Binding of c-Raf1 kinase to a conserved acidic sequence within the carboxyl-terminal region of the HIV-1 Nef protein
| Autor: | Kenneth Samuel, K. Joyce Dunn, Gou Kui Pei, James A. Lautenberger, Mrinal K. Chakrabarty, David R. Hodge, G. Heidecker |
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| Rok vydání: | 1998 |
| Předmět: |
Cell signaling
viruses Mutant Molecular Sequence Data Biology Biochemistry DNA-binding protein Peptide Mapping Gene Products nef Tumor Cells Cultured Humans Amino Acid Sequence nef Gene Products Human Immunodeficiency Virus Molecular Biology Conserved Sequence Binding Sites Effector virus diseases Cell Biology Proto-Oncogene Proteins c-raf Amino Acid Substitution Cytoplasm Phosphoprotein HIV-1 Mutagenesis Site-Directed Signal transduction Sequence motif |
| Zdroj: | The Journal of biological chemistry. 273(25) |
| ISSN: | 0021-9258 |
| Popis: | Nef is a membrane-associated cytoplasmic phosphoprotein that is well conserved among the different human (HIV-1 and HIV-2) and simian immunodeficiency viruses and has important roles in down-regulating the CD4 receptor and modulating T-cell signaling pathways. The ability to modulate T-cell signaling pathways suggests that Nef may physically interact with T-cell signaling proteins. In order to identify Nef binding proteins and map their site(s) of interaction, we targeted a highly conserved acidic sequence at the carboxyl-terminal region of Nef sharing striking similarity with an acidic sequence at the c-Raf1-binding site within the Ras effector region. Here, we used deletion and site-specific mutagenesis to generate mutant Nef proteins fused to bacterial glutathione S-transferase in in vitro precipitation assays and immunoblot analysis to map the specific interaction between the HIV-1LAI Nef and c-Raf1 to a conserved acidic sequence motif containing the core sequence Asp-Asp-X-X-X-Glu (position 174-179). Significantly, we demonstrate that substitution of the nonpolar glycine residue for either or both of the conserved negatively charged aspartic acid residues at positions 174 and 175 in the full-length recombinant Nef protein background completely abrogated binding of c-Raf1 in vitro. In addition, lysates from a permanent CEM T-cell line constitutively expressing the native HIV-1 Nef protein was used to coimmunoprecipitate a stable Nef-c-Raf1 complex, suggesting that molecular interactions between Nef and c-Raf1, an important downstream transducer of cell signaling through the c-Raf1-MAP kinase pathway, occur in vivo. This interaction may account for the Nef-induced perturbations of T-cell signaling and activation pathways in vitro and in vivo. |
| Databáze: | OpenAIRE |
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