David V. Goeddel

Dissection of TNF Receptor 1 Effector Functions: JNK Activation Is Not Linked to Apoptosis While NF-κB Activation Prevents Cell Death

Through its type 1 receptor (TNFR1), the cytokine TNF elicits an unusually wide range of biological responses, including inflammation, tumor necrosis, cell proliferation, differentiation, and apoptosis. We investigated how TNFR1 activates different effector functions; the protein kinase JNK, transcription factor NF-kappaB, and apoptosis. We found that the three responses are mediated through separate pathways. Recruitment of the signal transducer FADD to the TNFR1 complex mediates apoptosis but not NF-kappaB or JNK activation. Two other signal transducers, RIP and TRAF2, mediate both JNK and NF-kappaB activation. These two responses, however, diverge downstream to TRAF2. Most importantly, JNK activation is not involved in induction of apoptosis, while activation of NF-kappaB protects against TNF-induced apoptosis.

TRADD-TRAF2 and TRADD-FADD Interactions Define Two Distinct TNF Receptor 1 Signal Transduction Pathways

Tumor necrosis factor (TNF) can induce apoptosis and activate NF-kappa B through signaling cascades emanating from TNF receptor 1 (TNFR1). TRADD is a TNFR1-associated signal transducer that is involved in activating both pathways. Here we show that TRADD directly interacts with TRAF2 and FADD, signal transducers that activate NF-kappa B and induce apoptosis, respectively. A TRAF2 mutant lacking its N-terminal RING finger domain is a dominant-negative inhibitor of TNF-mediated NF-kappa B activation, but does not affect TNF-induced apoptosis. Conversely, a FADD mutant lacking its N-terminal 79 amino acids is a dominant-negative inhibitor of TNF-induced apoptosis, but does not inhibit NF-kappa B activation. Thus, these two TNFR1-TRADD signaling cascades appear to bifurcate at TRADD.

The TNF receptor 1-associated protein TRADD signals cell death and NF-κB activation

Abstract Many diverse activities of tumor necrosis factor (TNF) are signaled through TNF receptor 1(TNFR1). We have identified a novel 34 kDa protein, designated TRADD, that specifically interacts with an intracellular domain of TNFR1 known to be essential for mediating programmed cell death. Overexpression of TRADD leads to two major TNF-induced responses, apoptosis and activation of NF-κB. The C-terminal 118 amino acids of TRADD are sufficient to trigger both of these activities and likewise sufficient for interaction with the death domain of TNFR1. TRADD-mediated cell death can be suppressed by the crmA gene, which encodes a specific inhibitor of the interleukin-1 β-converting enzyme. However, NF-κB activation by TRADD is not inhibited by crmA expression, demonstrating that the signaling pathways for TNF-induced cell death and NF-κB activation are distinct.

A novel domain within the 55 kd TNF receptor signals cell death

Deletion mutagenesis of the intracellular region of the 55 kd TNF receptor (TNF-R1) identified an approximately 80 amino acid domain near the C-terminus responsible for signaling cytotoxicity. This domain shows weak homology with the intracellular domain of Fas antigen, a transmembrane polypeptide that can also initiate a signal for cytotoxicity. Alanine-scanning mutagenesis of TNF-R1 confirmed that many of the amino acids conserved with Fas antigen are critical for the cytotoxic signal. This region of TNF-R1-Fas homology is therefore likely to define a novel domain (death domain) that signals programmed cell death. Mutations within the death domain of TNF-R1 also disrupted its ability to signal anti-viral activity and nitric oxide (NO) synthase induction. In addition, large deletions in the membrane-proximal half of the intracellular domain did not block signaling of cytotoxicity or anti-viral activity but did block induction of NO synthase.

The TNFR2-TRAF signaling complex contains two novel proteins related to baculoviral inhibitor of apoptosis proteins

The 75 kDa tumor necrosis factor receptor (TNFR2) transduces extracellular signals via receptor-associated cytoplasmic proteins. Two of these signal transducers, TRAF1 and TRAF2, were isolated and characterized previously. We report here the biochemical purification and subsequent molecular cloning of two novel TNFR2-associated proteins, designated c-IAP1 and c-IAP2, that are closely related mammalian members of the inhibitor of apoptosis protein (IAP) family originally identified in baculoviruses. The viral and cellular IAPs contain N-terminal baculovirus IAP repeat (BIR) motifs and a C-terminal RING finger. The c-IAPs do not directly contact TNFR2, but rather associate with TRAF1 and TRAF2 through their N-terminal BIR motif-comprising domain. The recruitment of c-IAP1 or c-IAP2 to the TNFR2 signaling complex requires a TRAF2-TRAF1 heterocomplex.

Identification and Characterization of an IκB Kinase

Abstract Activation of the transcription factor NF-κB by tumor necrosis factor (TNF) and interleukin-1 (IL-1) requires the NF-κB-inducing kinase (NIK). In a yeast two-hybrid screen for NIK-interacting proteins, we have identified a protein kinase previously known as CHUK. Overexpression of CHUK activates a NF-κB-dependent reporter gene. A catalytically inactive mutant of CHUK is a dominant-negative inhibitor of TNF-, IL-1-, TRAF-, and NIK-induced NF-κB activation. CHUK associates with the NF-κB inhibitory protein, IκB-α, in mammalian cells. CHUK specifically phosphorylates IκB-α on both serine 32 and serine 36, modifications that are required for targeted degradation of IκB-α via the ubiquitin-proteasome pathway. This phosphorylation of IκB-α is greatly enhanced by NIK costimulation. Thus, CHUK is a NIK-activated IκB-α kinase that links TNF- and IL-1-induced kinase cascades to NF-κB activation.

Two TNF receptors.

In the past few years, considerable progress has been made on the identification and characterization of tumor necrosis factor (TNF) receptors. The relative roles played by the two receptor types in signaling the diverse functions of TNF are less clear. Here, Louis Tartaglia and David Goeddel summarize progress to date and propose a model of TNF receptor triggering that reconciles the seemingly conflicting data.

A novel family of putative signal transducers associated with the cytoplasmic domain of the 75 kDa tumor necrosis factor receptor

Mutational analysis identified a C-terminal region of 78 amino acids within the cytoplasmic domain of the human 75 kDa tumor necrosis factor receptor (TNF-R2) that is required for signal transduction. This region was subsequently shown to mediate the interaction of cytoplasmic factors with TNF-R2. Two of these factors were isolated and molecularly cloned using biochemical purification and the yeast two-hybrid system. TNF receptor-associated factor 1 (TRAF1) and TRAF2 are the first two members of a novel protein family containing a novel C-terminal homology region, the TRAF domain. In addition, TRAF2 contains an N-terminal RING finger motif. TRAF1 and TRAF2 can form homo- and heterotypic dimers. Our analysis indicates that TRAF1 and TRAF2 are associated with the cytoplasmic domain of TNF-R2 in a heterodimeric complex in which TRAF2 contacts the receptor directly. TRAF1 interacts with TNF-R2 indirectly through heterodimer formation with TRAF2.

Molecular cloning and expression of a receptor for human tumor necrosis factor

A human tumor necrosis factor (TNF) binding protein from serum of cancer patients was purified to homogeneity and partially sequenced. Synthetic DNA probes based on amino acid sequence information were used to isolate cDNA clones encoding a receptor for TNF. The TNF receptor (TNF-R) is a 415 amino acid polypeptide with a single membrane-spanning region. The extracellular cysteine-rich domain of the TNF-R is homologous to the nerve growth factor receptor and the B cell activation protein Bp50. Human embryonic kidney cells transfected with a TNF-R expression vector specifically bind both 125I-labeled and biotinylated TNF-alpha. Unlabeled TNF-alpha and TNF-beta were equally effective at displacing the binding of labeled TNF-alpha to TNF-R expressing cells. Northern analysis indicates a single species of mRNA for the TNF-R in a variety of cell types. Therefore, the soluble TNF binding protein found in human serum is probably proteolytically derived from the TNF-R.

TNF-Dependent Recruitment of the Protein Kinase RIP to the TNF Receptor-1 Signaling Complex

The death domain of tumor necrosis factor (TNF) receptor-1 (TNFR1) triggers distinct signaling pathways leading to apoptosis and NF-kappa B activation through its interaction with the death domain protein TRADD. Here, we show that TRADD interacts strongly with RIP, another death domain protein that was shown previously to associate with Fas antigen. We also show that RIP is a serine-threonine kinase that is recruited by TRADD to TNFR1 in a TNF-dependent process. Overexpression of the intact RIP protein induces both NF-kappa B activation and apoptosis. However, expression of the death domain of RIP Induces apoptosis, but potently inhibits NF-kappa B activation by TNF. These results suggest that distinct domains of RIP participate in the TNF signaling cascades leading to apoptosis and NF-kappa B activation.