title: Translation and phosphorylation of wheat germ lysate: phosphorylation of wheat germ initiation factor 2 by casein kinase II and in N-ethylmaleimide-treated lysates.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Hyderabad, Hyderabad 500 046, Andhra Pradesh, India.
authors.forename: Burela
authors.initials: B
authors.lastname: Laxminarayana
authors.forename: Vattem M
authors.initials: VM
authors.lastname: Krishna
authors.forename: Narahari
authors.initials: N
authors.lastname: Janaki
authors.forename: Kolluru V A
authors.initials: KV
authors.lastname: Ramaiah
mesh_terms: D047390:Casein Kinase II; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D006358:Hot Temperature; D010100:Oxygen / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D014176:Protein Biosynthesis; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry; D012697:Serine Endopeptidases / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors; D014908:Triticum / Q000378:metabolism*; D019892:eIF-2 Kinase / Q000378:metabolism
chemical_list: D010455:Peptides; D012694:Serine; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D005978:Glutathione; D005033:Ethylmaleimide; D010100:Oxygen; D004229:Dithiothreitol