Physical and functional association between GADD153 and CCAAT/enhancer-binding protein beta during cellular stress
Physical and functional association between GADD153 and CCAAT/enhancer-binding protein beta during cellular stress
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DOI：
10.1074/jbc.271.24.14285
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发表时间：
1996-06-14
影响因子：
4.8
通讯作者：
Holbrook, NJ
中科院分区：
文献类型：
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作者：
Fawcett, TW;Eastman, HB;Holbrook, NJ
GADD153是CCAAT/增强子结合蛋白(C/EBP)家族中普遍表达的成员，受多种生长抑制和DNA损伤因子的诱导。在功能上，GADD153被认为是C/EBPs的显性-负性调节因子。因此，我们试图获得GADD153和其他C/EBPs在细胞对应激反应过程中相互作用的证据。在这篇报告中，我们证明了亚砷酸钠处理大鼠嗜铬细胞瘤PC12细胞后，C/ERP-β和GADD153(生长停滞和DNA损伤诱导基因153)的表达增加，但其他C/EBPs没有。免疫共沉淀实验为亚砷酸盐处理的细胞内源性GADD153-C/EBP-β复合体的形成提供了证据。此外，还进行了其他实验，以确定这些复合体在调节GADD153表达方面的作用。本实验室前期研究表明，GADD153启动子含有一个C/EBP结合部位，其他C/EBPs通过该结合部位反式激活GADD153在肝癌细胞中的表达。在这里，我们证明了从亚砷酸盐处理的PC12细胞制备的提取物同样显示出能够与GADD153-C/EBP位点结合的因子数量增加，并且这些复合体至少部分包含C/EBP-β。在共表达GADD153报告基因和C/EBP-β蛋白的PC12细胞中，强制表达C/EBP-β能够反式激活GADD153启动子。然而，GADD153的过表达抑制了C/EBP-β对GADD153启动子的反式激活。这些发现为应激诱导的GADD153在细胞对应激反应过程中反馈减弱GADD153表达的自动调节环路提供了证据。
GADD153, a ubiquitously expressed member of the CCAAT/enhancer-binding protein (C/EBP) family is induced by a wide variety of growth-arresting and DNA-damaging agents. Functionally, GADD153 has been postulated to act as a dominant-negative regulator of C/EBPs. Therefore we sought to gain evidence for interactions between GADD153 and other C/EBPs during cellular responses to stress. In this report we have demonstrated that treatment of rat pheochromocytoma PC12 cells with sodium arsenite leads to enhanced expression of C/ERP-beta and GADD153 (growth arrest and DNA damage inducible gene 153) but not other C/EBPs. Coimmunoprecipitation experiments provided evidence for the formation of endogenous GADD153-C/EBP-beta complexes in arsenite-treated cells. Additional experiments were performed to determine the role of such complexes in regulating GADD153 expression. Previous studies in our laboratory demonstrated that the GADD153 promoter contains a C/EBP binding site through which other C/EBPs interact to transactivate GADD153 expression in liver hepatoma cells. Here, we demonstrate that extracts prepared from arsenite-treated PC12 cells likewise show increased amounts of factors capable of binding to the GADD153-C/EBP site and that these complexes are comprised at least in part of C/EBP-beta. Forced expression of C/EBP-beta was found to be capable of transactivating the GADD153 promoter in PC12 cells cotransfected with plasmids expressing a GADD153 reporter gene and C/EBP-beta protein. However, overexpression of GADD153 inhibited the transactivation of the GADD153 promoter by C/EBP-beta. These findings provide evidence for an autoregulatory loop in which stress-induced GADD153 feeds back to attenuate GADD153 expression during the cellular response to stress.