Under basal conditions, mixed lineage kinase domain-like protein (MLKL) and receptor-interacting serine/threonine-protein kinase 3 (RIPK3) are pre-associated in the cytoplasm (Meng Y et al. 2021). Upon induction of necroptosis the RIPK3:MLKL complex is recruited to the necrosome where RIPK3 phosphorylates and activates MLKL (Meng Y et al. 2021). Studies in human cell lines suggest that phosphorylated MLKL shifts to the plasma membrane and membranous organelles such as mitochondria, lysosome, endosome and ER (Wang H et al. 2014). Its trafficking via a Golgi-microtubule-actin-dependent mechanism facilitates plasma membrane translocation, where membrane disruption causes cell death (Samson AL et al. 2020). However, MLKL also exerts non-necroptotic functions such as regulation of endosomal trafficking or MLKL-induced activation of the NLRP3 inflammasome (Yoon S et al. 2017; Shlomovitz I et al. 2020; Yoon S et al. 2022). In addition, MLKL was shown to mediate myelin breakdown in diabetic neuropathy (Guo J et al. 2022). Activation of MLKL triggers its K63-linked ubiquitination (Yoon S et al. 2022). Conjugation of K63-linked polyubiquitin (pUb) chains in the N-terminal HeLo domain of phosphorylated MLKL targets MLKL to endosomes where MLKL is involved in endosomal compartment trafficking and generation of intraluminal and extracellular vesicles (EV) (Yoon S et al. 2017; Shlomovitz I et al. 2020; Yoon S et al. 2022). Endosome-bound MLKL is excluded from the cell within EV (Yoon S et al. 2022). Mass spectroscopic and western blot assays found that HECT-type E3 ubiquitin-protein ligase Itchy homolog (ITCH) is released within EVs alongside MLKL from human epithelial HT-29 cells in response to combined treatment with the cytokine TNF, the inhibitor of apoptosis proteins (IAP) antagonist BV6, and the caspase inhibitor z-VAD-fmk (TBZ) (Yoon S et al. 2017; 2022). Overexpression of ITCH also dramatically enhanced the association of MLKL with ESCRT proteins. Further, ITCH was shown to interact with MLKL to catalyze K63-linked pUb of MLKL at K50 targeting it to the endosomal membrane (Yoon S et al. 2022). Overexpression of ITCH in TBZ-treated HT-29 cells enhanced the ubiquitination of MLKL, but not of K50R MLKL and also enhanced the association of MLKL with ESCRT proteins (Yoon S et al. 2017; 2022). These data suggest that ITCH catalyzes K63-linked polyubiquitination of MLKL at K50 targeting it to the endosomal membrane, where MLKL is no longer involved in necroptosis but instead has a role in vesicle-mediated trafficking. Further, K63-linked polyUb-MLKL contributes to the host defense against pathogenic bacteria by targeting intracellular bacteria, such as Listeria monocytogenes, Yersinia enterocolitica and Escherichia coli, to endosomal-lysosome trafficking system (Yoon S et al. 2022).

This Reactome event shows ITCH-mediated K63-linked polyubiquitination of MLKL at K50.