Targeting of endothelial nitric-oxide synthase to the cytoplasmic face of the Golgi complex or plasma membrane regulates Akt- versus calcium-dependent mechanisms for nitric oxide release
Targeting of endothelial nitric-oxide synthase to the cytoplasmic face of the Golgi complex or plasma membrane regulates Akt- versus calcium-dependent mechanisms for nitric oxide release
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DOI：
10.1074/jbc.m402155200
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发表时间：
2004-07-16
影响因子：
4.8
通讯作者：
Sessa, WC
中科院分区：
文献类型：
--
作者：
Fulton, D;Babbitt, R;Sessa, WC
内皮型一氧化氮合酶（eNOS）在高尔基复合体上的异质性定位与质膜相比，使得很难剖析每个酶池的调节。在这里，我们产生的融合蛋白，专门针对质膜或高尔基复合体的细胞质方面，并评估eNOS激活。质膜靶向eNOS结构组成型活性，磷酸化，并响应跨膜钙通量，但不敏感Akt介导的磷酸化进一步激活。与此相反，顺式高尔基体复合体靶向eNOS的行为类似于野生型eNOS，是不太敏感的钙依赖性激活和高度响应Akt依赖性磷酸化相比，质膜版本。在质膜和高尔基复合体靶向的构建体中，Ser（1179）对NO的产生至关重要。这项研究为质膜和高尔基复合体定位的eNOS的功能作用提供了明确的证据，并支持了这样的概念：被认为仅在细胞质膜中调节和发挥功能的蛋白质确实可以在内部高尔基膜中发出信号并受到调节。
The heterogeneous localization of endothelial nitric-oxide synthase (eNOS) on the Golgi complex versus the plasma membrane has made it difficult to dissect the regulation of each pool of enzyme. Here, we generated fusion proteins that specifically target the plasma membrane or cytoplasmic aspects of the Golgi complex and have assessed eNOS activation. Plasma membrane-targeted eNOS constructs were constitutively active, phosphorylated, and responsive to transmembrane calcium fluxes, yet were insensitive to further activation by Akt-mediated phosphorylation. In contrast, cis-Golgi complex-targeted eNOS behaved similarly to wild-type eNOS and was less sensitive to calcium-dependent activation and highly responsive to Akt-dependent phosphorylation compared with plasma membrane versions. In plasma membrane- and Golgi complex-targeted constructs, Ser(1179) is critical for NO production. This study provides clear evidence for functional roles of plasma membrane- and Golgi complex-localized eNOS and supports the concept that proteins thought to be regulated and to function exclusively in the plasma membrane of cells can indeed signal and be regulated in internal Golgi membranes.