Hyperosmolarity and CD95L trigger CD95/EGFR association and tyrosine phosphorylation of CD95 as prerequisites for CD95 membrane trafficking and DISC formation

Abstract

The mechanisms underlying CD95 ligand (CD95L)- and hyperosmolarity-induced activation of the CD95 system [Reinehr, R., Graf, D., Fischer, R., Schliess, F., and Haussinger, D. (2002) Hepatology 36, 602-614] as initial steps of apoptosis were studied. Hyperosmotic exposure (405 mosmol/l) of rat hepatocytes induced within 1 min oxidative stress and antioxidant-sensitive activation of the epidermal growth factor receptor (EGFR) and c-Jun-N-terminal-kinase (JNK). After 30 min of hyperosmotic exposure EGFR associated with CD95 and CD95 became tyrosine phosphorylated. Inhibition of JNK or protein kinase C (PKC) had no effect on EGFR phosphorylation but abolished CD95/EGFR association, CD95-tyrosine phosphorylation, membrane targeting, and Fas-associated death domain/caspase 8 recruitment to CD95 [death-inducing signaling complex (DISC) formation]. Inhibition of EGFR tyrosine kinase activity prevented CD95 tyrosine phosphorylation and DISC formation but not hyperosmolarity-induced EGFR phosphorylation and EGFR association with CD95. Tyrosine-phosphorylated CD95 was enriched in the plasma membrane. All maneuvers preventing CD95 tyrosine phosphorylation inhibited CD95 membrane trafficking and DISC formation. Stimulation of EGFR by EGF induced EGFR phosphorylation but no association with CD95 or CD95 phosphorylation. Addition of CD95L also induced EGFR and JNK activation, EGFR/CD95 association, CD95 tyrosine phosphorylation, DISC formation, and CD95 membrane targeting with an inhibitor sensitivity profile similar to that of hyperosmotic CD95 activation, except that inhibition of PKC was ineffective. The data suggest that moderate hyperosmolarity or CD95L trigger oxidative stress and EGFR activation followed by a JNK-dependent EGFR/CD95association and CD95 tyrosine phosphorylation, probably through EGFR tyrosine kinase activity. This provides a signal for CD95 membrane trafficking and DISC formation.

Carbon Monoxide Protects against Hyperoxia-induced Endothelial Cell Apoptosis by Inhibiting Reactive Oxygen Species Formation

“…In this model, EGFR-mediated tyrosine phosphorylation of Fas was required for the trafficking of Fas to the membrane, for DISC formation, and for the initiation of the extrinsic apoptotic pathway (33). Consistent with this model, we observed that hyperoxia treatment promoted EGFR phosphorylation and its association with Fas in MLEC, in parallel with DISC activation (Fig.…”

“…Recent studies in hepatocytes show that FasL-induced apoptosis involved ROS generation and, consequently, the c-Jun NH 2 -terminal kinase-dependent tyrosine phosphorylation and activation of EGFR and its subsequent association with Fas (33). In this model, EGFR-mediated tyrosine phosphorylation of Fas was required for the trafficking of Fas to the membrane, for DISC formation, and for the initiation of the extrinsic apoptotic pathway (33).…”

Stress-induced corneal epithelial apoptosis mediated by K+ channel activation

“…Hyperosmotic stress induces cell apoptosis by activating JNK and p38 signaling pathways in various cell types (Qin et al, 1997;Bilney and Murray, 1998;Malek et al, 1998;Hoover et al, 2000;Lu et al, 2000;Morrison et al, 2003;Reinehr et al, 2003). In corneal epithelial cells, the effect of hyperosmotic stress on apoptosis is characterized by measuring cell viability and activations of JNK and p38 signaling pathways.…”

The Src Family Kinase Yes Triggers Hyperosmotic Activation of the Epidermal Growth Factor Receptor and CD95

“…Apart from effects on metabolism and gene expression (1)(2)(3)(4)(5)(6), hyperosmotic hepatocyte shrinkage triggers a rapid translocation of intracellular CD95 to the plasma membrane, which is accompanied by DISC 1 formation and sensitizes hepatocytes toward CD95 ligand (CD95L)-induced apoptosis (9,10). Hyperosmotic membrane targeting and activation of CD95 involves rapid activation of the epidermal growth factor receptor (EGFR) and of c-Jun-N-terminal kinases (JNK) (10), an association of activated EGFR and CD95, and subsequent CD95 tyrosine phosphorylation by the EGFR tyrosine kinase activity. CD95-Tyr phosphorylation is a prerequisite for CD95 membrane trafficking and formation of the death-inducing signaling complex (DISC).…”

“…CD95-Tyr phosphorylation is a prerequisite for CD95 membrane trafficking and formation of the death-inducing signaling complex (DISC). The mechanisms underlying the hyperosmotic EGFR activation in hepatocytes has remained unclear, however, the process is antioxidant-and genistein-sensitive, suggestive of the involvement of tyrosine kinases as upstream events (10).…”