title: Type 1 phosphatase inhibitors reduce the restoration of guanine nucleotide exchange activity of eukaryotic initiation factor 2B inhibited reticulocyte lysates rescued by hemin.
abstract: In heme-deficient reticulocyte lysates, the alpha-subunit of eukaryotic initiation factor-2 (eIF-2alpha) is phosphorylated due to the activation of the heme-regulated eIF-2alpha kinase (HRI). Phosphorylation of eIF-2alpha impairs the guanine nucleotide exchange activity of eIF-2B and thereby inhibits or shuts off protein synthesis. Delayed addition of hemin to shut-off lysates inhibits the eIF-2alpha kinase activity of HRI and restores protein synthesis; under those conditions, the endogenous phosphatase of the lysate dephosphorylates phosphorylated eIF-2alpha and restores eIF-2B activity. In this report we present evidence that the restoration of eIF-2B activity is dependent on the concentration of added hemin and is related to HRI activity in lysates. The recovery of eIF-2B activity is not affected by protein synthesis inhibitors such as cycloheximide, pactamycin and puromycin, which do not affect the eIF-2alpha phosphorylation. Also, the functional eIF-2B activity that is available in hemin-supplemented lysates is not affected by phosphatase inhibitors such as okadaic acid and heat-stable inhibitor-2. However, the recovery of eIF-2B activity that is observed by the delayed addition of hemin to inhibited heme-deficient lysates is reduced by inhibitor-2 and high concentrations of okadaic acid. These findings suggest that a type 1 phosphatase is involved in the recovery of eIF-2B activity and protein synthesis upon delayed addition of hemin to heme-deficient lysates.
authors.affiliation: Department of Biochemistry, School of Life Sciences, University of Hyderabad, India.
authors.forename: S V
authors.initials: SV
authors.lastname: Babu
authors.forename: K V
authors.initials: KV
authors.lastname: Ramaiah
mesh_terms: D000818:Animals; D002474:Cell-Free System; D003513:Cycloheximide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004988:Ethers, Cyclic / Q000494:pharmacology*; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000097:blood; D006418:Heme / Q000494:pharmacology*; D007700:Kinetics; D019319:Okadaic Acid; D010142:Pactamycin / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000097:blood*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000097:blood; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D019892:eIF-2 Kinase
chemical_list: D004791:Enzyme Inhibitors; D004988:Ethers, Cyclic; D015852:Eukaryotic Initiation Factor-2; D006153:Guanosine Diphosphate; D019319:Okadaic Acid; D010142:Pactamycin; D006418:Heme; D003513:Cycloheximide; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase; D010749:Phosphoprotein Phosphatases; D019204:GTP-Binding Proteins