Reconstitution of nuclear factor kappa B activation induced by tumor necrosis factor requires membrane-associated components. Comparison with pathway activated by ceramide

• Published in: The Journal of biological chemistry 1994-10, Vol.269 (41), p.25369-25372
• Main Authors: Reddy, S A, Chaturvedi, M M, Darnay, B G, Chan, H, Higuchi, M, Aggarwal, B B
• Format: Article
• Language: English
• Subjects: Cell Line; Cell-Free System; Ceramides - metabolism; DNA-Binding Proteins - metabolism; Histiocytes; Humans; Hydroquinones - pharmacology; I-kappa B Proteins; Membranes - metabolism; NF-kappa B - metabolism; NF-KappaB Inhibitor alpha; Protein-Tyrosine Kinases - antagonists & inhibitors; Receptors, Tumor Necrosis Factor - metabolism; Signal Transduction - drug effects; Subcellular Fractions - metabolism; Tumor Necrosis Factor-alpha - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)47258-4

Tumor necrosis factor (TNF) is known to induce the activation of a nuclear transcription factor, nuclear factor kappa B (NF-kappa B), in a wide variety of cell types. The post-receptor binding events that culminate in TNF-dependent NF-kappa B activation are not understood. To dissect this pathway, we developed a reconstitution system consisting of membrane, cytosolic, and post-nuclear fractions. Our results indicate that when incubated with the post-nuclear fraction derived from TNF-untreated cells, the membrane fraction from TNF-treated cells causes the activation of NF-kappa B with kinetics similar to that observed in intact cells. Under these conditions, the cytosolic fraction has no effect. This activation is tyrosine kinase-dependent since erbstatin completely abolished the effect. Furthermore, as revealed by immunoblotting, no degradation of the inhibitory subunit of NF-kappa B was observed. In this reconstitution system, we can also demonstrate the activation of NF-kappa B by ceramide, but this activation is not tyrosine kinase-dependent. Overall, our results indicate that intermediates required for NF-kappa B activation by TNF or ceramide are membrane-bound, but the mechanism of activation by TNF is most likely different from that of ceramide.