Regulation of eIF-2 α-subunit phosphorylation in reticulocyte lysate

Arup Chakraborty, Debabrata Saha, Avirup Bose, Madhumita Chatterjee, Naba K. Gupta

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Abstract

An eIF-2 associated 67-kDa protein (p67) protects eIF-2 α-subunit from eIF-2 kinase(s) catalyzed phosphorylation and promotes protein synthesis in the presence of active eIF-2 kinase(s). p67 is a glycoprotein and contains multiple O-linked GlcNAc moieties. We have now studied the roles of hemin, p67, and the glycosyl residues on p67 in the regulation of eIF-2 α-subunit phosphorylation in reticulocyte lysates. The results are as follows: (i) Both hemin and p67 inhibited HRI (heme-regulated protein synthesis inhibitor) and dsI (double-stranded RNA activated protein synthesis inhibitor) catalyzed phosphorylation of eIF-2 α-subunit in vitro. However, only hemin, and not p67, inhibited casein kinase catalyzed phosphorylation of eIF-2 β-subunit. (ii) Only p67, and not hemin, inhibited eIF-2 α-subunit phosphorylation by eIF-2 kinase(s) in reticulocyte lysate. Significant eIF-2 α-subunit phosphorylation was observed even in the presence of hemin when p67 in the reticulocyte lysate was removed by treatment with p67 antibodies, (iii) Reticulocyte lysate contains a p67-deglycosylase in latent form, and hemin prevents activation of this deglycosylase. In the absence of hemin, this p67-deglycosylase is activated. Once activated in the absence of hemin, the activated deglycosylase deglycosylates p67, even in the presence of hemin. This inactivates p67 and allows eIF-2 kinase to phosphorylate eIF-2 α-subunit and inhibit protein synthesis. Protein synthesis in reticulocyte lysate is thus regulated by two novel cascades of covalent modifications: protein deglycosylation leading to protein phosphorylation.

Original languageEnglish (US)
Pages (from-to)6700-6706
Number of pages7
JournalBiochemistry®
Publication statusPublished - 1994

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ASJC Scopus subject areas

  • Biochemistry

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Chakraborty, A., Saha, D., Bose, A., Chatterjee, M., & Gupta, N. K. (1994). Regulation of eIF-2 α-subunit phosphorylation in reticulocyte lysate. Biochemistry®, 6700-6706.