MLKL oligomerizes

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R-HSA-5357927
Type
Reaction [transition]
Species
Homo sapiens
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Mixed lineage kinase domain-like protein (MLKL) was found to form oligomers (detected as homotrimers) when it was overexpressed in human embryonic kidney 293 (HEK293) cells (Cai Z et al. 2014). The oligomerization of MLKL was also observed in multiple human (colon adenocarcinoma HT29, FADD-null Jurkat cells, leukemic monocyte lymphoma U937) and mouse cells upon (TNF+Smac mimetic+caspase inhibitor z-VAD-FMK)-induced necroptosis (Cai Z et al. 2014; Chen X et al. 2014). Low-resolution techniques including cross-linking and deuterium exchange mass spectrometry and small angle X-ray scattering (SAXS) showed that MLKL exists in equilibrium between a monomer and a daisy chain tetramer with the N-terminal execution domain (NED) of one monomer binding to the pseudokinase domain (psKD) of another monomer (Petrie EJ et al. 2018). Cys-oxidation under nonreducing conditions and crosslinking analyses detected tetramers and octamers in L929 murine fibroblast and HEK293 cells undergoing TNF-mediated necroptosis (Huang D et al. 2017). Whether MLKL forms trimers, tetramers or higher molecular structures, remains to be clarified (Cai Z et al. 2014; Chen X et al. 2014; Dondelinger Y et al. 2014; Wang H et al. 2014; Petrie EJ et al. 2018). The oligomerization of MLKL was blocked by reducing agents such as dithiothreitol or beta-mercaptoethanol, suggesting that oligomeric forms are probably stabilized by disulphide bonds (Cai Z et al. 2014; Wang H et al. 2014). Oligomeric MLKL was not detected in cell lysates of HEK293 cells that were transfected with kinase-dead RIPK3 and MLKL (Cai Z et al. 2014). Mutations in phosphorylation sites of MLKL also blocked MLKL oligomerization (Cai Z et al. 2014; Wang H et al. 2014). Moreover, gel filtration chromatography analysis of recombinant wild-type or phosphorylation site mutant MLKL proteins is in agreement with immunoblotting data, confirming that RIPK3-mediated phosphorylation of MLKL promotes oligomerization of MLKL (Wang H et al. 2014). RIPK3-mediated phosphorylation of MLKL’s pseudokinase domain leads to MLKL switching from an inert to activated state, where exposure of the N-terminal four-helix bundle (4HB) ‘executioner’ domain leads to cell death (Hildebrand JM et al 2014; Petrie EJ et al. 2018). Following activation, toggling within the MLKL pseudokinase domain promotes 4HB domain disengagement from the pseudokinase domain αC helix and pseudocatalytic loop, to enable formation of a necroptosis-inducing tetramer (Petrie EJ et al. 2018). The two interdomain helices, termed the ‘brace’ helices, contribute to MLKL oligomerization by connecting phosphorylation of the pseudokinase domain to the release or activation of the 4HB domain executioner function to enable its participation in membrane localisation, permeabilisation and cell death (Davies KA et al. 2018). In addition, the autoinhibited N-terminal execution domain of human MLKL is activated by inositol phosphate metabolites IP4, IP5 and IP6 produced by the IPKs inositol phosphate multikinase (IPMK), inositol tetrakisphosphate kinase 1 (ITPK1) and inositol pentakisphosphate 2-kinase (IPPK) (Dovey CM et al. 2018; McNamara DE et al. 2019). Oligomers of MLKL translocate to membrane compartments(Cai Z et al. 2014; Dondelinger Y et al. 2014; Wang H et al. 2014).

Even though the stoichiometry of the MLKL oligomerization in the Reactome event depicts MLKL homotetramer, it remains unclear whether MLKL forms trimers, tetramers or higher molecular structure (Cai Z et al. 2014; Chen X et al. 2014; Dondelinger Y et al. 2014; Wang H et al. 2014; Huang D et al. 2017; Petrie EJ et al. 2018).

Literature References
PubMed ID Title Journal Year
24703947 Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3

Wang, H, Sun, L, Su, L, Rizo, J, Liu, L, Wang, LF, Wang, FS, Wang, X

Mol. Cell 2014
24316671 Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis

Cai, Z, Jitkaew, S, Zhao, J, Chiang, HC, Choksi, S, Liu, J, Ward, Y, Wu, LG, Liu, ZG

Nat. Cell Biol. 2014
29930286 Conformational switching of the pseudokinase domain promotes human MLKL tetramerization and cell death by necroptosis

Petrie, EJ, Sandow, JJ, Jacobsen, AV, Smith, BJ, Griffin, MDW, Lucet, IS, Dai, W, Young, SN, Tanzer, MC, Wardak, A, Liang, LY, Cowan, AD, Hildebrand, JM, Kersten, WJA, Lessene, G, Silke, J, Czabotar, PE, Webb, AI, Murphy, JM

Nat Commun 2018
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