Tài liệu Báo cáo khoa học: Expression and function of Noxo1c, an alternative splicing form of the

Expression and function of Noxo1c, an alternative splicing

form of the NADPH oxidase organizer 1

Ryu Takeya1,2, Masahiko Taura1

, Tomoko Yamasaki1

, Seiji Naito3 and Hideki Sumimoto1,2

1 Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan

2 CREST, Japan Science and Technology Agency, Saitama, Japan

3 Department of Urology, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan

Members of the NADPH oxidase (Nox) family pro￾duce superoxide from molecular oxygen in conjunction

with oxidation of NADPH [1–10]. Superoxide gener￾ated serves as a precursor of other reactive oxygen spe￾cies, which are currently considered to be involved

in various physiological processes. The founder Nox

enzyme gp91phox, also termed Nox2, is predominantly

expressed in professional phagocytes, and plays a cru￾cial role in host defense; superoxide generation by

gp91phox leads to subsequent formation of microbicidal

reactive oxygen species such as hydroxyl radical and

hypochlorous acid. Nox1, the second member of the

Nox family, is abundant in the colon and vascular

tissues [11,12] and considered to participate in host

defense at the colon and signaling to cell proliferation

[11,13,14]. Recent studies have revealed that Nox1,

expressed heterologously, associates with the mem￾brane-integrated protein p22phox to form a functional

heterodimer [15,16].

Activation of gp91phox, also complexed with p22phox,

absolutely requires the two cytosolic proteins p47phox

and p67phox. Nox organizer 1 (Noxo1) and Nox

Keywords

NADPH oxidase (Nox1); Nox organiser 1

(Noxo1); PX domain; phosphoinositide

Correspondence

H. Sumimoto, Medical Institute of

Bioregulation, Kyushu University,

Fukuoka 812-8582, Japan

Fax: +81 92 642 6807

Tel: +81 92 642 6806

E-mail: [email protected]

(Received 31 May 2006, accepted 12 June

2006)

doi:10.1111/j.1742-4658.2006.05371.x

Activation of the superoxide-producing NADPH oxidase Nox1 requires

both the organizer protein Noxo1 and the activator protein Noxa1. Here

we describe an alternative splicing form of Noxo1, Noxo1c, which is

expressed in the testis and fetal brain. The Noxo1c protein contains an

additional five amino acids in the N-terminal PX domain, a phosphoinosi￾tide-binding module; the domain plays an essential role in supporting

superoxide production by NADPH oxidase (Nox) family oxidases including

Nox1, gp91phox ⁄ Nox2, and Nox3, as shown in this study. The PX domain

isolated from Noxo1c shows a lower affinity for phosphoinositides than

that from the classical splicing form Noxo1b. Consistent with this, in rest￾ing cells, Noxo1c is poorly localized to the membrane, and thus less effect￾ive in activating Nox1 than Noxo1b, which is constitutively present at the

membrane. On the other hand, cell stimulation with phorbol 12-myristate

13-acetate (PMA), an activator of Nox1–3, facilitates membrane transloca￾tion of Noxo1c; as a result, Noxo1c is equivalent to Noxo1b in Nox1 acti￾vation in PMA-stimulated cells. The effect of the five-amino-acid insertion

in the Noxo1 PX domain appears to depend on the type of Nox; in activa￾tion of gp91phox ⁄ Nox2, Noxo1c is less active than Noxo1b even in the

presence of PMA, whereas Noxo1c and Noxo1b support the superoxide￾producing activity of Nox3 to the same extent in a manner independent of

cell stimulation.

Abbreviations

CHO, Chinese hamster ovary; GST, glutathione S-transferase; HA, hemaglutinin; Nox, NADPH oxidase; Noxo1, Nox organizer 1; Noxa1,

Nox activator 1; PMA, phorbol 12-myristate 13-acetate; PtdIns(3)P, phosphatidylinositol 3-phosphate; PtdIns(4)P, phosphatidylinositol

4-phosphate; PtdIns(3,5)P2, phosphatidylinositol 3,5-bisphosphate; PX, phox homology.

FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS 3663