Upon necroptosis, receptor-interacting serine/threonine protein kinase 3 (RIPK3) phosphorylates the mixed lineage kinase domain-like (MLKL) protein at Thr357 and Ser358 located in the activation loop of pseudokinase domain. The RIPK3-mediated phosphorylation relieves the inhibitory effect of the pseudokinase domain of MLKL, thus allowing the activated MLKL to oligomerize and translocate from the cytosol to cell membranes to cause membrane disintegration. Various studies showed that the endosomal sorting complexes required for transport (ESCRT) pathway can remove phosphorylated MLKL-containing membrane vesicles from cells undergoing necroptosis, thereby attenuating the cell death process (Gong YN et al. 2017; Yoon S et al. 2017; Fan W et al. 2019). The ESCRT-associated proteins, programmed cell death 6-interacting protein (PDCD6IP or ALG-2-interacting protein X, ALIX) and syntenin-1 (SDCBP), were found to mediate exocytosis of MLKL antagonizing MLKL-mediated plasma membrane alteration (Fan W et al. 2019). Protein cross-linking followed by affinity purification assays detected PDCD6IP and SDCBP in immunoprecipitates of membrane-localized MLKL isolated from human HT-29 colon cancer cells expressing a 3xFlag-HA (hemagglutinin)-tagged MLKL (Fan W et al. 2019). Knockdown of either PDCD6IP or SDCBP reduced the levels of phospho-MLKL in the exosome fractions collected from the culture medium of caspase inhibitor z-VAD-fmk-treated HT-29 cells, suggesting that phosphorylated MLKL was eventually removed from membranes through PDCD6IP:SDCBP-mediated exocytosis. Mass spectrometry and immunoblotting showed that MLKL was associated with some components of ESCRT system within extracellular vesicles (EVs) released from caspase inhibitor z-VAD-fmk-treated HT-29 cells (Yoon S et al. 2017). Further, PDCD6IP was shown to bind tumor susceptibility gene 101 (TSG101
also known as VPS23, vacuolar protein sorting 23), the ESCRT-I subunit protein (von Schwedler UK et al. 2003; Okumura M et al. 2009). Knockdown of TSG101
prevented the exocytosis of phosphorylated MLKL in HT-29 cells, further confirming that mediated exocytosis of phospho-MLKL depends on the ESCRT pathway (Fan W et al. 2019).
SDCBP (syntenin-1) interacts directly with PDCD6IP (ALIX) through three LYPX(n)L motifs located in its N-terminus and with the conserved cytoplasmic domains of the syndecans, via its PDZ domains (Baietti MF et al. 2012). Syndecans are a family of proteins that by virtue of their extracellular heparan sulfate chains interact with a plethora of signaling and adhesion molecules (Sarrazin S et al. 2011). Since PDCD6IP binds several ESCRT proteins, PDCD6IP:SDCBP adapts syndecans and syndecan cargo to the ESCRT budding machinery, playing a role in membrane budding and scission at the endosome and generating intraluminal vesicles (ILVs) that are released as exosomes when multivesicular endosomes fuse with the plasma membrane (Baietti MF et al. 2012 ). Exocytosis was proposed to counteract the effector phase of necroptosis via ESCRT-, PDCD6IP:SDCBP- or RAB27A/B-mediated expulsion of MLKL-containing bubbles to diminish the MLKL residing at the plasma membrane (Gong YN et al. 2017; Yoon S et al. 2017; Fan W et al. 2019). However, the other study suggests that membrane bubbling during necroptosis is an incredibly dynamic and heterogenous phenomenon with protrusions variously extending, retracting and shedding in a fashion seemingly independent of the primary sites of MLKL accumulation and membrane damage (Samson AL et al. 2020).