Okadaic acid induces tyrosine phosphorylation of IκBα 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.

Double-stranded RNA-dependent protein kinase is required for bone calcification in MC3T3-E1 cells in vitro

“…In previous reports, we demonstrated that okadaic acid, an inhibitor of protein phosphatase types 1 and 2A, inhibited protein synthesis and stimulated phosphorylation of PKR and eIF-2a, and induced Runx2 protein in osteoblastic cells [32 -34]. However, okadaic acid did not phosphorylate eIF-2a in PKR-K/R mutant cells [34].…”

Okadaic acid activates the PKR pathway and induces apoptosis through PKR stimulation in MG63 osteoblast-like cells

“…IκBα was degraded upon OA treatment in MG63 cells. We previously demonstrated that IκBα was phosphorylated on tyrosine residues by the OA-treatment (36). In the present study IκBα was phosphorylated at least on serine residues at 32 position, because the anti-phospho IκBα (Ser32) recognized the phosphorylated form of IκBα.…”

Impact of Protein Kinase PKR in Cell Biology: from Antiviral to Antiproliferative Action

“…Osteoblast differentiation and calcification through modulation of STAT1␣ and/or Runx2 expression by PKR has been also described (249).…”