Rabbit reticulocyte double-stranded RNA-activated protein kinase and the hemin-controlled translational repressor phosphorylate the same Mr 1500 peptide of eukaryotic initiation factor 2α

Abstract

The inhibition of polypeptide chain initiation that occurs when rabbit reticulocyte lysate is incubated in the absence of hemin [ l--5] or in the presence of a low level (1 O-l 00 ng/ml) of dsRNA [ 1,6-81 appears to be mediated by the phosphorylation of the iW, 35 000 (a) subunit of eIF-2, the initiation factor that promotes binding of Met-tRNAf to 40 S ribosomal subunits [9-l 31, but involves the action of two distinct protein kinases [14]. The inhibitor formed in the absence of hemin (HCR) occurs in the post-ribosomal supernate, is rapidly activated by incubation with NEM [ 151, and is associated with the autophosphorylation of a M, 90 000-100 000 protein [1,16-191, whereas the dsRNA-activated protein kinase (ds1) is found in the ribosomal fraction,is activated by dsRNA, and is associated with the phosphorylation of a M, 67 000 protein(s) [ 1,7,8,20]. Peptide analyses of elF-2cu, phosphorylated either by HCR or dsI, have shown a similar pattern of phosphopeptides suggesting that these two protein kinases phosphorylate the same site or sites [20-231. When eIF-2ol, phosphorylated by HCR, is subjected to exhaustive trypsin digestion, almost all the phosphate can be localized to a single, M, 1500 peptide [24];we report here that this same single peptide is phosphorylated by dsl.