Tumor necrosis factor (TNF)-alpha activates c-raf-1 kinase via the p55 TNF receptor engaging neutral sphingomyelinase
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1Abteilung für Medizinische Onkologie und Angewandte Molekularbiologie, Freie Universität Berlin, Germany.
Abstract:
TNF-alpha mediates proliferation, functional activation and apoptotic death of cells depending upon its concentration and target cell type. The signaling pathways used by TNF-alpha to mount these responses are, at present, not completely understood. We report here that TNF-alpha promotes dose- and time-dependent phosphorylation and activation of the c-raf-1 kinase engaging the type I p55 TNF receptor (TNF-R). c-raf-kinase activation was duplicated by an agonistic monoclonal antibody directed against the p55 TNF-R. Moreover, ectopic expression of the human p55 TNF-R in murine pre-B 70Z/3 cells was sufficient to confer c-raf-1-kinase activation by human TNF-alpha. By inhibiting intracellular activation of acidic sphingomyelinase (SMase) and by using deleted forms of the type I TNF-R it was shown that the neutral, but not the acidic SMase, participated in TNF-alpha-mediated phosphorylation and activation of the c-raf kinase. TNF-alpha-induced transcriptional activation of a heterologous promoter construct harboring the AP-1 binding site was also mediated by the type I p55 TNF-R. In this case the initiation of transcription required the same cytoplasmic domain as that responsible for activation of c-raf-1 kinase and was liberated in the presence of a dominant negative mutant of c-raf-1.
Insights
Tumor Necrosis Factor-alpha (TNF-alpha) activates c-raf-1 kinase and gene transcription via the p55 TNF receptor. Neutral sphingomyelinase is crucial for this TNF-alpha signaling pathway.
Area of Science:
- Cellular Biology
- Molecular Biology
- Immunology
Background:
- Tumor Necrosis Factor-alpha (TNF-alpha) elicits diverse cellular responses, including proliferation, activation, and apoptosis.
- The precise signaling pathways governing TNF-alpha's effects are not fully elucidated.
Purpose of the Study:
- To investigate the role of c-raf-1 kinase and sphingomyelinase in TNF-alpha-mediated signaling.
- To identify the TNF receptor type involved in TNF-alpha-induced c-raf-1 activation and transcriptional changes.
Main Methods:
- Dose- and time-dependent analysis of c-raf-1 phosphorylation and activation.
- Utilizing agonistic monoclonal antibodies against the p55 TNF receptor.
- Ectopic expression of the human p55 TNF receptor in murine cells.
- Inhibition of sphingomyelinase activity and use of deleted TNF receptor forms.
- Assessing TNF-alpha-induced transcriptional activation using AP-1 binding site reporter constructs.
Main Results:
- TNF-alpha induces dose- and time-dependent phosphorylation and activation of c-raf-1 kinase through the type I p55 TNF receptor (TNF-R).
- Activation of neutral sphingomyelinase (SMase), but not acidic SMase, is essential for TNF-alpha-mediated c-raf-1 activation.
- TNF-alpha-induced transcriptional activation of an AP-1 promoter construct relies on the same cytoplasmic domain as c-raf-1 activation.
Conclusions:
- The type I p55 TNF receptor is critical for TNF-alpha-mediated c-raf-1 kinase activation and subsequent transcriptional responses.
- Neutral sphingomyelinase plays a key role in the TNF-alpha signaling cascade leading to c-raf-1 activation.