title: Novel phosphorylation and ubiquitination sites regulate reactive oxygen species-dependent degradation of anti-apoptotic c-FLIP protein.
abstract: The cytosolic protein c-FLIP (cellular Fas-associated death domain-like interleukin 1β-converting enzyme inhibitory protein) is an inhibitor of death receptor-mediated apoptosis that is up-regulated in a variety of cancers, contributing to apoptosis resistance. Several compounds found to restore sensitivity of cancer cells to TRAIL, a TNF family death ligand with promising therapeutic potential, act by targeting c-FLIP ubiquitination and degradation by the proteasome. The generation of reactive oxygen species (ROS) has been implicated in c-FLIP protein degradation. However, the mechanism by which ROS post-transcriptionally regulate c-FLIP protein levels is not well understood. We show here that treatment of prostate cancer PPC-1 cells with the superoxide generators menadione, paraquat, or buthionine sulfoximine down-regulates c-FLIP long (c-FLIP(L)) protein levels, which is prevented by the proteasome inhibitor MG132. Furthermore, pretreatment of PPC-1 cells with a ROS scavenger prevented ubiquitination and loss of c-FLIP(L) protein induced by menadione or paraquat. We identified lysine 167 as a novel ubiquitination site of c-FLIP(L) important for ROS-dependent degradation. We also identified threonine 166 as a novel phosphorylation site and demonstrate that Thr-166 phosphorylation is required for ROS-induced Lys-167 ubiquitination. The mutation of either Thr-166 or Lys-167 was sufficient to stabilize c-FLIP protein levels in PPC-1, HEK293T, and HeLa cancer cells treated with menadione or paraquat. Accordingly, expression of c-FLIP T166A or K167R mutants protected cells from ROS-mediated sensitization to TRAIL-induced cell death. Our findings reveal novel ROS-dependent post-translational modifications of the c-FLIP protein that regulate its stability, thus impacting sensitivity of cancer cells to TRAIL.
authors.affiliation: Sanford-Burnham Medical Research Institute, Program on Apoptosis and Cell Death Research, La Jolla, California 92037, USA.
authors.forename: Rachel P
authors.initials: RP
authors.lastname: Wilkie-Grantham
authors.forename: Shu-Ichi
authors.initials: S
authors.lastname: Matsuzawa
authors.forename: John C
authors.initials: JC
authors.lastname: Reed
mesh_terms: D000963:Antimetabolites / Q000494:pharmacology; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000235:genetics / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects / Q000235:genetics; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects; D057809:HEK293 Cells; D006367:HeLa Cells; D006540:Herbicides / Q000494:pharmacology; D006801:Humans; D007976:Leupeptins; D020125:Mutation, Missense; D010269:Paraquat / Q000494:pharmacology; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D017382:Reactive Oxygen Species / Q000378:metabolism*; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination; D024483:Vitamin K 3 / Q000494:pharmacology; D014815:Vitamins / Q000494:pharmacology
chemical_list: D000963:Antimetabolites; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; D015853:Cysteine Proteinase Inhibitors; D006540:Herbicides; D007976:Leupeptins; D017382:Reactive Oxygen Species; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014815:Vitamins; D019328:Buthionine Sulfoximine; D024483:Vitamin K 3; D010269:Paraquat; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde
keywords: Apoptosis; Post-translational Modification; ROS; Reactive Oxygen Species (ROS); Trail; Ubiquitin; Ubiquitination; Ubiquitylation; c-FLIP