Intracellular location regulates calcium-calmodulin-dependent activation of organelle-restricted eNOS
Intracellular location regulates calcium-calmodulin-dependent activation of organelle-restricted eNOS
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DOI：
10.1152/ajpcell.00162.2005
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发表时间：
2005-10-01
影响因子：
5.5
通讯作者：
Fulton, D
中科院分区：
文献类型：
--
作者：
Jagnandan, D;Sessa, WC;Fulton, D
内皮型一氧化氮合酶（eNOS）对氧化低密度脂蛋白、胆固醇耗竭、血压升高和结合eNOS相互作用蛋白/NOS交通诱导剂的应答定位错误与通过未知机制减少的NO释放有关。eNOS对质膜或细胞内细胞器的适当靶向是控制酶活性的重要调节步骤。以往的研究表明，质膜eNOS是组成性磷酸化的丝氨酸1179和高活性。相比之下，eNOS靶向细胞内细胞器的活性更为复杂。顺式高尔基体eNOS被Akt依赖性磷酸化完全激活。然而，eNOS针对反式高尔基体是决定性的不太活跃的所有模式的激活，包括突变的磷酸模拟天冬氨酸。在这项研究中，我们确定，当表达在其他细胞内的细胞器，如线粒体和细胞核，eNOS的活性也大大降低。为了解决这些位置内eNOS催化活性受损的机制，我们产生了表达钙非依赖性NOS亚型iNOS的亚细胞靶向结构。使用细胞器特异性（质膜，顺式与反式高尔基体，质膜，高尔基体，细胞核，和线粒体）靶向基序融合到野生型iNOS，我们测量NO从完整的细胞释放。除高尔基体腔，我们的研究结果表明，没有损害的能力，有针对性的iNOS合成NO。确认正确的目标是通过共聚焦显微镜使用相同的结构融合到绿色荧光蛋白。我们的结论是，减少激活的eNOS离散的细胞质区域内的高尔基体，线粒体和细胞核主要是由于钙-钙调蛋白的访问不足。
Mislocalization of endothelial nitric oxide (NO) synthase (eNOS) in response to oxidized low-density lipoprotein, cholesterol depletion, elevated blood pressure, and bound eNOS interacting protein/NOS traffic inducer is associated with reduced NO release via unknown mechanisms. The proper targeting of eNOS to the plasma membrane or intracellular organelles is an important regulatory step controlling enzyme activity. Previous studies have shown that plasma membrane eNOS is constitutively phosphorylated on serine 1179 and highly active. In contrast, the activity of eNOS targeted to intracellular organelles is more complex. The cis-Golgi eNOS is fully activated by Akt-dependent phosphorylation. However, eNOS targeted to the trans-Golgi is decidedly less active in response to all modes of activation, including mutation to the phosphomimetic aspartic acid. In this study, we establish that when expressed within other intracellular organelles, such as the mitochondria and nucleus, the activity of eNOS is also greatly reduced. To address the mechanisms underlying the impaired catalytic activity of eNOS within these locations, we generated subcellular-targeted constructs that express a calcium-independent NOS isoform, iNOS. With the use of organelle specific (plasma membrane, cis- vs. trans-Golgi, plasma membrane, and Golgi, nucleus, and mitochondria) targeting motifs fused to the wild-type iNOS, we measured NO release from intact cells. With the exception of the Golgi lumen, our results showed no impairment in the ability of targeted iNOS to synthesize NO. Confirmation of correct targeting was obtained through confocal microscopy using identical constructs fused to the green fluorescent protein. We conclude that the reduced activation of eNOS within discrete cytoplasmic regions of the Golgi, the mitochondria and the nucleus is primarily due to insufficient access to calcium-calmodulin.