Cell
Volume 148, Issues 1–2, 20 January 2012, Pages 213-227
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Article
Mixed Lineage Kinase Domain-like Protein Mediates Necrosis Signaling Downstream of RIP3 Kinase

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Summary

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)acrylamidehereafter referred to as necrosulfonamidethat 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.

Highlights

► Mixed lineage kinase domain-like protein (MLKL) functions in programmed necrosis ► MLKL is a functional substrate of RIP3 kinase ► MLKL interaction with RIP3 depends on autophosphorylation of RIP3 ► A small molecule blocks programmed necrosis by specifically targeting MLKL

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