Characterization of RIPK3-mediated Phosphorylation of the Activation Loop of MLKL During Necroptosis

Overview

Journal Cell Death Differ

Specialty Cell Biology

Date 2015 May 30

PMID 26024392

Citations 207

Authors

D A Rodriguez

R Weinlich

S Brown

C Guy

P Fitzgerald

C P Dillon

A Oberst

G Quarato

J Low

J G Cripps

T Chen

D R Green

Affiliations

Soon will be listed here.

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.

Citing Articles

Parkin deficiency aggravates inflammation-induced acute lung injury by promoting necroptosis in alveolar type II cells.

Quan M, Guo Q, Yan X, Yu C, Yang L, Zhang Y Chin Med J Pulm Crit Care Med. 2025; 2(4):265-278.

PMID: 39834583 PMC: 11742354. DOI: 10.1016/j.pccm.2024.11.004.

Therapeutic implications of necroptosis activation in Alzheimer´s disease.

Carrazana E, Salvadores N Alzheimers Res Ther. 2024; 16(1):275.

PMID: 39726013 PMC: 11670415. DOI: 10.1186/s13195-024-01649-8.

Exploration and breakthrough in the mode of intervertebral disc cell death may lead to significant advances in treatments for intervertebral disc degeneration.

Chen H, Tang T, Xue C, Liu X, Xi Z, Xie L J Orthop Surg Res. 2024; 19(1):825.

PMID: 39639370 PMC: 11619685. DOI: 10.1186/s13018-024-05280-z.

Regulated cell death in chronic kidney disease: current evidence and future clinical perspectives.

Giuliani K, Adams B, Healy H, Kassianos A Front Cell Dev Biol. 2024; 12:1497460.

PMID: 39544363 PMC: 11560912. DOI: 10.3389/fcell.2024.1497460.

The Evolution and Biological Activity of Metazoan Mixed Lineage Kinase Domain-Like Protein (MLKL).

Wang Q, Yuan Z, Xu H, Chen Y, Sun L Int J Mol Sci. 2024; 25(19).

PMID: 39408954 PMC: 11476962. DOI: 10.3390/ijms251910626.

References

Weinert B, Scholz C, Wagner S, Iesmantavicius V, Su D, Daniel J . Lysine succinylation is a frequently occurring modification in prokaryotes and eukaryotes and extensively overlaps with acetylation. Cell Rep. 2013; 4(4):842-51. DOI: 10.1016/j.celrep.2013.07.024. View

Li J, McQuade T, Siemer A, Napetschnig J, Moriwaki K, Hsiao Y . The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis. Cell. 2012; 150(2):339-50. PMC: 3664196. DOI: 10.1016/j.cell.2012.06.019. View

Murphy J, Czabotar P, Hildebrand J, Lucet I, Zhang J, Alvarez-Diaz S . The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism. Immunity. 2013; 39(3):443-53. DOI: 10.1016/j.immuni.2013.06.018. View

Green D . Pseudokiller, qu'est-ce que c'est?. Immunity. 2013; 39(3):421-2. PMC: 4095818. DOI: 10.1016/j.immuni.2013.08.020. View

Xie T, Peng W, Yan C, Wu J, Gong X, Shi Y . Structural insights into RIP3-mediated necroptotic signaling. Cell Rep. 2013; 5(1):70-8. DOI: 10.1016/j.celrep.2013.08.044. View

Kaiser W, Sridharan H, Huang C, Mandal P, Upton J, Gough P . Toll-like receptor 3-mediated necrosis via TRIF, RIP3, and MLKL. J Biol Chem. 2013; 288(43):31268-79. PMC: 3829437. DOI: 10.1074/jbc.M113.462341. View

Weinlich R, Oberst A, Dillon C, Janke L, Milasta S, Lukens J . Protective roles for caspase-8 and cFLIP in adult homeostasis. Cell Rep. 2013; 5(2):340-8. PMC: 3843376. DOI: 10.1016/j.celrep.2013.08.045. View

Cai Z, Jitkaew S, Zhao J, Chiang H, Choksi S, Liu J . Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis. Nat Cell Biol. 2013; 16(1):55-65. PMC: 8369836. DOI: 10.1038/ncb2883. View

Chen X, Li W, Ren J, Huang D, He W, Song Y . Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death. Cell Res. 2013; 24(1):105-21. PMC: 3879712. DOI: 10.1038/cr.2013.171. View

10.

Tait S, Oberst A, Quarato G, Milasta S, Haller M, Wang R . Widespread mitochondrial depletion via mitophagy does not compromise necroptosis. Cell Rep. 2013; 5(4):878-85. PMC: 4005921. DOI: 10.1016/j.celrep.2013.10.034. View

11.

Remijsen Q, Goossens V, Grootjans S, Van den Haute C, Vanlangenakker N, Dondelinger Y . Depletion of RIPK3 or MLKL blocks TNF-driven necroptosis and switches towards a delayed RIPK1 kinase-dependent apoptosis. Cell Death Dis. 2014; 5:e1004. PMC: 4040672. DOI: 10.1038/cddis.2013.531. View

12.

Vitner E, Salomon R, Farfel-Becker T, Meshcheriakova A, Ali M, Klein A . RIPK3 as a potential therapeutic target for Gaucher's disease. Nat Med. 2014; 20(2):204-8. DOI: 10.1038/nm.3449. View

13.

Wang H, Sun L, Su L, Rizo J, Liu L, Wang L . Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3. Mol Cell. 2014; 54(1):133-146. DOI: 10.1016/j.molcel.2014.03.003. View

14.

Dondelinger Y, Declercq W, Montessuit S, Roelandt R, Goncalves A, Bruggeman I . MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates. Cell Rep. 2014; 7(4):971-81. DOI: 10.1016/j.celrep.2014.04.026. View

15.

Rickard J, ODonnell J, Evans J, Lalaoui N, Poh A, Rogers T . RIPK1 regulates RIPK3-MLKL-driven systemic inflammation and emergency hematopoiesis. Cell. 2014; 157(5):1175-88. DOI: 10.1016/j.cell.2014.04.019. View

16.

Dillon C, Weinlich R, Rodriguez D, Cripps J, Quarato G, Gurung P . RIPK1 blocks early postnatal lethality mediated by caspase-8 and RIPK3. Cell. 2014; 157(5):1189-202. PMC: 4068710. DOI: 10.1016/j.cell.2014.04.018. View

17.

Li J, Feng J, Wang Y, Li X, Chen X, Su Y . The B-Raf(V600E) inhibitor dabrafenib selectively inhibits RIP3 and alleviates acetaminophen-induced liver injury. Cell Death Dis. 2014; 5:e1278. PMC: 4611716. DOI: 10.1038/cddis.2014.241. View

18.

Kaiser W, Daley-Bauer L, Thapa R, Mandal P, Berger S, Huang C . RIP1 suppresses innate immune necrotic as well as apoptotic cell death during mammalian parturition. Proc Natl Acad Sci U S A. 2014; 111(21):7753-8. PMC: 4040608. DOI: 10.1073/pnas.1401857111. View

19.

Cook W, Moujalled D, Ralph T, Lock P, Young S, Murphy J . RIPK1- and RIPK3-induced cell death mode is determined by target availability. Cell Death Differ. 2014; 21(10):1600-12. PMC: 4158685. DOI: 10.1038/cdd.2014.70. View

20.

Orozco S, Yatim N, Werner M, Tran H, Gunja S, Tait S . RIPK1 both positively and negatively regulates RIPK3 oligomerization and necroptosis. Cell Death Differ. 2014; 21(10):1511-21. PMC: 4158689. DOI: 10.1038/cdd.2014.76. View