Evolutionary divergence of the necroptosis effector MLKL

Abstract

The pseudokinase, MLKL (mixed-lineage kinase domain-like), is the most terminal obligatory component of the necroptosis cell death pathway known. Phosphorylation of the MLKL pseudokinase domain by the protein kinase, receptor interacting protein kinase-3 (RIPK3), is known to be the key step in MLKL activation. This phosphorylation event is believed to trigger a molecular switch, leading to exposure of the N-terminal four-helix bundle (4HB) domain of MLKL, its oligomerization, membrane translocation and ultimately cell death. To examine how well this process is evolutionarily conserved, we analysed the function of MLKL orthologues. Surprisingly, and unlike their mouse, horse and frog counterparts, human, chicken and stickleback 4HB domains were unable to induce cell death when expressed in murine fibroblasts. Forced dimerization of the human MLKL 4HB domain overcame this defect and triggered cell death in human and mouse cell lines. Furthermore, recombinant proteins from mouse, frog, human and chicken MLKL, all of which contained a 4HB domain, permeabilized liposomes, and were most effective on those designed to mimic plasma membrane composition. These studies demonstrate that the membrane-permeabilization function of the 4HB domain is evolutionarily conserved, but reveal that execution of necroptotic death by it relies on additional factors that are poorly conserved even among closely related species. Necroptosis is a form of programmed cell death that can be induced following ligation of death ligand and Toll-like receptors (TLRs). Most experimental work has focused on necroptosis induced by tumour necrosis factor (TNF). The key effectors in the pathway are the protein kinases, receptor interacting protein kinase (RIPK)-1 and RIPK3, 1-4 and the mixed-lineage kinase domain-like (MLKL) pseudokinase. [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. [10][11][12][13][14] Nonetheless, phosphorylation appears to be a key cue for MLKL activation 15 and, as the most terminal known post-translational modification in the pathway, could potentially be utilized as a biomarker in pathologies arising from necroptotic cell death. 14,16 A model whereby RIPK3-mediated phosphorylation of the MLKL pseudokinase domain activation loop (S345 in mouse;...

Active MLKL triggers the NLRP3 inflammasome in a cell-intrinsic manner

“…3C), which we have previously documented to be a hallmark for MLKL activation in fibroblasts treated with necroptotic stimuli TNF/QVD/Cp.A ( Fig. 3C) (15). Indeed, accumulation of activated MLKL at membranes correlated with both macrophage cell death and the secretion of IL-1β ( Fig.…”

“…Inducible lentiviral MLKL constructs were published previously (7,14,15) and used to stably infect THP1 cells. NF-κB reporter THP1 monocytic cells were made by infection with the lentiviral reporter vector (pTRH1-NF-κB-dscGFP, TR503PA; System Bioscience) and sorted by flow cytometry for GFP expression.…”

“…Although MLKL lacks enzymatic activity, phosphorylation of the pseudokinase domain of MLKL is thought to cause a conformational change that exposes the killer N-terminal four-helix bundle (4HB) domain (5,14). Recombinant MLKL (and the 4HB domain) have been shown to permeabilize artificial liposomes in vitro (12,13,15). However, if MLKL alone suffices to induce cell death through membrane disruption, or whether MLKL-induced killing requires other cellular factors, such as ion channel opening (10,11), remains to be clearly defined.…”

The pseudokinase MLKL mediates programmed hepatocellular necrosis independently of RIPK3 during hepatitis

“…This is supported by studies investigating the evolutionary origin of necroptosis that uncovered that RIPK3 is absent in many organisms, such as birds and in mammals of the superorder Xenarthra and in the Monotremata, whereas MLKL is present and RIPK1 retains its RHIM domain (44,45). Interestingly, one of the phosphorylation sites in MLKL identified thus far and the intrinsic membrane-permeabilization function of MLKL seem to be conserved, even in species that have lost RIPK3, indicating that other kinases, such as RIPK1, are likely to be additional modulators of MLKL activation (44,46). However, in our study we did not observe that RIPK1 directly phosphorylates MLKL during ConA hepatitis, indicating that, under these experimental conditions, an as-yet-unidentified kinase is likely to activate MLKL by phosphorylation and promote MLKL jci.org Volume 126 Number 11 November 2016…”

An evolutionary perspective on the necroptotic pathway

“…Oligomerized MLKL was suggested to activate sodium or calcium channels in the plasma membrane, thereby disrupting cellular ion homeostasis and resulting in osmotic cell swelling [20,21]. MLKL was also suggested to directly induce plasma membrane permeabilization; a model supported by the fact that recombinant MLKL can induce leakage from phosphatidylinositol phosphate-containing liposomes [22,23,73,74]. Recently, it was proposed that MLKL could form a cation selective channel in the plasma membrane thereby unifying both models [24].…”