Reconstitution of nuclear factor kappa B activation induced by tumor necrosis factor requires membrane-associated components. Comparison with pathway activated by ceramide.
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.
Reddy
S A
SA
Department of Clinical Immunology and Biological Therapy, University of Texas M. D. Anderson Cancer Center, Houston 77030.
Chaturvedi
M M
MM
Darnay
B G
BG
Chan
H
H
Higuchi
M
M
Aggarwal
B B
BB
D002460:Cell Line; D002474:Cell-Free System; D002518:Ceramides / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D006644:Histiocytes; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology; D020672:I-kappa B Proteins; D008566:Membranes / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013347:Subcellular Fractions / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D002518:Ceramides; D004268:DNA-Binding Proteins; D006873:Hydroquinones; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D011505:Protein-Tyrosine Kinases; C048212:erbstatin