title: Cellular inhibitor of apoptosis (cIAP)-mediated ubiquitination of phosphofurin acidic cluster sorting protein 2 (PACS-2) negatively regulates tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity.
abstract: Lysosomal membrane permeabilization is an essential step in TRAIL-induced apoptosis of liver cancer cell lines. TRAIL-induced lysosomal membrane permeabilization is mediated by the multifunctional sorting protein PACS-2 and repressed by the E3 ligases cIAP-1 and cIAP-2. Despite the opposing roles for PACS-2 and cIAPs in TRAIL-induced apoptosis, an interaction between these proteins has yet to be examined. Herein, we report that cIAP-1 and cIAP-2 confer TRAIL resistance to hepatobiliary cancer cell lines by reducing PACS-2 levels. Under basal conditions, PACS-2 underwent K48-linked poly-ubiquitination, resulting in PACS-2 proteasomal degradation. Biochemical assays showed cIAP-1 and cIAP-2 interacted with PACS-2 in vitro and co-immunoprecipitation studies demonstrated that the two cIAPs bound PACS-2 in vivo. More importantly, both cIAP-1 and cIAP-2 directly mediated PACS-2 ubiquitination in a cell-free assay. Single c-Iap-1 or c-Iap-2 gene knock-outs in mouse hepatocytes did not lead to PACS-2 accumulation. However, deletion of both cIAP-1 and cIAP-2 reduced PACS-2 ubiquitination, which increased PACS-2 levels and sensitized HuH-7 cells to TRAIL-induced lysosomal membrane permeabilization and apoptosis. Correspondingly, deletion of cIAPs sensitized wild-type, but not PACS-2-deficient hepatocarcinoma cells or Pacs-2-/- mouse hepatocytes to TRAIL-induced apoptosis. Together, these data suggest cIAPs constitutively downregulate PACS-2 by polyubiquitination and proteasomal degradation, thereby restraining TRAIL-induced killing of liver cancer cells.
authors.affiliation: Division of Gastroenterology and Hepatology, College of Medicine, Mayo Clinic, Rochester, Minnesota, United States of America.
authors.forename: Maria Eugenia
authors.initials: ME
authors.lastname: Guicciardi
authors.affiliation: Division of Gastroenterology and Hepatology, College of Medicine, Mayo Clinic, Rochester, Minnesota, United States of America.
authors.forename: Nathan W
authors.initials: NW
authors.lastname: Werneburg
authors.affiliation: Division of Gastroenterology and Hepatology, College of Medicine, Mayo Clinic, Rochester, Minnesota, United States of America.
authors.forename: Steven F
authors.initials: SF
authors.lastname: Bronk
authors.affiliation: Division of Gastroenterology and Hepatology, College of Medicine, Mayo Clinic, Rochester, Minnesota, United States of America.
authors.forename: Adrian
authors.initials: A
authors.lastname: Franke
authors.affiliation: Department of Immunology, School of Medicine, Juntendo University, Tokyo, Japan.
authors.forename: Hideo
authors.initials: H
authors.lastname: Yagita
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Gary
authors.initials: G
authors.lastname: Thomas
authors.affiliation: Division of Gastroenterology and Hepatology, College of Medicine, Mayo Clinic, Rochester, Minnesota, United States of America.
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Gores
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D000075747:Baculoviral IAP Repeat-Containing 3 Protein; D040761:Biomimetic Materials / Q000494:pharmacology; D045744:Cell Line, Tumor; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000378:metabolism*; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000293:injuries; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism; D010539:Permeability / Q000187:drug effects; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D059748:Proteolysis / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000494:pharmacology / Q000633:toxicity*; D044767:Ubiquitin-Protein Ligases; D054875:Ubiquitination / Q000187:drug effects*; D033921:Vesicular Transport Proteins / Q000378:metabolism*
chemical_list: D051034:Inhibitor of Apoptosis Proteins; D024101:Mitochondrial Proteins; C499638:PACS2 protein, human; D053221:TNF-Related Apoptosis-Inducing Ligand; D033921:Vesicular Transport Proteins; C494832:BIRC3 protein, human; D000075747:Baculoviral IAP Repeat-Containing 3 Protein; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex