The binding between p67 and eukaryotic initiation factor 2 plays important roles in the protection of eIF2α from phosphorylation by kinases

Phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 is the major regulatory step in the initiation of protein synthesis in mammals. P67, a cellular glycoprotein, protects phosphorylation of eIF2 alpha( from kinases. Previously, we reported that the 136/2 mutant of p67 has higher levels of protection of eIF2 alpha phosphorylation (POEP) activity. In this study, we report that the 136/2 mutant and its double mutants containing second-site alanine substitutions at the five conserved amino acid residues (13251, D262, H331, E364, and E459) show increased POEP activity in serum-starved rat tumor hepatoma cells. Serum-restoration to those cells did not abolish their increased POEP activity except the D6/2+H331 A double mutant. The latter mutant shows slight inhibition of POEP activity during serum starvation and this inhibition increased significantly during serum restoration. KRC-7 cells constitutively expressing the 136/2 mutant showed slightly decreased levels of PKR phosphorylation and significantly low level of phosphorylation of ERKs 1 and 2. The D6/2 mutant also showed increased binding with eIF2 alpha and eIF2 gamma and almost similar binding with ERKs 1 and 2 as compared to wild type p67. Altogether, our data demonstrate that the increased binding of the 136/2 mutant with the subunits of eIF2 may be in part the cause for its high POEP activity. (c) 2006 Elsevier Inc. All rights reserved.