Mixed Lineage Kinase Domain-like Protein Mediates Necrosis Signaling Downstream of RIP3 Kinase

Abstract

The receptor-interacting serine-threonine kinase 3 (RIP3) is a key signaling molecule in the programmed necrosis (necroptosis) pathway. This pathway plays important roles in a variety of physiological and pathological conditions, including development, tissue damage response, and antiviral immunity. Here, we report the identification of a small molecule called (E)-N-(4-(N-(3-methoxypyrazin-2-yl)sulfamoyl)phenyl)-3-(5-nitrothiophene-2-yl)acrylamide--hereafter referred to as necrosulfonamide--that specifically blocks necrosis downstream of RIP3 activation. An affinity probe derived from necrosulfonamide and coimmunoprecipitation using anti-RIP3 antibodies both identified the mixed lineage kinase domain-like protein (MLKL) as the interacting target. MLKL was phosphorylated by RIP3 at the threonine 357 and serine 358 residues, and these phosphorylation events were critical for necrosis. Treating cells with necrosulfonamide or knocking down MLKL expression arrested necrosis at a specific step at which RIP3 formed discrete punctae in cells. These findings implicate MLKL as a key mediator of necrosis signaling downstream of the kinase RIP3.

Necroptosis is a key mediator of enterocytes loss in intestinal ischaemia/reperfusion injury

“…In addition, RIP3 controls necroptosis by initiating the pro‐necrotic kinase cascade and assembling of the RIP1/RIP3 complex, thus leading to the regulation of programmed necrosis 39. Mixed lineage kinase domain‐like protein, the direct substrate of RIP3, is essential for the TNFR‐mediated formation of necrosome and targeting appropriate downstream effectors in the necroptosis‐inducing process 40, 41, 42. In our current study, RIP1 and RIP3 expression were increased in the intestinal response to I/R (Fig.…”

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.…”

The phospholipid code: a key component of dying cell recognition, tumor progression and host–microbe interactions

“…When apoptosis is blocked, for example, using the pan caspase inhibitor zVAD-fmk, assembly of the 'necrosome' is triggered within the cytosol, a complex formed by receptor-interacting serine/threonine-protein kinase-1 and -3 (RIPK1 and RIPK3, respectively), 24 followed by recruitment and activation of mixed lineage kinase domain-like (MLKL) protein. 25,26 Recently, it has been proposed that during necroptosis, MLKL is translocated to the plasma membrane where it oligomerizes upon interaction with PI(4,5)P 2 and PI(5)P via its N-terminal four helical bundle domain to induce membrane permeabilization 27 (Figure 2c). Interestingly, whether necroptotic, primary necrotic and secondary necrotic cells are recognized by the same phospholipid detector(s) to mediate their removal is unclear.…”