CD95 tyrosine phosphorylation is required for CD95 oligomerization.
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 (CD95(Y232,291F)), revealed that at least one tyrosine (Y(232,291))-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.
Eberle
Andrea
A
Clinic for Gastroenterology, Hepatology and Infectiology, Heinrich-Heine-University Düsseldorf, D-40225 Düsseldorf, Germany.
Reinehr
Roland
R
Becker
Stephan
S
Keitel
Verena
V
Häussinger
Dieter
D
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D045744:Cell Line, Tumor; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D053222:Fas Ligand Protein / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D010766:Phosphorylation; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*; D019014:fas Receptor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D053222:Fas Ligand Protein; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D019014:fas Receptor; D014443:Tyrosine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors