Parker R, Phan T, Baumeister P, Roy B, Cheriyath V, Roy A L, Lee A S
Department of Biochemistry and Molecular Biology and the USC/Norris Comprehensive Cancer Center, Keck School of Medicine of the University of Southern California, Los Angeles, California 90089-9176, USA.
Mol Cell Biol. 2001 May;21(9):3220-33. doi: 10.1128/MCB.21.9.3220-3233.2001.
When mammalian cells are subjected to stress targeted to the endoplasmic reticulum (ER), such as depletion of the ER Ca(2+) store, the transcription of a family of glucose-regulated protein (GRP) genes encoding ER chaperones is induced. The GRP promoters contain multiple copies of the ER stress response element (ERSE), consisting of a unique tripartite structure, CCAAT(N(9))CCACG. Within a subset of mammalian ERSEs, N(9) represents a GC-rich sequence of 9 bp that is conserved across species. A novel complex (termed ERSF) exhibits enhanced binding to the ERSE of the grp78 and ERp72 promoters using HeLa nuclear extracts prepared from ER-stressed cells. Optimal binding of ERSF to ERSE and maximal ERSE-mediated stress inducibility require the conserved GGC motif within the 9-bp region. Through chromatographic purification and subsequent microsequencing, we have identified ERSF as TFII-I. Whereas TFII-I remains predominantly nuclear in both nontreated NIH 3T3 cells and cells treated with thapsigargin (Tg), a potent inducer of the GRP stress response through depletion of the ER Ca(2+) store, the level of TFII-I transcript was elevated in Tg-stressed cells, correlating with an increase in TFII-I protein level in the nuclei of Tg-stressed cells. Purified recombinant TFII-I isoforms bind directly to the ERSEs of grp78 and ERp72 promoters. The stimulation of ERSE-mediated transcription by TFII-I requires the consensus tyrosine phosphorylation site of TFII-I and the GGC sequence motif of the ERSE. We further discovered that TFII-I is an interactive protein partner of ATF6 and that optimal stimulation of ERSE by ATF6 requires TFII-I.
当哺乳动物细胞受到针对内质网（ER）的应激时，如内质网Ca²⁺储存的耗竭，编码内质网伴侣蛋白的葡萄糖调节蛋白（GRP）基因家族的转录就会被诱导。GRP启动子包含内质网应激反应元件（ERSE）的多个拷贝，其由独特的三联体结构CCAAT(N⁹)CCACG组成。在哺乳动物ERSE的一个子集中，N⁹代表一个富含GC的9个碱基对的序列，该序列在物种间保守。使用从内质网应激细胞制备的HeLa细胞核提取物，一种新型复合物（称为ERSF）与grp78和ERp72启动子的ERSE的结合增强。ERSF与ERSE的最佳结合以及ERSE介导的最大应激诱导性需要9个碱基对区域内保守的GGC基序。通过色谱纯化和随后的微测序，我们已将ERSF鉴定为TFII-I。在未处理的NIH 3T3细胞和用毒胡萝卜素（Tg）处理的细胞中，TFII-I主要保留在细胞核中，毒胡萝卜素是通过耗竭内质网Ca²⁺储存来强力诱导GRP应激反应的物质，在Tg应激细胞中TFII-I转录本水平升高，这与Tg应激细胞核中TFII-I蛋白水平的增加相关。纯化的重组TFII-I异构体直接与grp78和ERp72启动子的ERSE结合。TFII-I对ERSE介导的转录的刺激需要TFII-I的共有酪氨酸磷酸化位点和ERSE的GGC序列基序。我们进一步发现TFII-I是ATF6的相互作用蛋白伴侣，并且ATF6对ERSE的最佳刺激需要TFII-I。
