Translation and Phosphorylation of Wheat Germ Lysate: Phosphorylation of Wheat Germ Initiation Factor 2 by Casein Kinase II and in N-Ethylmaleimide-Treated Lysates

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 α, the p41–42 doublet subunit, and β, 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 eIF2α but not the α subunit (p41–42 doublet) of WG eIF2. A phosphospecific anti-eIF2α antibody recognizes the WG eIF2α(P) that is phosphorylated by an authentic eIF2α kinase such as double-stranded RNA-dependent protein kinase, but it is unable to recognize the eIF2α that is phosphorylated in NEM-treated lysates. These findings therefore suggest that phosphorylation of WG eIF2α in NEM-treated lysates occurs on a site different from the serine 51 residue that is phosphorylated by authentic eIF2α kinases. In addition, it also suggests that WG eIF2α, unlike reticulocyte eIF2α, is phosphorylated by eIF2α kinases and also by other kinases. Consistent with this idea, it has been observed here that casein kinase II (CKII) phosphorylates WG eIF2α and the phosphorylation is enhanced by NEM in vitro and in lysates. The phosphopeptide analysis suggests that WG eIF2α has separate phosphorylation sites for CKII and heme-regulated eIF2α kinase (a well-characterized mammalian eIF2α kinase), and NEM-induced phosphorylation in WG lysates resembles CKII-mediated phosphorylation.