Phosphorylated MLKL causes plasma membrane rupture

paper type paper type COMMeNtary Necroptosis, cell death caused by uncontrolled swelling (oncosis) and rapid plasma membrane rupture, exerts detrimental pathogenic effects. According to recent reports by Wang et al. 3 in Molecular Cell and Dondelinger et al. 4 in Cell Reports, the underlying mechanism of necroptosis might involve the assembly of pore-forming oligomers of phosphory-lated mixed lineage kinase domain-like protein (pMLKL). Wang et al. 3 followed a straight line of investigation after their group had identified phosphorylation of MLKL as a downstream target of receptor-interacting protein kinase 3 (RIPK3). 5 Within a supramolecular platform referred to as the necrosome RIPK3 phosphorylates a number of proteins to exert currently uncharacterized functions, but the only one shown to be critically involved in the execution of necroptosis to date is MLKL. The authors report the first tool to directly detect an active necroptosis pathway: a highly specific rabbit anti-human pMLKL antibody that was raised against the MLKL phosphorylation motif. In addition to its value as a scientific tool, this antibody may potentially be used for clinical diagnostics. In the commonly used necroptosis-sensitive human adeno-carcinoma cell line HT29, the pMLKL-antibody detected a specific signal within 6 h after induction of necroptosis by the classic combination of TNFα, Smac mimetics, and the pan-caspase inhibi-tor zVAD-fmk. Because the execution of necroptosis includes cellular swelling (oncosis), the authors hypothesized that pMLKL itself might form pores in membranes or might associate with membranes as preformed complexes. In an elegant series of centrifugation and phase shift assays, Wang et al. demonstrate that pMLKL, in contrast to cytosolic non-phosphorylated MLKL, is almost exclusively detected in membrane fractions such as plasma membranes and heavy or light intracellular organelles, but not in the cytosol. Furthermore, protein-lipid overlay assays revealed association of the MLKL coiled-coil region with phospha-tidylinositol phosphates (phosphoinositi-des) and with high concentrations of cardiolipids. Importantly, recombinant MLKL phosphomimic mutants were sufficient to disintegrate the membranes of liposomes, especially when the liposomes contained PtdIns(4,5)P2, raising the possibility that MLKL itself, which forms supramolecular clusters, creates a hole in the lipid bilayer to facilitate oncosis. Certainly, this is not a cellular model and is far from direct in vivo proof, but this report proposes a novel concept for the downstream execution of necropto-sis (Fig. 1). Importantly, necrostatin-1, a compound that keeps RIPK1 in a closed configuration and thereby inhibits tumor necrosis factor (TNF)-mediated RIPK3 activation, 6,7 prevents phosphorylation of MLKL and bursting …