Although the molecular mechanisms of TNF signaling have been largely elucidated, the principle that regulates the balance of life and death is still unknown. We report here that the death domain kinase RIP, a key component of the TNF signaling complex, was cleaved by Caspase-8 in TNF-induced apoptosis. The cleavage site was mapped to the aspartic acid at position 324 of RIP. We demonstrated that the cleavage of RIP resulted in the blockage of TNF-induced NF-B activation. RIPc, one of the cleavage products, enhanced interaction between TRADD and FADD/MORT1 and increased cells' sensitivity to TNF. Most importantly, the Caspase-8 resistant RIP mutants protected cells against TNF-induced apopotosis. These results suggest that cleavage of RIP is an important process in TNF-induced apoptosis. Further more, RIP cleavage was also detected in other death receptor-mediated apoptosis. Therefore, our study provides a potential mechanism to convert cells from life to death in death receptor-mediated apoptosis.

During the last decades a considerable amount of research has been focused on cancer. Recently, tumor cell metabolism has been considered as a possible target for cancer therapy. It is widely accepted that tumors display enhanced glycolytic activity and impaired oxidative phosphorylation (Warburg effect). Therefore, it seems reasonable that disruption of glycolysis might be a promising candidate for specific anti-cancer therapy. Nevertheless, the concept of aerobic glycolysis as the paradigm of tumor cell metabolism has been challenged, as some tumor cells exhibit high rates of oxidative phosphorylation. Mitochondrial physiology in cancer cells is linked to the Warburg effect. Besides, its central role in apoptosis makes this organelle a promising "dual hit target" to selectively eliminate tumor cells. From a metabolic point of view, the fermenting yeast Saccharomyces cerevisiae and tumor cells share several features. In this paper we will review these common metabolic properties as well as the possible origins of the Crabtree and Warburg effects.

The death domain kinase, receptor interacting protein (RIP), is one of the major components of the tumor necrosis factor receptor 1 (TNFR1) complex and plays an essential role in tumor necrosis factor (TNF)-mediated nuclear factor B (NF-B) activation. The activation of NF-B protects cells against TNF-induced apoptosis. Heat-shock proteins (Hsps) are chaperone molecules that confer protein stability and help to restore protein native folding following heat shock and other stresses. The most abundant Hsp, Hsp90, is also involved in regulating the stability and function of a number of cell-signaling molecules. Here we report that RIP is a novel Hsp90-associated kinase and that disruption of Hsp90 function by its specific inhibitor, geldanamycin (GA), selectively causes RIP degradation and the subsequent inhibition of TNF-mediated IB kinase and NF-B activation. MG-132, a specific proteasome inhibitor, abrogated GA-induced degradation of RIP but failed to restore the activation of IB kinase by TNF, perhaps because, in the presence of GA and MG-132, RIP accumulated in a detergent-insoluble subcellular fraction. Most importantly, the degradation of RIP sensitizes cells to TNF-induced apoptosis. These data indicate that Hsp90 plays an important role in TNF-mediated NF-B activation by modulating the stability and solubility of RIP. Thus, inhibition of NF-B activation by GA may be a critical component of the anti-tumor activity of this drug.

Mitochondria are the main source of reactive oxygen species in the cell. These reactive oxygen species have long been known as being involved in oxidative stress. This is a review of the mechanisms involved in reactive oxygen species generation by the respiratory chain and some of the dehydrogenases and the control by thermodynamic and kinetic constraints. Mitochondrial ROS produced at the level of the bc1 complex as well at the level of complex I are discussed. It was recognized more than a decade ago that they can also function as signaling molecules. This signaling role will be developed both in terms of mechanism and in terms of mitochondrial ROS signaling. The notion that hydrogen peroxide acts not only as a damaging oxidant but also as a signaling molecule was proposed more than a decade ago. Hydrogen peroxide signaling can be either direct (oxidation of its target) or indirect (involving peroxiredoxins, for example). The consequences of ROS signaling on crucial biologic processes such as cell proliferation and differentiation are discussed.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) (Apo2 ligand [Apo2L]) is a member of the TNF superfamily and has been shown to have selective antitumor activity. Although it is known that TRAIL (Apo2L) induces apoptosis and activates NF-B and Jun N-terminal kinase (JNK) through receptors such as TRAIL-R1 (DR4) and TRAIL-R2 (DR5), the components of its signaling cascade have not been well defined. In this report, we demonstrated that the death domain kinase RIP is essential for TRAIL-induced IB kinase (IKK) and JNK activation. We found that ectopic expression of the dominant negative mutant RIP, RIP(559-671), blocks TRAIL-induced IKK and JNK activation. In the RIP null fibroblasts, TRAIL failed to activate IKK and only partially activated JNK. The endogenous RIP protein was detected by immunoprecipitation in the TRAIL-R1 complex after TRAIL treatment. More importantly, we found that RIP is not involved in TRAIL-induced apoptosis. In addition, we also demonstrated that the TNF receptor-associated factor 2 (TRAF2) plays little role in TRAIL-induced IKK activation although it is required for TRAIL-mediated JNK activation. These results indicated that the death domain kinase RIP, a key factor in TNF signaling, also plays a pivotal role in TRAIL-induced IKK and JNK activation.

The death domain kinase RIP and the TNF receptor-associated factor 2 (TRAF2) are essential effectors in TNF signaling. To understand the mechanism by which RIP and TRAF2 regulate TNF-induced activation of the transcription factor NF-kappaB, we investigated their respective roles in TNF-R1-mediated IKK activation using both RIP-/- and TRAF2-/- fibroblasts. We found that TNF-R1-mediated IKK activation requires both RIP and TRAF2 proteins. Although TRAF2 or RIP can be independently recruited to the TNF-R1 complex, neither one of them alone is capable of transducing the TNF signal that leads to IKK activation. Moreover, we demonstrated that IKK is recruited to the TNF-R1 complex through TRAF2 upon TNF treatment and that IKK activation requires the presence of RIP in the same complex.

The activation of IB kinase (IKK) is a key step in the nuclear translocation of the transcription factor NF-B. IKK is a complex composed of three subunits: IKK␣, IKK␤, and IKK␥ (also called NEMO). In response to the proinflammatory cytokine tumor necrosis factor (TNF), IKK is activated after being recruited to the TNF receptor 1 (TNF-R1) complex via TNF receptor-associated factor 2 (TRAF2). We found that the IKK␣ and IKK␤ catalytic subunits are required for IKK-TRAF2 interaction. This interaction occurs through the leucine zipper motif common to IKK␣, IKK␤, and the RING finger domain of TRAF2, and either IKK␣ or IKK␤ alone is sufficient for the recruitment of IKK to TNF-R1. Importantly, IKK␥ is not essential for TNF-induced IKK recruitment to TNF-R1, as this occurs efficiently in IKK␥-deficient cells. Using TRAF2؊/؊ cells, we demonstrated that the TNF-induced interaction between IKK␥ and the death domain kinase RIP is TRAF2 dependent and that one possible function of this interaction is to stabilize the IKK complex when it interacts with TRAF2.The transcription factor NF-B plays a critical role in regulating the expression of many cytokines and immunoregulatory proteins (1, 2, 3). NF-B is composed of homo-or heterodimers of Rel and NF-B proteins (1). The transcription activity of NF-B can be elevated by various stimuli, including the proinflammatory cytokine tumor necrosis factor (TNF) (24). When bound to their specific inhibitors, referred to as IBs, NF-B dimers are sequestered in the cytoplasm and are therefore inactive (1, 32). In response to various stimuli, IBs are phosphorylated by the IB kinase complex (IKK) and are then rapidly degraded by the proteasome after their polyubiquitination (1). The degradation of IBs allows NF-B to translocate into the nucleus and activate its target genes (1).The three proteins IKK␣, IKK␤, and IKK␥ (also called NEMO) were identified as the components of the IKK complex (6,23,26,29,36,37,39,40). IKK␣ and IKK␤ are two related catalytic subunits sharing about 52% identity, both containing an N-terminal kinase domain, a leucine zipper, and C-terminal helix-loop-helix motifs (12). IKK␣ and IKK␤ can form homo-or heterodimers via their leucine zipper motif, but the predominant IKK complex appears to contain mostly IKK␣ and IKK␤ heterodimers (29). The recent generation of IKK␣ Ϫ/Ϫ and IKK␤ Ϫ/Ϫ mice has established that IKK␣ and IKK␤ are required for the activation of NF-B, although the absence of IKK␣ has a much smaller effect due to a compensatory effect of IKK␤ (11,15,17,18,34). In IKK␣ and IKK␤ double-knockout cells, TNF-induced NF-B activation is completely abolished (16). Interestingly, however, IKK␣ and IKK␤ knockout mice exhibit completely different phenotypes (11,15,18,34). It has also been suggested that IKK␣ plays a role in the activation of IKK␤ (25). However, IKK activation by TNF or interleukin-1 is barely affected in IKK␣ Ϫ/Ϫ cells (11). Meanwhile, IKK␥ is the regulatory subunit of the complex, and it binds to the C termini of IKK␣ and IKK␤ (22,29,37). Studies with IKK...

The death-domain kinase RIP (receptor-interacting protein) is an important effector of tumour necrosis factor (TNF) signalling and is essential for TNF-induced nuclear factor-κB activation. However, the function of RIP in the TNF-induced activation of mitogen-activated protein kinases (MAPKs) has not been fully investigated. In this report, using Rip null (Rip -/-) mouse fibroblast cells, we investigated whether RIP is required for TNF-induced activation of the MAPKs extracellular-signal-related kinase (ERK), p38 and c-Jun amino-terminal kinase (JNK). We found that TNFinduced activation of ERK, p38 and JNK is decreased in Rip -/-cells. The activation of these kinases by interleukin-1 is normal in Rip -/-cells. More importantly, we showed that the kinase activity of RIP is needed for ERK activation.