The proline-rich domain of dynamin-2 is responsible for dynamin-dependent in vitro potentiation of endothelial nitric-oxide synthase activity via selective effects on reductase domain function
The proline-rich domain of dynamin-2 is responsible for dynamin-dependent in vitro potentiation of endothelial nitric-oxide synthase activity via selective effects on reductase domain function
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DOI：
10.1074/jbc.m212546200
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发表时间：
2003-02-21
影响因子：
4.8
通讯作者：
Shah, V
中科院分区：
文献类型：
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作者：
Cao, S;Yao, J;Shah, V
GTPase dynamin-2 （dyn-2）结合并正向调节一氧化氮生成酶内皮一氧化氮合成酶（eNOS） (Cao， S., Yao， Y., McCabe， T., Yao， Q., Katusic， Z., Sessa， W., and Shah， V. (2001) J. Biol。276年化学,14249 - 14256)。在这里，我们使用纯化蛋白证明，这是通过dyn-2脯氨酸丰富结构域（dyn-2 PRD）对eNOS还原酶结构域的选择性影响发生的。体外研究表明，与谷胱甘肽s转移酶（GST）融合的dyn-2 PRD可以特异性结合重组eNOS蛋白，其结合动力学与dyn-2全长与eNOS的结合动力学相当。此外，GST-dyn-2 PRD结合体外转录的S-35-eNOS还原酶结构域，但不结合35 S-eNOS加氧酶结构域。此外，GST-dyn-2 PRD结合s -35标记的eNOS还原酶结构域片段（氨基酸645-850），该片段与eNOS的FAD结合结构域部分重叠。含sh3蛋白Fyn的重组形式竞争重组eNOS蛋白与dyn-2 PRD的结合，从而暗示该还原酶结构域片段中包含的sh3样区域是dyn-2结合区域。哺乳动物双杂交实验也证实了细胞间的相互作用。功能研究表明，dyn-2 PRD以浓度依赖的方式选择性地增强eNOS活性，其数量级与dyn-2全长所观察到的相似，并且需要钙调素。虽然dyn-2 PRD不影响eNOS加氧酶结构域功能或铁氰化物还原，但它确实增强了重组eNOS降低细胞色素c的能力，支持dyn-2 PRD对FAD和FMN之间电子转移的影响。（这些数据表明dyn-2和eNOS的结合域分别位于dyn-2 PRD结构域和eNOS还原酶结构域的FAD结合区，dyn-2 PRD足以介导eNOS活性的dyn-2依赖性增强，至少部分是通过增强电子转移。）
The GTPase dynamin-2 (dyn-2) binds and positively regulates the nitric oxide-generating enzyme, endothelial nitric-oxide synthase (eNOS) (Cao, S., Yao, Y., McCabe, T., Yao, Q., Katusic, Z., Sessa, W., and Shah, V. (2001) J. Biol. Chem. 276,14249-14256). Here we demonstrate, using purified proteins, that this occurs through a selective influence of the dyn-2 proline-rich domain (dyn-2 PRD) on the eNOS reductase domain. In vitro studies demonstrate that dyn-2 PRD fused with glutathione S-transferase (GST) binds recombinant eNOS protein specifically and with binding kinetics comparable with that observed between dyn-2 full-length and eNOS. Additionally, GST-dyn-2 PRD binds the in vitro transcribed S-35-eNOS reductase domain but not the 35 S-eNOS oxygenase domain. Furthermore GST-dyn-2 PRD binds a S-35-labeled eNOS reductase domain fragment (amino acids 645-850) that partially overlaps with the FAD binding domain of eNOS. A recombinant form of the SH3-containing protein Fyn competes the binding of recombinant eNOS protein with dyn-2 PRD, thereby implicating the SH3-like region contained within this reductase domain fragment as the dyn-2 binding region. Mammalian two-hybrid screen corroborates these interactions in cells as well. Functional studies demonstrate that dyn-2 PRD selectively potentiates eNOS activity in a concentration-dependent manner in an order of magnitude similar to that observed with dyn-2 full-length and in a manner that requires calmodulin. Although dyn-2 PRD does not influence eNOS oxygenase domain function or ferricyanide reduction, it does potentiate the ability of recombinant eNOS to reduce cytochrome c, supporting an influence of dyn-2 PRD on electron transfer between FAD and FMN. (These data indicate that the binding domains of dyn-2 and eNOS reside within the dyn-2 PRD domain and the FAD binding region of the eNOS reductase domains, respectively, and that dyn-2 PRD is sufficient to mediate dyn-2-dependent potentiation of eNOS activity, at least in part, by potentiating electron transfer.).