Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death

Abstract

Mixed lineage kinase domain-like protein (MLKL) was identified to function downstream of receptor interacting protein 3 (RIP3) in tumor necrosis factor-α (TNF)-induced necrosis (also called necroptosis). However, how MLKL functions to mediate necroptosis is unknown. By reconstitution of MLKL function in MLKL-knockout cells, we showed that the N-terminus of MLKL is required for its function in necroptosis. The oligomerization of MLKL in TNF-treated cells is essential for necroptosis, as artificially forcing MLKL together by using the hormone-binding domain (HBD*) triggers necroptosis. Notably, forcing together the N-terminal domain (ND) but not the C-terminal kinase domain of MLKL causes necroptosis. Further deletion analysis showed that the four-α-helix bundle of MLKL (1-130 amino acids) is sufficient to trigger necroptosis. Both the HBD*-mediated and TNF-induced complexes of MLKL(ND) or MLKL are tetramers, and translocation of these complexes to lipid rafts of the plasma membrane precedes cell death. The homo-oligomerization is required for MLKL translocation and the signal sequence for plasma membrane location is located in the junction of the first and second α-helices of MLKL. The plasma membrane translocation of MLKL or MLKL(ND) leads to sodium influx, and depletion of sodium from the cell culture medium inhibits necroptosis. All of the above phenomena were not seen in apoptosis. Thus, the MLKL oligomerization leads to translocation of MLKL to lipid rafts of plasma membrane, and the plasma membrane MLKL complex acts either by itself or via other proteins to increase the sodium influx, which increases osmotic pressure, eventually leading to membrane rupture.

GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death

“…Binding of the FHBD to negatively charged phosphatidylinositol phosphates has been proposed to recruit MLKL to the plasma membrane allowing it to directly permeabilize the membrane by forming a pore (Dondelinger et al , 2014; Su et al , 2014; Wang et al , 2014). Alternatively, it has been also proposed that plasma membrane‐bound MLKL recruits Ca 2+ or Na + ion channels to permeabilize the membrane (Cai et al , 2014; Chen et al , 2014). Thus, although the steps leading up to necroptosis are markedly different from pyroptosis, the execution of both types of lytic cell death involves the formation of plasma membrane pores.…”

RIP kinases: key decision makers in cell death and innate immunity

“…Phosphorylation of MLKL promotes its oligomerization and translocation to the plasma membrane where it interacts with phospholipids and compromises membrane integrity ultimately resulting in cell rupture. [88][89][90][91] MLKL also promotes the generation of reactive oxygen species (ROS) and late phase activation of JNK. 92 The increased production of ROS, especially by the mitochondria, has been strongly linked with mediating TNFinduced necrosis.…”

Evolutionary divergence of the necroptosis effector MLKL

“…[5][6][7][8] RIPK3 phosphorylates the pseudokinase domain of MLKL, the most terminal known essential component of the pathway, 5,6 which is believed to induce a conformational change and unleash the N-terminal four-helix bundle (4HB) domain of MLKL: an executioner domain. 5,9,10 Several models have been proposed for how this 4HB domain might induce cell death, including activation of downstream effectors, such as ion channels, 11,12 direct permeabilization of membranes and/or formation of a transmembrane pore, 13,14 all of which remain the subject of debate. The consensus from these and other studies is that in order to kill, MLKL must translocate to membranes and assemble into high molecular weight signalling complexes, which are likely to be MLKL oligomers, although the stoichiometry of these MLKL oligomers remains an open question.…”