Autoproteolysis of rat p67 generates several peptide fragments: the N-terminal fragment, p26, is required for the protection of eIF2alpha from phosphorylation.

Eukaryotic initiation factor 2-associated glycoprotein, p67, plays important roles in the regulation of eIF2alpha phosphorylation and thus maintains cell growth and proliferation. The p67 sequence can be divided into two segments, the N-terminal segment of amino acids 1-107 (p26) and the downstream segment of amino acids 108-480 (p52). Comparison of the amino acid sequences of p67 from lower to higher organisms suggests that there is a progressive addition of several unique domains at the N-terminus of p67, and these unique domains, which are present in p26, play important roles in the modulation of eIF2alpha phosphorylation in mammalian cells. To test the hypothesis that the p26 segment is generated from p67 due to its autoproteolysis and whether p26 is required for the protection of eIF2alpha from phosphorylation, we have analyzed the time-dependent cleavage of functionally active rat recombinant p67 purified from either baculovirus-infected insect cells or transiently transfected mammalian cells. We noticed a regulated cleavage of p67 that generates several peptides along with the most stable p26 and p52 fragments. The p52 fragment has a low level of autoproteolysis activity that possibly increases the autoproteolysis of full-length p67. This activity could not be inhibited by a serine protease inhibitor, PMSF, but could be inhibited by a cocktail of protease inhibitors that includes bestatin, leupeptin, E64, AEBSF, and aprotinin. To provide evidence that the fragmentation of p67 is not due to the presence of any contaminant protease(s), we fractionated purified rat p67 with molecular sieve, anion exchange, and cation exchange chromatographic steps performed in the presence of different K+ ion concentrations. Our data show that the extent of cleavage of p67 into different fragments is higher in the presence of 0.75 M K+ ion and in samples stored at -80 degrees C. Under parallel conditions, p67's mutants, D251A and D262A, exhibited very little to no cleavage, whereas the H231E mutant exhibited extensive cleavage that generated a large amount of p26 fragment. The p26 fragment exhibited protection of eIF2alpha phosphorylation both in vivo and in vitro. Altogether, our data provide evidence that rat p67 has autoproteolytic activity that generates p26, which is required to block eIF2alpha from phosphorylation.

The N-terminal lysine residue-rich domain II and the 340-430 amino acid segment of eukaryotic initiation factor 2-associated glycoprotein p67 are the binding sites for the gamma-subunit of eIF2.

Eukaryotic initiation factor 2 (eIF2)-associated glycoprotein, p67, plays an important role in protecting eIF2alpha from phosphorylation by eIF2alpha-specific kinases. To understand the molecular details of interaction between p67 and the subunits of eIF2, we applied several biochemical and mutational analyses to identify interacting domains within p67 and eIF2gamma. These studies were combined with functional in vivo and in vitro assays to address the importance of the interactions between p67 and eIF2gamma in eIF2alpha phosphorylation. Studies from yeast two-hybrid assays show that p67 interacts strongly with eIF2gamma, relatively weakly with eIF2alpha, and no interaction with eIF2beta. Further mutational analyses provided evidence that the N-terminal lysine-rich domain II and the 340-430 amino acid segment of p67 interact strongly with the C-terminal 409-472 amino acid segment of eIF2gamma. GST pull-down assays show that the interaction between p67 and eIF2gamma is direct. From co-immunoprecipitation studies, we find that the interaction between p67 and eIF2gamma could not only be detected in mammalian cells growing in growth medium, it could also be detected in transiently transfected cells with expression plasmids encoding p67 and eIF2gamma. However, this interaction could not be detected in p67 mutants lacking lysine-rich domain II and the 340-430 amino acid segment. We also find a very good correlation between p67 binding to eIF2gamma and the protection of eIF2alpha from phosphorylation. Altogether, our data provide genetic evidence for the interaction between p67 and eIF2gamma and that this interaction modulates the phosphorylation of eIF2alpha.

The binding between p67 and eukaryotic initiation factor 2 plays important roles in the protection of eIF2alpha 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 eIF2alpha from kinases. Previously, we reported that the D6/2 mutant of p67 has higher levels of protection of eIF2alpha 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 eIF2alpha and eIF2gamma 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.

The stability of eukaryotic initiation factor 2-associated glycoprotein, p67, increases during skeletal muscle differentiation and that inhibits the phosphorylation of extracellular signal-regulated kinases 1 and 2.

Eukaryotic initiation factor 2-associated glycoprotein, p67, protects eIF2 from phosphorylation by its kinases. To understand the roles of p67 during skeletal muscle differentiation of mouse C2C12 myoblasts, we measured the level of p67 during myotube formation. We noticed that the level of p67 increases during myoblast differentiation and this increased level is controlled at the translational stage. The stability of p67 in the myotubes is due to its low turnover rate. The phosphorylation of the extracellular signal-regulated kinases (ERKs 1 and 2) is high in growth-factor-mediated cycling of C2C12 myoblasts and this phosphorylation decreases at 96 h when these myoblasts are grown in differentiation medium. At this time of differentiation, the level of p67 is higher compared to 0 h of differentiation. p67 binds to ERK2 and inhibits its activity in vitro. Taken together, these results suggest that the stability of p67 increases during myotube formation while inhibiting the phosphorylation of ERKs 1 and 2.

Treatment of cells with the angiogenic inhibitor fumagillin results in increased stability of eukaryotic initiation factor 2-associated glycoprotein, p67, and reduced phosphorylation of extracellular signal-regulated kinases.

Fumagillin, an angiogenic inhibitor, binds to methionine aminopeptidase 2, which is the same as eukaryotic initiation factor 2-associated glycoprotein, p67. p67 protects eIF2alpha from phosphorylation by its kinases. To understand the importance of fumagillin binding to p67, we measured the level of p67 in mouse C2C12 myoblasts treated with fumagillin. We show that fumagillin increases the stability of p67 by decreasing its turnover rate. The increased levels of p67 result in inhibition of phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERKs 1 and 2). p67 binds to these ERKs, and the 108-480 amino acid segment is sufficient for this binding. p67's affinity to ERKs 1 and 2 also increases in fumagillin-treated myoblasts while its affinity for eIF2alpha remains unchanged. A mutant at the conserved amino acid residue D251A increases the phosphorylation of ERKs 1 and 2 without affecting the binding to p67, thus indicating the importance of this residue in the regulation of the phosphorylation of these ERKs. These results suggest that fumagillin increases the stability of p67 and its affinity to ERKs 1 and 2 and causes the inhibition of the phosphorylation of ERKs 1 and 2.

Eukaryotic initiation factor 2-associated glycoprotein, p67, shows differential effects on the activity of certain kinases during serum-starved conditions.

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 eIF2alpha from kinases. P67 has five conserved amino acid residues at the D251, D262, H331, E364, and E459 positions. To determine the roles of these conserved amino acid residues in eIF2alpha phosphorylation during serum-starved conditions, we constitutively expressed D251A, D262A, H331A, E364A, and E459A mutants in rat tumor hepatoma cells. We find that the point mutants D251A, H331A, and E364A lower the levels of eIF2alpha phosphorylation. These low levels of phosphorylation decrease when serum-starved cells are grown in medium containing serum. To understand the mechanism of action of the p67 mutants in eIF2alpha phosphorylation during serum-starvation, we performed detailed biochemical analyses with the D251A mutant. We find that neither the O-GlcNAc modification on the D251A mutant nor the binding of D251A mutant with eIF2gamma has significant effects on eIF2alpha phosphorylation during serum-starved conditions. However, the D251A mutant inhibits p67's activity to suppress the activity of ERK1/2. Our data suggest that both p67 and the D251A mutant bind to ERK1, thus strengthening the idea that p67 regulates the activity of ERK1. During serum-starvation conditions, both PKR and PERK are phosphorylated and the D251A mutant shows increased stability of PERK as well as a slight decrease in its activity. Altogether, our data provide evidence to suggest that p67 modulates the expression and activity of certain eIF2alpha-specific kinases.

A glycosylation site, 60SGTS63, of p67 is required for its ability to regulate the phosphorylation and activity of eukaryotic initiation factor 2alpha.

Eukaryotic initiation factor 2- (eIF2-) associated glycoprotein p67 blocks eIF2alpha phosphorylation by kinases, and its N-terminal 1-97 amino acid segment can induce efficient translation. To investigate whether glycosylation at the serine/threonine clusters at this region is important in protein synthesis, we selected (27)TSST(30) and (60)SGTS(63) clusters for further analysis. By site-directed mutagenesis, (27)TSST(30) and (60)SGTS(63) clusters were substituted with (27)AAGA(30) and (60)AGAA(63) amino acid residues in full-length p67, and their EGFP fusions were constitutively expressed in rat tumor hepatoma cells (KRC-7). The (60)AGAA(63) mutant blocked eIF2alpha phosphorylation less than either wild-type p67 or the (27)AAGA(30) mutant. The (60)AGAA(63) mutant also showed a low level of protein synthesis rate, a lower level of glycosylation, increased turnover rate, and weaker binding to eIF2alpha. These results suggest that glycosylation within the (60)SGTS(63) sequence of p67 plays an important role in its stability and thus its regulation of protein synthesis by modulating the phosphorylation of the alpha-subunit of eIF2.

Mutation at the acidic residue-rich domain of eukaryotic initiation factor 2 (eIF2alpha)-associated glycoprotein p67 increases the protection of eIF2alpha phosphorylation during heat shock.

Eukaryotic initiation factor 2 (eIF2)-associated glycoprotein p67 protects eIF2alpha phosphorylation from kinases. The N-terminal lysine-rich domains increase this activity and the acidic residue-rich domain inhibits it. Conserved amino acid residues D251, D262, E364, and E459 are involved in this inhibition. During heat shock, the overall protein synthesis rate decreases due to the increased levels of eIF2alpha phosphorylation. In this study, we examined whether the above inhibition is also found during heat shock. Indeed, the acidic residue-rich domain mutant (D6/2) showed a decreased level of eIF2alpha phosphorylation, and its second-site alanine substitutions at D251, D262, and E459 reversed this effect, whereas second-site alanine substitution at H331 and E364 residues further augmented it. A high-molecular-weight phosphoprotein and at least two faster-migrating phosphoproteins were detected by the monospecific polyclonal antibody against eIF2alpha(P) form in rat tumor hepatoma cells constitutively expressing the double mutant D6/2+D251A. Although the levels of p67 mutants were unaffected during heat shock, those of p67 and p67-deactivating enzyme varied. Furthermore, the overall rate of protein synthesis correlated with the level of eIF2alpha phosphorylation. Taken together, these results suggest that the lysine-rich domains and conserved amino acid residues of p67 are involved in the regulation of eIF2alpha phosphorylation during heat shock.

Negative regulation of the protection of eIF2alpha phosphorylation activity by a unique acidic domain present at the N-terminus of p67.

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.