RIPK1:RIPK3 complex formation further potentiates kinase activation through autophosphorylation and/or cross-phosphorylation, propagating the pronecrotic signal. RIPK1, RIPK3 and their kinase activities were shown to be essential for regulated necrosis (Degterev A et al. 2008; Cho YS et al. 2009). RIPK3 kinase-dead mutant (K50A) was found to function as a dominant negative mutant, which blocked TNF-alpha induced necrotic pathway in human colorectal adenocarcinoma HT-29 cells (He S et al. 2009). Mutation of the RIP homotypic interaction motif (RHIM) of RIP3 abrogated RIP1 phosphorylation by RIPK3, suggesting that RIPK1 phosphorylation by RIP3 is dependent on the formation of a RIP:RIP3 complex (Sun X et al. 2001).
Phosphorylation on Ser227 is thought to mediate recruitment and activation of mixed-lineage kinase domain-like (MLKL), a crucial downstream substrate of RIP3 in the necrosis pathway (Sun et al. 2012; Chen et al. 2013).