Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan.
FEBS J. 2013 Oct;280(20):5145-59. doi: 10.1111/febs.12489. Epub 2013 Sep 12.
Superoxide production by Nox1, a member of the Nox family NAPDH oxidases, requires expression of its regulatory soluble proteins Noxo1 (Nox organizer 1) and Noxa1 (Nox activator 1) and is markedly enhanced upon cell stimulation with phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C (PKC). The mechanism underlying PMA-induced enhancement of Nox1 activity, however, remains to be elucidated. Here we show that, in response to PMA, Noxo1 undergoes phosphorylation at multiple sites, which is inhibited by the PKC inhibitor GF109203X. Among them, Thr341 in Noxo1 is directly phosphorylated by PKC in vitro, and alanine substitution for this residue reduces not only PMA-induced Noxo1 phosphorylation but also PMA-dependent enhancement of Nox1-catalyzed superoxide production. Phosphorylation of Thr341 allows Noxo1 to sufficiently interact with Noxa1, an interaction that participates in Nox1 activation. Thus phosphorylation of Noxo1 at Thr341 appears to play a crucial role in PMA-elicited activation of Nox1, providing a molecular link between PKC-mediated signal transduction and Nox1-catalyzed superoxide production. Furthermore, Ser154 in Noxo1 is phosphorylated in both resting and PMA-stimulated cells, and the phosphorylation probably participates in a PMA-independent constitutive activity of Nox1. Ser154 may also be involved in protein kinase A (PKA) mediated regulation of Nox1; this serine is the major residue that is phosphorylated by PKA in vitro. Thus phosphorylation of Noxo1 at Thr341 and at Ser154 appears to regulate Nox1 activity in different manners.
Noxo1 binds to p22phox by pull down (1, 2, 3) Noxo1 binds to Noxo1 by pull down (View interaction) Noxa1 binds to Noxo1 by pull down (1, 2, 3, 4, 5).
Nox1 是 NAPDH 氧化酶 Nox 家族的成员，其超氧化物的产生需要其调节可溶性蛋白 Noxo1（Nox 组织蛋白 1）和 Noxa1（Nox 激活蛋白 1）的表达，并在细胞受到佛波醇 12-肉豆蔻酸 13-乙酸酯（PMA）刺激时明显增强，PMA 是蛋白激酶 C（PKC）的有效激活剂。然而，PMA 诱导的 Nox1 活性增强的机制仍有待阐明。在这里，我们表明，在 PMA 作用下，Noxo1 在多个位点发生磷酸化，PKC 抑制剂 GF109203X 可抑制该磷酸化。其中，Noxo1 中的 Thr341 可被 PKC 在体外直接磷酸化，且该残基的丙氨酸取代不仅降低了 PMA 诱导的 Noxo1 磷酸化，还降低了 PMA 依赖性 Nox1 催化的超氧化物产生的增强。Thr341 的磷酸化使 Noxo1 与 Noxa1 充分相互作用，该相互作用参与了 Nox1 的激活。因此，Noxo1 的 Thr341 磷酸化似乎在 PMA 诱导的 Nox1 激活中起着至关重要的作用，为 PKC 介导的信号转导与 Nox1 催化的超氧化物产生之间提供了分子联系。此外，Noxo1 中的 Ser154 在静止和 PMA 刺激的细胞中均被磷酸化，且磷酸化可能参与了 Nox1 的 PMA 非依赖性组成性活性。Ser154 也可能参与蛋白激酶 A（PKA）对 Nox1 的调节；该丝氨酸是 PKA 在体外磷酸化的主要残基。因此，Noxo1 的 Thr341 和 Ser154 磷酸化似乎以不同的方式调节 Nox1 的活性。
