Direct visualization of the translocation of the gamma-subspecies of protein kinase C in living cells using fusion proteins with green fluorescent protein.
Direct visualization of the translocation of the gamma-subspecies of protein kinase C in living cells using fusion proteins with green fluorescent protein.
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使用带有绿色荧光蛋白的融合蛋白在活细胞中蛋白激酶C的伽马脉冲的转运直接可视化。
DOI：
10.1083/jcb.139.6.1465
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发表时间：
1997-12-15
影响因子：
7.8
通讯作者：
Saito, N
中科院分区：
文献类型：
--
作者：
Sakai, N;Sasaki, K;Ikegaki, N;Shirai, Y;Ono, Y;Saito, N
我们在不同的细胞系中表达了与绿色荧光蛋白融合的蛋白激酶C的γ亚种(γ-PKC)，并在激光共聚焦扫描荧光显微镜下观察了该融合蛋白在活细胞中的运动。γ-PKC-GFP融合蛋白具有与天然γ-PKC非常相似的酶学性质。γ-PKC-GFP在瞬时转染的COS7细胞胞浆内可观察到荧光。活性佛波酯(12-O-十四酰佛波醇13-乙酸酯)刺激γ-PKC-GFP，而非活性佛波酯(4-佛波酯-12，13-十二酸佛波酯)则不刺激，可将PKC-GFP从细胞质转移到细胞膜。钙离子载体A23187诱导γ-PKC-GFP的转位比TPA更快。A23187诱导的易位可通过消除细胞外和细胞内的钙离子而被取消。TPA诱导的γ-PKC-GFP转位是单向的，而钙离子载体诱导的转位是可逆的，即γ-PKC-GFP转位到细胞膜，返回细胞质，最后以斑点状聚集在质膜上。为了探讨γ-PKC的C1和C2结构域在易位中的意义，我们表达了突变的γ-PKC-GFP融合蛋白，其中C1区的两个富含半胱氨酸区被破坏(命名为BS238)或C2区被缺失(BS239)。突变体BS238可被钙离子载体转运，但不能被TPA转运。突变体BS239可被TPA转位，但不能被钙离子载体转位。为了研究γ-PKC-GFP在生理条件下的转位情况，我们在NG-108细胞、N-甲基-d-天冬氨酸受体转导的COS7细胞和表达代谢型谷氨酸受体1的CHO细胞中进行了表达。在NG-108细胞中，K+去极化引起γ-PKC-GFP的快速易位。在转导N-甲基-D-天冬氨酸受体的COS-7细胞中，N-甲基-D-天冬氨酸加甘氨酸也使γ-PKC-GFP易位。此外，在CHO/mGluR1细胞中观察到γ-PKC-GFP在受体刺激下的快速转位和顺序转位。细胞松弛素D和秋水仙碱均不影响γ-PKC-GFP的易位，说明γ-PKC的易位不依赖于肌动蛋白和微管。γ-PKC-GFP融合蛋白是研究γ-PKC易位的分子机制以及γ-PKC在中枢神经系统中的作用的有用工具。
We expressed the γ-subspecies of protein kinase C (γ-PKC) fused with green fluorescent protein (GFP) in various cell lines and observed the movement of this fusion protein in living cells under a confocal laser scanning fluorescent microscope. γ-PKC–GFP fusion protein had enzymological properties very similar to that of native γ-PKC. The fluorescence of γ-PKC– GFP was observed throughout the cytoplasm in transiently transfected COS-7 cells. Stimulation by an active phorbol ester (12-O-tetradecanoylphorbol 13-acetate [TPA]) but not by an inactive phorbol ester (4α-phorbol 12, 13-didecanoate) induced a significant translocation of γ-PKC–GFP from cytoplasm to the plasma membrane. A23187, a Ca2+ ionophore, induced a more rapid translocation of γ-PKC–GFP than TPA. The A23187-induced translocation was abolished by elimination of extracellular and intracellular Ca2+. TPA- induced translocation of γ-PKC–GFP was unidirected, while Ca2+ ionophore–induced translocation was reversible; that is, γ-PKC–GFP translocated to the membrane returned to the cytosol and finally accumulated as patchy dots on the plasma membrane. To investigate the significance of C1 and C2 domains of γ-PKC in translocation, we expressed mutant γ-PKC–GFP fusion protein in which the two cysteine rich regions in the C1 region were disrupted (designated as BS 238) or the C2 region was deleted (BS 239). BS 238 mutant was translocated by Ca2+ ionophore but not by TPA. In contrast, BS 239 mutant was translocated by TPA but not by Ca2+ ionophore. To examine the translocation of γ-PKC–GFP under physiological conditions, we expressed it in NG-108 cells, N-methyl-d-aspartate (NMDA) receptor–transfected COS-7 cells, or CHO cells expressing metabotropic glutamate receptor 1 (CHO/mGluR1 cells). In NG-108 cells , K+ depolarization induced rapid translocation of γ-PKC–GFP. In NMDA receptor–transfected COS-7 cells, application of NMDA plus glycine also translocated γ-PKC–GFP. Furthermore, rapid translocation and sequential retranslocation of γ-PKC–GFP were observed in CHO/ mGluR1 cells on stimulation with the receptor. Neither cytochalasin D nor colchicine affected the translocation of γ-PKC–GFP, indicating that translocation of γ-PKC was independent of actin and microtubule. γ-PKC–GFP fusion protein is a useful tool for investigating the molecular mechanism of γ-PKC translocation and the role of γ-PKC in the central nervous system.
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DOI：
10.1083/jcb.108.2.553
发表时间：
1989-02
期刊：
The Journal of cell biology
影响因子：
--
作者：
Papadopoulos V;Hall PF
通讯作者：
Hall PF
影响因子：
64.5
作者：
ABELIOVICH, A;CHEN, C;TONEGAWA, S
通讯作者：
TONEGAWA, S
DOI：
10.1073/pnas.78.2.1237
发表时间：
1981-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
ATLAS, D;ADLER, M
通讯作者：
ADLER, M
影响因子：
--
作者：
CHENEY, RE;RILEY, MA;MOOSEKER, MS
通讯作者：
MOOSEKER, MS
DOI：
10.1091/mbc.1.9.693
发表时间：
1990-08-01
期刊：
CELL REGULATION
影响因子：
--
作者：
MOCHLYROSEN, D;HENRICH, CJ;SIMPSON, PC
通讯作者：
SIMPSON, PC