Fig. 2: Identification of human RIPK3 phosphorylation sites using mass spectrometry.

a and b RIPK3 phosphorylation sites were detected in the FLAG-immunoprecipitates of HT29 RIPK3^−/− cells containing doxycycline-inducible FLAG-RIPK3 (wild-type and D142N kinase-inactive mutant). Cells were treated with doxycycline (500 ng/mL) overnight, before treatment with dTSI (d, doxycycline; T, TNF; S, Smac mimetic Compound A; I, pan-caspase inhibitor IDN-6556) for 3 h, before lysis and FLAG-immunoprecipitation. a Heatmap of the detected RIPK3 phosphorylation sites in cells with wild-type and D142N kinase-inactive mutant FLAG-RIPK3. Numbers indicate the percentage of replicates in each sample in which the phosphorylation was detected (n = 5). Color was assigned as a gradient accordingly, where darker red color corresponds to detection of this phosphorylation in more replicates. b Scatter plot of log₂-based peptide intensity values for the pS227 or pT224 single phosphorylation, and pT224/pS227 double phosphorylation tryptic peptide (EVELPTEPSLVYEAVCNR) in RIPK3^−/− HT29 cells with doxycycline-inducible wild-type FLAG-RIPK3, comparing cells stably expressing FLAG-RIPK3 (dox) with those stimulated to undergo necroptosis (dTSI) (n = 5). c Schematic of human RIPK3 domain architecture and the phosphorylation sites identified. Phosphorylation sites with proposed functions are shown on the top. pT224 and pS227 positively regulates necroptosis (green) by recruiting MLKL. pS164 and pT165 negatively regulate necroptosis by inhibiting RIPK3 kinase activity (red) [38]. Although not detected in our mass spectrometry study, phosphorylation of T182 (grey) was proposed to promote RIPK3 kinase activity and to recruit PELI1 to mediate proteasomal degradation of RIPK3 [54]. Phosphorylation sites with unknown functions are shown on the bottom (white). Asterisks (*) denotes multiple serine/threonine on the same peptide, as such the exact site of phosphorylation could not be unambiguously identified.