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Function of Calcium-Dependent Protease in Down Regulation of Protein Dinase C

钙依赖性蛋白酶在蛋白激酶C下调中的作用

基本信息

批准号：
01480526
负责人：
SUZUKI Koichi
金额：
$ 3.9万
依托单位：
Tokyo Metropolitan Institute of Medical Science
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-01480526/
关键词：
protein kinase C calcium protease phorbol ester カルシウムイオン

项目摘要

1. Phosphorylation of protein kinase C (PKC) is essential for its down-regulationPKCalpha cDNA was expressed transiently in COS cells or stably in rat fibroblast 3Yl cells. The down regulation of PKCalpha was examined using these cells. A point mutation where Lys-368 at the putative ATP-binding site is replaced with arginine confers enhanced phorbol ester binding activity to both COS and 3Yl cells. Like endogenous and exogenously expressed wild type PKCalpha, the mutant is translocated from the cytosol to the particulate fraction when cells are treated with a phorbol ester (TPA). The mutant PKC is, however, not degraded after the TPA treatment, making a clear contrast to wild type PKC. The mutant is resistant to TPA-induced down-regulation. The mutant lacks kinase activity as expected, as judged by autophosphorylation and by a kinase assay. Autophosphorylation of PKC is a requisite for proteolytic cleavage associated with down-regulation of PKC.2. Novel calcium independent protein kinase (nPKC) also transduce physiological signalIn GH4Cl cells, nPKCepsilon is expressed together with PKCalpha and beta II. nPKC epsilon is translocated to the cell membrane from the cytosol and down regulated when cells are treated with Thyrotropin Releasing Hormone (TRH), whereas other conventional PKC's are not down-regulated. Thus in GH4Cl cells, nPKCepsilon plays a crucial role distinct from that meadiated by calcium-dependent PKCalpha and beta II in cellular response elicited by both TRH and phorbol esters.

1. 蛋白激酶C （PKC）的磷酸化是其下调的关键。pkcalpha cDNA在COS细胞中短暂表达，在大鼠成纤维细胞3Yl细胞中稳定表达。使用这些细胞检测PKCalpha的下调。当假定的atp结合位点上的Lys-368被精氨酸取代时，一个点突变会增强COS和3Yl细胞的磷酸酯结合活性。与内源性和外源性表达的野生型PKCalpha一样，当细胞用磷酸酯（TPA）处理时，突变体从细胞质转移到颗粒部分。然而，突变型PKC在TPA处理后没有降解，与野生型PKC形成明显对比。突变体对tpa诱导的下调具有抗性。正如预期的那样，突变体缺乏激酶活性，可以通过自磷酸化和激酶测定来判断。PKC的自磷酸化是与PKC.2下调相关的蛋白水解裂解的必要条件。在GH4Cl细胞中，nPKCepsilon与PKCalpha和β II一起表达。nPKC epsilon从细胞质转移到细胞膜上，当细胞接受促甲状腺激素释放激素（TRH）处理时，nPKC epsilon下调，而其他常规PKC epsilon则不下调。因此，在GH4Cl细胞中，nPKCepsilon在TRH和磷酯引发的细胞反应中起着与钙依赖性PKCalpha和β II介导的作用不同的关键作用。