The pseudokinase MLKL (mixed lineage kinase domain-like), has recently emerged as a critical component of the necroptosis cell death pathway. Although it is clear that phosphorylation of the activation loop in the MLKL pseudokinase domain by the upstream protein kinase RIPK3 (receptor-interacting protein kinase-3), is crucial to trigger MLKL activation, it has remained unclear whether other phosphorylation events modulate MLKL function. By reconstituting Mlkl−/−, Ripk3−/− and Mlkl−/−Ripk3−/− cells with MLKL phospho-site mutants, we compared the function of known MLKL phosphorylation sites in regulating necroptosis with three phospho-sites that we identified by MS, Ser158, Ser228 and Ser248. Expression of a phosphomimetic S345D MLKL activation loop mutant-induced stimulus-independent cell death in all knockout cells, demonstrating that RIPK3 phosphorylation of the activation loop of MLKL is sufficient to induce cell death. Cell death was also induced by S228A, S228E and S158A MLKL mutants in the absence of death stimuli, but was most profound in Mlkl−/−Ripk3−/− double knockout fibroblasts. These data reveal a potential role for RIPK3 as a suppressor of MLKL activation and indicate that phosphorylation can fine-tune the ability of MLKL to induce necroptosis.
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Research Article|
October 02 2015
Necroptosis signalling is tuned by phosphorylation of MLKL residues outside the pseudokinase domain activation loop
Maria C. Tanzer;
Maria C. Tanzer
*The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia
†Department of Medical Biology, University of Melbourne, Parkville, Victoria 3050, Australia
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Anne Tripaydonis;
Anne Tripaydonis
*The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia
†Department of Medical Biology, University of Melbourne, Parkville, Victoria 3050, Australia
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Andrew I. Webb;
Andrew I. Webb
*The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia
†Department of Medical Biology, University of Melbourne, Parkville, Victoria 3050, Australia
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Samuel N. Young;
Samuel N. Young
*The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia
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Leila N. Varghese;
Leila N. Varghese
*The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia
†Department of Medical Biology, University of Melbourne, Parkville, Victoria 3050, Australia
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Cathrine Hall;
Cathrine Hall
*The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia
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Warren S. Alexander;
Warren S. Alexander
*The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia
†Department of Medical Biology, University of Melbourne, Parkville, Victoria 3050, Australia
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Joanne M. Hildebrand;
Joanne M. Hildebrand
1
*The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia
†Department of Medical Biology, University of Melbourne, Parkville, Victoria 3050, Australia
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John Silke;
*The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia
†Department of Medical Biology, University of Melbourne, Parkville, Victoria 3050, Australia
2Correspondence may be addressed to either of these authors (email [email protected] or [email protected]
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James M. Murphy
*The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia
†Department of Medical Biology, University of Melbourne, Parkville, Victoria 3050, Australia
2Correspondence may be addressed to either of these authors (email [email protected] or [email protected]
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Publisher: Portland Press Ltd
Received:
June 09 2015
Revision Received:
August 03 2015
Accepted:
August 17 2015
Accepted Manuscript online:
August 17 2015
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2015 Authors; published by Portland Press Limited
2015
Biochem J (2015) 471 (2): 255–265.
Article history
Received:
June 09 2015
Revision Received:
August 03 2015
Accepted:
August 17 2015
Accepted Manuscript online:
August 17 2015
Citation
Maria C. Tanzer, Anne Tripaydonis, Andrew I. Webb, Samuel N. Young, Leila N. Varghese, Cathrine Hall, Warren S. Alexander, Joanne M. Hildebrand, John Silke, James M. Murphy; Necroptosis signalling is tuned by phosphorylation of MLKL residues outside the pseudokinase domain activation loop. Biochem J 15 October 2015; 471 (2): 255–265. doi: https://doi.org/10.1042/BJ20150678
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