Functional interaction of bZIP proteins and the large subunit of replication factor C in liver and adipose cells
Functional interaction of bZIP proteins and the large subunit of replication factor C in liver and adipose cells
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DOI：
10.1074/jbc.m010912200
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发表时间：
2001-07-27
影响因子：
4.8
通讯作者：
Cheong, JH
中科院分区：
文献类型：
--
作者：
Hong, SH;Park, SJ;Cheong, JH
转录因子 CCAAT/增强子结合蛋白-vt (C/EBP α) 在细胞生长和分化中起着至关重要的作用。为了进一步描述 C/EBP α 介导的分化机制，我们通过远西方筛选筛选了 C/EBP α 相互作用蛋白。最强的相互作用之一是与 RFC140（复制因子 C 复合物的大亚基）的相互作用。通过亲和层析和免疫共沉淀的组合测定，C/EBPα与来自大鼠肝核提取物的RFC140特异性相互作用。随后的远蛋白印迹显示C/EBPα的bZIP结构域与RFC140的DNA结合区相互作用。 RFC140在哺乳动物细胞中的过表达增加了C/EBPα对最小启动子和天然启动子的反式激活活性。与增强的反式激活相一致，在体外检测到了 C/EBP α 和 RFC140 蛋白与同源 DNA 元件的复合物。在 3T3-LI 前脂肪细胞向脂肪细胞的终末分化中检测到 C/EBP α 和 RFC140 之间的特异性相互作用。这两种蛋白的协同转录效应增加了过氧化物酶体增殖物激活受体-γ的启动子活性和蛋白表达，这是脂肪细胞分化的主要调节因子。我们的结果表明，特定转录因子 C/EBPa 和通用 DNA 复制因子 RFC140 在功能和物理上相互作用。这一观察结果强调了一种独特的机制，通过该机制，通用复制因子的水平可以强烈调节特定转录因子作为共激活因子的功能活性。
The transcription factor CCAAT/enhancer-binding protein-vt (C/EBP alpha) has a vital role in cell growth and differentiation. To delineate further a mechanism for C/EBP alpha -mediated differentiation, we screened C/EBP alpha -interacting proteins through far-Western screening. One of the strongest interactions was with RFC140, the large subunit of the replication factor C complex. C/EBPa specifically interacted with RFC140 from rat liver nuclear extract as determined by a combination of affinity chromatography and co-immunoprecipitation. Subsequent far-Western blotting showed that the bZIP domain of C/EBPa interacted with the DNA-binding region of RFC140. Overexpression of RFC140 in mammalian cells increased the transactivation activity of C/EBPa on both minimal and native promoters. Consistent with the enhanced transactivation, a complex of C/EBP alpha and RFC140 proteins with the cognate DNA element was detected in vitro. The specific interaction between C/EBP alpha and RFC140 was detected in the terminal differentiation of 3T3-LI preadipocytes to adipocytes. The synergistic transcription effect of these two proteins increased the promoter activity and protein expression of peroxisome proliferator-activated receptor-gamma, which is a main regulator of adipocyte differentiation. Our results demonstrate that the specific transcription factor C/EBPa and the general DNA replication factor RFC140 interact functionally and physically. This observation highlights a unique mechanism by which the levels of the general replication factor can strongly modulate the functional activity of the specific transcription factor as a coactivator.