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Proc Natl Acad Sci U S A. 2014 Oct 21;111(42):15072-7. doi: 10.1073/pnas.1408987111. Epub 2014 Oct 6.

Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death.

Author information

1
The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Departments of Medical Biology and.
2
The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Departments of Medical Biology and Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, VIC 3800, Australia;
3
The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia;
4
Biomolecular Interactions Centre, School of Biological Sciences, University of Canterbury, Christchurch, New Zealand; Department of Biochemistry, Bio21 Institute, University of Melbourne, Parkville, VIC 3052, Australia;
5
Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia; and.
6
SynThesis MedChem, Parkville, VIC 3052, Australia.
7
The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Departments of Medical Biology and Pharmacology and Therapeutics, University of Melbourne, Parkville, VIC 3050, Australia;
8
The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Departments of Medical Biology and jamesm@wehi.edu.au silke@wehi.edu.au.

Abstract

Necroptosis is considered to be complementary to the classical caspase-dependent programmed cell death pathway, apoptosis. The pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) is an essential effector protein in the necroptotic cell death pathway downstream of the protein kinase Receptor Interacting Protein Kinase-3 (RIPK3). How MLKL causes cell death is unclear, however RIPK3-mediated phosphorylation of the activation loop in MLKL trips a molecular switch to induce necroptotic cell death. Here, we show that the MLKL pseudokinase domain acts as a latch to restrain the N-terminal four-helix bundle (4HB) domain and that unleashing this domain results in formation of a high-molecular-weight, membrane-localized complex and cell death. Using alanine-scanning mutagenesis, we identified two clusters of residues on opposing faces of the 4HB domain that were required for the 4HB domain to kill cells. The integrity of one cluster was essential for membrane localization, whereas MLKL mutations in the other cluster did not prevent membrane translocation but prevented killing; this demonstrates that membrane localization is necessary, but insufficient, to induce cell death. Finally, we identified a small molecule that binds the nucleotide binding site within the MLKL pseudokinase domain and retards MLKL translocation to membranes, thereby preventing necroptosis. This inhibitor provides a novel tool to investigate necroptosis and demonstrates the feasibility of using small molecules to target the nucleotide binding site of pseudokinases to modulate signal transduction.

KEYWORDS:

ATP mimetic; RIP kinase; programmed necrosis; pseudoenzyme

PMID:
25288762
PMCID:
PMC4210347
DOI:
10.1073/pnas.1408987111
[Indexed for MEDLINE]
Free PMC Article

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