The mixed lineage kinase domain-like protein (MLKL) has recently been identified as a key RIP3 (receptor interacting protein 3) downstream component of tumour necrosis factor (TNF)-induced necroptosis. MLKL is phosphorylated by RIP3 and is recruited to the necrosome through its interaction with RIP3. However, it is still unknown how MLKL mediates TNF-induced necroptosis. Here, we report that MLKL forms a homotrimer through its amino-terminal coiled-coil domain and locates to the cell plasma membrane during TNF-induced necroptosis. By generating different MLKL mutants, we demonstrated that the plasma membrane localization of trimerized MLKL is critical for mediating necroptosis. Importantly, we found that the membrane localization of MLKL is essential for Ca(2+) influx, which is an early event of TNF-induced necroptosis. Furthermore, we identified that TRPM7 (transient receptor potential melastatin related 7) is a MLKL downstream target for the mediation of Ca(2+) influx and TNF-induced necroptosis. Hence, our study reveals a crucial mechanism of MLKL-mediated TNF-induced necroptosis.

Activation of IkappaB kinase (IKK) is the key step in stimulation of the transcription factor NF-kappaB, which regulates many genes in the inflammatory response pathway. The molecular mechanism that underlies IKK activation in response to tumor necrosis factor (TNF) is still unknown. Using mitogen-activated protein kinase kinase kinase 3 (MEKK3)-deficient fibroblast cells, we found that MEKK3 plays a critical role in TNF-induced NF-kappaB activation. We have shown that MEKK3 is required for IKK activation and functions downstream of receptor-interacting protein (RIP) and TNF receptor- associated factor 2. We have also shown that MEKK3 interacts with RIP and directly phosphorylates IKK. The kinase activity of MEKK3 is pivotal to its function and, therefore, MEKK3 links RIP and IKK in TNF-induced NF-kappaB activation.

Poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation-induced necrosis has been implicated in several pathophysiological conditions. Although mitochondrial dysfunction and apoptosis-inducing factor translocation from the mitochondria to the nucleus have been suggested to play very important roles in PARP-1-mediated cell death, the signaling events downstream of PARP-1 activation in initiating mitochondria dysfunction are not clear. Here we used the DNA alkylating agent N-methyl-N-nitro-N-nitrosoguanidine, a potent PARP-1 activator, to study PARP-1 activation-mediated cell death. We found, based on genetic knockouts and pharmacological inhibition, that c-Jun N-terminal kinase (JNK), especially JNK1, but not the other groups of mitogen-activated protein kinase, is required for PARP-1-induced mitochondrial dysfunction, apoptosis-inducing factor translocation, and subsequent cell death. We reveal that receptor-interacting protein 1 (RIP1) and tumor necrosis factor receptor-associated factor 2 (TRAF2), are upstream of JNK in PARP-1 hyperactivated cells, because PARP-1-induced JNK activation was attenuated in RIP1؊/؊ and TRAF2؊/؊ mouse embryonic fibroblast cells. Consistently, knockouts of RIP1 and TRAF2 caused a resistance to PARP-1-induced cell death. Therefore, our study uncovers that RIP1, TRAF2, and JNK comprise a pathway to mediate the signaling from PARP-1 overactivation to mitochondrial dysfunction.Poly(ADP-ribose) polymerase-1 (PARP-1) 2 is a nuclear enzyme activated by DNA strand breaks that catalyzes the covalent attachment of long branched chains of poly(ADP-ribose) with NAD ϩ as its substrate to a variety of nuclear DNA-binding proteins, including PARP-1 itself (1). PARP-1 activation plays an essential role in DNA repair under moderate stress (2); however, in several pathological situations that involve massive DNA damage, extensive activation of PARP-1 depletes cellular NAD ϩ and its precursor ATP, leading to irreversible cellular energy failure and necrotic cell death (3-5). The pathophysiological importance of PARP-1-mediated cell death has been suggested by the observation that genetic ablation of PARP-1 and pharmacological inhibition of PARP-1 activity elicit strong protection in several disease models, including ischemia-reperfusion injury after cerebral ischemia and myocardial infarction, inflammatory injury, reactive oxygen species-induced injury, and glutamate excitotoxicity (6 -10). Apoptosis and necrosis are two major forms of cell death with distinct morphological features. Apoptosis is an ordered and regulated process in which the cell actively destroys itself while maintaining plasma membrane integrity, thus permitting non-inflammatory phagocytosis of the dying cell. Necrosis, on the other hand, has traditionally been regarded as a passive and unregulated form of cell death with morphology of cell swelling, loss of plasma membrane integrity, and the release of cellular contents into the extracellular environment, thus triggering an inflammatory response (11). PARP-1-mediated cell death is c...

Tumor necrosis factor (TNF) is a proinflammatory cytokine that plays a critical role in diverse cellular events, including cell proliferation, differentiation and apoptosis. TNF is also involved in many types of diseases. In recent years, the molecular mechanisms of TNF functions have been intensively investigated. Studies from many laboratories have demonstrated that the TNF-mediated diverse biological responses are achieved through activating multiple signaling pathways. Especially the activation of transcription factors NF-κB and AP-1 plays a critical role in mediating these cellular responses. Several proteins, including FADD, the death domain kinase RIP and the TNF receptor associated factor TRAF2 have been identified as the key effectors of TNF signaling. Recently, we found that the effector molecules of TNF signaling, such as RIP and TRAF2, are also involved in other cellular responses. These finding suggests that RIP and TRAF2 serve a broader role than as just an effector of TNF signaling.

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase that plays a key role in maintaining focal adhesion function and cell survival and is implicated in cell migration, adhesion, and cell cycle control (9,13,18,20,33,44). Overexpression of FAK is a common event in numerous tumor systems, including breast, colon, and thyroid carcinomas (2,24,32,41), and occurs at early stages of tumorigenesis, before a tumor has developed the capacity for invasion and metastasis (2). Importantly, FAK has been shown to be one of the critical factors protecting cells from apoptosis, but the exact mechanism is unknown (8,9,12,19,37,43). Attenuation of FAK expression by antisense oligonucleotides led to apoptosis in tumor cells (42), and the treatment of cells with anti-FAK antibody (18, 26) or overexpression of the focal adhesion targeting (FAT) domain of FAK led to cell rounding, detachment, and apoptosis (19,21,40). We have created a model system for the attenuation of FAK function by adenoviral gene transduction of the carboxy-terminal domain of FAK (FAK-CD) and have demonstrated a loss of adhesion and apoptosis in breast cancer cells with this treatment (43). Both anchoragedependent and anchorage-independent apoptotic signaling required Fas-associated death domain protein (FADD) and caspase 8, suggesting an important role for FAK in inhibiting death receptor-related apoptosis (43). This finding provided additional evidence that a death receptor-mediated apoptotic pathway or death receptor-related death domain proteins are involved in the apoptotic process triggered by the expression of FAK-CD.The loss of adhesion and induction of apoptosis upon attenuation of FAK function by the expression of FAK-CD is similar to the phenomenon of anoikis (7-9). Intriguingly, there is evidence for the involvement of death receptor-related, death domain-containing proteins in anoikis (7, 35), whereby the silencer of death domain (SODD) and dominant-negative FADD efficiently inhibited anoikis in Madin-Darby canine kidney (MDCK) cells and in a number of untransformed epithelial cell lines. In these studies, it was also shown that cell matrix detachment activated caspase 8. However, the linkage of the signaling pathways to the death receptors remains unknown.RIP is a serine/threonine kinase that contains a death domain (17, 38) and is named for its association with the death receptor complex. RIP interacts with the death domains of cell surface receptors of the tumor necrosis factor (TNF) superfamily and death domain adaptor proteins (3,5,17) and plays an indispensable role in 39). Recently, it was shown that TNF alpha-mediated activation of NF-B depends on the association of RIP and FAK (11). TNF-induced NF-B DNA binding activity and activation of IB kinases were markedly impaired in FAK Ϫ/Ϫ cells (11). However, it has been well established that RIP has a dual function and is capable of either inducing apoptosis or activating cellular survival signals (14,17,23,27,38,39). Similarly, it has been proposed that RIP is one of the switch...

Epstein-Barr virus (EBV) is associated with several human diseases including infectious mononucleosis and nasopharyngeal carcinoma. EBV-encoded latent membrane protein 1 (LMP1) is oncogenic and indispensablefor cellular transformation caused by EBV. Expression of LMP1 in host cells constitutively activates both the c-Jun N-terminal kinase (JNK) and NF-B pathways, which contributes to the oncogenic effect of LMP1. However, the underlying signaling mechanisms are not very well understood. Based mainly on overexpression studies with various dominant-negative constructs, LMP1 was generally thought to functionally mimic members of the tumor necrosis factor (TNF) receptor superfamily in signaling. In contrast to the prevailing paradigm, using embryonic fibroblasts from different knockout mice and the small interfering RNA technique, we find that the LMP1-mediated JNK pathway is distinct from those mediated by either TNF-␣ or interleukin-1. Moreover, we have further elucidated the LMP1-mediated JNK pathway by demonstrating that LMP1 selectively utilizes TNF receptor-associated factor 6, TAK1/TAB1, and c-Jun N-terminal kinase kinases 1 and 2 to activate JNK.Epstein-Barr virus (EBV) is a human ␥-herpesvirus causally linked with several different human diseases including nasopharyngeal carcinoma (NPC), Hodgkin's lymphoma, Burkitt's lymphoma, and infectious mononucleosis (15, 31). The incidence of NPC in southern China, including Guangdong province and Hong Kong, is among the highest in the world (28). A clear understanding of the molecular mechanisms underlying EBV-associated pathogenesis is of paramount importance in formulating an effective therapy.EBV can readily transform primary resting B lymphocytes into immortalized lymphoblastoid cell lines (LCLs) (15, 31). EBV-encoded latent membrane protein 1 (LMP1) is indispensable for establishment of lymphoblastoid cell lines by EBV (23). Among several latent viral genes expressed in NPC and EBV-positive Hodgkin's disease, LMP1 is the only one with oncogenic properties (31). When introduced into rodent fibroblasts, LMP1 causes oncogenic transformation (43). LMP1 also promotes a higher incidence of lymphoma in transgenic mice when specifically introduced into lymphocytes (27). These results establish a critical role for LMP1 in EBV-associated malignances.LMP1 is a membrane protein of 386 amino acids containing six transmembrane domains. Both the amino (amino acids [aa] 1 to 24) and carboxyl (aa 186 to 386) termini of LMP1 are located in the cytoplasm (Fig. 1A). While the short amino terminus of LMP1 is implicated in anchoring LMP1 on the plasma membrane, its carboxyl terminus is implicated in both cellular transformation and activation of intracellular signaling pathways. Two subregions in the carboxyl tail of LMP1 are critical in cell transformation and signaling: carboxyl-terminal activating region 1 (CTAR1, aa 194 to 231) and CTAR2 (aa 351 to 386) (Fig. 1A). CTAR1 is capable of binding several tumor necrosis factor (TNF) receptor-associated factors (TRAFs) and activating ...

Although the p53 tumor suppressor/transcription factor often accumulates in the cytoplasm of healthy cells, limited information is available on the cytoplasmic function of p53. Here, we show that cytoplasmic p53 suppresses cell invasion by reducing mitochondrial reactive oxygen species (ROS) levels. Analysis revealed that this function is mediated by Bcl-2 family proteins: Cytoplasmic p53 binds Bcl-w, liberating Bax, which then binds ND5, a subunit of respiratory complex-I, thereby suppressing complex-I activity and thus ROS production. The G13289A mutation of ND5, identified in cancer patients, prevents Bax/ND5 interactions and promotes ROS production and cell invasion. We also showed that Bcl-XL and Bak can substitute for Bcl-w and Bax, respectively, regulating complex-I activity and supporting the cytoplasmic function of p53; nuclear p53 also suppresses complex-I activity by inducing Bax expression. Studies in animal models support the notion that p53 and Bcl-2 family proteins exhibit these functions in vivo. This study demonstrates a link between p53 and Bcl-2 proteins as regulators of ROS production and cellular invasiveness, and reveals complex-I, especially ND5, as their functional target.

Constitutive NF-κB activation in cancer cells is caused by defects in the signalling network responsible for terminating the NF-κB response. Here we report that plant homeodomain finger protein 20 maintains NF-κB in an active state in the nucleus by inhibiting the interaction between PP2A and p65. We show that plant homeodomain finger protein 20 induces canonical NF-κB signalling by increasing the DNA-binding activity of NF-κB subunit p65. In plant homeodomain finger protein 20-overexpressing cells, the termination of tumour necrosis factor-induced p65 phosphorylation is impaired whereas upstream signalling events triggered by tumour necrosis factor are unaffected. This effect strictly depends on the interaction between plant homeodomain finger protein 20 and methylated lysine residues of p65, which hinders recruitment of PP2A to p65, thereby maintaining p65 in a phosphorylated state. We further show that plant homeodomain finger protein 20 levels correlate with p65 phosphorylation levels in human glioma specimens. Our work identifies plant homeodomain finger protein 20 as a novel regulator of NF-κB activation and suggests that elevated expression of plant homeodomain finger protein 20 may drive constitutive NF-κB activation in some cancers.