In 2012, Mixed lineage kinase domain-like (MLKL), a catalytically-dead (“zombie”) cousin of conventional protein kinases, termed a pseudokinase, was implicated as the key effector in the programmed necrosis (or necroptosis) cell death pathway. This pathway has been implicated in innate immunity, the pathogenesis of inflammatory diseases, and tissue injury arising from ischemia-reperfusion. As a result, an improved fundamental knowledge of MLKL’s activation mechanism is of enormous interest as we and others look to target the pathway therapeutically.
Here, we describe our recent work dissecting the chronology of events in this pathway using novel tools, structural biology, biochemistry, microscopy and proteomics. These studies have uncovered a series of regulated events that govern the pathway that we term checkpoints. Death is (i) initiated by recruitment of dormant MLKL from the cytoplasm to a large molecular weight platform termed the necrosome where RIPK3-mediated phosphorylation triggers (ii) a conformation change in MLKL, release from the necrosome and assembly into large MLKL clusters. MLKL oligomers are subsequently (iii) trafficked to the plasma membrane where (iv) MLKL accumulates into hotspots that exceed a critical threshold to enable membrane permeabilization and cell death.