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Molecular evidence for the nuclear localization of FADD
M Gómez-Angelats1, J A Cidlowski
1The Laboratory of Signal Transduction, Molecular Endocrinology Group, National Institute of Environmental Health Sciences, National Institutes of Health, 111 Alexander Drive, Research Triangle Park, NC 27709, USA.
Abstract:
The Fas-associated death domain (FADD) adaptor protein FADD/Mort-1 is recruited by several members of the tumor necrosis factor receptor (TNFR) superfamily during cell death activated via death receptors. Since most studies have focused on the interaction of FADD with plasma membrane proteins, FADD's subcellular location is thought to be confined to the cytoplasm. In this report, we show for the first time that FADD is present in both the cytoplasm and the nucleus of cells, and that its nuclear localization relies on strong nuclear localization and nuclear export signals (NLS and NES, respectively) that reside in the death-effector domain (DED) of the protein. Specifically, we found that a conserved basic KRK35 sequence of the human protein is necessary for FADD's nuclear localization, since disruption of this motif leads to the confinement of FADD in the cytoplasm. Furthermore, we show that the leucine-rich motif LTELKFLCL28 in the DED is necessary for FADD's nuclear export. Functionally, mutation of the NES of FADD and its seclusion in the nucleus reduces the cell death-inducing efficacy of FADD reconstituted in FADD-deficient T cells.
Insights
The Fas-associated death domain (FADD) protein is found in both the cytoplasm and nucleus. Nuclear FADD localization impacts cell death signaling, revealing new insights into its function.
Area of Science:
- Cell Biology
- Molecular Biology
- Immunology
Background:
- The Fas-associated death domain (FADD) adaptor protein is crucial for tumor necrosis factor receptor (TNFR) superfamily-mediated cell death.
- Previous research suggested FADD is primarily cytoplasmic due to its interactions with plasma membrane proteins.
Purpose of the Study:
- To investigate the subcellular localization of FADD.
- To identify the signals governing FADD's nuclear import and export.
- To determine the functional significance of FADD's nuclear localization in cell death signaling.
Main Methods:
- Immunofluorescence microscopy to visualize FADD localization.
- Site-directed mutagenesis to disrupt putative nuclear localization signals (NLS) and nuclear export signals (NES).
- Functional assays in FADD-deficient T cells to assess cell death induction.
Main Results:
- FADD is localized in both the cytoplasm and nucleus.
- Nuclear localization of FADD depends on NLS and NES within its death-effector domain (DED).
- A specific basic KRK35 motif is essential for nuclear import, while a leucine-rich LTELKFLCL28 motif mediates nuclear export.
- Nuclear retention of FADD impairs its cell death-inducing capacity.
Conclusions:
- FADD exhibits dual cytoplasmic and nuclear localization.
- Specific NLS and NES within the DED regulate FADD's nucleocytoplasmic shuttling.
- Nuclear FADD localization plays a role in modulating cell death signaling efficacy.