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Type 1 phosphatase inhibitors reduce the restoration of guanine nucleotide exchange activity of eukaryotic initiation factor 2B inhibited reticulocyte lysates rescued by hemin.

Authors
Type
Published Article
Journal
Archives of biochemistry and biophysics
Publication Date
Volume
327
Issue
2
Pages
201–208
Identifiers
PMID: 8619603
Source
Medline
License
Unknown

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.

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