Involvement of a putative protein-tyrosine phosphatase and IκB-α serine phosphorylation in nuclear factor κB activation by tumor necrosis factor

Abstract

Inhibitors of phosphotyrosyl protein phosphatases, pervanadate and phenylarsine oxide, abrogate tumor necrosis factor (TNF)-induced nuclear factor κB (NF-κB) nuclear translocation in transformed cell lines (U-937 and Jurkat) and primary fibroblasts (MRC-5 and REF). The inhibitors also abrogate NF-κB activation by the phosphoseryl/threonyl protein phosphatase inhibitor okadaic acid in U-937 cells. Inhibition of NF-κB activation is not due to a general inhibitory effect since neither pervanadate nor phenylarsine oxide treatment affected the constitutive DNA-binding activity of the transcription factors octamer-1 and cAMP response element-binding protein in U-937 cells, nor did these compounds inhibit the TNF-induced phosphorylation of proteins, viz. hsp-27, eukaryotic initiation factor 4e, and pp19, in MRC- 5 fibroblasts. Overexpression of the protein-tyrosine phosphatase HPTPα resulted in a constitutive nuclear NF-κB-like DNA-binding activity in REF cells. Conversely, treatment of human protein-tyrosine phosphatase α- overexpressing cells with phenylarsine oxide led to a loss of the constitutive NF-κB activity. The presence of a tyrosine phosphorylation site on the inhibitor of NF-κB (IκB-α) suggested that it could be a target for TNF/okadaic acid-induced tyrosine dephosphorylation. However, no tyrosine phosphorylation was detected on IκB-α from unstimulated cells, while TNF/okadaic acid-treated cells showed increased phosphorylation of IκB-α exclusively at serine residue(s). Treatment of cells with pervanadate inhibited TNF-induced IκB-α phosphorylation and degradation, whereas the serine protease inhibitors tosylphenylalanyl chloromethyl ketone and N(α)- p-tosyl-L-lysine chloromethyl ketone prevented TNF-induced IκB-α degradation and NF-κB nuclear translocation, but not the TNF-induced phosphorylation of IκB-α. The data suggest that TNF and okadaic acid induce the activation of a putative protein-tyrosine phosphatase(s), leading to IκB-α serine phosphorylation and degradation and NF-κB nuclear translocation.