Abstract
Fas ligand (FasL) is a type II transmembrane protein belonging to the tumor necrosis factor family. Its binding to the cognate Fas receptor triggers the apoptosis that plays a pivotal role in the maintenance of immune system homeostasis. The cell death-inducing property of FasL has been associated with its extracellular domain, which can be cleaved off by metalloprotease activity to produce soluble FasL. The fate of the remaining membrane-anchored N-terminal part of the FasL molecule has not been determined. Here we show that post-translational processing of overexpressed and endogenous FasL in T-cells by the disintegrin and metalloprotease ADAM10 generates a 17-kDa N-terminal fragment, which lacks the receptor-binding extracellular domain. This FasL remnant is membrane anchored and further processed by SPPL2a, a member of the signal peptide peptidase-like family of intramembrane-cleaving proteases. SPPL2a cleavage liberates a smaller and highly unstable fragment mainly containing the intracellular FasL domain (FasL ICD). We show that this fragment translocates to the nucleus and is capable of inhibiting gene transcription. With ADAM10 and SPPL2a we have identified two proteases implicated in FasL processing and release of the FasL ICD, which has been shown to be important for retrograde FasL signaling.
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Abbreviations
- FasL:
-
Fas ligand
- sFasL:
-
soluble Fas ligand
- APL:
-
ADAM10-processed FasL form
- SPA:
-
SPPL2a-processed APL
- (N)ICD:
-
(Notch) intracellular domain
- PS:
-
presenilins
- MMP:
-
matrix metalloprotease
- TACE:
-
tumor necrosis factor-alpha (TNF-α)-converting enzyme
- ADAM:
-
a disintegrin and metalloproteinase
- SPPL:
-
signal peptide peptidase-like
- RIP:
-
regulated intramembrane proteolysis
- I-CliP:
-
intramembrane-cleaving protease
- kDa:
-
kilo Dalton
- CKI:
-
casein kinase I
- FCH domain:
-
Fes/CIP4 homology domain
- SH3 domain:
-
Src homology 3 domain
- NCoR:
-
nuclear receptor coreppressor
- NLS:
-
nuclear localization signal
- PHA:
-
phytohemagglutinin
- DMEM:
-
Dulbecco's Modified Eaglés Medium
- HEK293 cells:
-
human embryonic kidney 293 cells
- ATCC:
-
American Type Culture Collection
- SDS-PAGE:
-
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- PVDF:
-
polyvinyliden difluoride
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Acknowledgements
We thank S Bösser for excellent technical assistance, and P Scherle (Incyte Corporation, Wilmington, Delaware, USA) for providing the small molecule inhibitors INCB-3619, INCB-3420, INCB-8765 and INCB-12881. This work was supported in part by grants from the DFG (ZO 110/2; MZ), the German National Genome Research Network (NGFN project N1KR-S12T23; VK and MZ), the Centre National de la Recherche Scientifique (AOH), the Ligue Nationale contre le Cancer (AOH), the Association pour la Recherche contre le Cancer (AOH), The Emerald Foundation (AOH) and the Canceropole-PACA ACI 2004 (AOH).
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Kirkin, V., Cahuzac, N., Guardiola-Serrano, F. et al. The Fas ligand intracellular domain is released by ADAM10 and SPPL2a cleavage in T-cells. Cell Death Differ 14, 1678–1687 (2007). https://doi.org/10.1038/sj.cdd.4402175
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DOI: https://doi.org/10.1038/sj.cdd.4402175
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