Activation of NADPH oxidase 1 in tumour colon epithelial cells.
In the plasma membrane fraction from Caco-2 human colon carcinoma cells, active Nox1 (NADPH oxidase 1) endogenously co-localizes with its regulatory components p22(phox), 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, O(2)(-)-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 O(2)(-)-producing activity in Caco-2 cells.
Nisimoto
Yukio
Y
Department of Biochemistry, Aichi Medical University, School of Medicine, Nagakute, Aichi 480-1195, Japan. nisiio@aichi-med-u.ac.jp
Tsubouchi
Ryoko
R
Diebold
Becky A
BA
Qiao
Shanlou
S
Ogawa
Hisamitsu
H
Ohara
Takuya
T
Tamura
Minoru
M
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism; D018938:Caco-2 Cells; D002462:Cell Membrane / Q000201:enzymology; D002474:Cell-Free System / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D018922:HL-60 Cells; D006801:Humans; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000378:metabolism; D009861:Onium Compounds / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; C476420:NOXA1 protein, human; C476419:NOXO1 protein, human; D009861:Onium Compounds; C107953:RAC1 protein, human; D011993:Recombinant Fusion Proteins; D013481:Superoxides; C007517:diphenyleneiodonium; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases; C402799:NOX1 protein, human; D020830:rac1 GTP-Binding Protein; D013755:Tetradecanoylphorbol Acetate