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Involvement of a putative protein-tyrosine phosphatase and I kappa B-alpha serine phosphorylation in nuclear factor
1Institute of Molecular and Cell Biology, National University of Singapore.
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.
Insights
Inhibitors of protein phosphatases block tumor necrosis factor (TNF)-induced nuclear factor kappa B (NF-kappa B) activation. This suggests a role for tyrosine phosphatases in regulating NF-kappa B signaling pathways.
Area of Science:
- Molecular Biology
- Cell Signaling
- Immunology
Background:
- Nuclear factor kappa B (NF-kappa B) is a key transcription factor regulating immune responses and inflammation.
- Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that activates NF-kappa B.
- Protein phosphatases play critical roles in cellular signaling by dephosphorylating proteins.
Purpose of the Study:
- To investigate the role of protein-tyrosine phosphatases in TNF-induced NF-kappa B activation.
- To elucidate the mechanism by which TNF activates NF-kappa B, focusing on protein phosphorylation and dephosphorylation events.
Main Methods:
- Utilized inhibitors of phosphotyrosyl protein phosphatases (pervanadate, phenylarsine oxide) and phosphoseryl/threonyl protein phosphatase (okadaic acid).
- Assessed NF-kappa B nuclear translocation and DNA-binding activity in various cell lines (U-937, Jurkat, MRC-5, REF).
- Examined protein phosphorylation and dephosphorylation of NF-kappa B inhibitor alpha (I kappa B-alpha) and other proteins using Western blotting and phosphatase overexpression studies.
Main Results:
- Pervanadate and phenylarsine oxide inhibited TNF-induced NF-kappa B nuclear translocation and activation.
- These inhibitors also abrogated NF-kappa B activation induced by okadaic acid.
- Overexpression of protein-tyrosine phosphatase HPTP alpha led to constitutive NF-kappa B activity, which was lost upon phenylarsine oxide treatment.
- TNF/okadaic acid induced serine phosphorylation of I kappa B-alpha, not tyrosine phosphorylation.
- Pervanadate inhibited TNF-induced I kappa B-alpha phosphorylation and degradation, while serine protease inhibitors blocked degradation and translocation but not phosphorylation.
Conclusions:
- TNF and okadaic acid activate a protein-tyrosine phosphatase that leads to I kappa B-alpha serine phosphorylation and degradation.
- This process culminates in NF-kappa B nuclear translocation and activation.
- Protein-tyrosine phosphatases are crucial regulators of the NF-kappa B signaling pathway initiated by TNF.