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

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Abstract

Phosphorylation of the α-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 eIF2α from kinases. Previously, we reported that the D6/2 mutant of p67 has higher levels of protection of eIF2α phosphorylation (POEP) activity. In this study, we report that the D6/2 mutant and its double mutants containing second-site alanine substitutions at the five conserved amino acid residues (D251, 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+H331A 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 D6/2 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 eIF2α and eIF2γ 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 D6/2 mutant with the subunits of eIF2 may be in part the cause for its high POEP activity.

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Materials and methods

All chemicals used in this study were obtained from Sigma Chemicals (St. Louis, MO), Merck (Darmstadt, Germany), ICN Biomedicals, Inc. (Aurora, OH), Fisher Chemicals (Fairlawn, NJ), or Gibco-BRL (Rockville, MD). All enzymes used in this study were purchased from New England Biolabs (Beverly, MA). [35S]Methionine was purchased from Amersham Corporation.

Results and discussion

Previously, we reported that the D6/2 mutant has increased protection of eIF2α phosphorylation activity during normal growth conditions [8]. We measured its effect and the effects of five double mutants D6/2 + D251A, D6/2 + D262A, D6/2 + H331A, D6/2 + E364A, and D6/2 + E459A on the levels of eIF2α(P) and total eIF2α in serum-starved cells. The results were compared with the control cells expressing wild type p67 and EGFP alone (Fig. 1A and B). Results were normalized with the total eIF2α. Wild type p67

Acknowledgment

Kent State University Research Council supports this work.

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