title: NK lytic-associated molecule, involved in NK cytotoxic function, is an E3 ligase.
abstract: NK lytic-associated molecule (NKLAM) is a protein involved in the cytolytic function of NK cells and CTLs. It has been localized to the cytolytic granules in NK cells and is up-regulated when cells are exposed to cytokines IL-2 or IFN-beta. We report in this study that NKLAM contains a cysteine-rich really interesting new gene (RING) in between RING-RING domain, and that this domain possesses strong homology to the RING domain of the known E3 ubiquitin ligase, Dorfin. To determine whether NKLAM functions as an E3 ligase, we performed coimmunoprecipitation binding assays with ubiquitin conjugates (Ubcs) UbcH7, UbcH8, and UbcH10. We demonstrated that both UbcH7 and UbcH8 bind to full-length NKLAM. We then performed a similar binding assay using endogenous NKLAM and UbcH8 expressed by human NK clone NK3.3 to show that the protein interaction occurs in vivo. Using the yeast two-hybrid system, we identified uridine kinase like-1 (URKL-1) protein as a substrate for NKLAM. We confirmed that NKLAM and URKL-1 interact in mammalian cells by using both immunoprecipitation and confocal microscopy. We demonstrated decreased protein expression and enhanced ubiquitination of URKL-1 in the presence of NKLAM. These data indicate that NKLAM is a RING finger protein that binds Ubcs and has as one of its substrates, URKL-1, thus defining this cytolytic protein as an E3 ubiquitin ligase.
authors.affiliation: Department of Pathology, St. Louis University School of Medicine, St. Louis, MO 63104, USA.
authors.forename: Julie M
authors.initials: JM
authors.lastname: Fortier
authors.forename: Jacki
authors.initials: J
authors.lastname: Kornbluth
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism; D003602:Cytotoxicity, Immunologic; D004268:DNA-Binding Proteins / Q000737:chemistry; D006801:Humans; D047468:Immunoprecipitation; D007694:Killer Cells, Natural / Q000201:enzymology* / Q000276:immunology*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000276:immunology; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000502:physiology*; D016335:Zinc Fingers
chemical_list: D004268:DNA-Binding Proteins; D008565:Membrane Proteins; C120864:NK lytic-associated molecule; C079437:UBE2L3 protein, human; C471622:UBE2L6 protein, human; D044763:Ubiquitin-Conjugating Enzymes; C423571:RNF19A protein, human; D044767:Ubiquitin-Protein Ligases; D003545:Cysteine