Morgavi P, Bonifaci N, Pagani M, Costigliolo S, Sitia R, Rubartelli A
National Institute of Cancer Research, 16132 Genova, Italy.
J Biol Chem. 1997 Apr 25;272(17):11256-60. doi: 10.1074/jbc.272.17.11256.
Human immunodeficiency virus-1 (HIV-1) Tat, a nuclear transcription factor, has been shown to function extracellularly, implying that some Tat molecules escape nuclear import and are secreted. This raises the question of what regulates, in HIV-1-infected cells, the nuclear targeting of the polypeptide. Here we show that cytosolic components activated by Ca2+ ions are required to reveal the karyophilic properties of Tat: in vitro translated Tat molecules do not associate with isolated nuclei unless preincubated with Ca2+. Moreover, Ca2+ ions induce karyophilicity of chemically synthesized Tat molecules only upon addition of cytosolic extracts. The Ca2+-induced karyophilicity is prevented by inhibitors of either tyrosine kinases (herbimycin A and genistein) or tyrosine phosphatases (vanadate), suggesting the involvement of Ca2+-dependent phosphorylation/dephosphorylation events. In line with these observations, the transcriptional activity of Tat is inhibited by treatment with either vanadate or genistein. The same occurs with Tat mutants lacking either one or both the two tyrosine residues (positions 26 and 47). Hence, Ca2+-dependent tyrosine kinase(s) and phosphatase(s) act on accessory cellular protein(s), which in turn are responsible of Tat karyophilicity.
人类免疫缺陷病毒1型（HIV-1）反式激活因子（Tat）是一种核转录因子，已被证明可在细胞外发挥作用，这意味着一些Tat分子逃脱了核输入并被分泌。这就提出了一个问题：在HIV-1感染的细胞中，是什么调节了该多肽的核靶向作用。在这里我们表明，由钙离子激活的胞质成分是揭示Tat亲核特性所必需的：体外翻译的Tat分子除非与钙离子预孵育，否则不会与分离的细胞核结合。此外，只有在添加胞质提取物后，钙离子才会诱导化学合成的Tat分子具有亲核性。酪氨酸激酶（除草霉素A和染料木黄酮）或酪氨酸磷酸酶（钒酸盐）的抑制剂可阻止钙离子诱导的亲核性，这表明钙离子依赖性磷酸化/去磷酸化事件的参与。与这些观察结果一致，用钒酸盐或染料木黄酮处理会抑制Tat的转录活性。缺乏两个酪氨酸残基（第26位和第47位）中的一个或两个的Tat突变体也会出现同样的情况。因此，钙离子依赖性酪氨酸激酶和磷酸酶作用于辅助细胞蛋白，而这些辅助细胞蛋白反过来又决定了Tat的亲核性。
