Exogenous stimuli provoke assembly of the receptor-interacting serine/threonine protein kinase RIPK1:RIPK3 oligomeric complex, termed the necrosome, which acts as a platform for recruiting and activating mixed lineage kinase domain-like protein (MLKL), the terminal effector pseudokinase in the necroptotic signaling pathway (reviewed by Murphy JM 2020). RIPK3-mediated phosphorylation is thought to initiate MLKL oligomerization, membrane translocation and membrane disruption (Sun L et al. 2012; Wang H et al. 2014; Petrie EJ et al. 2018; Samson AL et al. 2020). Studies in human cell lines suggest that upon induction of necroptosis 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 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). 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). Further, ITCH co-immunoprecipitated with MLKL upon co-expression of tagged proteins in human embryonic kidney 293T (HEK293T) cells (Yoon S et al. 2022). ITCH also associated with MLKL in MLKL knocked-down HT-29 cells that inducibly expressed wild-type MLKL. Immunocytochemistry assay showed that MLKL colocalized with ITCH in both the early and the late endosomes in TBZ-treated HT-29 cells. Mutagenesis analysis using HEK293T cells revealed that ITCH binds the pseudokinase domain of MLKL. WW domain of ITCH facilitates this binding. ITCH was shown to catalyze K63-linked pUb of MLKL at K50 targeting it to the endosomal membrane (Yoon S et al. 2022).