Growth arrest and DNA damage-inducible protein GADD34 targets protein phosphatase 1α to the endoplasmic reticulum and promotes dephosphorylation of the α subunit of eukaryotic translation initiation factor 2
Growth arrest and DNA damage-inducible protein GADD34 targets protein phosphatase 1α to the endoplasmic reticulum and promotes dephosphorylation of the α subunit of eukaryotic translation initiation factor 2
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DOI：
10.1128/mcb.23.4.1292-1303.2003
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发表时间：
2003-02-01
影响因子：
5.3
通讯作者：
Shenolikar, S
中科院分区：
文献类型：
--
作者：
Brush, MH;Weiser, DC;Shenolikar, S
生长阻滞和DNA损伤诱导蛋白GADD34与蛋白磷酸酶1 （PP1）相关，并促进真核翻译起始因子2 (eIF-2alpha) α亚基的体外去磷酸化。在本报告中，我们发现在培养细胞中表达人GADD34逆转了由thapsigargin和tunicamycin诱导的eIF-2alpha磷酸化，这两种药物促进内质网（ER）中蛋白质的展开。GADD34的表达也逆转了冈田酸诱导的eIF-2alpha磷酸化，但不逆转另一种磷酸酶抑制剂calyculin A （CA）诱导的eIF-2alpha磷酸化，这与PP1是GADD34组装的eIF-2alpha磷酸酶的一个组成部分的结果一致。结构-功能研究在GADD34中发现了一个双部c端结构域，该结构域包含一个典型的PP1结合基序KVRF和一个新的RARA序列，两者都是PP1结合所必需的。GADD34的n端缺失证实了PP1结合是必要的，但并不足以促进细胞中eIF-2alpha的去磷酸化。绿色荧光蛋白(GFP)-GADD34蛋白的成像显示，n端180个残基将GADD34定位于内质网，GADD34将PP1的a亚型靶向内质网。这些数据为GADD34在组装er相关的eIF-2alpha磷酸酶的作用模式提供了新的见解，该磷酸酶调节哺乳动物细胞中的蛋白质翻译。
The growth arrest and DNA damage-inducible protein, GADD34, associates with protein phosphatase 1 (PP1) and promotes in vitro dephosphorylation of the alpha subunit of eukaryotic translation initiation factor 2, (eIF-2alpha). In this report, we show that the expression of human GADD34 in cultured cells reversed eIF-2alpha phosphorylation induced by thapsigargin and tunicamycin, agents that promote protein unfolding in the endoplasmic reticulum (ER). GADD34 expression also reversed eIF-2alpha phosphorylation induced by okadaic acid but not that induced by another phosphatase inhibitor, calyculin A (CA), which is a result consistent with PP1 being a component of the GADD34-assembled eIF-2alpha phosphatase. Structure-function studies identified a bipartite C-terminal domain in GADD34 that encompassed a canonical PP1-binding motif, KVRF, and a novel RARA sequence, both of which were required for PP1 binding. N-terminal deletions of GADD34 established that while PP1 binding was necessary, it was not sufficient to promote eIF-2alpha dephosphorylation in cells. Imaging of green fluorescent protein (GFP)-GADD34 proteins showed that the N-terminal 180 residues directed the localization of GADD34 at the ER and that GADD34 targeted the a isoform of PP1 to the ER. These data provide new insights into the mode of action of GADD34 in assembling an ER-associated eIF-2alpha phosphatase that regulates protein translation in mammalian cells.