15-lipoxygenase-1 activates tumor suppressor p53 independent of enzymatic activity.
15-LOX-1 and its metabolites are involved in colorectal cancer. Recently, we reported that 15-LOX-1 overexpression in HCT-116 human colorectal cancer cells inhibited cell growth by induction of p53 phosphorylation (4). To determine whether the 15-LOX-1 protein or its metabolites are responsible for phosphorylation of p53 in HCT-116 cells, we used HCT-116 cells that expressed a mutant 15-LOX-1. The mutant 15-LOX-1 enzyme, with a substitution of Leu at residue His361, was devoid of enzymatic activity. HCT-116 cells transiently transfected with either native or mutant 15-LOX-1 showed an increase in p53 phosphorylation and an increase in the expression of downstream genes. Thus, 15-LOX-1 induces p53 phosphorylation independent of enzymatic activity. Treatment of A549 human lung carcinoma cells with IL-4 increased the expression of 15-LOX-1 and also increased the expression of downstream targets of p53. This confirmed that the activation of p53 was also observed in wild-type cells expressing physiological 15-LOX-1. Immunoprecipitation experiments revealed that 15-LOX-1 interacts with, and binds to, DNA-dependent protein kinase (DNA-PK). The binding of 15-LOX-1 to DNA-PK caused an approximate 3.0-fold enhancement in kinase activity, resulting in increased p53 phosphorylation at Ser15. Knockdown of DNA-PK by small interfering RNA (siRNA) significantly reduced p53 phosphorylation. Furthermore, confocal microscopy demonstrated a colocalization of 15-LOX and DNA-PK in the cells. We propose that the 15-LOX-1 protein binds to DNA-PK, increasing its kinase activity and results in downstream activation of the tumor suppressor p53, thus revealing a new mechanism by which lipoxygenases (LOX) may influence the phenotype of tumor cells.
Zhu
Hong
H
Eicosanoid Biochemistry Section, Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC, USA.
Glasgow
Wayne
W
George
Margaret D
MD
Chrysovergis
Kali
K
Olden
Kenneth
K
Roberts
John D
JD
Eling
Thomas
T
D001093:Arachidonate 15-Lipoxygenase / Q000378:metabolism*; D015153:Blotting, Western; D015179:Colorectal Neoplasms / Q000201:enzymology* / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015972:Gene Expression Regulation, Neoplastic; D045325:HCT116 Cells; D006801:Humans; D047468:Immunoprecipitation; D019787:Linoleic Acid / Q000378:metabolism; D008175:Lung Neoplasms / Q000201:enzymology* / Q000378:metabolism; D010641:Phenotype; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics; D034741:RNA, Small Interfering; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D034741:RNA, Small Interfering; D016159:Tumor Suppressor Protein p53; D004247:DNA; D019787:Linoleic Acid; D001093:Arachidonate 15-Lipoxygenase; D011494:Protein Kinases
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