title: Okadaic acid induces tyrosine phosphorylation of IkappaBalpha that mediated by PKR pathway in human osteoblastic MG63 cells.
abstract: Treatment of human osteosarcoma cell line MG 63 cells with okadaic acid stimulated phosphorylation of IkappaBalpha, as judged from the results of Western blot analysis and a lambda protein phosphatase dephosphorylation assay. The stimulated phosphorylation of IkappaBalpha was both time- and dose-dependent. The phosphorylation sites of IkappaBalpha were taken to be tyrosine residues because the anti-phospho-tyrosine antibody bound to the samples immunoprecipitated with the anti-IkappaBalpha antibody. In the cells treated with 100 nM okadaic acid consequential translocation of NF-kappaB p65 from the cytosol to the nucleus occurred. Double-stranded RNA-dependent protein kinase (PKR) is a player in the cellular antiviral response and is involved in transcriptional stimulation through activation of NF-kappaB. We investigated the functional relationship between PKR and IkappaBalpha phosphorylation by constructing MG 63 PKR K/R cells that produced a catalytically inactive mutant PKR. NF-kappaB p65 was detected in the nucleus of these cells, even in the unstimulated cells. Although IkappaBalpha was degraded phosphorylation of eIF-2 alpha, a substrate of PKR, did not occur in the mutant cells treated with okadaic acid. Our results suggest that okadaic acid-induced tyrosine phosphorylation of IkappaBalpha was mediated by PKR kinase activity, thus indicating the involvement of this kinase in the control mechanism governing the activation of NF-kappaB.
authors.affiliation: Department of Oral and Maxillofacial Anatomy, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15, Kuramoto, Tokushima 770-8504, Japan. morimoto@dent.tokushima-u.ac.jp
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Morimoto
authors.forename: Akiko
authors.initials: A
authors.lastname: Ozaki
authors.forename: Hirohiko
authors.initials: H
authors.lastname: Okamura
authors.forename: Kaya
authors.initials: K
authors.lastname: Yoshida
authors.forename: Seiichiro
authors.initials: S
authors.lastname: Kitamura
authors.forename: Tatsuji
authors.initials: T
authors.lastname: Haneji
mesh_terms: D045744:Cell Line, Tumor; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism*; D000072000:NF-KappaB Inhibitor alpha; D019319:Okadaic Acid / Q000494:pharmacology*; D010006:Osteoblasts / Q000187:drug effects*; D012516:Osteosarcoma / Q000201:enzymology / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism*; D019892:eIF-2 Kinase / Q000378:metabolism*
chemical_list: D020672:I-kappa B Proteins; C000606204:NFKBIA protein, human; D000072000:NF-KappaB Inhibitor alpha; D019319:Okadaic Acid; D019000:Phosphotyrosine; D019892:eIF-2 Kinase