title: Phosphorylation of wheat germ initiation factor 2 (eIF-2) by N-ethylmaleimide-treated wheat germ lysates and by purified casein kinase II does not affect the guanine nucleotide exchange on eIF-2.
abstract: Phosphorylation of the small subunit of eukaryotic initiation factor-2 (eIF-2 alpha) impairs protein synthesis in mammalian systems. It is not known, however, if a similar regulatory mechanism exists in plants. Previous reports indicate that one of the wheat germ eIF-2 subunits, the p40-41 doublet, is phosphorylated by heterologous eIF-2 alpha kinases. Here we report that phosphorylation of the small subunit in wheat germ eIF-2, p36, occurs in translating wheat germ lysates which are pretreated with N-ethylmaleimide (NEM) and dithiothreitol. Also, a purified sea star casein kinase II (CKII) phosphorylates the p41-42 doublet and p36 subunits of wheat germ eIF-2. While heme-regulated eIF-2 alpha kinase from reticulocyte lysates does not inhibit wheat germ protein synthesis, CKII and NEM are found to be inhibitory. To determine whether phosphorylation of the small subunit (p36) is the cause for protein synthesis inhibition, we have further studied the exchange of labeled GDP for unlabeled GDP in the preformed eIF-2. [3H]GDP complex in vitro in the presence of CKII and ATP. The GDP exchange in eIF-2.GDP complex can occur without the addition of any protein factor and the exchange reaction is marginally inhibited by CKII. A 0-70% ammonium sulfate cut fraction, prepared from NEM-treated wheat germ lysate, also does not inhibit the guanine nucleotide exchange reaction. These findings suggest that the protein synthesis inhibition in these cases is not mediated by eIF-2 phosphorylation.
authors.affiliation: Department of Biochemistry, School of Life Sciences, University of Hyderabad, Andhra Pradesh, India.
authors.forename: N
authors.initials: N
authors.lastname: Janaki
authors.forename: V M
authors.initials: VM
authors.lastname: Krishna
authors.forename: K V
authors.initials: KV
authors.lastname: Ramaiah
mesh_terms: D047390:Casein Kinase II; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology*; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D010766:Phosphorylation; D014176:Protein Biosynthesis; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000494:pharmacology*; D012156:Reticulocytes / Q000201:enzymology; D012639:Seeds / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014908:Triticum / Q000378:metabolism*; D019892:eIF-2 Kinase
chemical_list: D015852:Eukaryotic Initiation Factor-2; D013439:Sulfhydryl Reagents; D006153:Guanosine Diphosphate; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase; D005033:Ethylmaleimide; D004229:Dithiothreitol