Protein kinase Cα targeting is regulated by temporal and spatial changes in intracellular free calcium concentration [Ca2+]i
Protein kinase Cα targeting is regulated by temporal and spatial changes in intracellular free calcium concentration [Ca2+]i
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蛋白激酶 Cα 靶向受细胞内游离钙浓度 [Ca2+]i 的时间和空间变化调节
DOI：
10.1096/fj.99-0403com
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
H. Haller
中科院分区：
文献类型：
--
作者：
C. Maasch;S. Wagner;C. Lindschau;G. Alexander;K. Buchner;M. Gollasch;F. Luft;H. Haller
蛋白激酶C （PKC）异构体在细胞内发挥特异性功能，但不同的异构体在体外表现出很少的底物特异性。选择性PKC异构体靶向可能是实现特异性的一种机制。我们使用绿色荧光融合蛋白（GFP）来验证[Ca2+]i的局部变化调节PKCα的易位，以及不同模式的Ca2+和Ca2+释放在PKCα靶向中发挥作用的假设。我们构建了PKCα的缺失突变体来分析Ca2+敏感结构域及其在靶向中的作用。使用共聚焦显微镜，用fluo‐3测定[Ca2+]i。融合蛋白PKCα‐GFP在血管平滑肌细胞中表达，其胞质分布与野生型PKCα蛋白相似。Ca2+离子载体离子霉素诱导细胞内PKCα - GFP分布呈斑点状，随后发生膜易位，而KCl去极化主要诱导膜易位。选择性电压操作的Ca2+通道打开导致PKCα - GFP在质膜附近的局部积累。用InsP3、thapsigargin或ryanodine打开Ca2+存储诱导特异性PKCα - GFP靶向不同的细胞内区域。G蛋白偶联受体激动剂凝血酶诱导融合蛋白快速易位到病灶结构域。酪氨酸激酶受体激动剂PDGF诱导Ca2+内流并导致PKCα - GFP膜线性关联。PKCα - GFP缺失突变体表明，C2结构域是Ca2+诱导的PKCα靶向所必需的，而不是催化亚基。当ATP结合位点被删除时，靶向性也被取消。我们得出结论，PKCα可以通过局部增加[Ca2+]i迅速易位到不同的细胞内或膜结构域。靶向机制依赖于酶的C2和ATP结合位点。局部[Ca2+]i的变化决定PKCα的时空靶向性。-Maasch， C, Wagner， S., Lindschau， C., Alexander， G., Buchner， K., Gollasch， M., Luft， F. C., Haller， H.细胞内游离钙浓度[Ca2+]时空变化对蛋白激酶Cα靶向的调控[j]。中华医学杂志14,1653-1663 （2000）
Protein kinase C (PKC) isoforms exert specific intracellular functions, but the different isoforms display little substrate specificity in vitro. Selective PKC isoform targeting may be a mechanism to achieve specificity. We used a green fluorescent fusion protein (GFP) to test the hypothesis that local changes in [Ca2+]i regulate translocation of PKCα and that different modes of Ca2+ and Ca2+ release play a role in PKCα targeting. We constructed deletion mutants of PKCα to analyze the Ca2+‐sensitive domains and their role in targeting. Confocal microscopy was used and [Ca2+]i was measured by fluo‐3. The fusion protein PKCα‐GFP was expressed in vascular smooth muscle cells and showed a cytosolic distribution similar to the wildtype PKCα protein. The Ca2+ ionophore ionomycin induced a speckled cytosolic PKCα‐GFP distribution, followed by membrane translocation, while depolarization by KCl induced primarily membrane translocation. Selective voltage‐operated Ca2+ channel opening led to a localized accumulation of PKCα‐GFP near the plasma membrane. Opening Ca2+ stores with InsP3, thapsigargin, or ryanodine induced a specific PKCα‐GFP targeting to distinct intracellular areas. The G‐protein‐coupled receptor agonist thrombin induced a rapid translocation of the fusion protein to focal domains. The tyrosine kinase receptor agonist PDGF induced Ca2+ influx and led to a linear PKCα‐GFP membrane association. PKCα‐GFP deletion mutants demonstrated that the C2 domain, but not the catalytic subunit, is necessary for Ca2+‐induced PKCα targeting. Targeting was also abolished when the ATP binding site was deleted. We conclude that PKCα can rapidly be translocated to distinct intracellular or membrane domains by local increases in [Ca2+]i. The targeting mechanism is dependent on the C2 and ATP binding site of the enzyme. Localized [Ca2+]i changes determine the spatial and temporal targeting of PKCα.–Maasch, C., Wagner, S., Lindschau, C., Alexander, G., Buchner, K., Gollasch, M., Luft, F. C., Haller, H. Protein kinase Cα targeting is regulated by temporal and spatial changes in intracellular free calcium concentration [Ca2+]i. FASEB J. 14, 1653–1663 (2000)
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牛主动脉内皮细胞中蛋白激酶 C 的免疫细胞化学表达和定位。
DOI：
10.1016/0006-291x(92)91522-r
发表时间：
1992
影响因子：
3.1
作者：
Rosales,OR;Isales,C;Nathanson,M;Sumpio,BE
通讯作者：
Sumpio,BE
DOI：
10.1096/fasebj.12.1.35
发表时间：
1998
期刊：
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
影响因子：
--
作者：
D. Mochly‐Rosen;A. Gordon
通讯作者：
D. Mochly‐Rosen;A. Gordon
DOI：
10.1016/s0021-9258(18)82231-1
发表时间：
1993-05
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
K. Ha;J. Exton
通讯作者：
K. Ha;J. Exton
影响因子：
3.4
作者：
CANNELL, MB;CHENG, H;LEDERER, WJ
通讯作者：
LEDERER, WJ