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CD95-tyrosine nitration inhibits hyperosmotic and CD95 ligand-induced CD95 activation in rat hepatocytes
Roland Reinehr1, Boris Görg, Andrea Höngen
1Clinic for Gastroenterology, Hepatology and Infectiology, Heinrich Heine University Düsseldorf, Germany.
Abstract:
Epidermal growth factor receptor-dependent CD95-tyrosine phosphorylation was recently identified as an early step in apoptosis induction via the CD95 system (Reinehr, R., Schliess, F., and Häussinger, D. (2003) FASEB J. 17, 731-733). The effect of peroxynitrite (ONOO(-)) on modulation of the hyperosmotic and CD95 ligand (CD95L)-induced CD95 activation process was studied. Pretreatment of hepatocytes with ONOO(-) inhibited CD95L- and hyperosmolarity-induced CD95 membrane trafficking and formation of the death-inducing signaling complex, but not epidermal growth factor receptor activation and its association with CD95. Under these conditions, however, no tyrosine phosphorylation of CD95 occurred; instead, CD95 was tyrosine-nitrated. When ONOO(-) was added after induction of CD95-tyrosine phosphorylation by CD95L or hyperosmolarity, tyrosine nitration of CD95 was largely prevented and death-inducing signaling complex formation occurred. CD95-tyrosine nitration abolished the hyperosmotic sensitization of hepatocytes toward CD95L-induced apoptosis. Additionally, in CD95-yellow fluorescent protein-transfected Huh7-hepatoma cells, ONOO(-) induced CD95 Tyr nitration and prevented CD95L-induced Tyr phosphorylation and apoptosis. Tyrosine-nitrated CD95 was also found in rat livers derived from an in vivo model of endotoxinemia. The data suggest that CD95-tyrosine nitration prevents CD95 activation by inhibiting CD95-tyrosine phosphorylation. Apparently, CD95-tyrosine phosphorylation and nitration are mutually exclusive. The data identify critical tyrosine residues of CD95 as another target of the anti-apoptotic action of NO.
Insights
Peroxynitrite (ONOO(-)) prevents CD95 activation by causing tyrosine nitration instead of phosphorylation. This CD95 tyrosine nitration inhibits apoptosis signaling, acting as an anti-apoptotic mechanism.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Epidermal growth factor receptor (EGFR)-dependent CD95 tyrosine phosphorylation is an early step in CD95-mediated apoptosis.
- The role of peroxynitrite (ONOO(-)) in modulating CD95 activation pathways remains to be fully elucidated.
Purpose of the Study:
- To investigate the effect of peroxynitrite (ONOO(-)) on CD95 activation induced by hyperosmolarity and CD95 ligand (CD95L).
- To determine whether CD95 tyrosine nitration or phosphorylation occurs under ONOO(-) influence and its impact on apoptosis.
Main Methods:
- Hepatocytes and Huh7-hepatoma cells were pretreated or treated post-induction with peroxynitrite (ONOO(-)).
- CD95 activation, membrane trafficking, death-inducing signaling complex (DISC) formation, and tyrosine phosphorylation/nitration were analyzed.
- EGFR activation and its association with CD95 were assessed.
- Experiments utilized CD95-yellow fluorescent protein-transfected cells and an in vivo endotoxemia model in rats.
Main Results:
- Peroxynitrite (ONOO(-)) pretreatment inhibited CD95L- and hyperosmolarity-induced CD95 membrane trafficking and DISC formation.
- ONOO(-) treatment led to CD95 tyrosine nitration, preventing CD95 tyrosine phosphorylation.
- CD95 tyrosine nitration abolished hyperosmotic sensitization to CD95L-induced apoptosis.
- Tyrosine-nitrated CD95 was detected in rat livers during endotoxemia.
Conclusions:
- CD95 tyrosine nitration by peroxynitrite (ONOO(-)) inhibits CD95 activation by preventing tyrosine phosphorylation.
- CD95 tyrosine phosphorylation and nitration are mutually exclusive events.
- CD95 tyrosine residues are a target for the anti-apoptotic effects of nitric oxide (NO).
