A single point mutation in the nuclear localization domain of Sam68 blocks the Rev/RRE-mediated transactivation
A single point mutation in the nuclear localization domain of Sam68 blocks the Rev/RRE-mediated transactivation
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DOI：
10.1038/sj.onc.1203637
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发表时间：
2000-06-22
期刊：
影响因子：
8
通讯作者：
Reddy, TR
中科院分区：
文献类型：
--
作者：
Reddy, TR
我们之前已经证明，在rre介导的基因表达和病毒复制中，Sam68的过表达在功能上替代HIV-1 Rev，并与之协同。此外，我们已经证明，Sam68的c端缺失突变体在HIV复制中具有跨显性阴性表型。此前，有报道称Sam68的c端结构域内的Arginine429突变对Sam68在细胞核中的定位至关重要。然而，这些研究是在截断蛋白的背景下进行的，其中Sam68的c端氨基酸420-443融合到GFP中。相比之下，我们现在报道了具有相同突变（Arginine429 -> Alanine）的全长Sam68蛋白完全定位于细胞核，而另一个Sam68 （Proline439- >Arginine）突变在细胞质中被发现。这些Sam68突变蛋白的定位也与其在rre介导的报告基因表达中的功能相关，即定位于细胞质中的Sam68突变蛋白未能增强rre介导的转激活。此外，我们发现Sam68 P439—>R抑制野生型Sam68和rev对rre介导的基因表达的反激活。这些结果表明，与429位置的精氨酸不同，位于439位置的脯氨酸残基可能在Sam68蛋白靶向细胞核中发挥关键作用。我们认为Sam68的这些阴性显性突变体可能有潜力成为对抗艾滋病的抗病毒药物。
We have previously demonstrated that overexpression of Sam68 functionally substitutes for, as well as synergizes with, HIV-1 Rev in RRE-mediated gene expression and virus replication. In addition, we have shown that the C-terminal deletion mutants of Sam68 act with a transdominant negative phenotype in HIV replication, Previously, an Arginine429 mutation within the C-terminal domain of Sam68 has been reported to be critical for the localization of Sam68 in the nucleus. However, these studies were done in the context of truncated protein in which C-terminal amino acids 420-443 of Sam68 were fused to GFP. In contrast, we now report that the full length Sam68 protein having the same mutation (Arginine429 --> Alanine) is completely localized in the nucleus while another Sam68 (Proline439-->Arginine) mutant is found in the cytoplasm. The localization of these Sam68 mutant proteins also correlates,vith their function in RRE-mediated reporter gene expression, i.e. Sam68 mutant protein that is localized in the cytoplasm failed to enhance RRE-mediated transactivation. Furthermore, we demonstrate that Sam68 P439-->R inhibited the transactivation of RRE-mediated gene expression by both wild type Sam68 and Rev. These results indicate that the proline residue at position 439 unlike arginine at position 429, mag play a critical role in targeting Sam68 protein to nucleus. We propose that these negative dominant mutants of Sam68 may have potential as anti-viral agents to combat AIDS.