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 kappa B (NF-kappa B) nuclear translocation in transformed cell lines (U-937 and Jurkat) and primary fibroblasts (MRC-5 and REF). The inhibitors also abrogate NF-kappa B activation by the phosphoseryl/threonyl protein phosphatase inhibitor okadaic acid in U-937 cells. Inhibition of NF-kappa B 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 HPTP alpha resulted in a constitutive nuclear NF-kappa B-like DNA-binding activity in REF cells. Conversely, treatment of human protein-tyrosine phosphatase alpha-overexpressing cells with phenylarsine oxide led to a loss of the constitutive NF-kappa B activity. The presence of a tyrosine phosphorylation site on the inhibitor of NF-kappa B (I kappa B-alpha) suggested that it could be a target for TNF/okadaic acid-induced tyrosine dephosphorylation. However, no tyrosine phosphorylation was detected on I kappa B-alpha fron unstimulated cells, while TNF/okadaic acid-treated cells showed increased phosphorylation of I kappa B-alpha exclusively at serine residue(s). Treatment of cells with pervanadate inhibited TNF-induced I kappa B-alpha phosphorylation and degradation, whereas the serine protease inhibitors tosylphenylalanyl chloromethyl ketone and N alpha-p-tosyl-L-lysine chloromethyl ketone prevented TNF-induced I kappa B-alpha degradation and NF-kappa B nuclear translocation, but not the TNF-induced phosphorylation of I kappa B-alpha. The data suggest that TNF and okadaic acid induce the activation of a putative protein-tyrosine phosphatase(s), leading to I kappa B-alpha serine phosphorylation and degradation and NF-kappa B nuclear translocation.