Critical residues of the homeodomain involved in contacting DNA bases also specify the nuclear accumulation of thyroid transcription factor-1
Critical residues of the homeodomain involved in contacting DNA bases also specify the nuclear accumulation of thyroid transcription factor-1
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DOI：
10.1046/j.1432-1327.1999.00796.x
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发表时间：
1999-10-01
期刊：
影响因子：
--
通讯作者：
Christophe, D
中科院分区：
文献类型：
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作者：
Christophe-Hobertus, C;Duquesne, V;Christophe, D
甲状腺转录因子-1（TTF-1）同源结构域的N末端由5个碱性氨基酸组成，在POU类和NK 2类同源结构域中都是保守的，构成了功能性的核定位信号。通过分析融合蛋白的细胞分布，由水母绿色荧光变体和TTF-1的不同部分组成，我们证明了这种基本序列的存在本身不足以赋予完整的核积累。通过诱变，我们确定了位于同源结构域的DNA识别螺旋中心的第二个区域，该区域也能够指定嵌合蛋白的主要核定位，独立于碱性NLS的存在。通过诱变破坏DNA识别螺旋中的基本伸展和基序导致核积累的完全丧失，表明TTF-1的完全核积累是这两种蛋白信号的协同作用的结果。参与核靶向的同源结构域的两个区域还包括负责DNA结合位点识别的关键氨基酸，如通过诱变后体外DNA结合活性的丧失所证明的。具体而言，在DNA识别螺旋的中心部分的残基参与接触DNA的大沟中的碱基，并且在同源结构域蛋白质中是最保守的，这表明同源结构域的这一部分可以在该蛋白质家族的成员的核定位中发挥一般作用。
The N-terminal end of thyroid transcription factor-1 (TTF-1) homeodomain is composed of a stretch of five basic amino-acids that is conserved in both POU- and NK2-class homeodomains and constitutes a functional nuclear localization signal. By analyzing the cellular distribution of fusion proteins, composed of a jellyfish green fluorescent variant and different parts of TTF-1, we show hers that the presence of this basic sequence is not sufficient by itself to confer complete nuclear accumulation. By mutagenesis, we identified a second region located in the center of the DNA recognition helix of the homeodomain that is also able to specify a predominantly nuclear localization of the chimeric proteins, independently of the presence of the basic NLS. The destruction, by mutagenesis, of both the basic stretch and the motif in the DNA recognition helix led to the total loss of nuclear accumulation, indicating that complete nuclear accumulation of TTF-1 results from the concerted action of these two proteic signals. Both of the regions of the homeodomain that are involved in nuclear targeting also encompass critical amino-acids responsible for DNA binding site recognition, as evidenced by the loss of DNA binding activity in vitro upon mutagenesis. Specifically, residues in the central part of the DNA recognition helix are involved in contacting bases in the major groove of DNA and are the most conserved in homeodomain proteins, suggesting that this part of the homeodomain could play a general role in the nuclear localization of members of this family of proteins.