TNF-induced necroptosis requires the plasma membrane localization of the MLKL protein

The transient, sequential targeting of multiple remodeler complexes to nucleosomes by specific DNA-bound factors results in the opening (right) and closing (left) of chromatin.

MLKL-shRNA cells—in which the gene for MLKL is silenced—were imaged with confocal microscopy immediately after and 4 hours after TSZ-induced necroptosis. Cells transfected with MLKL conjugated to the fluorescent tag DsRed showed more localization of the protein to the cell’s plasma membrane, away from the the cytosol (blue clumps) than cells transfected with mutant forms of MLKL (CC1 and CC2) that do not form trimers.

The cell signaling protein tumor necrosis factor (TNF), produced by white blood cells, promotes inflammation and immunity processes such as fever and is involved in tumorigenesis and apoptosis (programmed cell death). However, dysregulation of TNF can also lead to another form of programmed cell death called necroptosis, which is characterized by a rise in intracellular Ca2+, generation of reactive oxygen species (ROS), intracellular acidity, depletion of ATP, and, eventually, plasma membrane rupture. TNF-induced necroptosis has been associated with a wide variety of diseases including neurodegenerative diseases, major depression, rheumatoid arthritis, and cancer. Whereas the signaling mechanisms underlying TNF-induced apoptosis have largely been determined, the events precipitating in TNF-initiated necroptosis are still unknown.

Zheng-Gang Liu, Ph.D., the head of CCR’s Molecular Mechanisms of Apoptosis Section, and his colleagues recently identified the mixed lineage kinase domain-like protein (MLKL) as a key downstream component of TNF-induced necroptosis in human colon adenocacinoma cultured cell lines (HT29 cells). Liu’s team found that MLKL was phosphorylated by RIP3 (receptor interacting protein 3) and, through its interactions with RIP3, MLKL was recruited into the RIP3 “necrosome,” the multiprotein-signaling complex that triggers TNF-induced necroptosis. However little is known about the activity of MLKL during necroptosis.

To determine MLKL’s role in necroptosis, Liu and his colleagues first sought to identify the form MLKL takes during necroptosis, which they initiated with treatment of the HT29 cells with the combination treatment of TSZ. Using SDS-polyacrylamide gel electrophoresis, the researchers demonstrated that MLKL forms a homotrimer—a macromolecule composed of three identical polypeptides—upon initiation of necroptosis. They also discovered that trimerization results from MLKL’s phosphorylation by RIP3. 

The team then used fluorescent markers conjugated to MLKL and RIP3 to image the proteins’ intracellular movements following the initiation of necroptosis. Both proteins were found to localize to the plasma membrane. Further tests indicated that phosphorylation of MLKL was essential for its translocation to the membrane, and that translocation of RIP3 to the membrane required the presence of MLKL.

Once MLKL was found to localize to the plasma membrane, the team investigated the protein’s function. Knowing that Ca2+ influx is an early event in TNF-induced necroptosis, the researchers explored whether MLKL played a role in triggering the ion’s influx. Short hairpin RNA-silencing of the gene that encodes MLKL resulted in complete blockage of Ca2+ influx, which was restored upon ectopic expression of wild type MLKL. Knocking down RIP3 also blocked Ca2+ influx.

Next the team sought to identify which type of Ca2+ channel was activated by MLKL to allow the influx of Ca2+ ions. They treated their HT29 cells with a variety of ion channel blockers, and found that only blockers of non-voltage-sensitive ion channel blockers spared the cells from TSZ-induced necroptosis. In a knockdown experiment in which the genetically encoded Ca2+ indicator GCaMP3 was used to track the ion’s movement, the team found that TRPM7, a non-voltage-sensitive ion channel that has previously been implicated in necroptosis, was the channel activated by MLKL. Importantly, knockdown of TRPM7 did not interfere with MLKL’s phosphorylation by RIP3, MLKL’s subsequent trimerization, or MLKL’s translocation to the plasma membrane.

In summary, Liu and his colleagues demonstrated that during TNF-induced necroptosis homotrimers of the protein MLKL facilitate the translocation of the RIP3 necrosome-signaling complex to the plasma membrane. MLKL then triggers the influx of Ca2+ through TRPM7 ion channels, a process that disrupts the integrity of the plasma membrane, ultimately leading to cell death.

Summary Posted: 01/2014


Cai Z, Jitkaew S, Zhao J, Chiang H-C, Choksi S, Liu J, Ward Y, Wu L-G, Liu Z-G. Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis. Nature Cell Biology, December 8, 2013 PubMed Link