title: Two-hybrid cloning of a gene encoding TNF receptor-associated protein 2, a protein that interacts with the intracellular domain of the type 1 TNF receptor: identity with subunit 2 of the 26S protease.
abstract: A protein that binds the intracellular domain of the type 1 TNFR (TNFR-1IC) has been identified by two-hybrid cloning. The 97-kDa TNFR-associated protein, TRAP2, shows sequence identity with internal amino acid sequences from subunit 2 of the 26S protease. TRAP2 antiserum recognizes subunit 2 of the 26S protease, which is consistent with the identity of these proteins. TRAP2 antiserum interacted with the 97-kDa protein in HeLa cell lysates and cytosol, the latter observation showing that TRAP2 resides in the same cellular compartment as TNFR-1IC. A fusion of glutathione-S-transferase and TNFR-1IC (GST-TNFR-1IC) precipitated TRAP2 from a HeLa cell lysate; conversely, GST-TRAP2 precipitated TNFR-1 from such a lysate. These observations show that the proteins interact in the cellular milieu. After in vitro transcription/translation and 35S labeling, TRAP2 was precipitated from a cellfree system by GST-TNFR-1IC, showing that TNFR-1IC and TRAP2 interact directly. TRAP2 was also precipitated from the cellfree translation system by a GST fusion containing the N-terminal half of TNFR-1IC, but not by a GST fusion containing the C-terminal half of TNFR-1IC that contains a "death domain" that plays an obligatory role in signaling cytotoxicity. The ability of deletion mutants of TNFR-1IC to interact with TRAP2 was tested using the two-hybrid system. This also showed that the amino acid sequences that mediate binding reside outside of the death domain in TNFR-1IC. The demonstration that a subunit of the 26S protease binds TNFR-1 may identify a novel TNF-signaling pathway.
authors.affiliation: Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis 46202, USA.
authors.forename: J D
authors.initials: JD
authors.lastname: Dunbar
authors.forename: H Y
authors.initials: HY
authors.lastname: Song
authors.forename: D
authors.initials: D
authors.lastname: Guo
authors.forename: L W
authors.initials: LW
authors.lastname: Wu
authors.forename: D B
authors.initials: DB
authors.lastname: Donner
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000737:chemistry*; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism*; D047992:TNF Receptor-Associated Factor 2; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins
chemical_list: D002352:Carrier Proteins; C101338:PSMD2 protein, human; D011506:Proteins; D012333:RNA, Messenger; D018124:Receptors, Tumor Necrosis Factor; D047992:TNF Receptor-Associated Factor 2; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease