Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis

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.