Abstract
Phosphorylation of the pseudokinase mixed lineage kinase domain-like protein (MLKL) by the protein kinase RIPK3 targets MLKL to the cell membrane, where it triggers necroptotic cell death. We report that conjugation of K63-linked polyubiquitin chains to distinct lysine residues in the N-terminal HeLo domain of phosphorylated MLKL (facilitated by the ubiquitin ligase ITCH that binds MLKL via a WW domain) targets MLKL instead to endosomes. This results in the release of phosphorylated MLKL within extracellular vesicles. It also prompts enhanced endosomal trafficking of intracellular bacteria such as Listeria monocytogenes and Yersinia enterocolitica to the lysosomes, resulting in decreased bacterial yield. Thus, MLKL can be directed by specific covalent modifications to differing subcellular sites, whence it signals either for cell death or for non-deadly defense mechanisms.
Highlights
Exploration of the mechanisms by which cytokines of the TNF family induce cell death has led to the identification of a form of programmed cell death called “necroptosis” in which the protein mixed lineage kinase domain-like molecule (MLKL), upon its phosphorylation by the protein kinase RIPK3, triggers rupture of the cellular membrane
Mutation of the lysine residue at Besides phosphorylating mixed lineage kinase domain-like protein (MLKL), treatment of cells by agents that position 50 in MEFs did not affect the extent of its the caspase inhibitor z-VAD-fmk (TBZ)-induced activate the protein kinase RIPK3 triggers a time-dependent MLKL ubiquitination, nor was the ubiquitination affected by mutation of ubiquitination [25]
Both in induced in human epithelial HT-29 cells by combined treatment MEFs and in the mouse L929 cells, ubiquitination was dramatically with the cytokine TNF, the inhibitor of apoptosis proteins (IAP) reduced when lysines 50 and 51 were each replaced by arginine antagonist BV6, and the caspase inhibitor z-VAD-fmk (TBZ)
Summary
Exploration of the mechanisms by which cytokines of the TNF family induce cell death has led to the identification of a form of programmed cell death called “necroptosis” in which the protein mixed lineage kinase domain-like molecule (MLKL), upon its phosphorylation by the protein kinase RIPK3, triggers rupture of the cellular membrane. Detailed mutational and structural analyses of the HeLo domains of human and mouse MLKL revealed evolutionary divergence, reflected in the involvement of different epitopes in these two species in death mediation, at differing locations in their HeLo domains and with differing abilities to bind lipids Studies of both species have so far yielded no clear notion of the way by which association of these epitopes with the cellular membrane dictates its rupture [reviewed in [9].
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