Evidence that the phosphorylation of eukaryotic initiation factor 2 alpha by the hemin-controlled translational repressor occurs at a single site.
We have investigated whether the phosphorylation of the Mr = 35,000 subunit of eukaryotic initiation factor 2 (eIF-2 alpha) by the hemin-controlled translational repressor (HCR), which appears to mediate the regulation of protein synthesis by hemin in rabbit reticulocytes, occurs at a single or multiple sites. Following phosphorylation of highly purified ribosomal or soluble eIF-2 with [gamma-32P]ATP and highly purified HCR, eIF-2 alpha was isolated by elution from a 7% sodium dodecyl sulfate-polyacrylamide gel, subjected to trypsin digestion, and analyzed on 20% sodium dodecyl sulfate-polyacrylamide gels. By 9 h of digestion none of the original, Mr = 35,000 subunit remained, and after 24 h the 32P radioactivity was distributed in multiple polypeptides in the range of Mr = 4,000 to 8,000. After 4 days of trypsinization, however, all of these peptides were reduced to a low level except for a minor band with Mr = 4,500, and a Mr = 1,500 component became the predominant 32P-labeled product. After 6 days of trypsin digestion the minor Mr = 4,500 component was much less but still detectable, and the Mr = 1,500 component represented 85% (ribosomal eIF-2 alpha) and 90% (soluble eIF-2 alpha) of the total radioactivity. The Mr = 1,500 component, when eluted fro 20% gels, showed a single spot when fingerprinted on thin layer cellulose sheets that was identical whether derived from ribosomal or soluble eIF-2 alpha. These findings suggest that HCR phosphorylates eIF-2 alpha at one specific site, although we cannot exclude the possibility that a second, minor site may also become phosphorylated to a very limited degree.
D000818:Animals; D001665:Binding Sites; D015852:Eukaryotic Initiation Factor-2; D006418:Heme / Q000031:analogs & derivatives*; D006427:Hemin / Q000378:metabolism* / Q000494:pharmacology*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D010448:Peptide Initiation Factors / Q000378:metabolism*; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D014176:Protein Biosynthesis / Q000187:drug effects*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D014357:Trypsin; D019892:eIF-2 Kinase
D015852:Eukaryotic Initiation Factor-2; D010446:Peptide Fragments; D010448:Peptide Initiation Factors; D010748:Phosphopeptides; D006418:Heme; D006427:Hemin; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase; D014357:Trypsin