Characterization of RIPK3-mediated phosphorylation of the activation loop of MLKL during necroptosis

Abstract

Mixed lineage kinase domain-like pseudokinase (MLKL) mediates necroptosis by translocating to the plasma membrane and inducing its rupture. The activation of MLKL occurs in a multimolecular complex (the 'necrosome'), which is comprised of MLKL, receptor-interacting serine/threonine kinase (RIPK)-3 (RIPK3) and, in some cases, RIPK1. Within this complex, RIPK3 phosphorylates the activation loop of MLKL, promoting conformational changes and allowing the formation of MLKL oligomers, which migrate to the plasma membrane. Previous studies suggested that RIPK3 could phosphorylate the murine MLKL activation loop at Ser345, Ser347 and Thr349. Moreover, substitution of the Ser345 for an aspartic acid creates a constitutively active MLKL, independent of RIPK3 function. Here we examine the role of each of these residues and found that the phosphorylation of Ser345 is critical for RIPK3-mediated necroptosis, Ser347 has a minor accessory role and Thr349 seems to be irrelevant. We generated a specific monoclonal antibody to detect phospho-Ser345 in murine cells. Using this antibody, a series of MLKL mutants and a novel RIPK3 inhibitor, we demonstrate that the phosphorylation of Ser345 is not required for the interaction between RIPK3 and MLKL in the necrosome, but is essential for MLKL translocation, accumulation in the plasma membrane, and consequent necroptosis.

Evolutionary divergence of the necroptosis effector MLKL

“…However, neither S345D mMLKL (1-464) nor mMLKL (1-180) were able to induce death of HT29 cells (Figures 2i and j), despite both being potent killers of mouse fibroblasts. 5,10,15,17 In contrast, expression of S345D mMLKL (1-464) in U937 cells led to a modest, but reproducible, amount of death ( Figure 2k). The killing activity of S345D mMLKL (1-464) corresponded with its capacity to translocate from the cytoplasmic (C) fraction to membranes (M) and assemble into a high molecular weight species (Figure 2l).…”

“…5,15,17 To determine whether a deficit in endogenous mouse RIPK3-mediated activation of hMLKL underpins the inability of hMLKL to reconstitute mouse fibroblasts, we tested whether full-length hMLKL bearing the phosphomimetic mutations, T357E/S358E (TSEE), was lethal to wild-type or Mlkl −/− MDFs (Figure 2f). Unlike the S345D mMLKL mutant, 5,15,17 the hMLKL TSEE mutant did not induce death of either wild-type or Mlkl −/− MDFs ( Figure 2f). Similarly, expression of the hMLKLTSEE mutant in the human cell lines, U937 and HT29, did not induce cell death (Figures 2g and h).…”

“…10 Second, cell death occurred to a similar extent when full-length mouse MLKL harbouring the S345D mutation, that mimics activation loop phosphorylation by RIPK3, was expressed in murine fibroblasts. 5,15,17 In addition, three studies have attributed a direct membrane-permeabilization function to recombinant human MLKL 4HB domain in in vitro liposome dye-release assays. 13,14,18 However, a number of questions remain unanswered.…”

The pseudokinase MLKL mediates programmed hepatocellular necrosis independently of RIPK3 during hepatitis

“…However, pretreatment of mice or cells with IFN-γ upregulated Mlkl in hepatocytes and increased their susceptibility toward cell death (Supplemental Figure 12). Similarly, it was recently reported that MEFs may require IFN pretreatment for effective necroptosis induction (49), indicating that sensitization of cells with IFNs may represent a general mechanism supporting programmed necrosis. IFN-induced necroptosis was described in macrophages in the presence of caspase inhibitors.…”

“…RIPK1 is known to regulate the RIPK3-dependent phosphorylation and activation of MLKL, which is a hallmark of the canonical necroptosis pathway (7,(31)(32)(33)(34)(35)(36)(37). However, the role of these RIP kinases in models of experimental induced liver injury remains controversial and incompletely understood.…”