Distinct roles of RIP1–RIP3 hetero- and RIP3–RIP3 homo-interaction in mediating necroptosis

Abstract

Necroptosis is mediated by a signaling complex called necrosome, containing receptor-interacting protein (RIP)1, RIP3, and mixed-lineage kinase domain-like (MLKL). It is known that RIP1 and RIP3 form heterodimeric filamentous scaffold in necrosomes through their RIP homotypic interaction motif (RHIM) domain-mediated oligomerization, but the signaling events based on this scaffold has not been fully addressed. By using inducible dimer systems we found that RIP1-RIP1 interaction is dispensable for necroptosis; RIP1-RIP3 interaction is required for necroptosis signaling, but there is no necroptosis if no additional RIP3 protein is recruited to the RIP1-RIP3 heterodimer, and the interaction with RIP1 promotes the RIP3 to recruit other RIP3; RIP3-RIP3 interaction is required for necroptosis and RIP3-RIP3 dimerization is sufficient to induce necroptosis; and RIP3 dimer-induced necroptosis requires MLKL. We further show that RIP3 oligomer is not more potent than RIP3 dimer in triggering necroptosis, suggesting that RIP3 homo-interaction in the complex, rather than whether RIP3 has formed homo polymer, is important for necroptosis. RIP3 dimerization leads to RIP3 intramolecule autophosphorylation, which is required for the recruitment of MLKL. Interestingly, phosphorylation of one of RIP3 in the dimer is sufficient to induce necroptosis. As RIP1-RIP3 heterodimer itself cannot induce necroptosis, the RIP1-RIP3 heterodimeric amyloid fibril is unlikely to directly propagate necroptosis. We propose that the signaling events after the RIP1-RIP3 amyloid complex assembly are the recruitment of free RIP3 by the RIP3 in the amyloid scaffold followed by autophosphorylation of RIP3 and subsequent recruitment of MLKL by RIP3 to execute necroptosis. Cell Death and Differentiation (2014) 21, 1709-1720; doi:10.1038/cdd.2014.77; published online 6 June 2014Necroptosis is a type of programmed necrosis characterized by necrotic morphological changes, including cellular organelle swelling, cell membrane rupture, 1-3 and dependence of receptor-interacting protein (RIP)1 4 and RIP3. [5][6][7] Physiological function of necroptosis has been illustrated in host defense, [8][9][10][11] inflammation, 12-16 tissue injury, 10,17,18 and development. [19][20][21] Necroptosis can be induced by a number of different extracellular stimuli such as tumor necrosis factor (TNF). TNF stimulation leads to formation of TNF receptor 1 (TNFR1) signaling complex (named complex I), and complex II containing RIP1, TRADD, FAS-associated protein with a death domain (FADD), and caspase-8, of which the activation initiates apoptosis. If cells have high level of RIP3, RIP1 recruits RIP3 to form necrosome containing FADD, [22][23][24] caspase-8, RIP1, and RIP3, and the cells undergo necroptosis. 25,26 Caspase-8 and FADD negatively regulates necroptosis, 27-30 because RIP1, RIP3, and CYLD are potential substrates of caspase-8. [31][32][33][34] Necrosome also suppresses apoptosis but the underlying mechanism has not been described yet. Mixed-lineage kinase domain-like (ML...

Tissue damage negatively regulates LPS-induced macrophage necroptosis

“…23,24 To further confirm that necroptosis was occurring in the Mϕ/ monocytes, cells were double stained with the RIPK1 and RIPK3. 26 Colocalization of RIPK1 and RIPK3 was visualized by confocal microscopy and represents the RIPK1 and RIPK3 association to form the necrosome. 27 Figure 1e shows that LPS induced necrosome formation, and prior PF decreased necrosome formation in PMϕ.…”

RIP kinases: key decision makers in cell death and innate immunity

“…In this process, the initial formation of a RIP1-RIP3 heterodimer is insufficient to trigger necroptosis and instead the RIP1-RIP3 amyloid structure must recruit more free RIP3 to the amyloid scaffold resulting in auto-phosphorylation of RIP3 and recruitment of mixed lineage kinase domain-like protein (MLKL) to trigger downstream necroptosis. 84 The recruitment of MLKL to the necrosome leads to RIP3-mediated phosphorylation of MLKL with the RIP3 inhibitor, necrosulfonamide, blocking necrosis downstream of RIP3 activation 85 and MLKL-deficient mice being resistant to necroptosis. 86,87 Various downstream effector mechanisms have been proposed to mediate necroptosis.…”

A Role for Tubular Necroptosis in Cisplatin-Induced AKI

“…In addition, we detected cisplatin-induced RIP3 phosphorylation, an essential event in necroptosis ( Figure 6C). Because the kinase activity of RIP3 and its RHIM domain are required to mediate necroptosis in other cell systems, 12,13 we reconstituted WT, RHIM mutant, or kinase dead (D143N) mutant of RIP3 in RIP3-KO PTCs ( Figure 6D) and compared their effects on cisplatin-induced necrosis. Only WT RIP3 rescued cisplatin-induced necroptosis in PTCs, while the two mutants did not ( Figure 6E), confirming that kinase activity and the RHIM domain of RIP3 are required for cisplatininduced necroptosis in PTCs.…”

“…Because RIP3 and MLKL are essential for the necroptosis pathway, 10,13,14 we investigated their role in cisplatin-induced Figure 1. Necroptosis contributes to cisplatin-induced nephrotoxity.…”

“…12 This RIP1-RIP3 hetero-interaction promotes RIP3-RIP3 homo-interactions, leading to the recruitment of mixed lineage kinase domainlike protein (MLKL) and phosphorylation of MLKL. 13 Phosphorylated MLKL forms tetramers and translocates onto the plasma membrane to form higher-ordered complexes, resulting in ion influx and eventual plasma membrane disruption. [14][15][16] Necroptosis is involved in various pathologicconditions, includingantiviralresponses, acute pancreatitis, atherosclerosis, and druginduced liver injury.…”