Structure-function analysis of the C-clamp of TCF/Pangolin in Wnt/ß-catenin signaling.
Structure-function analysis of the C-clamp of TCF/Pangolin in Wnt/ß-catenin signaling.
复制标题
Wnt/ß-catenin信号传导中TCF/PANGOLIN的C夹的结构 - 功能分析。
DOI：
10.1371/journal.pone.0086180
复制
发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Cadigan KM
中科院分区：
文献类型：
--
作者：
Ravindranath AJ;Cadigan KM
进化上保守的 Wnt/ß-catenin (Wnt/ß-cat) 途径在后生动物的发育中发挥着重要作用。许多 Wnt 靶标受转录因子 TCF/LEF1 (TCF) 家族成员的调节。所有 TCF 均包含结合特定 DNA 序列的高迁移率组 (HMG) 结构域。无脊椎动物 TCF 和一些脊椎动物 TCF 亚型还包含另一个称为 C 夹的结构域，它允许 TCF 识别称为辅助位点的附加 DNA 基序。虽然 C 夹已被证明对于调节细胞培养中的多个 Wnt 报告基因非常重要，但其在调节 Wnt 靶标中的生理作用尚不清楚。此外，除了四个保守半胱氨酸中的两个具有重要功能外，对该结构域知之甚少。在这里，我们对果蝇 TCF/穿山甲 (TCF/Pan) 蛋白的 C 夹进行了系统的诱变和功能分析。我们发现 C 夹是一个锌结合结构域，足以与辅助位点结合。除了这种 DNA 结合活性之外，C 夹还抑制 HMG 结构域结合其同源 DNA 位点。点突变被鉴定为特异性影响 DNA 结合或降低抑制效果的点突变。这些突变体在 TCF/Pan 拯救试验中进行了表征。 C 夹的特异性 DNA 结合活性对于细胞培养和果蝇胚胎表皮图案化中的 TCF/Pan 功能至关重要，证明了这种 C 夹活性在调节 Wnt 靶基因表达中的重要性。相比之下，抑制性突变对细胞培养有微妙的影响，并且对胚胎中的 TCF/Pan 活性没有影响。这些结果提供了有关 C 夹功能域的重要信息，并强调了其对果蝇中 Wnt/ß-cat 信号传导的重要性。
The evolutionarily conserved Wnt/ß-catenin (Wnt/ß-cat) pathway plays an important role in animal development in metazoans. Many Wnt targets are regulated by members of the TCF/LEF1 (TCF) family of transcription factors. All TCFs contain a High Mobility Group (HMG) domain that bind specific DNA sequences. Invertebrate TCFs and some vertebrate TCF isoforms also contain another domain, called the C-clamp, which allows TCFs to recognize an additional DNA motif known as the Helper site. While the C-clamp has been shown to be important for regulating several Wnt reporter genes in cell culture, its physiological role in regulating Wnt targets is less clear. In addition, little is known about this domain, except that two of the four conserved cysteines are functionally important. Here, we carried out a systematic mutagenesis and functional analysis of the C-clamp from the Drosophila TCF/Pangolin (TCF/Pan) protein. We found that the C-clamp is a zinc-binding domain that is sufficient for binding to the Helper site. In addition to this DNA-binding activity, the C-clamp also inhibits the HMG domain from binding its cognate DNA site. Point mutations were identified that specifically affected DNA-binding or reduced the inhibitory effect. These mutants were characterized in TCF/Pan rescue assays. The specific DNA-binding activity of the C-clamp was essential for TCF/Pan function in cell culture and in patterning the embryonic epidermis of Drosophila, demonstrating the importance of this C-clamp activity in regulating Wnt target gene expression. In contrast, the inhibitory mutation had a subtle effect in cell culture and no effect on TCF/Pan activity in embryos. These results provide important information about the functional domains of the C-clamp, and highlight its importance for Wnt/ß-cat signaling in Drosophila.
登录
查看更多内容
DOI：
10.1126/science.1162327
发表时间：
2009-06-26
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Badis G;Berger MF;Philippakis AA;Talukder S;Gehrke AR;Jaeger SA;Chan ET;Metzler G;Vedenko A;Chen X;Kuznetsov H;Wang CF;Coburn D;Newburger DE;Morris Q;Hughes TR;Bulyk ML
通讯作者：
Bulyk ML
影响因子：
7.5
作者：
Holland, Jane D.;Klaus, Alexandra;Birchmeier, Walter
通讯作者：
Birchmeier, Walter
影响因子：
5.3
作者：
Hoverter, Nate P.;Ting, Ju-Hui;Waterman, Marian L.
通讯作者：
Waterman, Marian L.
影响因子：
64.5
作者：
Hallikas, O;Palin, K;Taipale, J
通讯作者：
Taipale, J
影响因子：
14.9
作者：
Grishin, NV
通讯作者：
Grishin, NV