Protection of translation initiation factor eIF2 phosphorylation correlates with eIF2-associated glycoprotein p67 levels and requires the lysine-rich domain I of p67.
The rate of protein synthesis in mammals is largely regulated by phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2) that is modulated by the cellular glycoprotein, p67, due to its protection of eIF2alpha phosphorylation (POEP) activity. At the N-terminus of p67, there are three unique domains, and at the C-terminus there is a conserved amino acid sequence. To analyze the importance of these domains, C-terminal deletion mutants of rat p67 were expressed constitutively in KRC-7 cells. In these cells, the phosphorylation level of the alpha-subunit of eIF2 was determined, and it was found that expression of the 1-97 amino acid segment of rat p67 increases POEP activity in vivo, and induces the endogenous levels of p67. These cells also show increased growth rate, and efficient translation of chloramphenicol acetyltransferase and beta-galactosidase reporter genes. At the N-terminus of p67, there are two unique domains: a lysine-rich domain I with the sequence (36)KKKRRKKKK(44), and an acidic residue-rich domain with the sequence (77)EEKEKDDDDEDGDGD(91). Substitution of lysine-rich domain I with (36)NMKSGNKTQ(44) in rat recombinant p67 resulted in the inhibition of its POEP activity, and substitution of the acidic residue-rich domain with (77)QNIQKALEPEAGDGA(91), resulted in no inhibition of POEP activity in KRC-7 cells. Taken together, our data suggest that protection of translation initiation factor eIF2 phosphorylation correlates with eIF2-associated glycoprotein p67 levels and requires the lysine-rich domain I of p67.
Datta
R
R
Department of Chemistry, Kent State University, OH 44242, USA.
Choudhury
P
P
Bhattacharya
M
M
Soto Leon
F
F
Zhou
Y
Y
Datta
B
B
D000595:Amino Acid Sequence; D000626:Aminopeptidases; D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D017124:Conserved Sequence / Q000235:genetics / Q000502:physiology; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D063208:Methionyl Aminopeptidases; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000096:biosynthesis / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured
D015852:Eukaryotic Initiation Factor-2; D006023:Glycoproteins; D008164:Luminescent Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; D000626:Aminopeptidases; C081311:METAP2 protein, human; C489894:Metap2 protein, mouse; C489895:Metap2 protein, rat; D063208:Methionyl Aminopeptidases