title: FLT3-driven redox-modulation of Ezrin regulates leukaemic cell migration.
abstract: The concept of reactive oxygen species (ROS) being produced via the activation of specific oncogenes provides a basis for generating genomic instability and pro-survival signalling in tumour cells. The purpose of this study was to identify downstream targets of NADPH oxidase (Nox)-derived ROS signalling in acute myeloid leukaemia cells, by performing a proteomic analysis utilizing two-dimensional phosphotyrosine immunoblotting. The majority of the targets identified were cytoskeletal-associated proteins including Ezrin, a known regulator of the cytoskeleton, which was examined further. The study demonstrated that inhibition of Nox enzymes, using diphenyleneiodonium chloride in the acute myeloid leukaemia cell line MOLM-13, resulted in a decrease in Ezrin tyrosine phosphorylation and also triggered a shift in Ezrin sub-cellular localization as detected by immunofluorescence. The change in Ezrin localization coincided with altered cell morphology, observed using scanning electron microscopy and a decreased ability to migrate through a polycarbonate transwell membrane. Similar effects were observed upon inhibition of the oncogenic receptor tyrosine kinase FLT3 using the staurosporine derivate PKC412, implicating a role for FLT3 as an upstream regulator of Ezrin. Our results indicate that FLT3 drives production of ROS by Nox, which stimulates changes in Ezrin tyrosine phosphorylation and localization via redox regulation of Src. Furthermore, inhibition of FLT3 signalling leads to alterations in MOLM-13 cell morphology and has a significant influence on cell motility.
authors.affiliation: Tumour Biology Laboratory, Biochemistry Department, Biosciences Research Institute, University College Cork, Cork, Ireland.
authors.forename: Aoife
authors.initials: A
authors.lastname: Corcoran
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Cotter
mesh_terms: D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D015479:Leukemia, Myelomonocytic, Acute / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009861:Onium Compounds / Q000494:pharmacology; D010084:Oxidation-Reduction; D010766:Phosphorylation; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D019311:Staurosporine / Q000031:analogs & derivatives / Q000494:pharmacology; D014162:Transfection; D051941:fms-Like Tyrosine Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000099149:Ezrin
chemical_list: D003598:Cytoskeletal Proteins; D019255:NADPH Oxidases; D009861:Onium Compounds; D017382:Reactive Oxygen Species; D019311:Staurosporine; D051941:fms-Like Tyrosine Kinase 3; D000099149:Ezrin; C007517:diphenyleneiodonium; C497970:FLT3 protein, human; C059539:midostaurin