Table 3.

Post-translational modifications of MLKL.

Domain	Residuea		Modification	Modifying enzyme	Effect on necroptosis	Proposed mechanism	Ortholog studied	Cell modelb	Mouse model	Recombinant protein	Reference
	Human	Mouse
4HB	S125	S124	Phosphorylation	Unknown	—Inconclusive	–	Human, mouse	293T, MDF	No	No	[47, 188, 189]
Brace	–	S158	Phosphorylation	Unknown	↓ Inhibits necroptosis	Unknown	Mouse	MDF	No	No	[47]
PsKDc	S239	S228	Phosphorylation	RIPK3	↑ Modestly enhances necroptosis	Unknown	Mouse	MDF	No	No	[47]
PsKD	S259	S248	Phosphorylation	RIPK3	↓ Inhibits necroptosis	Unknown	Mouse	MDF	No	No	[47]
PsKD	T357/S358	S345	Phosphorylation	RIPK3	↑Promotes necroptosis	Promotes MLKL oligomerization and membrane translocation	Human, mouse	HeLa, NIH 3T3, HT29	No	Yes	[28, 30, 40, 47, 48]
PsKD	S360	S347	Phosphorylation	RIPK3	↑Promotes necroptosis slightly	Unknown	Mouse	MEF	No	No	[30, 48]
PsKD	–	T349	Phosphorylation	RIPK3	No effect	–	Mouse	MEF	No	No	[30, 48]
PsKD	T374	S360	Phosphorylation	Unknown	Unknown	–	Human	HeLa	No	No	[188, 189]
PsKD	Y376	Y363	Phosphorylation	TAM kinases	↑Promotes necroptosis	Promotes MLKL oligomerization at the plasma membrane	Human, mouse	HT29, MEF, Jurkat, L929	Yes	No	[187]
PsKD	S454	S441	Phosphorylation	Unknown	No effect	Mediates myelin breakdown independent from necroptosis pathway	Mouse	Schwann cells	Yes	No	[190]
4HBc	C18	C18	Disulfide bond	Unknown	↑Promotes necroptosis	Mediates polymer formation	Human, mouse	HT29, L929	No	Yes	[193, 195]
4HB	C24	C24	Disulfide bond	Unknown	↑Promotes necroptosis	Mediates polymer formation	Human, mouse	HT29, L929	No	Yes	[193, 195]
4HB	C28	C28	Disulfide bond	Unknown	↑Promotes necroptosis	Mediates polymer formation	Human, mouse	HT29, L929	No	Yes	[193, 195]
4HB	C18/C24/C28	C18/C24/C28	Disulfide bond	Unknown	↑Promotes necroptosis	Possible structural function	Mouse	MDF	No	No	[43]
4HB	C86	–	Disulfide bond	Unknown	↑Promotes necroptosis	Mediates polymer formation	Human, mouse	HT29, L929	No	Yes	[193]
		–	Disulfide bond reduction	Thioredoxin-1	↓ Suppress necroptosis	Prevents polymer formation	Human	HeLa, HT29, 293T	No	Yes	[194]

^aEquivalent residues in human and mouse orthologs are inferred by sequence homology. Bold indicates the ortholog residue with direct evidence of such post-translational modification.

^bCell line abbreviations: MDF mouse dermal fibroblasts, HeLa human adenocarcinoma cells, NIH-3T3 immortalized mouse embryonic fibroblasts, HT29 human adenocarcinoma cells, MEF mouse embryonic fibroblasts, Jurkat human T lymphocyte Jurkat cells, L929 mouse fibrosarcoma cells, 293T human embryonic kidney cell line HEK293T.

^c4HB four-helix bundle domain, PsKD pseudokinase domain.