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 nuc lear transcription factor, nuclear factor kappa B (NF-kappa B), in a w ide variety of cell types. The post-receptor binding events that culmi nate in TNF-dependent NF-kappa B activation are not understood. To dis sect 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-un treated cells, the membrane fraction from TNF-treated cells causes the activation of NF-kappa B with kinetics similar to that observed in in tact cells. Under these conditions, the cytosolic fraction has no effe ct. This activation is tyrosine kinase-dependent since erbstatin compl etely 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 t he mechanism of activation by TNF is most likely different from that o f ceramide.