Laxminarayana Burela, Krishna Vattem M, Janaki Narahari, Ramaiah Kolluru V A
Department of Biochemistry, University of Hyderabad, Hyderabad 500 046, Andhra Pradesh, India.
Arch Biochem Biophys. 2002 Apr 1;400(1):85-96. doi: 10.1006/abbi.2002.2763.
Previously, we observed that N-ethylmaleimide (NEM), a thiol-alkylating agent, was found to stimulate the phosphorylation of several proteins in translating wheat germ (WG) lysates, including the phosphorylation of alpha, the p41-42 doublet subunit, and beta, the p36 subunit, of the WG initiation factor 2 (eIF2). We find now that NEM increases phosphorylation of several proteins significantly in lysates which are moderate or low in their translation compared to optimally active lysates. Heat treatment, which stimulates oxidation of protein sulfhydryls, decreases the translation and phosphorylation ability of WG lysates. The decrease in phosphorylation, but not translation, that occurs in heat-treated lysates is prevented very efficiently by NEM and partially by reducing agents such as dithiothreitol (DTT) and GSH. DTT prevents, however, completely the loss of sulfhydryl content of heat-treated WG lysates and does not at all prevent heat-induced inhibition of translation. In contrast, DTT prevents completely the diamide-induced translational inhibition and also the loss of sulfhydryl content. These findings therefore suggest that in addition to the maintenance of sulfhydryl groups, heat-labile proteins and their interactions with other proteins play an important role in overall translation and phosphorylation. It is also observed here that heat treatment stimulates the phosphorylation of rabbit reticulocyte eIF2 alpha but not the alpha subunit (p41-42 doublet) of WG eIF2. A phosphospecific anti-eIF2 alpha antibody recognizes the WG eIF2 alpha(P) that is phosphorylated by an authentic eIF2 alpha kinase such as double-stranded RNA-dependent protein kinase, but it is unable to recognize the eIF2 alpha that is phosphorylated in NEM-treated lysates. These findings therefore suggest that phosphorylation of WG eIF2 alpha in NEM-treated lysates occurs on a site different from the serine 51 residue that is phosphorylated by authentic eIF2 alpha kinases. In addition, it also suggests that WG eIF2 alpha, unlike reticulocyte eIF2 alpha, is phosphorylated by eIF2 alpha kinases and also by other kinases. Consistent with this idea, it has been observed here that casein kinase II (CKII) phosphorylates WG eIF2 alpha and the phosphorylation is enhanced by NEM in vitro and in lysates. The phosphopeptide analysis suggests that WG eIF2 alpha has separate phosphorylation sites for CKII and heme-regulated eIF2 alpha kinase (a well-characterized mammalian eIF2 alpha kinase), and NEM-induced phosphorylation in WG lysates resembles CKII-mediated phosphorylation.
此前，我们观察到硫醇烷基化剂N - 乙基马来酰亚胺（NEM）可刺激小麦胚（WG）裂解物中几种蛋白质的磷酸化，包括WG起始因子2（eIF2）的α亚基（p41 - 42双峰亚基）和β亚基（p36亚基）的磷酸化。我们现在发现，与活性最佳的裂解物相比，NEM可显著增加翻译活性中等或较低的裂解物中几种蛋白质的磷酸化。热处理可刺激蛋白质巯基的氧化，降低WG裂解物的翻译和磷酸化能力。NEM能非常有效地阻止热处理裂解物中发生的磷酸化降低，但不能阻止翻译降低，而二硫苏糖醇（DTT）和谷胱甘肽（GSH）等还原剂能部分阻止。然而，DTT能完全阻止热处理WG裂解物中巯基含量的损失，且根本不能阻止热诱导的翻译抑制。相比之下，DTT能完全阻止二酰胺诱导的翻译抑制以及巯基含量的损失。因此，这些发现表明，除了维持巯基基团外，热不稳定蛋白及其与其他蛋白的相互作用在整体翻译和磷酸化中起重要作用。在此还观察到，热处理可刺激兔网织红细胞eIF2α的磷酸化，但不能刺激WG eIF2的α亚基（p41 - 42双峰）的磷酸化。一种磷酸特异性抗eIF2α抗体可识别由真正的eIF2α激酶（如双链RNA依赖性蛋白激酶）磷酸化的WG eIF2α(P)，但无法识别在NEM处理的裂解物中磷酸化的eIF2α。因此，这些发现表明，在NEM处理的裂解物中WG eIF2α的磷酸化发生在与真正的eIF2α激酶磷酸化的丝氨酸51残基不同的位点。此外，这也表明与网织红细胞eIF2α不同，WG eIF2α可被eIF2α激酶以及其他激酶磷酸化。与此想法一致，在此观察到酪蛋白激酶II（CKII）可磷酸化WG eIF2α，且在体外和裂解物中NEM可增强这种磷酸化。磷酸肽分析表明，WG eIF2α有针对CKII和血红素调节的eIF2α激酶（一种特征明确的哺乳动物eIF2α激酶）的不同磷酸化位点，且NEM诱导的WG裂解物中的磷酸化类似于CKII介导的磷酸化。
