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.

Tumor necrosis factor (TNF) is an important inflammatory cytokine and induces many cellular responses, including inflammation, cell proliferation, apoptosis, and necrosis. It is known that receptor interacting protein (RIP) kinases, RIP1 and RIP3, are key effectors of TNF-induced necrosis, but little is known about how these two RIP kinases mediate this process, although reactive oxygen species (ROS) generation and JNK activation have been suggested to be two downstream events of RIP kinases. Here we report the identification of mixed lineage kinase domain-like, MLKL, as a key RIP3 downstream component of TNF-induced necrosis. Through screening a kinase/phosphatase shRNA library in human colon adenocarcinoma HT-29 cells, we found that knockdown of MLKL blocked TNF-induced necrosis. Our data suggest that MLKL functions downstream of RIP1 and RIP3 and is recruited to the necrosome through its interaction with RIP3. Finally, we found that MLKL is required for the generation of ROS and the late-phase activation of JNK during TNF-induced necrosis. However, because these two events are not involved in TNF-induced necrosis in HT-29 cells, the target of MLKL during TNF-induced necrosis remains elusive. Taken together, our study suggests that MLKL is a key RIP3 downstream component of TNF-induced necrotic cell death.T umor necrosis factor (TNF) is a pleiotropic inflammatory cytokine and plays a critical role in diverse cellular events, including cell proliferation, differentiation, apoptosis, and necrosis (1, 2). TNF is also a major mediator of both inflammation and immunity and is involved in many pathological conditions and autoimmune diseases, such as rheumatoid arthritis and Crohn disease (3). Since its tumoricidal activity was discovered, the TNF pathway has been one of the most studied signaling pathways (4). In almost all types of cells treated with TNF, the transcription factor NF-κB and three MAP kinases, ERK, JNK, and p38, are activated and, occasionally, apoptotic or necrotic cell death can be induced as well (5, 6).The molecular mechanisms of TNF signaling have been significantly worked out. It is known that the binding of TNF homotrimer to TNF-receptor 1 (TNF-R1) initiates the formation of TNF-R1 signaling complex by recruiting several adaptor/ effector proteins. TRADD (TNF-R1-associated death domain protein) is the first protein to interact with the receptor through its death domain and recruits other effector proteins, such as RIP1 (receptor interacting protein) and TRAF2 (TNFR-associated factor 2) to form the TNF-R1 signaling complex leading to the activation of several pathways, including NF-κB and MAP kinases (1, 2). Both TRAF2 and RIP1 are necessary for the activation of NF-κB and MAP kinase pathways through recruiting IKK (IκB kinase) and MAP3Ks to the complex (7). Under certain conditions, the complex of TRADD, RIP1, and TRAF2 proteins dissociates from the receptor and recruits other proteins to form different secondary complexes to mediate apoptosis and necrosis (8, 9). Apoptosis is primarily initia...

Summary The regulation of apoptosis is critical for controlling tissue homeostasis and preventing tumor formation and growth. Reactive Oxygen Species (ROS) generation plays a key role in such regulation. Here, we describe a HIF-1 target, ATIA (anti-TNFα-induced apoptosis), which protects cells against TNFα- and hypoxia-induced apoptosis. Through the generation of ATIA knockout mice, we show that ATIA protects cells from apoptosis through regulating the function of the mitochondrial antioxidant, thioredoxin-2, and ROS generation. ATIA is highly expressed in human glioblastoma and ATIA knockdown in glioblastoma cells renders them sensitive to hypoxia-induced apoptosis. Therefore, ATIA is not only a HIF-1 target that regulates mitochondrial redox pathways but a potentially diagnostic marker and therapeutic target in human glioblastoma.