Simultaneous visualization of the translocation of protein kinase Cα-green fluorescent protein hybrids and intracellular calcium concentrations
Simultaneous visualization of the translocation of protein kinase Cα-green fluorescent protein hybrids and intracellular calcium concentrations
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DOI：
10.1042/0264-6021:3370211
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发表时间：
1999-01-15
影响因子：
4.1
通讯作者：
Tullin, S
中科院分区：
文献类型：
--
作者：
Almholt, K;Arkhammar, POG;Tullin, S
蛋白激酶C （PKC α）的α亚型是一种普遍存在的蛋白激酶，一经激活，可迅速从细胞质转运到质膜。为了跟踪这种易位，PKC α被绿色荧光蛋白的高荧光衍生物标记，并在过表达毒蕈碱1型受体的幼鼠肾细胞中稳定表达。添加激动剂氨甲酰胆碱可在1 s内触发易位。半最大易位和最大易位分别发生在3 s和15 s左右。融合蛋白与质膜的结合是短暂的，约45 s内蛋白返回细胞质。一项高分辨率研究显示，标记PKC α的细胞质分布几乎均匀。在未受刺激的细胞中，几乎完全转移到质膜，以响应磷脂酯，PMA。同时可视化单细胞内钙浓度（[Ca2+](i)）和PKC α易位显示，在中、高浓度激动剂下，这些参数之间存在良好的相关性。在低激动剂浓度下，观察到[Ca2+](i)的少量增加，没有检测到PkC α的易位。相比之下，PMA诱导PKC α易位而不增加[Ca2+](i)。细胞松弛素D和秋碱都不影响标记PKC α的分布或钙依赖性易位，这表明PKC α易位可能独立于肌动蛋白丝和微管。PKC α易位的时间过程与该蛋白从细胞质定位到质膜的扩散是一致的。
The alpha isoform of protein kinase C (PKC alpha) is a ubiquitous protein kinase, which, upon activation, translocates rapidly from the cytoplasm to the plasma membrane. To follow this translocation, PKC alpha was tagged with a highly fluorescent derivative of green fluorescent protein and stably expressed in baby hamster kidney cells overexpressing the muscarinic type 1 receptor. Addition of the agonist carbamylcholine triggered the onset of translocation within 1 s. Half-maximal and maximal translocation occurred after about 3 and 15 s respectively. Plasma membrane association of the fusion protein was transient and the protein returned to the cytoplasm within about 45 s. A high-resolution study showed an almost homogeneous cytoplasmic distribution of tagged PKC alpha. in unstimulated cells and virtually complete translocation to the plasma membrane in response to the phorbol ester, PMA. Simultaneous visualization of intracellular calcium concentration ([Ca2+](i)) and PKC alpha translocation in single cells showed a good correlation between these parameters at intermediate and high concentrations of agonist. At low agonist concentration, a small increase in [Ca2+](i) was observed, without detectable translocation of PkC alpha. In contrast, PMA induced translocation of PKC alpha without any increase in [Ca2+](i). Neither cytochalasin D nor colcemid influenced the distribution or calcium-dependent translocation of tagged PKC alpha, indicating that PKC alpha translocation may be independent of both actin filaments and microtubules. The time course of PKC alpha translocation is compatible with diffusion of the protein from its cytoplasmic localization to the plasma membrane.