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

Joanne M Hildebrand, Maria C Tanzer, Isabelle S Lucet, Samuel N Young, Sukhdeep K Spall, Pooja Sharma, Catia Pierotti, Jean-Marc Garnier, Renwick C J Dobson, Andrew I Webb, Anne Tripaydonis, Jeffrey J Babon, Mark Mulcair, Martin J Scanlon, Warren S Alexander, Andrew F Wilks, Peter E Czabotar, Guillaume Lessene, James M Murphy, John Silke

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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.
Original languageEnglish
Pages (from-to)15072 - 15077
Number of pages6
JournalProceedings of the National Academy of Sciences
Volume111
Issue number42
DOIs
Publication statusPublished - 2014

Cite this

Hildebrand, Joanne M ; Tanzer, Maria C ; Lucet, Isabelle S ; Young, Samuel N ; Spall, Sukhdeep K ; Sharma, Pooja ; Pierotti, Catia ; Garnier, Jean-Marc ; Dobson, Renwick C J ; Webb, Andrew I ; Tripaydonis, Anne ; Babon, Jeffrey J ; Mulcair, Mark ; Scanlon, Martin J ; Alexander, Warren S ; Wilks, Andrew F ; Czabotar, Peter E ; Lessene, Guillaume ; Murphy, James M ; Silke, John. / Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death. In: Proceedings of the National Academy of Sciences. 2014 ; Vol. 111, No. 42. pp. 15072 - 15077.
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title = "Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death",
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.",
author = "Hildebrand, {Joanne M} and Tanzer, {Maria C} and Lucet, {Isabelle S} and Young, {Samuel N} and Spall, {Sukhdeep K} and Pooja Sharma and Catia Pierotti and Jean-Marc Garnier and Dobson, {Renwick C J} and Webb, {Andrew I} and Anne Tripaydonis and Babon, {Jeffrey J} and Mark Mulcair and Scanlon, {Martin J} and Alexander, {Warren S} and Wilks, {Andrew F} and Czabotar, {Peter E} and Guillaume Lessene and Murphy, {James M} and John Silke",
year = "2014",
doi = "10.1073/pnas.1408987111",
language = "English",
volume = "111",
pages = "15072 -- 15077",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
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Hildebrand, JM, Tanzer, MC, Lucet, IS, Young, SN, Spall, SK, Sharma, P, Pierotti, C, Garnier, J-M, Dobson, RCJ, Webb, AI, Tripaydonis, A, Babon, JJ, Mulcair, M, Scanlon, MJ, Alexander, WS, Wilks, AF, Czabotar, PE, Lessene, G, Murphy, JM & Silke, J 2014, 'Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death', Proceedings of the National Academy of Sciences, vol. 111, no. 42, pp. 15072 - 15077. https://doi.org/10.1073/pnas.1408987111

Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death. / Hildebrand, Joanne M; Tanzer, Maria C; Lucet, Isabelle S; Young, Samuel N; Spall, Sukhdeep K; Sharma, Pooja; Pierotti, Catia; Garnier, Jean-Marc; Dobson, Renwick C J; Webb, Andrew I; Tripaydonis, Anne; Babon, Jeffrey J; Mulcair, Mark; Scanlon, Martin J; Alexander, Warren S; Wilks, Andrew F; Czabotar, Peter E; Lessene, Guillaume; Murphy, James M; Silke, John.

In: Proceedings of the National Academy of Sciences, Vol. 111, No. 42, 2014, p. 15072 - 15077.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death

AU - Hildebrand, Joanne M

AU - Tanzer, Maria C

AU - Lucet, Isabelle S

AU - Young, Samuel N

AU - Spall, Sukhdeep K

AU - Sharma, Pooja

AU - Pierotti, Catia

AU - Garnier, Jean-Marc

AU - Dobson, Renwick C J

AU - Webb, Andrew I

AU - Tripaydonis, Anne

AU - Babon, Jeffrey J

AU - Mulcair, Mark

AU - Scanlon, Martin J

AU - Alexander, Warren S

AU - Wilks, Andrew F

AU - Czabotar, Peter E

AU - Lessene, Guillaume

AU - Murphy, James M

AU - Silke, John

PY - 2014

Y1 - 2014

N2 - 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.

AB - 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.

UR - http://www.pnas.org/content/111/42/15072.full.pdf+html

U2 - 10.1073/pnas.1408987111

DO - 10.1073/pnas.1408987111

M3 - Article

VL - 111

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EP - 15077

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 42

ER -