Binding of c-Raf1 kinase to a conserved acidic sequence within the carboxyl-terminal region of the HIV-1 Nef protein
Binding of c-Raf1 kinase to a conserved acidic sequence within the carboxyl-terminal region of the HIV-1 Nef protein
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DOI：
10.1074/jbc.273.25.15727
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发表时间：
1998-06-19
影响因子：
4.8
通讯作者：
Samuel, KP
中科院分区：
文献类型：
--
作者：
Hodge, DR;Dunn, KJ;Samuel, KP
Nef是一种膜相关的细胞质磷酸化蛋白，在不同的人类（HIV-1和HIV-8）和猿猴免疫缺陷病毒中被很好地保守，在下调CD4受体和调节t细胞信号通路中起重要作用。调节t细胞信号通路的能力表明Nef可能与t细胞信号蛋白发生物理相互作用。为了鉴定Nef结合蛋白并绘制其相互作用位点，我们在Nef的羧基末端区域定位了一个高度保守的酸性序列，该序列与Ras效应区c- raf1结合位点的酸性序列有着惊人的相似性。在这里，我们使用了缺失和位点特异性诱变的方法，在体外沉淀试验和免疫印迹分析中产生了与细菌谷胱甘肽s转移酶融合的突变Nef蛋白，将HIV-1(LAI) Nef和c-Raf1之间的特异性相互作用映射到一个保守的酸性序列基序，该基序包含核心序列Asp-Asp-X-X-X-Glu（位置174-179）。值得注意的是，我们证明非极性甘氨酸残基取代全长重组Nef蛋白背景中位置174和175的保守带负电荷的天冬氨酸残基中的一个或两个，完全消除了c-Raf1在体外的结合。此外，研究人员利用表达天然HIV-1 Nef蛋白的永久性CEM t细胞系的裂解物共免疫沉淀稳定的Nef c-Raf1复合物，这表明Nef和c-Raf1之间的分子相互作用在体内发生。c-Raf1是通过c-Raf1- map激酶途径传递细胞信号的重要下游传感器。这种相互作用可能解释了nef诱导的t细胞信号传导和激活途径在体外和体内的扰动。
Nef is a membrane-associated cytoplasmic phosphoprotein that is well conserved among the different human (HIV-1 and HIV-8) 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-1(LAI) 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.