Identification of the TBX5 transactivating domain and the nuclear localization signal
Identification of the TBX5 transactivating domain and the nuclear localization signal
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DOI：
10.1016/j.gene.2004.01.017
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发表时间：
2004-04-14
期刊：
影响因子：
3.5
通讯作者：
Huang, TS
中科院分区：
文献类型：
--
作者：
Zaragoza, MV;Lewis, LE;Huang, TS
TBX 5是T-box基因家族的成员，编码一种参与心脏和肢体发育的转录因子。TBX 5的突变导致Holt-Oram综合征（HOS），这是一种常染色体显性疾病，伴有先天性心脏缺陷和前肢畸形。在这里，我们使用GAL 4-TBX 5融合蛋白在一个修改的酵母单杂交系统，以阐明TBX 5的反式激活结构域。利用TBX 5的一系列缺失突变，我们将其功能结构域缩小到其C-末端一半的氨基酸339-379，点突变分析然后显示氨基酸349-351的缺失取消了反式激活。这一结果在哺乳动物细胞中得到证实。此外，野生型TBX 5.而不是在氨基酸349-351处具有突变的TBX 5，具有抑制NCI-H1299细胞生长的能力，这也表明这些氨基酸对于TBX 5在哺乳动物细胞中的功能是至关重要的。此外，为了鉴定TBX 5的核定位信号，我们搜索了碱性氨基酸簇。我们发现在氨基酸325-327处的KRK序列的缺失使TBX 5错误定位于细胞质。这表明，这些氨基酸作为核定位信号。这些研究加深了我们对TBX 5结构与功能关系的理解，并提示TBX 5截短突变可能通过其反式激活结构域和/或核定位信号的丢失引起HOS。(C)2004 Elsevier B. V.保留所有权利。
TBX5 is a member of the T-box gene family and encodes a transcription factor involved in cardiac and limb development. Mutations of TBX5 cause Holt-Oram syndrome (HOS), an autosomal-dominant condition with congenital cardiac defects and forelimb anomalies. Here, we used a GAL4-TBX5 fusion protein in a modified yeast-one hybrid system to elucidate the TBX5 transactivating domain. Using a series of deletion mutations of TBX5, we narrowed down its functional domain to amino acids 339-379 of its C-terminal half, point mutagenesis analysis then showed that the loss of amino acids 349-351 abolished transactivation. This result was confirmed in mammalian cells. Furthermore, wild-type TBX5. but not TBX5 with mutations at the amino acids 349-351, has ability to inhibit NCI-H1299 cell growth also Suggesting that these amino acids are crucial for the TBX5 function in mammalian cells. In addition, to identify the nuclear localization signal of TBX5, we searched for cluster of basic amino acids. We found that the deletion of the KRK sequence at amino acids 325-327 mislocalizes TBX5 to cytoplasm. suggesting, that these amino acids serve as a nuclear localization signal. These studies enhance Our understanding of the structure-function relationship of TBX5 and suggest that truncation mutations of TBX5 could cause HOS through the loss of its transactivating domain and/or the nuclear localization signal. (C) 2004 Elsevier B.V. All rights reserved.