Yerlikaya Azmi, Kimball Scot R, Stanley Bruce A
Department of Biology, Faculty of Science and Arts, University of Dumlupinar, Kutahya, Turkey.
Biochem J. 2008 Jun 15;412(3):579-88. doi: 10.1042/BJ20080324.
The present study demonstrates that even brief inhibition of degradation by the 26S proteasome inhibits global protein synthesis, mediated through increased phosphorylation of eIF2alpha (eukaryotic translational initiation factor 2alpha) by the HRI (haem-regulated inhibitor) kinase. Exposure of COS-7 cells to the proteasome inhibitor MG-132 (the proteasome inhibitor carbobenzoxy-L-leucyl-L-leucyl-leucinal) for 4 h resulted in a 55-60% decrease in protein synthesis rate compared with control cells. This repression of protein synthesis after treatment with MG-132 is not due to induction of apoptosis, which is known to occur after longer periods of 26S inhibition. Instead, we observed a significantly increased phosphorylation of eIF2alpha, which is known to repress global protein synthesis. In three MEF (mouse embryonic fibroblast) knockout cell lines lacking one of the four kinases known to phosphorylate eIF2alpha, increased phosphorylation of eIF2alpha still occurred after inhibition of the 26S proteasome. These three cell lines included a deletion of the PKR (double-stranded-RNA-dependent protein kinase); a deletion of the PERK (PKR-like endoplasmic reticulum resident kinase); or a deletion of the GCN2 (positive general control of transcription-2) kinase, indicating that none of these kinases was primarily responsible for the observed phosphorylation of eIF2alpha. In contrast, in a fourth MEF knockout cell line, HRI(-/-) cells lacking the HRI kinase failed to increase eIF2alpha phosphorylation upon proteasome inhibitor treatment (MG-132 or various doses of Bortezomib), indicating that the HRI kinase is the primary kinase activated by brief treatment of MEFs with 26S proteasome inhibitors.
本研究表明，即使26S蛋白酶体介导的降解受到短暂抑制，也会抑制整体蛋白质合成，这是通过血红素调节抑制剂（HRI）激酶增加真核翻译起始因子2α（eIF2α）的磷酸化来介导的。与对照细胞相比，COS-7细胞暴露于蛋白酶体抑制剂MG-132（蛋白酶体抑制剂苄氧羰基-L-亮氨酰-L-亮氨酰-亮氨酸）4小时后，蛋白质合成速率降低了55%-60%。MG-132处理后蛋白质合成的这种抑制不是由于诱导凋亡，已知凋亡会在26S抑制较长时间后发生。相反，我们观察到eIF2α的磷酸化显著增加，已知这会抑制整体蛋白质合成。在三种缺乏已知可磷酸化eIF2α的四种激酶之一的小鼠胚胎成纤维细胞（MEF）基因敲除细胞系中，26S蛋白酶体抑制后eIF2α的磷酸化仍会增加。这三种细胞系包括双链RNA依赖性蛋白激酶（PKR）缺失；蛋白激酶R样内质网激酶（PERK）缺失；或转录-2正调控因子（GCN2）激酶缺失，这表明这些激酶都不是观察到的eIF2α磷酸化的主要原因。相反，在第四个MEF基因敲除细胞系中，缺乏HRI激酶的HRI（-/-）细胞在蛋白酶体抑制剂处理（MG-132或不同剂量的硼替佐米）后未能增加eIF2α磷酸化，这表明HRI激酶是MEF经26S蛋白酶体抑制剂短暂处理后激活的主要激酶。
