Transactivation and growth suppression by the gut-enriched Kruppel-like factor (Kruppel-like factor 4) are dependent on acidic amino acid residues and protein-protein interaction
Transactivation and growth suppression by the gut-enriched Kruppel-like factor (Kruppel-like factor 4) are dependent on acidic amino acid residues and protein-protein interaction
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DOI：
10.1093/nar/28.5.1106
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发表时间：
2000-03-01
影响因子：
14.9
通讯作者：
Yang, VW
中科院分区：
文献类型：
--
作者：
Geiman, DE;Han, TT;Yang, VW
外富集Kruppel样因子（GKLF或KLF 4）是一种多效性（激活和抑制）转录因子。本研究的特点的反式激活GKLF的机制。使用GAL 4融合测定，小鼠GKLF的活化结构域定位于N-末端的109个氨基酸残基。定点突变表明，该区域内的两个相邻簇的酸性残基负责的激活效果。GKLF的反式激活涉及分子间相互作用，如野生型而非突变型GKLF与N-末端激活结构域竞争的能力所证明的。此外，野生型腺病毒EIA，但不是一个突变的E1 A，不能结合p300/CBP，抑制反式激活的N-末端109个氨基酸的GKLF，表明p300/CBP是GKLF的相互作用的合作伙伴。谷胱甘肽-S转移酶下拉和体内免疫共沉淀实验证明GKLF和CBP之间的物理相互作用。我们还表明，这两个酸性氨基酸簇是必不可少的这种相互作用，因为GKLF与这些残基的突变未能与CBP免疫共沉淀。重要的是，相同的突变消除了GKLF抑制细胞生长的能力，如通过集落抑制测定所确定的。因此，这些研究为GKLF的反式激活和生长抑制作用之间的结构和功能相关性提供了合理的证据。
Out-enriched Kruppel-like factor (GKLF or KLF4) is a pleiotropic (activating and repressive) transcription factor. This study characterizes the mechanisms of transactivation by GKLF. Using a GAL4 fusion assay, the activating domain of murine GKLF was localized to the 109 amino acid residues in the N-terminus. Site-directed mutagenesis showed that two adjacent clusters of acidic residues within this region are responsible for the activating effect. Transactivation by GKLF involves intermolecular interactions as demonstrated by the ability of wild-type, but not mutated, GKLF to compete with the N-terminal activation domain. In addition, wild-type adenovirus EIA, but not a mutated E1A that failed to bind p300/CBP, inhibited transactivation by the N-terminal 109 amino acids of GKLF, suggesting that p300/CBP are GKLF's interacting partners. A physical interaction between GKLF and CBP was demonstrated by glutathione-S transferase pull-down and by in vivo co-immunoprecipitation experiments. We also showed that the two acidic amino acid clusters are essential for this interaction, since GKLF with mutations in these residues failed to co-immunoprecipitate with CBP. Importantly, the same mutations abrogated the ability of GKLF to suppress cell growth as determined by a colony suppression assay. These studies therefore provide plausible evidence for a structural and functional correlation between the transactivating and growth-suppressing effects of GKLF.