Oncogenic Ras upregulates NADPH oxidase 1 gene expression through MEK-ERK-dependent phosphorylation of GATA-6.
Ras oncogene upregulates the expression of nicotinamide adenine dinucleotide phosphate oxidase (Nox) 1 via the Raf/MEK/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/ERK pathway, Ras signaling enhances the activity of the functional Nox1 promoter (nt -321 to -1) in colon cancer CaCo-2 cells and thereby induces the formation of the specific protein-DNA complexes in the two GATA-binding site-containing regions (nt -161 to -136 and -125 to -100). Supershift assays with GATA antibodies, protein analyses and chromatin immunoprecipitation revealed that GATA-6 is a component of the specific protein-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 (PYS(120)P to PYA(120)P) 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.
Adachi
Y
Y
Department of Molecular Biology and Biochemistry, Shinshu University School of Medicine, Nagano, Japan.
Shibai
Y
Y
Mitsushita
J
J
Shang
W H
WH
Hirose
K
K
Kamata
T
T
D001483:Base Sequence; D018938:Caco-2 Cells; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D050993:GATA6 Transcription Factor / Q000378:metabolism*; D017930:Genes, Reporter; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics*; D000074624:NADPH Oxidase 1; D015689:Oncogene Protein p21(ras) / Q000502:physiology*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D015398:Signal Transduction; D015854:Up-Regulation / Q000502:physiology*
D017931:DNA Primers; D050993:GATA6 Transcription Factor; D012333:RNA, Messenger; D009247:NADH, NADPH Oxidoreductases; D000074624:NADPH Oxidase 1; D020928:Mitogen-Activated Protein Kinases; D015689:Oncogene Protein p21(ras)