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

作者：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 D; Scanlon Martin J; Alexander Warren S; Wilks Andrew F; Czabotar Peter E; Lessene Guillaume; Murphy James M^*; Silke John

来源：Proceedings of the National Academy of Sciences, 2014, 111(42): 15072-15077.

DOI：10.1073/pnas.1408987111

• pseudoenzyme
• RIP kinase
• ATP mimetic
• programmed necrosis

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 alaninescanning 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.

• 出版日期2014-10-21
• 单位河北医科大学