Receptor interacting protein 3 (RIP3) is a protein kinase essential for 
TNF-induced necroptosis. Phosphorylation on Ser-227 in human RIP3 (hRIP3) is 
required for its interaction with human mixed lineage kinase domain-like (MLKL) 
in the necrosome, a signaling complex induced by TNF stimulation. RIP1 and RIP3 
mediate necrosome aggregation leading to the formation of amyloid-like signaling 
complexes. We found that TNF induces Thr-231 and Ser-232 phosphorylation in 
mouse RIP3 (mRIP3) and this phosphorylation is required for mRIP3 to interact 
with mMLKL. Ser-232 in mRIP3 corresponds to Ser-227 in hRIP3, whereas Thr-231 is 
not conserved in hRIP3. Although the RIP3-MLKL interaction is required for 
necroptosis in both human and mouse cells, hRIP3 does not interact with mMLKL 
and mRIP3 cannot bind to hMLKL. The species specificity of the RIP3-MLKL 
interaction is primarily determined by the sequence differences in the 
phosphorylation sites and the flanking sequence around the phosphorylation sites 
in hRIP3 and mRIP3. It appears that the RIP3-MLKL interaction has been selected 
as an evolutionarily conserved mechanism in mediating necroptosis signaling 
despite that differing structural and mechanistic bases for this interaction 
emerged simultaneously in different organisms. In addition, we further revealed 
that the interaction of RIP3 with MLKL prevented massive abnormal RIP3 
aggregation, and therefore should be crucial for formation of the amyloid 
signaling complex of necrosomes. We also found that the interaction between RIP3 
and MLKL is required for the translocation of necrosomes to 
mitochondria-associated membranes. Our data demonstrate the importance of the 
RIP3-MLKL interaction in the formation of functional necrosomes and suggest that 
translocation of necrosomes to mitochondria-associated membranes is essential 
for necroptosis signaling.