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

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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.