Biochem. J. (2008) 415
(57–65) (Printed in Great Britain)
Activation of NADPH oxidase 1 in tumour colon epithelial cells
Yukio NISIMOTO*1, Ryoko TSUBOUCHI*, Becky A. DIEBOLD†, Shanlou QIAO‡, Hisamitsu OGAWA§, Takuya OHARA¶ and Minoru TAMURA¶
*Department of Biochemistry, Aichi Medical University, School of Medicine, Nagakute, Aichi 480-1195, Japan, †Department of Pathology and Laboratory Medicine, Emory University Medical School, Atlanta, GA 30322, U.S.A., ‡Department of Biomedical Sciences, Chubu University, College of Life and Health Sciences, Kasugai, Aichi 487-8501, Japan, §Department of Biology, Fujita Health University, School of Medicine, Toyoake, Aichi 470-1192, Japan, and ¶Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama, Ehime 790-8577, Japan
In the plasma membrane fraction from Caco-2 human colon carcinoma cells, active Nox1 (NADPH oxidase 1) endogenously co-localizes with its regulatory components p22phox, NOXO1, NOXA1 and Rac1. NADPH-specific superoxide generating activity was reduced by 80% in the presence of either a flavoenzyme inhibitor DPI (diphenyleneiodonium) or NADP+. The plasma membranes from PMA-stimulated cells showed an increased amount of Rac1 (19.6 pmol/mg), as compared with the membranes from unstimulated Caco-2 cells (15.1 pmol/mg), but other components did not change before and after the stimulation by PMA. Spectrophotometric analysis found approx. 36 pmol of FAD and 43 pmol of haem per mg of membrane and the turnover of superoxide generation in a cell-free system consisting of the membrane and FAD was 10 mol/s per mol of haem. When the constitutively active form of Rac, Rac1(Q61L) or GTP-bound Rac1 was added exogenously to the membrane, O2−-producing activity was enhanced up to 1.5-fold above the basal level, but GDP-loaded Rac1 did not affect superoxide-generating kinetics. A fusion protein [NOXA1N–Rac1(Q61L)] between truncated NOXA1(1–211) and Rac1-(Q61L) exhibited a 6-fold increase of the basal Nox1 activity, but NOXO1N(1–292) [C-terminal truncated NOXO1(1–292)] alone showed little effect on the activity. The activated forms of Rac1 and NOXA1 are essentially involved in Nox1 activation and their interactions might be responsible for regulating the O2−-producing activity in Caco-2 cells.
Key words: Caco-2 cells, Nox1, Rac1, reactive oxygen species, small GTPase, superoxide.
Abbreviations used: ATRA, all-trans-retinoic acid, DPI, diphenyleneiodonium; GST, glutathione transferase; GTP[S], guanosine 5′-[γ-thio]triphosphate; HEK-293 cell, human embryonic kidney 293 cell; IOD, integral optical density; IPTG, isopropyl β-D-thiogalactoside; Nox, NADPH oxidase; NOXA1N, C-terminal truncated NOXA1(1–211); NOXO1N, C-terminal truncated NOXO1(1–292); PX domain, Phox homology domain; ROS, reactive oxygen species; SOD, superoxide dismutase; TPR domain, tetratricopeptide repeat domain.
1To whom correspondence should be addressed (email nisiio@aichi-med-u.ac.jp).
Received 4 February 2008/15 May 2008; accepted 2 June 2008
Published as BJ Immediate Publication 2 June 2008, doi:10.1042/BJ20080300
© The Authors Journal compilation © 2008 Biochemical Society
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