Structure and regulation of eukaryotic initiation factor eIF-2. Sequence of the site in the alpha subunit phosphorylated by the haem-controlled repressor and by the double-stranded RNA-activated inhibitor

Abstract

Eukaryotic protein synthesis initiation factor 2 (eIF-2) can be phosphorylated on its tl subunit by two wellcharacterised protein kinases, termed the haem-controlled repressor (HCR) and the double-stranded RNAactivated inhibitor (dsI). Phosphorylation of eIF-2 by these kinases is thought to be important in the regulation of peptide-chain initiation. We report the location of the serine residue in the a subunit, which is phosphorylated by both these enzymes. Limited tryptic digestion and subsequent cyanogen bromide treatment of rat liver eIF-2 phosphorylated by HCR yielded one major phosphopeptide. This peptide had the sequence Ile-Leu-Leu-Ser-Glu-Leu-Ser(P)-Arg-Arg .The same major phosphopeptide was obtained from rabbit reticulocyte eIF-2 phosphorylated by HCR or dsl as judged by its behaviour on two-dimensional mapping and reverse-phase chromatography. In all cases the phosphorylated residue was found to be serine-7, and not serine-4, of the above sequence as determined from sequence analysis and by subdigestion of the peptide with Staphylococcus aureus V8 proteinase.Protein synthesis initiation factor-2 (eIF-2) mediates the binding of the initiator tRNA (Met-tRNAi) to the 40s ribosomal subunit during peptide-chain initiation. The activity of eIF-2 is believed to be important in controlling the overall rate of chain initiation and is regulated by phosphorylation of its smallest (a) subunit (reviewed in [l-31). Briefly, phosphorylation of eIF-2a impairs the recycling of eIF-2 which is required to regenerate active eIF-2 from the inactive [eIF-2 . GDP] complexes which are released from the ribosome after each round of chain initiation [4]. This recycling is mediated by an additional initiation factor [5 -91 termed GEF (guanine-nucleotide-exchange factor). This factor promotes the exchange of bound GDP for GTP and yields the active [eIF-2 . GTP] species, which can bind the initiator Met-tRNA, and participate in a further round of chain initiation.The role of the phosphorylation of eIF-2a in the control of translation has been studied extensively in reticulocytes, where haem deprivation rapidly leads to inhibition of chain initiation associated with increased phosphorylation of eIF-2a. This results from the activation of a haem-regulated

Heme: a versatile signaling molecule controlling the activities of diverse regulators ranging from transcription factors to MAP kinases

“…Under heme-deficient conditions, HRI inhibits protein synthesis at the level of translational initia- [52,54]. During heme deficiency, HRI is activated, permitting it to phosphorylate serine 51 of the a-subunit of the eIF2 [52,54,58]. Phosphorylation of eIF2a prevents it from being recycled for protein translation and results in the inhibition of protein synthesis [52,54].…”

Regulation of polypeptide‐chain initiation in rat skeletal muscle Starvation does not alter the activity or phosphorylation state of initiation factor eIF‐2

“…Nylon membranes (Biodyne A) were from Pall Ultrafine Filtration Corp., Glen Cove, NY, USA. The eIF-2cu kinase HCR and eIF-2 were purified from reticulocyte lysates as described [13].…”

Proteomic Analysis of Wild-type and Mutant Huntingtin-associated Proteins in Mouse Brains Identifies Unique Interactions and Involvement in Protein Synthesis

“…Stress granule assembly depends on the inhibition of mRNA translation. This inhibition can occur through the activity of the protein kinase PKR (58), which is present and active at elevated levels in HD patient brains (59). PKR activity is directly regulated by Prkra (41), which was present in Htt purifications and more abundant in 140Q samples than in 7Q samples.…”