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CD95 (APO-1/Fas) induces activation of SAP kinases downstream of ICE-like proteases
M A Cahill1, M E Peter, F C Kischkel
1Institute for Molecular Biology, Hannover Medical School, Germany.
Abstract:
Triggering of CD95 (APO-1/Fas) on different T- and B-cell lines resulted in the induction of a number of kinases (35 kDa, 38 kDa, 46 kDa and 54 kDa) that phosphorylate c-Jun and to a lesser extent Histone H1. Activation of these kinases was independent of protein biosynthesis and preceded apoptotic DNA degradation. The kinase activation pattern was specific for CD95 triggering since a variety of physical or chemical inducers of T- and B-cell apoptosis activated different kinases. The kinase activities at 46 and 54 kDa contained members of the stress-activated family of protein kinases (JNK/SAPK). Activation of the CD95-specific set of kinases was prevented by treating cells with the ICE-inhibiting peptide N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) or by overexpression of the cow pox virus serpin CrmA. However, despite inhibition of ICE-like proteases the death signal was readily initiated at the cell membrane since a CD95 death-inducing signaling complex (DISC) was formed. Thus, our results demonstrate that ICE-like proteases in the CD95 pathway function downstream of the DISC but upstream of SAP kinases.
Insights
CD95 (APO-1/Fas) triggering activates specific kinases, including stress-activated protein kinases (SAP kinases), independent of protein synthesis. These kinases function downstream of the CD95 death-inducing signaling complex but upstream of ICE-like proteases in apoptosis.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- CD95 (APO-1/Fas) is a key receptor involved in apoptosis.
- Understanding the signaling pathways downstream of CD95 is crucial for cell death research.
Purpose of the Study:
- To identify kinases activated by CD95 triggering.
- To elucidate the role of these kinases and ICE-like proteases in the CD95-mediated apoptosis pathway.
Main Methods:
- Triggering CD95 on T- and B-cell lines.
- Kinase activity assays (Western blot for phosphorylated c-Jun and Histone H1).
- Inhibition studies using zVAD-fmk and CrmA, and analysis of DISC formation.
Main Results:
- CD95 triggering induced specific kinases (35, 38, 46, 54 kDa) phosphorylating c-Jun and Histone H1.
- Kinase activation was independent of protein biosynthesis and preceded DNA degradation.
- 46 and 54 kDa kinases included stress-activated protein kinases (JNK/SAPK).
- ICE-like protease inhibition (zVAD-fmk, CrmA) blocked kinase activation but not DISC formation.
Conclusions:
- CD95-specific kinase activation is an early event in apoptosis.
- ICE-like proteases act downstream of the CD95 DISC but upstream of SAP kinases.