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

1.蛋白激酶C（PKC）的磷酸化是其下调的必要条件。使用这些细胞检查PKCalpha的下调。其中在推定的ATP结合位点的Lys-368被精氨酸取代的点突变赋予COS和3 Y1细胞增强的佛波酯结合活性。与内源性和外源性表达的野生型PKCalpha一样，当用佛波醇酯（TPA）处理细胞时，突变体从胞质溶胶易位到颗粒部分。然而，突变型PKC在TPA处理后不降解，与野生型PKC形成鲜明对比。该突变体对TPA诱导的下调具有抗性。突变体缺乏激酶活性，如预期的，通过自磷酸化和激酶测定判断。PKC的自磷酸化是与PKC下调相关的蛋白水解切割的必要条件。新的钙非依赖性蛋白激酶（nPKC）也参与生理信号。在GH4 C1细胞中，nPKC α与PKCalpha和β II一起表达。当细胞用促甲状腺激素释放激素（TRH）处理时，nPKC β从胞质溶胶易位到细胞膜并下调，而其它常规PKC不下调。因此，在GH4 C1细胞中，nPKC α在TRH和佛波醇酯引起的细胞反应中起着与钙依赖性PKCalpha和β II不同的关键作用。

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.