1. ¹Department of Molecular Biology and Biochemistry, Shinshu University School of Medicine, Nagano, Japan
2. ²Biomedical Research Institute, Kureha Chemical Industry Co. Ltd, Tokyo, Japan

Correspondence: Professor T Kamata, Department of Molecular Biology and Biochemistry, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan. E-mail: kamatat@sch.md.shinshu-u.ac.jp

Received 28 September 2007; Revised 13 February 2008; Accepted 25 March 2008; Published online 5 May 2008.

Abstract

Ras oncogene upregulates the expression of nicotinamide adenine dinucleotide phosphate oxidase (Nox) 1 via the Raf|[sol]|MEK|[sol]|ERK pathway, leading to the elevated production of reactive oxygen species that is essential for maintenance of Ras-transformation phenotypes. However, the precise transcriptional control mechanism underlying Ras-induced Nox1 expression remains to be elucidated. Here we demonstrated that via the MEK|[sol]|ERK pathway, Ras signaling enhances the activity of the functional Nox1 promoter (nt |[minus]|321 to |[minus]|1) in colon cancer CaCo-2 cells and thereby induces the formation of the specific protein|[ndash]|DNA complexes in the two GATA-binding site-containing regions (nt |[minus]|161 to |[minus]|136 and |[minus]|125 to |[minus]|100). Supershift assays with GATA antibodies, protein analyses and chromatin immunoprecipitation revealed that GATA-6 is a component of the specific protein|[ndash]|DNA complexes at the Nox1 promoter. GATA-6 was able to trans-activate the Nox1 promoter but not a promoter in which the GATA-binding sites are mutated. Moreover, GATA-6 was phosphorylated at serine residues by MEK-activated ERK, which increased GATA-6 DNA binding, correlating with suppression of the Nox1 promoter activity by an MEK inhibitor PD98059. Finally, the site-directed mutation of the consensus ERK phosphorylation site (PYS¹²⁰P to PYA¹²⁰P) of GATA-6 abolished its trans-activation activity, suppressing of the growth of CaCo-2 cells. On the basis of these results, we propose that oncogenic Ras signaling upregulates the transcription of Nox1 through MEK-ERK-dependent phosphorylation of GATA-6.