title: Negative regulation of the protection of eIF2alpha phosphorylation activity by a unique acidic domain present at the N-terminus of p67.
abstract: Eukaryotic initiation factor 2 (eIF2)-associated glycoprotein, p67, has protection of eIF2alpha phosphorylation (POEP) activity, and this activity requires lysine-rich domains I and II of p67. Another unique acidic residue-rich domain is also present at the N-terminus of p67. In this study we analyzed the role of this acidic residue-rich domain in POEP activity. Our data revealed that constitutive expression of a mutant form of p67 (D6/2) in mammalian cells resulted in increased POEP activity, and this activity was partially inhibited when second-site alanine substitutions at the conserved amino acids D251, D262, E364, and E459 were introduced in the D6/2 mutant. In contrast, a similar mutation at the conserved H331 position did not show any effect on POEP activity. Individual alanine substitutions at the above conserved amino acids in wild-type p67 did not show any significant effect on POEP activity except the E459 position where alanine substitution caused approximately 50% increase in POEP activity as compared to the wild type. Although, the levels of endogenous p67 and p67-deglycosylase did not correlate with the POEP activity, we found that the D6/2 mutant of p67 was glycosylated at a higher level in mammalian cells as compared to wild-type p67. The increased POEP activity of the D6/2 mutant also correlated with the higher rate of overall protein synthesis in mammalian cells constitutively expressing this mutant form of p67. Taken together, these data suggest that the acidic residue-rich domain present at the N-terminus of p67 may have a negative role in POEP activity.
authors.affiliation: Department of Chemistry, Kent State University, Kent, OH 44242, USA.
authors.forename: Rekha
authors.initials: R
authors.lastname: Datta
authors.forename: Ravinder
authors.initials: R
authors.lastname: Tammali
authors.forename: Bansidhar
authors.initials: B
authors.lastname: Datta
mesh_terms: D000409:Alanine; D019943:Amino Acid Substitution; D000626:Aminopeptidases; D000818:Animals; D001483:Base Sequence; D017124:Conserved Sequence; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D006023:Glycoproteins / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D006801:Humans; D063208:Methionyl Aminopeptidases; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation / Q000187:drug effects; D014176:Protein Biosynthesis / Q000187:drug effects; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D015852:Eukaryotic Initiation Factor-2; D006023:Glycoproteins; D011993:Recombinant Fusion Proteins; D000626:Aminopeptidases; C081311:METAP2 protein, human; C489894:Metap2 protein, mouse; C489895:Metap2 protein, rat; D063208:Methionyl Aminopeptidases; D000409:Alanine