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.

蛋白激酶和蛋白质磷酸酶已被认为在细胞信号转导中起关键作用。分子生物学技术的最新进展表明，这些酶中的许多具有不同基因编码的亚型。在这项研究中，我们旨在建立细胞系，这些细胞系特别表达了这些酶的亚型，以阐明这些亚型的生理作用。获得的结果如下：1）在大肠杆菌中表达钙调神经磷酸酶（α和β）催化亚基的两个亚型，并通过将它们注入兔子来制备特定的抗体。使用这些特定抗体研究了这两种亚型在大鼠脑中的分布。还使用这些抗体使用免疫沉淀样品研究了FK506/FKBP复合物对含有这些亚型钙调神经蛋白活性的影响。 2）我们克隆了一个cDNA，该cDNA编码了钙调蛋白调节亚基的新型睾丸特异性亚型，提供了一个证据，表明至少有两个不同的基因编码钙调蛋白的调节亚基。 3）我们研究了蛋白激酶A（alpha和beta）催化亚基的两个亚型，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的各种亚型在Nigro - 纹状体途径中的分布。 5）研究了在神经组织发育过程中蛋白激酶C亚型表达的变化，并建议每个亚型的个体发育。 6）在异爪蟾卵母细胞中表达了GABAB受体和蛋白激酶C，并表明GABA_B受体的功能受蛋白激酶C的调节。