Compensatory phosphorylation and protein-protein interactions revealed by loss of function and gain of function mutants of multiple-serine phosphorylation sites in endothelial nitric-oxide synthase
Compensatory phosphorylation and protein-protein interactions revealed by loss of function and gain of function mutants of multiple-serine phosphorylation sites in endothelial nitric-oxide synthase
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DOI：
10.1074/jbc.m211926200
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发表时间：
2003-04-25
影响因子：
4.8
通讯作者：
Sessa, WC
中科院分区：
文献类型：
--
作者：
Bauer, PM;Fulton, D;Sessa, WC
我们研究了内皮一氧化氮合酶（eNOS）中单个丝氨酸磷酸化位点对基础和刺激NO释放、协同磷酸化以及与hsp90和Akt的共关联的影响。丝氨酸磷酸化位点116、617和1179对丙氨酸的突变影响了至少一个其他位点的磷酸化状态，表明多个磷酸化事件之间存在合作关系，而丝氨酸635对丙氨酸的突变没有引起补偿。丝氨酸116和617突变为丙氨酸，促进了与hsp90和Akt之间更大的蛋白相互作用，丝氨酸1179 （Akt磷酸化的主要位点）磷酸化。更重要的是，使用丙氨酸取代，Ser-116对激动剂很重要，但不是基础的NO释放，Ser-635对基础的NO释放很重要，但不是刺激的，Ser-617负调节基础和刺激的NO释放，Ser-1179磷酸化对基础和激动剂介导的NO释放都有刺激作用。使用假定的“功能增益”突变体（丝氨酸到天冬氨酸），丝氨酸635和1179是基础和刺激NO释放的重要正调节因子。S635D eNOS是最有效的，使细胞的基础NO释放增加5倍，刺激NO释放增加2倍。然而，在NOS活性测定中，S617A和S617D eNOS均增加了NO释放，但作用相反。因此，多个丝氨酸磷酸化事件调节基础和刺激NO释放，Ser-635和Ser-1179是重要的正调控位点，Ser-116是负调控位点。Ser-617可能对直接调节NO释放并不重要，但作为其他位点磷酸化和蛋白-蛋白相互作用的调节剂很重要。
We examined the influence of individual serine phosphorylation sites in endothelial nitric-oxide synthase (eNOS) on basal and stimulated NO release, cooperative phosphorylation, and co-association with hsp90 and Akt. Mutation of the serine phosphorylation sites 116, 617, and 1179 to alanines affected the phospho-state of at least one other site, demonstrating cooperation between multiple phosphorylation events, whereas mutation of serine 635 to alanine did not cause compensation. Mutation of serines 116 and 617 to alanine promoted a greater protein-protein interaction with hsp90 and Akt and greater phosphorylation on serine 1179, the major site for Akt phosphorylation. More importantly, using alanine substitutions, Ser-116 is important for agonist, but not basal NO release, Ser-635 is important for basal, but not stimulated, Ser-617 negatively regulates basal and stimulated NO release, and Ser-1179 phosphorylation is stimulatory for both basal and agonist-mediated NO release. Using putative "gain of function" mutants (serine to aspartate) serines 635 and 1179 are important positive regulators of basal and stimulated NO release. S635D eNOS is the most efficacious, yielding 5-fold increases in basal and 2-fold increases in stimulated NO release from cells. However, S617A and S617D eNOS both increased NO release with opposite actions in NOS activity assays. Thus, multiple serine phosphorylation events regulate basal and stimulate NO release with Ser-635 and Ser-1179 being important positive regulatory sites and Ser-116 as a negative regulatory. Ser-617 may not be important for directly regulating NO release but is important as a modulator of phosphorylation at other sites and protein-protein interactions.