Degradation of protein kinase C alpha and its free catalytic subunit, protein kinase M, in intact human neuroblastoma cells and under cell-free conditions. Evidence that PKM is degraded by mM calpain-mediated proteolysis at a faster rate than PKC.
Degradation of protein kinase C alpha and its free catalytic subunit, protein kinase M, in intact human neuroblastoma cells and under cell-free conditions. Evidence that PKM is degraded by mM calpain-mediated proteolysis at a faster rate than PKC.
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在完整的人神经母细胞瘤细胞和无细胞条件下，蛋白激酶 C α 及其游离催化亚基蛋白激酶 M 的降解。
DOI：
10.1016/0014-5793(94)00769-1
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发表时间：
1994
期刊：
影响因子：
3.5
通讯作者：
Leli,U
中科院分区：
文献类型：
--
作者：
Shea,TB;Beermann,ML;Griffin,WR;Leli,U
蛋白激酶C(PKC)在无细胞条件下的蛋白水解性裂解产生一个不依赖于辅因子的自由催化亚基(PKM)。然而，在完整细胞中可视化PKM的困难已经引起了关于它的生理学相关性的争议。在本研究中，用2-O-十四酰佛波醇13-乙酸酯处理SH-SY-5Y细胞，可在24小时内完全下调PKC，而未检测到PKM。相反，在没有引起可检测到的PKC丢失的条件下，在离子载体介导的钙内流后，瞬时检测到低水平的PKM。部分纯化的SH-SY-5YPKCα经纯化的人脑Mm calain在快速无细胞降解过程中未检测到PKM。然而，当通过降低钙蛋白酶水平来减缓PKC的降解动力学时，PKM被瞬时检测到。在Calain介导的纯化的大鼠脑内PKCα的降解过程中，也只观察到一过性的PKM。密度分析表明，一旦形成，PKM的降解速度大约是PKC的10倍。这些数据解释了为什么PKM很难在原位观察到，并表明PKM不应该被认为是一种‘无调控’的激酶，因为它的持久性显然受到蛋白降解的严格调控。
Proteolytic cleavage of protein kinase C (PKC) under cell-free conditions generates a co-factor independent, free catalytic subunit (PKM). However, the difficulty in visualizing PKM in intact cells has generated controversy regarding its physiological relevance. In the present study, treatment of SH-SY-5Y cells with 2-O-tetradecanoylphorbol 13-acetate resulted in complete down-regulation of PKC within 24 h without detection of PKM. By contrast, low levels of PKM were transiently detected following ionophore-mediated calcium influx under conditions which induced no detectable PKC loss. PKM was not detected during rapid cell-free degradation of partially purified SH-SY-5Y PKCα by purified human brain mM calpain. However, when the kinetics of PKC degradation were slowed by lowering levels of calpain, PKM was transiently detected. PKM was also only transiently observed following calpain-mediated degradation of purified rat brain PKCα. Densitometric analyses indicated that, once formed, PKM was degraded approximately 10 times faster than PKC. These data provide an explanation as to why PKM is difficult to observe in situ, and indicate that PKM should not be considered as an ‘unregulated’ kinase, since its persistence is apparently strictly regulated by proteolysis.