title: 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.
authors.affiliation: Department of Immunology, St Jude Children's Research Hospital, Memphis, TN 38105, USA.
authors.forename: D A
authors.initials: DA
authors.lastname: Rodriguez
authors.affiliation: Department of Immunology, St Jude Children's Research Hospital, Memphis, TN 38105, USA.
authors.forename: R
authors.identifier: http://orcid.org/0000-0001-6822-7330
authors.initials: R
authors.lastname: Weinlich
authors.affiliation: Department of Immunology, St Jude Children's Research Hospital, Memphis, TN 38105, USA.
authors.forename: S
authors.initials: S
authors.lastname: Brown
authors.affiliation: Department of Immunology, St Jude Children's Research Hospital, Memphis, TN 38105, USA.
authors.forename: C
authors.initials: C
authors.lastname: Guy
authors.affiliation: Department of Immunology, St Jude Children's Research Hospital, Memphis, TN 38105, USA.
authors.forename: P
authors.initials: P
authors.lastname: Fitzgerald
authors.affiliation: Department of Immunology, St Jude Children's Research Hospital, Memphis, TN 38105, USA.
authors.forename: C P
authors.initials: CP
authors.lastname: Dillon
authors.affiliation: Department of Immunology, University of Washington, Seattle, WA 98109, USA.
authors.forename: A
authors.initials: A
authors.lastname: Oberst
authors.affiliation: Department of Immunology, St Jude Children's Research Hospital, Memphis, TN 38105, USA.
authors.forename: G
authors.identifier: http://orcid.org/0000-0003-1167-3422
authors.initials: G
authors.lastname: Quarato
authors.affiliation: Department Chemical Biology & Therapeutics, St Jude Children's Research Hospital, Memphis, TN 38105, USA.
authors.forename: J
authors.initials: J
authors.lastname: Low
authors.affiliation: Institute for Cellular Therapeutics, University of Louisville, Louisville, KY 40202, USA.
authors.forename: J G
authors.initials: JG
authors.lastname: Cripps
authors.affiliation: Department Chemical Biology & Therapeutics, St Jude Children's Research Hospital, Memphis, TN 38105, USA.
authors.forename: T
authors.initials: T
authors.lastname: Chen
authors.affiliation: Department of Immunology, St Jude Children's Research Hospital, Memphis, TN 38105, USA.
authors.forename: D R
authors.initials: DR
authors.lastname: Green
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D016923:Cell Death / Q000235:genetics*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D051379:Mice; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D009336:Necrosis / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D046912:Multiprotein Complexes; C570694:MLKL protein, mouse; D011494:Protein Kinases; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; C506251:Ripk1 protein, mouse; C485615:Ripk3 protein, mouse