ABSTRACT
NRADD (neurotrophin receptor alike death domain protein) is a novel protein with transmembrane and cytoplasmic regions highly homologous to death receptors, particularly p75(NTR). However, the short N-terminal domain is unique. Expression of NRADD induced apoptosis in a number of cell lines. The apoptotic mechanism involved the activation of caspase-8 and execution of apoptosis without requiring mitochondrial components. The activation of this death receptor-like mechanism required the N-terminal domain, which is N-glycosylated and needed for subcellular targeting. Deletion of the N-terminal domain produced a dominant-negative form of NRADD that protected neurons and Schwann cells from a variety of endoplasmic reticulum (ER) stressors. NRADD may therefore be a necessary component for generating an ER-induced proapoptotic signal.
Subject(s)
Apoptosis/physiology , Endoplasmic Reticulum/metabolism , Membrane Glycoproteins/metabolism , Membrane Proteins/metabolism , Amino Acid Sequence , Animals , Animals, Newborn , Binding Sites/genetics , Caspases/metabolism , Cell Line , Cells, Cultured , Gene Expression , Gene Expression Regulation, Developmental , Glycosylation , HeLa Cells , Humans , JNK Mitogen-Activated Protein Kinases , Male , Membrane Glycoproteins/genetics , Membrane Proteins/genetics , Mice , Mitogen-Activated Protein Kinases/metabolism , Molecular Sequence Data , PC12 Cells , Rats , Receptors, Death Domain , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Signal Transduction , TransfectionABSTRACT
A20, a TNF inducible gene, inhibits TNF-mediated apoptosis as well as NF-kappa B induced by this cytokine. Reporter assay experiments revealed that A20 is a very effective inhibitor of NF-kappa B signaling induced by TRAFs and several Map3 kinases, including NIK, MEKK1, COT, and TAK1. Similarly, the NF-kappa B inducing activity of TAX, an activator of the I kappa B kinase complex, is also abrogated by A20. Inhibition of NF-kappa B is specific as A20 has no effect on TNF-alpha-induced JNK activation. These results suggest that the molecular target of A20 is more distal to the receptor than TRAFs as previously proposed. A20 inhibits NF-kappa B-dependent transcription without a concomitant decrease in nuclear NF-kappa B DNA binding activity or nuclear translocation of p65. This apparent discrepancy between transcriptional readout and gel shift experiments is observed with a variety of stimuli, including expression of IKK beta. Therefore, in addition to the phosphorylation of I kappa B, another signal is needed for transcriptional activation of NF-kappa B. A20 inhibits this non-redundant signal. The observation that A20 associates with IKK alpha and is phosphorylated upon IKK beta co-expression may suggest that A20 interferes with some aspects of signalosome function.
Subject(s)
Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/metabolism , Plant Proteins , Proteins/metabolism , Proteins/physiology , Active Transport, Cell Nucleus , Cell Line , Cytoplasm/metabolism , DNA-Binding Proteins , Dose-Response Relationship, Drug , Enzyme Activation , Humans , I-kappa B Proteins/metabolism , Immunohistochemistry , Intracellular Signaling Peptides and Proteins , Luciferases/metabolism , Mitogen-Activated Protein Kinase 8 , Nuclear Proteins , Phosphoric Monoester Hydrolases/metabolism , Phosphorylation , Plasmids/metabolism , Precipitin Tests , Protein Binding , Signal Transduction , Transcription Factor RelA , Transcription, Genetic , Transcriptional Activation , Transfection , Tumor Necrosis Factor alpha-Induced Protein 3 , Tumor Necrosis Factor-alpha/metabolism , p38 Mitogen-Activated Protein KinasesABSTRACT
The p75 neurotrophin receptor (p75(NTR)) belongs to the tumor necrosis factor receptor/nerve growth factor receptor superfamily. In some cells derived from neuronal tissues it causes cell death through a poorly characterized pathway. We developed a neuronal system using conditionally immortalized striatal neurons, in which the expression of p75(NTR) is inducibly controlled by the ecdysone receptor. In these cells p75(NTR) induces apoptosis through its death domain in a nerve growth factor-independent manner. Caspases 9, 6, and 3 are activated by receptor expression indicating the activation of the common effector pathway of apoptosis. Cell death is blocked by a dominant negative form of caspase 9 and Bcl-X(L) consistent with a pathway that involves mitochondria. Significantly, the viral flice inhibitory protein E8 protects from p75(NTR)-induced cell death indicating that death effector domains are involved. A p75(NTR) construct with a deleted death domain dominantly interferes with p75(NTR) signaling, implying that receptor multimerization is required. However, in contrast to the other receptors of the family, p75(NTR)-mediated apoptosis does not involve the adaptor proteins Fas-associated death domain protein or tumor necrosis factor-associated death domain protein, and the apical caspase 8 is not activated. We conclude that p75(NTR) signals apoptosis by similar mechanisms as other death receptors but uses different adaptors and apical caspases.
