不同基因编码的几种亚型。在这项研究中，我们的目的是建立特异性表达这些酶的亚型的细胞系，以阐明这些亚型的生理作用。所得结果如下： 1)在大肠杆菌中表达钙调神经磷酸酶催化亚基的两种亚型（α和β），并注射至家兔体内制备特异性抗体。使用这些特异性抗体通过免疫组织化学研究了这两种亚型在大鼠大脑中的分布。还使用这些抗体的免疫沉淀样品研究了 FK506/FKBP 复合物对含有这些亚型的钙调神经磷酸酶活性的影响。 2)我们克隆了编码钙调磷酸酶调节亚基的新型睾丸特异性亚型的cDNA，提供了至少有两个不同的基因编码钙调磷酸酶调节亚基的证据。 3）我们克隆了蛋白激酶A催化亚基的两种亚型（α和β），并研究了它们在各种大鼠组织和各种细胞系中的分布。 4)研究了蛋白激酶C各亚型在黑质-纹状体通路中的分布。 5)研究了神经组织发育过程中蛋白激酶C亚型表达的变化，提示各亚型的个体发育不同。 6) GABAB受体和蛋白激酶C在非洲爪蟾卵母细胞中表达，表明GABA_B受体的功能受蛋白激酶C的调节。