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Oligomerisation-driven MLKL ubiquitylation antagonises necroptosis

ABSTRACT: Mixed lineage kinase domain-like (MLKL) is the executioner in the caspase-independent form of programmed cell death called necroptosis. Receptor-interacting serine/threonine protein kinase 3 (RIPK3) phosphorylates MLKL, triggering MLKL oligomerization, membrane translocation and membrane disruption. MLKL also undergoes ubiquitylation during necroptosis, yet neither the mechanism nor the significance of this event has been demonstrated. Here we show that necroptosis-specific multi-mono-ubiquitylation of MLKL occurs following its activation and oligomerization. Ubiquitylated MLKL accumulates in a digitonin-insoluble cell fraction comprising organellar and plasma membranes and protein aggregates. Appearance of this ubiquitylated MLKL form can be reduced by expression of a plasma membrane-located deubiquitylating enzyme. Oligomerization-induced MLKL ubiquitylation occurs on at least four separate lysine residues, and correlates with its proteasome- and lysosome-dependent turnover. Using a MLKL-DUB fusion strategy, we show that constitutive removal of ubiquitin from MLKL licences MLKL auto-activation independent of necroptosis signalling in mouse and human cells. Therefore, in addition to the role of ubiquitylation in the kinetic regulation of MLKL-induced death following an exogenous necroptotic stimulus, it also contributes to restraining basal levels of activated MLKL to avoid unwanted cell death.

SUBMITTER: Mr. Zikou Liu

PROVIDER: S-SCDT-EMBOJ-2019-103718 | biostudies-other |

REPOSITORIES: biostudies-other

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Oligomerization-driven MLKL ubiquitylation antagonizes necroptosis.

Project description:Mixed lineage kinase domain-like (MLKL) is the executioner in the caspase-independent form of programmed cell death called necroptosis. Receptor-interacting serine/threonine protein kinase 3 (RIPK3) phosphorylates MLKL, triggering MLKL oligomerization, membrane translocation and membrane disruption. MLKL also undergoes ubiquitylation during necroptosis, yet neither the mechanism nor the significance of this event has been demonstrated. Here, we show that necroptosis-specific multi-mono-ubiquitylation of MLKL occurs following its activation and oligomerization. Ubiquitylated MLKL accumulates in a digitonin-insoluble cell fraction comprising organellar and plasma membranes and protein aggregates. Appearance of this ubiquitylated MLKL form can be reduced by expression of a plasma membrane-located deubiquitylating enzyme. Oligomerization-induced MLKL ubiquitylation occurs on at least four separate lysine residues and correlates with its proteasome- and lysosome-dependent turnover. Using a MLKL-DUB fusion strategy, we show that constitutive removal of ubiquitin from MLKL licences MLKL auto-activation independent of necroptosis signalling in mouse and human cells. Therefore, in addition to the role of ubiquitylation in the kinetic regulation of MLKL-induced death following an exogenous necroptotic stimulus, it also contributes to restraining basal levels of activated MLKL to avoid unwanted cell death.

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Oligomerisation-driven MLKL ubiquitylation antagonises necroptosis

Project description:Mixed lineage kinase domain-like (MLKL) is the executioner in the caspase 8-independent form of programmed cell death called necroptosis. Once Receptor Interacting serine/threonine Protein Kinase 3 (RIPK3) is activated by upstream cell death signals, it phosphorylates MLKL and triggers the oligomerization and membrane translocation required for MLKL induced membrane disruption. Besides phosphorylation, MLKL also undergoes ubiquitylation during the early stages of necroptosis, yet neither the mechanism nor the significance of this event has been demonstrated. Here we show that necroptosis-specific, multi-mono-ubiquitylation of MLKL occurs on biological membranes, and requires its activation and oligomerisation. Inactive MLKL mutants recruited to membranes during necroptosis are ubiquitylated but this results in their proteasome and lysosome dependent turnover. We identified several ubiquitylated lysines however mutation of these did not affect MLKL ubiquitylation in response to a necroptotic stimulus.

2021-04-28 | PXD015537 | Pride

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