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.     

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.     

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.     

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.     

MAPs and POEP of the roads from prokaryotic to eukaryotic kingdoms

Methionine aminopeptidases (MAPs) play important roles in protein processing. MAPs from various organisms, for example E. coli, S. typhimurium, P. furiosus, Saccharomyces cerevisiae, and porcine have been purified to homogeneity and their MAP activities have been tested in vitro and in vivo. The DNA sequence analyses of MAP genes from the above organisms reveal sequence homologies with other prokaryotic MAPs as well as with various eukaryotic homologues of rat p67. The cellular glycoprotein, p67 protects the alpha-subunit of eukaryotic initiation factor 2 (eIF2) from phosphorylation by its kinases. We call this POEP (protection of eIF2alpha phosphorylation) activity of p67. The POEP activity of p67 is observed in different stress-related situations such as during heme-deficiency of reticulocytes, serum starvation and heat-shock of mammalian cells, vaccinia virus infection of mammalian cells, baculovirus infection of insect cells, mitosis, apoptosis, and possibly during normal cell growth. The POEP activity of p67 is regulated by an enzyme, called p67-deglycosylase (p67-DG). When active, p67-DG inactivates p67 by removing its carbohydrate moieties. Remarkable amino acid sequence similarities at the C-terminus of rat p67 with its eukaryotic and prokaryotic homologues which have MAP activities, raise several important questions: i) does rat p67 have MAP activity?; and ii) if it does have MAP activity, how the two activities (POEP and MAP) of p67 are used by mammalian cells during their growth and differentiation. In this review, discussions have been made to evaluate both POEP and MAP activities of p67 and their possible involvement during normal growth and cancerous growth of mammalian cells.     

Inhibition of host and viral translation during vesicular stomatitis virus infection. eIF2 is responsible for the inhibition of viral but not host translation

In cells that allow replication of vesicular stomatitis virus (VSV), there are two phases of translation inhibition: an early block of host translation and a later inhibition of viral translation. We investigated the phosphorylation of the alpha subunit of the eIF2 complex during these two phases of viral infection. In VSV-infected cells, the accumulation of phosphorylated (inactivated) eIF2alpha did not begin until well after host protein synthesis was inhibited, suggesting that it only plays a role in blocking viral translation later after infection. Consistent with this, cells expressing an unphosphorylatable eIF2alpha showed prolonged viral protein synthesis without an effect on host protein synthesis inhibition. Induction of eIF2alpha phosphorylation at early times of viral infection by treatment with thapsigargin showed that virus and host translation are similarly inhibited, demonstrating that viral and host messages are similarly sensitive to eIF2alpha phosphorylation. A recombinant virus that expresses a mutant matrix protein and is defective in the inhibition of host and virus protein synthesis showed an altered phosphorylation of eIF2alpha, demonstrating an involvement of viral protein function in inducing this antiviral response. This analysis of eIF2alpha phosphorylation, coupled with earlier findings that the eIF4F complex is modified earlier during VSV infection, supports a temporal/kinetic model of translation control, where at times soon after infection, changes in the eIF4F complex result in the inhibition of host protein synthesis; at later times, inactivation of the eIF2 complex blocks VSV protein synthesis.     

In vivo regulation of the dsRNA-dependent protein kinase PKR by the cellular glycoprotein p67

Regulation of eIF2alpha phosphorylation is critical to the maintenance of cellular homeostasis, and eIF2alpha kinases are subject to complex and multidimensional controls. A cellular 67 kDa glycoprotein (p67) has been proposed to have an important role in regulating the activity of eIF2alpha kinases including the interferon-induced, dsRNA-stimulated protein kinase PKR. To dissect p67-PKR interactions and evaluate their significance in vivo, we have used a vaccinia virus (VV) expression system that successfully mimics PKR control pathways. Recombinant VV were constructed that constitutively express p67 and inducibly express PKR in BSC-40 cells. Stable expression of p67 reduced the PKR-mediated antiviral response and apoptosis. These effects correlated with decreased eIF2alpha phosphorylation, with rescue of PKR-mediated inhibition of protein synthesis, and with partial inhibition of PKR-triggered activation of NF-kappaB. The direct interaction between PKR and p67 was suggested by in vivo and in vitro analyses. These data demonstrate that in vivo p67 is an important modulator of PKR-mediated signal transduction pathways and may provide a useful tool to dissect the relative contributions of PKR to cell growth and stress response.     

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.     

Translation initiation factor modifications and the regulation of protein synthesis in apoptotic cells

The rate of protein synthesis is rapidly down-regulated in mammalian cells following the induction of apoptosis. Inhibition occurs at the level of polypeptide chain initiation and is accompanied by the phosphorylation of the alpha subunit of initiation factor eIF2 and the caspase-dependent cleavage of initiation factors eIF4G, eIF4B, eIF2alpha and the p35 subunit of eIF3. Proteolytic cleavage of these proteins yields characteristic products which may exert regulatory effects on the translational machinery. Inhibition of caspase activity protects protein synthesis from long-term inhibition in cells treated with some, but not all, inducers of apoptosis. This review describes the initiation factor modifications and the possible signalling pathways by which translation may be regulated during apoptosis. We discuss the significance of the initiation factor cleavages and other changes for protein synthesis, and the implications of these events for our understanding of the cellular changes associated with apoptosis.     

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.     

Caerulein-induced acute pancreatitis inhibits protein synthesis through effects on eIF2B and eIF4F

Acute pancreatitis (AP) has been shown in some studies to inhibit total protein synthesis in the pancreas, whereas in other studies, protein synthesis was not affected. Previous in vitro work has shown that high concentrations of cholecystokinin both inhibit protein synthesis and inhibit the activity of the guanine nucleotide exchange factor eukaryotic initiation factor (eIF)2B by increasing the phosphorylation of eIF2alpha. We therefore evaluated in C57BL/6 mice the effects of caerulein-induced AP on pancreatic protein synthesis, eIF2B activity and other protein translation regulatory mechanisms. Repetitive hourly injections of caerulein were administered at 50 microg/kg ip. Pancreatic protein synthesis was reduced 10 min after the initial caerulein administration and was further inhibited after three and five hourly injections. Caerulein inhibited the two major regulatory points of translation initiation: the activity of the guanine nucleotide exchange factor eIF2B (with an increase of eIF2alpha phosphorylation) and the formation of the eIF4F complex due, in part, to degradation of eIF4G. This inhibition was not accounted for by changes in the upstream stimulatory pathway, because caerulein activated Akt as well as phosphorylating the downstream effectors of mTOR, 4E-BP1, and ribosomal protein S6. Caerulein also decreased the phosphorylation of the eukaryotic elongation factor 2, implying that this translation factor was not inhibited in AP. Thus the inhibition of pancreatic protein synthesis in this model of AP most likely results from the inhibition of translation initiation as a result of increased eIF2alpha phosphorylation, reduction of eIF2B activity, and the inhibition of eIF4F complex formation.     

Regulation of protein synthesis by hypoxia via activation of the endoplasmic reticulum kinase PERK and phosphorylation of the translation initiation factor eIF2alpha

Hypoxia profoundly influences tumor development and response to therapy. While progress has been made in identifying individual gene products whose synthesis is altered under hypoxia, little is known about the mechanism by which hypoxia induces a global downregulation of protein synthesis. A critical step in the regulation of protein synthesis in response to stress is the phosphorylation of translation initiation factor eIF2alpha on Ser51, which leads to inhibition of new protein synthesis. Here we report that exposure of human diploid fibroblasts and transformed cells to hypoxia led to phosphorylation of eIF2alpha, a modification that was readily reversed upon reoxygenation. Expression of a transdominant, nonphosphorylatable mutant allele of eIF2alpha attenuated the repression of protein synthesis under hypoxia. The endoplasmic reticulum (ER)-resident eIF2alpha kinase PERK was hyperphosphorylated upon hypoxic stress, and overexpression of wild-type PERK increased the levels of hypoxia-induced phosphorylation of eIF2alpha. Cells stably expressing a dominant-negative PERK allele and mouse embryonic fibroblasts with a homozygous deletion of PERK exhibited attenuated phosphorylation of eIF2alpha and reduced inhibition of protein synthesis in response to hypoxia. PERK(-/-) mouse embryo fibroblasts failed to phosphorylate eIF2alpha and exhibited lower survival after prolonged exposure to hypoxia than did wild-type fibroblasts. These results indicate that adaptation of cells to hypoxic stress requires activation of PERK and phosphorylation of eIF2alpha and suggest that the mechanism of hypoxia-induced translational attenuation may be linked to ER stress and the unfolded-protein response.     

Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) induces initiation factor 2 alpha phosphorylation and translation inhibition in PC12 cells

We have investigated the effect of the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) on protein synthesis rate and initiation factor 2 (eIF2) phosphorylation in PC12 cells differentiated with nerve growth factor. FCCP treatment induced a very rapid 2-fold increase in intracellular Ca(2+) concentration that was accompanied by a strong protein synthesis rate inhibition (68%). The translation inhibition correlated with an increased phosphorylation of the alpha subunit of eIF2 (eIF2 alpha) (25% vs. 7%, for FCCP-treated and control cells, respectively) and a 1.7-fold increase in the double-stranded RNA-dependent protein kinase activity. No changes in the PKR endoplasmic reticulum-related kinase or eIF2 alpha phosphatase were found. Translational regulation may play a significant role in the process triggered by mitochondrial calcium mobilization.     

Acute and chronic ethanol consumption differentially impact pathways limiting hepatic protein synthesis

This review identifies the various pathways responsible for modulating hepatic protein synthesis following acute and chronic alcohol intoxication and describes the mechanism(s) responsible for these changes. Alcohol intoxication induces a defect in global protein synthetic rates that is localized to impaired translation of mRNA at the level of peptide-chain initiation. Translation initiation is regulated at two steps: formation of the 43S preinitiation complex [controlled by eukaryotic initiation factors 2 (eIF2) and 2B (eIF2B)] and the binding of mRNA to the 40S ribosome (controlled by the eIF4F complex). To date, alcohol-induced alterations in eIF2 and eIF2B content and activity are best investigated. Ethanol decreases eIF2B activity when ingested either acutely or chronically. The reduced eIF2B activity most likely is a consequence of twofold increased phosphorylation of the alpha-subunit of eIF2 on Ser(51) following acute intoxication. The increase in eIF2alpha phosphorylation after chronic alcohol consumption is the same as that induced by acute ethanol intoxication, and protein synthesis is not further reduced by long-term alcohol ingestion despite additional reduced expression of initiation factors and elongation factors. eIF2alpha phosphorylation alone appears sufficient to maximally inhibit hepatic protein synthesis. Indeed, pretreatment with Salubrinal, an inhibitor of eIF2alpha(P) phosphatase, before ethanol treatment does not further inhibit protein synthesis or increase eIF2alpha phosphorylation, suggesting that acute ethanol intoxication causes maximal eIF2alpha phosphorylation elevation and hepatic protein synthesis inhibition. Ethanol-induced inhibition of hepatic protein synthesis is not rapidly reversed by cessation of ethanol consumption. In conclusion, sustained eIF2alpha phosphorylation is a hallmark of excessive alcohol intake leading to inhibition of protein synthesis. Enhanced phosphorylation of eIF2alpha represents a unique response of liver to alcohol intoxication, because the ethanol-induced elevation of eIF2alpha(P) is not observed in skeletal muscle or heart.     

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.     

Nitric oxide mediates NMDA-induced persistent inhibition of protein synthesis through dephosphorylation of eukaryotic initiation factor 4E-binding protein 1 and eukaryotic initiation factor 4G proteolysis

Cerebral ischaemia causes long-lasting protein synthesis inhibition that is believed to contribute to brain damage. Energy depletion promotes translation inhibition during ischaemia, and the phosphorylation of eIF (eukaryotic initiation factor) 2alpha is involved in the translation inhibition induced by early ischaemia/reperfusion. However, the molecular mechanisms underlying prolonged translation down-regulation remain elusive. NMDA (N-methyl-D-aspartate) excitotoxicity is also involved in ischaemic damage, as exposure to NMDA impairs translation and promotes the synthesis of NO (nitric oxide), which can also inhibit translation. In the present study, we investigated whether NO was involved in NMDA-induced protein synthesis inhibition in neurons and studied the underlying molecular mechanisms. NMDA and the NO donor DEA/NO (diethylamine-nitric oxide sodium complex) both inhibited protein synthesis and this effect persisted after a 30 min exposure. Treatments with NMDA or NO promoted calpain-dependent eIF4G cleavage and 4E-BP1 (eIF4E-binding protein 1) dephosphorylation and also abolished the formation of eIF4E-eIF4G complexes; however, they did not induce eIF2alpha phosphorylation. Although NOS (NO synthase) inhibitors did not prevent protein synthesis inhibition during 30 min of NMDA exposure, they did abrogate the persistent inhibition of translation observed after NMDA removal. NOS inhibitors also prevented NMDA-induced eIF4G degradation, 4E-BP1 dephosphorylation, decreased eIF4E-eIF4G-binding and cell death. Although the calpain inhibitor calpeptin blocked NMDA-induced eIF4G degradation, it did not prevent 4E-BP1 dephosphorylation, which precludes eIF4E availability, and thus translation inhibition was maintained. The present study suggests that eIF4G integrity and hyperphosphorylated 4E-BP1 are needed to ensure appropriate translation in neurons. In conclusion, our data show that NO mediates NMDA-induced persistent translation inhibition and suggest that deficient eIF4F activity contributes to this process.     

A potential role for extracellular signal-regulated kinases in prostaglandin F2alpha-induced protein synthesis in smooth muscle cells

To understand the mechanisms of prostaglandin F2alpha (PGF2alpha)-induced protein synthesis in vascular smooth muscle cells (VSMC), we have studied its effect on two major signal transduction pathways: mitogen-activated protein kinases and phosphatidylinositol 3-kinase (PI3-kinase) and their downstream targets ribosomal protein S6 kinase (p70(S6k)) and eukaryotic initiation factor eIF4E and its regulator 4E-BP1. PGF2alpha induced the activities of extracellular signal-regulated kinase 2 (ERK2) and Jun N-terminal kinase 1 (JNK1) groups of mitogen-activated protein kinases, PI3-kinase, and p70(S6k) in a time-dependent manner in growth-arrested VSMC. PGF2alpha also induced eIF4E and 4E-BP1 phosphorylation, global protein synthesis, and basic fibroblast growth factor-2 (bFGF-2) expression in VSMC. Whereas inhibition of PI3-kinase by wortmannin completely blocked the p70(S6k) activation, it only partially decreased the ERK2 activity, and had no significant effect on global protein synthesis and bFGF-2 expression induced by PGF2alpha. Rapamycin, a potent inhibitor of p70(S6k), also failed to prevent PGF2alpha-induced global protein synthesis and bFGF-2 expression, although it partially decreased ERK2 activity. In contrast, inhibition of ERK2 activity by PD 098059 led to a significant loss of PGF2alpha-induced eIF4E and 4E-BP1 phosphorylation, global protein synthesis, and bFGF-2 expression. PGF2alpha-induced phosphorylation of eIF4E and 4E-BP1 was also found to be sensitive to inhibition by both wortmannin and rapamycin. These findings demonstrate that 1) PI3-kinase-dependent and independent mechanisms appear to be involved in PGF2alpha-induced activation of ERK2; 2) PGF2alpha-induced eIF4E and 4E-BP1 phosphorylation appear to be mediated by both ERK-dependent and PI3-kinase-dependent rapamycin-sensitive mechanisms; and 3) ERK-dependent eIF4E phosphorylation but not PI3-kinase-dependent p70(S6k) activation correlates with PGF2alpha-induced global protein synthesis and bFGF-2 expression in VSMC.