title: The p60 tumor necrosis factor (TNF) receptor-associated kinase (TRAK) binds residues 344-397 within the cytoplasmic domain involved in TNF signaling.
abstract: The p60 form of the tumor necrosis factor (TNF) receptor lacks motifs characteristic of tyrosine or serine/threonine protein kinases. Our recent observations have indicated that a p60 TNF receptor-associated kinase (p60-TRAK) from U-937 cells physically interacts with and causes the phosphorylation of the cytoplasmic domain of the TNF receptor. To define which region of the cytoplasmic domain is necessary for physical interaction with p60-TRAK, we constructed a series of deletions (grouped into three sets delta 1-delta 5, delta 6-delta 12, and delta 13-delta 16) of the p60 cytoplasmic domain, expressed them as glutathione S-transferase (GST) fusion proteins, and used them in affinity precipitations, followed by in vitro kinase assays. Our detailed analysis indicated that a serine-, threonine-, and proline-rich region (residues 243-274, delta 2) and the N-terminal half of the cytoplasmic domain (residues 243-323, delta 3) neither associated with p60-TRAK nor underwent phosphorylation. We found that out of 222 residues (205-426) in the cytoplasmic domain, only 54 (344-397, delta 12) were sufficient for binding p60-TRAK and for phosphorylation of the cytoplasmic domain. A region of approximately 30 residues (397-426) at the C-terminal end was found to interfere with optimal binding of the p60-TRAK activity. Thus, our results indicate that the minimal region of the cytoplasmic domain necessary for interacting with p60-TRAK and for phosphorylation resides within the domain previously reported to be needed for signaling the cytotoxic effect of TNF.
authors.affiliation: Department of Molecular Oncology, University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
authors.forename: B G
authors.initials: BG
authors.lastname: Darnay
authors.forename: S
authors.initials: S
authors.lastname: Singh
authors.forename: M M
authors.initials: MM
authors.lastname: Chaturvedi
authors.forename: B B
authors.initials: BB
authors.lastname: Aggarwal
mesh_terms: D001665:Binding Sites; D002460:Cell Line; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D016296:Mutagenesis; D010766:Phosphorylation; D011392:Proline; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D012694:Serine; D015398:Signal Transduction; D013912:Threonine; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D013912:Threonine; D012694:Serine; D011392:Proline; D005982:Glutathione Transferase; C094313:p60 tumor necrosis factor receptor-associated kinase; D017346:Protein Serine-Threonine Kinases