title: Stable expression and function of the inositol 1,4,5-triphosphate receptor requires palmitoylation by a DHHC6/selenoprotein K complex.
abstract: Calcium (Ca(2+)) is a secondary messenger in cells and Ca(2+) flux initiated from endoplasmic reticulum (ER) stores via inositol 1,4,5-triphosphate (IP3) binding to the IP3 receptor (IP3R) is particularly important for the activation and function of immune cells. Previous studies demonstrated that genetic deletion of selenoprotein K (Selk) led to decreased Ca(2+) flux in a variety of immune cells and impaired immunity, but the mechanism was unclear. Here we show that Selk deficiency does not affect receptor-induced IP3 production, but Selk deficiency through genetic deletion or low selenium in culture media leads to low expression of the IP3R due to a defect in IP3R palmitoylation. Bioinformatic analysis of the DHHC (letters represent the amino acids aspartic acid, histidine, histidine, and cysteine in the catalytic domain) family of enzymes that catalyze protein palmitoylation revealed that one member, DHHC6, contains a predicted Src-homology 3 (SH3) domain and DHHC6 is localized to the ER membrane. Because Selk is also an ER membrane protein and contains an SH3 binding domain, immunofluorescence and coimmunoprecipitation experiments were conducted and revealed DHHC6/Selk interactions in the ER membrane that depended on SH3/SH3 binding domain interactions. DHHC6 knockdown using shRNA in stably transfected cell lines led to decreased expression of the IP3R and impaired IP3R-dependent Ca(2+) flux. Mass spectrophotometric and bioinformatic analyses of the IP3R protein identified two palmitoylated cysteine residues and another potentially palmitoylated cysteine, and mutation of these three cysteines to alanines resulted in decreased IP3R palmitoylation and function. These findings reveal IP3R palmitoylation as a critical regulator of Ca(2+) flux in immune cells and define a previously unidentified DHHC/Selk complex responsible for this process.
authors.affiliation: Departments of Cell and Molecular Biology.
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Fredericks
authors.affiliation: Departments of Cell and Molecular Biology.
authors.forename: FuKun W
authors.initials: FW
authors.lastname: Hoffmann
authors.affiliation: Departments of Cell and Molecular Biology.
authors.forename: Aaron H
authors.initials: AH
authors.lastname: Rose
authors.affiliation: Biotechnology Department, University of Applied Sciences Mannheim, 68163 Mannheim, Germany.
authors.forename: Hanna J
authors.initials: HJ
authors.lastname: Osterheld
authors.affiliation: Medicine, and.
authors.forename: Franz M
authors.initials: FM
authors.lastname: Hess
authors.affiliation: Tropical Medicine, Medical Microbiology and Pharmacology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI 96813; and.
authors.forename: Frederic
authors.initials: F
authors.lastname: Mercier
authors.affiliation: Departments of Cell and Molecular Biology, peterrh@hawaii.edu.
authors.forename: Peter R
authors.initials: PR
authors.lastname: Hoffmann
mesh_terms: D000217:Acyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000502:physiology*; D000818:Animals; D001854:Bone Marrow Cells / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D003545:Cysteine / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000201:enzymology; D057809:HEK293 Cells; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D019169:Jurkat Cells; D054878:Lipoylation; D051379:Mice; D018345:Mice, Knockout; D046912:Multiprotein Complexes; D016297:Mutagenesis, Site-Directed; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012643:Selenium / Q000502:physiology; D051140:Selenoproteins / Q000737:chemistry / Q000172:deficiency / Q000502:physiology*; D016176:T-Lymphocyte Subsets / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection; D018909:src Homology Domains
chemical_list: C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; C025389:Itpr1 protein, mouse; C488910:Itpr1 protein, rat; D046912:Multiprotein Complexes; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D051140:Selenoproteins; C572507:ZDHHC6 protein, human; C000597151:ZDHHC6 protein, mouse; C000597149:selenoprotein K, mouse; D019284:Thapsigargin; D000217:Acyltransferases; D012643:Selenium; D003545:Cysteine
keywords: calcium; endoplasmic reticulum; immune; palmitoylation; selenium