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CD95 tyrosine phosphorylation is required for CD95 oligomerization

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Abstract

Proapoptotic stimuli, such as CD95 ligand and hydrophobic bile acids induce an epidermal growth factor receptor (EGFR)-catalyzed tyrosine phosphorylation of CD95-death receptor in hepatocytes, as a prerequisite for CD95-translocation to the plasma membrane, formation of the death-inducing signalling complex and execution of apoptotic cell death. However, the molecular role played by CD95 tyrosine phosphorylation remained unclear. The present study shows that CD95-tyrosine phosphorylation is required for CD95-oligomerization. Fluorescence resonance energy transfer (FRET)-analysis in Huh7 hepatoma cells, which were cotransfected with CD95-YFP/CD95-CFP revealed that stimulation of these cells with CD95 ligand, proapoptotic bile acids or hyperosmolarity resulted within 30 min in an intracellular FRET-signal, suggestive for CD95/CD95-oligomerization. After 120 min the FRET-signal was detected in the plasma membrane, indicating translocation of the CD95/CD95-oligomer to the plasma membrane. CD95/CD95-oligomerization was abolished in presence of AG1478 or a JNK-inhibitory peptide, i.e. maneuvers known to prevent EGFR-catalyzed CD95-tyrosine phosphorylation. Transfection studies with YFP/CFP-coupled CD95-mutants, which contain tyrosine/phenylalanine-exchanges in positions 232 and 291 (CD95Y232,291F), revealed that at least one tyrosine (Y232,291)-phosphorylated CD95 is required for CD95/CD95-oligomerization. FRET-studies in mouse embryonic fibroblasts, which in contrast to Huh7 express endogenous CD95, revealed that EGF, but not CD95L induced EGFR-homomerization, whereas CD95 ligand, but not EGF resulted in EGFR/CD95-heteromerization. These findings suggest that EGFR-catalyzed CD95-tyrosine phosphorylation is involved in the CD95/CD95-oligomerization process, which is induced by proapoptotic stimuli and is required for apoptosis induction.

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Acknowledgments

The authors are grateful to Prof. D. Arndt-Jovin, Ph.D. (Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany) for kindly providing the EGFR-GFP construct. This study was supported by Deutsche Forschungsgemeinschaft through Sonderforschungsbereich 575 “Experimentelle Hepatologie” (Düsseldorf).

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  1. Clinic for Gastroenterology, Hepatology and Infectiology, Heinrich-Heine-University Düsseldorf, D-40225, Düsseldorf, Germany

    Andrea Eberle, Roland Reinehr, Stephan Becker, Verena Keitel & Dieter Häussinger

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  1. Andrea Eberle
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  2. Roland Reinehr
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  3. Stephan Becker
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  4. Verena Keitel
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  5. Dieter Häussinger
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Correspondence to Dieter Häussinger.

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Eberle, A., Reinehr, R., Becker, S. et al. CD95 tyrosine phosphorylation is required for CD95 oligomerization. Apoptosis 12, 719–729 (2007). https://doi.org/10.1007/s10495-006-0003-2

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  • DOI: https://doi.org/10.1007/s10495-006-0003-2

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