Striking multiplicity of eIF4E-BP1 phosphorylated isoforms identified by 2D gel electrophoresis regulation by heat shock.

Eukaryotic initiation factor eIF4E-binding protein 1 (eIF4E-BP1), or PHAS-I, is multiply phosphorylated by insulin-stimulated protein kinase(s). Estimates for the number of phosphorylation sites range from two to greater than eight. IEF/SDS/PAGE can precisely differentiate protein isoforms based on their differences in charge (phosphorylation) and molecular mass. In this study, the diversity of eIF4E-BP1 isoforms was determined using IEF/SDS/PAGE/immunoblotting of unfractionated cell lysates. To investigate the molecular regulation of phosphorylation, alterations in eIF4E-BP1 in response to heat shock in HeLa cells were determined. In exponentially growing cells, 8-10 prominent eIF4E-BP1 isoforms were detected. Following heat shock, a rapid, temperature-dependent dephosphorylation of eIF4E-BP1 occurs roughly concurrent with protein synthesis inhibition; during recovery from heat shock rephosphorylation of eIF4E-BP1 parallels restoration of protein synthesis. However, eIF4E-BP1 and eIF4E kinases remain highly active during heat shock, as okadaic acid treatment restores phosphorylation of both factors in heat shocked cells. eIF4E-BP1 dephosphorylation is associated with eIF4E dissociation from large molecular mass complexes and increased binding to eIF4E-BP1. The amount of eIF4E-BP1 converted to the dephosphorylated state is sufficient to titrate all the eIF4E present. eIF4E-BP1 phosphorylation changes regulated by heat shock also occur in Drosophila. Of the 10 isoforms of eIF4E-BP1 resolved by IEF/SDS/PAGE, at least seven are labelled with [32P] and all 10 are recognized by (eIF4E-BP1)-specific antibodies. These results identify a complex set of eIF4E-BP1 phosphorylation isoforms; changes in the expression of these isoforms in response to stresses such as heat shock may contribute to translation repression.     

Translation initiation factors are differentially regulated in cereals during development and following heat shock

The level of protein synthetic activity varies greatly in plants during development or following many environmental stresses. In order to determine whether these global changes in protein synthesis involve changes in the expression of the translational machinery itself, the expression patterns of the initiation factors (eIFs) during cereal seed development, germination, and in leaves following a heat shock were investigated. The expression patterns of initiation factors during seed development fell into four classes: those whose levels were high during early to mid-development but decreased during late seed development (eIF4 A, eIFiso4E, eIFiso4G, and most eIF3 subunits); those whose levels increased from early to mid-development followed by a decrease during late seed development (eIF4B, eIF4E, eIF2α, eIF2β, and PABP); those whose levels steadily increased throughout seed development (eIF4G); and those whose levels remained constant (the p34 subunit of eIF3). In contrast to these observations, the expression patterns of the factors appeared to be coordinately regulated during early germination although differences were observed at later stages. Following a heat shock, the changes in initiation factor expression in wheat leaves fell into two classes, those whose level increased (eIF4G, eIFiso4G, eIF3, and PABP) and those whose level remained unchanged (eIF4 A, eIF4B, eIF4E, eIFiso4E). These observations reveal the complexity of the expression patterns of the initiation factors during seed development, germination, and following thermal stress and may have mechanistic importance for the selective translation of certain mRNAs under these conditions.     

Amino acids and insulin are both required to regulate assembly of the eIF4E. eIF4G complex in rat skeletal muscle

The respective roles of insulin and amino acids in regulation of skeletal muscle protein synthesis and degradation after feeding were examined in rats fasted for 17 h and refed over 1 h with either a 25 or a 0% amino acid/protein meal. In each nutritional condition, postprandial insulin secretion was either maintained (control groups: C(25) and C(0)) or blocked with diazoxide injections (diazoxide groups: DZ(25) and DZ(0)). Muscle protein metabolism was examined in vitro in epitrochlearis muscles. Only feeding the 25% amino acid/protein meal in the presence of increased plasma insulin concentration (C(25) group) stimulated protein synthesis and inhibited proteolysis in skeletal muscle compared with the postabsorptive state. The stimulation of protein synthesis was associated with increased phosphorylation of eukaryotic initiation factor (eIF)4E binding protein-1 (4E-BP1), reduced binding of eIF4E to 4E-BP1, and increased assembly of the active eIF4E. eIF4G complex. The p70 S6 kinase (p70(S6k)) was also hyperphosphorylated in response to the 25% amino acid/protein meal. Acute postprandial insulin deficiency induced by diazoxide injections totally abolished these effects. Feeding the 0% amino acid/protein meal with or without postprandial insulin deficiency did not stimulate muscle protein synthesis, reduce proteolysis, or regulate initiation factors and p70(S6k) compared with fasted rats. Taken together, our results suggest that both insulin and amino acids are required to stimulate protein synthesis, inhibit protein degradation, and regulate the interactions between eIF4E and 4E-BP1 or eIF4G in response to feeding.     

Phosphorylation of eukaryotic initiation factor 4E markedly reduces its affinity for capped mRNA.

In eukaryotes, a key step in the initiation of translation is the binding of the eukaryotic initiation factor 4E (eIF4E) to the cap structure of the mRNA. Subsequent recruitment of several components, including the small ribosomal subunit, is thought to allow migration of initiation complexes and recognition of the initiation codon. Mitogens and cytokines stimulate the phosphorylation of eIF4E at Ser(209), but the functional consequences of this modification have remained a major unresolved question. Using fluorescence spectroscopy and surface plasmon resonance techniques, we show that phosphorylation of eIF4E markedly reduces its affinity for capped RNA, primarily due to an increased rate of dissociation. Variant eIF4E proteins harboring negatively charged acidic residues at position 209 also showed decreased binding to capped RNA. Furthermore, a basic residue at position 159 was shown to be essential for cap binding. Although eIF4E-binding protein 1 greatly stabilized binding of phosphorylated eIF4E to capped RNA, in the presence of eIF4E-binding protein 1 the phosphorylated form still dissociated faster compared with nonphopshorylated eIF4E. The implications of our findings for the mechanism of translation initiation are discussed.     

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.     

Phosphorylation of eukaryotic initiation factor (eIF) 4E is not required for de novo protein synthesis following recovery from hypertonic stress in human kidney cells

Previous work has suggested that increased phosphorylation of eukaryotic initiation factor (eIF) 4E at Ser-209 in the C-terminal loop of the protein often correlates with increased translation rates. However, the functional consequences of phosphorylation have remained contentious with our understanding of the role of eIF4E phosphorylation in translational control far from complete. To investigate the role for eIF4E phosphorylation in de novo translation, we studied the recovery of human kidney cells from hypertonic stress. Results show that hypertonic shock caused a rapid inhibition of protein synthesis and the disaggregation of polysomes. These changes were associated with the dephosphorylation of eIF4G, eIF4E, 4E-binding protein 1 (4E-BP1), and ribosomal protein S6. In addition, decreased levels of the eIF4F complex and increased association of 4E-BP1 with eIF4E were observed over a similar time course. The return of cells to isotonic medium rapidly promoted the phosphorylation of these initiation factors, increased levels of eIF4F complexes, promoted polysome assembly, and increased rates of translation. However, by using a cell-permeable, specific inhibitor of eIF4E kinase, Mnk1 (CGP57380), we show that de novo initiation of translation and eIF4F complex assembly during this recovery phase did not require eIF4E phosphorylation.     

Chaperone-dependent E3 ligase CHIP ubiquitinates and mediates proteasomal degradation of soluble guanylyl cyclase

The nitric oxide receptor soluble guanylyl cyclase (sGC) exists in multimeric protein complexes, including heat shock protein (HSP) 90 and endothelial nitric oxide synthase. Inhibition of HSP90 by geldanamycin causes proteasomal degradation of sGC protein. In this study, we have investigated whether COOH terminus of heat shock protein 70-interacting protein (CHIP), a co-chaperone molecule that is involved in protein folding but is also a chaperone-dependent ubiquitin E3 ligase, could play a role in the process of degradation of sGC. Transient overexpression of CHIP in COS-7 cells degraded heterologous sGC in a concentration-related manner; this downregulation of sGC was abrogated by the proteasome inhibitor MG-132. Transfection of tetratricopeptide repeats and U-box domain CHIP mutants attenuated sGC degradation, suggesting that both domains are indispensable for CHIP function. Results from immunoprecipitation and indirect immunofluorescent microscopy experiments demonstrated that CHIP is associated with sGC, HSP90, and HSP70 in COS-7 cells. Furthermore, CHIP increased the association of HSP70 with sGC. In in vitro ubiquitination assays using purified proteins and ubiquitin enzymes, E3 ligase CHIP directly ubiquitinated sGC; this ubiquitination was potentiated by geldanamycin in COS-7 cells, followed by proteasomal degradation. In rat aortic smooth muscle cells, endogenous sGC was also degraded by adenovirus-infected wild-type CHIP but not by the chaperone interaction-deficient K30A CHIP, whereas CHIP, but not K30A, attenuated sGC expression in, and nitric oxide donor-induced relaxation of, rat aortic rings, suggesting that CHIP plays a regulatory role under physiological conditions. This study reveals a new mechanism for the regulation of sGC, an important mediator of cellular and vascular function.     

Regulation of translation initiation by insulin and amino acids in skeletal muscle of neonatal pigs

Previous studies have shown that intravenous infusion of insulin and/or amino acids reproduces the feeding-induced stimulation of muscle protein synthesis in neonates and that insulin and amino acids act independently to produce this effect. The goal of the present study was to delineate the regulatory roles of insulin and amino acids on muscle protein synthesis in neonates by examining translational control mechanisms, specifically the eukaryotic translation initiation factors (eIFs), which enable coupling of initiator methionyl-tRNAi and mRNA to the 40S ribosomal subunit. Insulin secretion was blocked by somatostatin in fasted 7-day-old pigs (n = 8-12/group), insulin was infused to achieve plasma levels of approximately 0, 2, 6, and 30 microU/ml, and amino acids were clamped at fasting or fed levels or, at the high insulin dose, below fasting. Both insulin and amino acids increased the phosphorylation of ribosomal protein S6 kinase (S6K1) and the eIF4E-binding protein (4E-BP1), decreased the binding of 4E-BP1 to eIF4E, increased eIF4E binding to eIF4G, and increased fractional protein synthesis rates but did not affect eIF2B activity. In the absence of insulin, amino acids had no effect on these translation initiation factors but increased the protein synthesis rates. Raising insulin from below fasting to fasting levels generally did not alter translation initiation factor activity but raised protein synthesis rates. The phosphorylation of S6K1 and 4E-BP1 and the amount of 4E-BP1 bound to eIF4E and eIF4E bound to eIF4G were correlated with insulin level, amino acid level, and protein synthesis rate. Thus insulin and amino acids regulate muscle protein synthesis in skeletal muscle of neonates by modulating the availability of eIF4E for 48S ribosomal complex assembly, although other processes also must be involved.     

Tristetraprolin Recruits Eukaryotic Initiation Factor 4E2 To Repress Translation of AU-Rich Element-Containing mRNAs

Tristetraprolin (TTP) regulates the expression of AU-rich element-containing mRNAs through promoting the degradation and repressing the translation of target mRNA. While the mechanism for promoting target mRNA degradation has been extensively studied, the mechanism underlying translational repression is not well established. Here, we show that TTP recruits eukaryotic initiation factor 4E2 (eIF4E2) to repress target mRNA translation. TTP interacted with eIF4E2 but not with eIF4E. Overexpression of eIF4E2 enhanced TTP-mediated translational repression, and downregulation of endogenous eIF4E2 or overexpression of a truncation mutant of eIF4E2 impaired TTP-mediated translational repression. Overexpression of an eIF4E2 mutant that lost the cap-binding activity also impaired TTP''s activity, suggesting that the cap-binding activity of eIF4E2 is important in TTP-mediated translational repression. We further show that TTP promoted eIF4E2 binding to target mRNA. These results imply that TTP recruits eIF4E2 to compete with eIF4E to repress the translation of target mRNA. This notion is supported by the finding that downregulation of endogenous eIF4E2 increased the production of tumor necrosis factor alpha (TNF-α) protein without affecting the mRNA levels in THP-1 cells. Collectively, these results uncover a novel mechanism by which TTP represses target mRNA translation.     

IGF-I and insulin regulate eIF4F formation by different mechanisms in muscle and liver in the ovine fetus

The mechanisms by which insulin-like growth factor I (IGF-I) and insulin regulate eukaryotic initiation factor (eIF)4F formation were examined in the ovine fetus. Insulin infusion increased phosphorylation of eIF4E-binding protein (4E-BP1) in muscle and liver. IGF-I infusion did not alter 4E-BP1 phosphorylation in liver. In muscle, IGF-I increased 4E-BP1 phosphorylation by 27%; the percentage in the gamma-form in the IGF-I group was significantly lower than that in the insulin group. In liver, only IGF-I increased eIF4G. Both IGF-I and insulin increased eIF4E. eIF4G binding in muscle, but only insulin decreased the amount of 4E-BP1 associated with eIF4E. In liver, only IGF-I increased eIF4E. eIF4G binding. Insulin increased the phosphorylation of p70 S6 kinase (p70(S6k)) in both muscle and liver and protein kinase B (PKB/Akt) in muscle, two indicative signal proteins in the phosphatidylinositol (PI) 3-kinase pathway. IGF-I increased PKB/Akt phosphorylation in muscle but had no effect on p70(S6k) phosphorylation in muscle or liver. We conclude that insulin and IGF-I modulate eIF4F formation; however, the two hormones have different regulatory mechanisms. Insulin increases phosphorylation of 4E-BP1 and eIF4E. eIF4G binding in muscle, whereas IGF-I regulates eIF4F formation by increasing total eIF4G. Insulin, but not IGF-I, decreased 4E-BP1 content associated with eIF4E. Insulin regulates translation initiation via the PI 3-kinase-p70(S6k) pathway, whereas IGF-I does so mainly via mechanisms independent of the PI 3-kinase-p70(S6k) pathway.     

eIF4E/4E-BP dissociation and 4E-BP degradation in the first mitotic division of the sea urchin embryo

The mRNA's cap-binding protein eukaryotic translation initiation factor (eIF)4E is a major target for the regulation of translation initiation. eIF4E activity is controlled by a family of translation inhibitors, the eIF4E-binding proteins (4E-BPs). We have previously shown that a rapid dissociation of 4E-BP from eIF4E is related with the dramatic rise in protein synthesis that occurs following sea urchin fertilization. Here, we demonstrate that 4E-BP is destroyed shortly following fertilization and that 4E-BP degradation is sensitive to rapamycin, suggesting that proteolysis could be a novel means of regulating 4E-BP function. We also show that eIF4E/4E-BP dissociation following fertilization is sensitive to rapamycin. Furthermore, while rapamycin modestly affects global translation rates, the drug strongly inhibits cyclin B de novo synthesis and, consequently, precludes the completion of the first mitotic cleavage. These results demonstrate that, following sea urchin fertilization, cyclin B translation, and thus the onset of mitosis, are regulated by a rapamycin-sensitive pathway. These processes are effected at least in part through eIF4E/4E-BP complex dissociation and 4E-BP degradation.     

Translational control by the poly(A) binding protein: a check for mRNA integrity

The eukaryotic mRNA 3' poly(A) tail and the 5' cap cooperate to synergistically enhance translation. This interaction is mediated by a ribonucleoprotein network that contains, at a minimum, the poly(A) binding protein (PABP), the capbinding protein eIF4E and a scaffolding protein, eIF4G. eIF4G, in turn, contains binding sites for eIF4A and eIF3, a 40S ribosome-associated initiation factor. The combined cooperative interactions within this "closed loop" mRNP among other effects enhance the affinity of eIF4E for the 5' cap by lowering its dissociation rate and, ultimately, facilitate the formation of 48S and 80S ribosome initiation complexes. The PABP-poly(A) interaction also stimulates initiation driven by picomavirus' internal ribosomal entry sites (IRESs), a process that requires eIF4G but not eIF4E. PABP, therefore, should be considered a canonical initiation factor, integral to initiation complex formation. Poly(A)-mediated translation is subjected to regulation by the PABP-interacting proteins Paip1 and Paip2. Paip1 acts as a translational enhancer. In contrast, Paip2 strongly inhibits translation by promoting dissociation of PABP from poly(A) and by competing with eIF4G for binding to PABP.     

The potyviral virus genome-linked protein VPg forms a ternary complex with the eukaryotic initiation factors eIF4E and eIF4G and reduces eIF4E affinity for a mRNA cap analogue

The virus protein linked to the genome (VPg) of plant potyviruses is a 25-kDa protein covalently attached to the genomic RNA 5' end. It was previously reported that VPg binds specifically to eIF4E, the mRNAcap-binding protein of the eukaryotic translation initiation complex. We performed a spectroscopic study of the interactions between lettuce eIF4E and VPg from lettuce mosaic virus (LMV). The cap analogue m7GDP and VPg bind to eIF4E at two distinct sites with similar affinity (K(d) = 0.3 microm). A deeper examination of the interaction pathway showed that the binding of one ligand induces a decrease in the affinity for the other by a factor of 15. GST pull-down experiments from plant extracts revealed that VPg can specifically trap eIF4G, the central component of the complex required for the initiation of protein translation. Our data suggest that eIF4G recruitment by VPg is indirectly mediated through VPg-eIF4E association. The strength of interaction between eIF4E and pep4G, the eIF4E-binding domain on eIF4G, was increased significantly by VPg. Taken together these quantitative data show that VPg is an efficient modulator of eIF4E biochemical functions.     

Role of eIF4E in stimulation of protein synthesis by IGF-I in perfused rat skeletal muscle

Insulin-like growth factor I (IGF-I) promotes anabolism by stimulating protein synthesis in skeletal muscle. In the present study, we have examined mechanisms by which IGF-I stimulates protein synthesis in skeletal muscle with a perfused rat hindlimb preparation. IGF-I (10 nM) stimulated protein synthesis over 2.7-fold. Total RNA content was unaffected, but translational efficiency was increased by IGF-I. We next examined the effect of IGF-I on eukaryotic initiation factor (eIF) 4E as a mechanism regulating translation initiation. IGF-I did not alter either the amount of eIF4E associated with the eIF4E binding protein 4E-BP1 or the phosphorylation state of 4E-BP1. Likewise, the phosphorylation state of eIF4E was unaltered by IGF-I. In contrast, the amount of eIF4E bound to eIF4G was increased threefold by IGF-I. We conclude that IGF-I regulates protein synthesis in skeletal muscle by enhancing formation of the active eIF4E x eIF4G complex.     

Repressor binding to a dorsal regulatory site traps human eIF4E in a high cap-affinity state.

Eukaryotic translation initiation involves recognition of the 5' end of cellular mRNA by the cap-binding complex known as eukaryotic initiation factor 4F (eIF4F). Initiation is a key point of regulation in gene expression in response to mechanisms mediated by signal transduction pathways. We have investigated the molecular interactions underlying inhibition of human eIF4E function by regulatable repressors called 4E-binding proteins (4E-BPs). Two essential components of eIF4F are the cap-binding protein eIF4E, and eIF4G, a multi-functional protein that binds both eIF4E and other essential eIFs. We show that the 4E-BPs 1 and 2 block the interaction between eIF4G and eIF4E by competing for binding to a dorsal site on eIF4E. Remarkably, binding of the 4E-BPs at this dorsal site enhances cap-binding via the ventral cap-binding slot, thus trapping eIF4E in inactive complexes with high affinity for capped mRNA. The binding contacts and affinities for the interactions between 4E-BP1/2 and eIF4E are distinct (estimated K(d) values of 10(-8) and 3x10(-9) for 4E-BP1 and 2, respectively), and the differences in these properties are determined by three amino acids within an otherwise conserved motif. These data provide a quantitative framework for a new molecular model of translational regulation.     

The proteasome inhibitor, MG132, promotes the reprogramming of translation in C2C12 myoblasts and facilitates the association of hsp25 with the eIF4F complex.

The eukaryotic translation initiation factor (eIF) 4E, is regulated by modulating both its phosphorylation and its availability to interact with the scaffold protein, eIF4G, to form the mature eIF4F complex. Here we show that treatment of C2C12 myoblasts with the proteasomal inhibitor, MG132 (N-carbobenzoxyl-Leu-Leu-leucinal), resulted in an early decrease in protein synthesis rates followed by a partial recovery, reflecting the reprogramming of translation. The early inhibition of protein synthesis was preceded by a transient increase in eIF2alpha phosphorylation, followed by a sustained increase in eIF4E phosphorylation. Inhibition of eIF4E phosphorylation with CGP57380 failed to prevent translational reprogramming or the moderate decrease in eIF4F complexes at later times. Prolonged incubation with MG132 resulted in the increased expression of heat shock protein (hsp)25, alphaB-crystallin and hsp70, with a population of hsp25 associating with the eIF4F complex in a p38 mitogen-activated protein kinase-dependent manner. Under these conditions, eIF4GI, and to a lesser extent eIF4E, re-localized from a predominantly cytoplasmic distribution to a more perinuclear and granular staining. Although MG132 had little effect on the colocalization of eIF4E and eIF4GI, it promoted the SB203580-sensitive association of eIF4GI and hsp25, an effect not observed with alphaB-crystallin. Addition of recombinant hsp25 to an in vitro translation assay resulted in stimulation of on-going translation and a moderate decrease in de novo translation, indicating that this modified eIF4F complex containing hsp25 has a role to play in recovery of mRNA translation following cellular stress.     

Use of the novel technique of analytical ultracentrifugation with fluorescence detection system identifies a 77S monosomal translation complex

A fundamental problem in proteomics is the identification of protein complexes and their components. We have used analytical ultracentrifugation with a fluorescence detection system (AU-FDS) to precisely and rapidly identify translation complexes in the yeast Saccharomyces cerevisiae. Following a one-step affinity purification of either poly(A)-binding protein (PAB1) or the large ribosomal subunit protein RPL25A in conjunction with GFP-tagged yeast proteins/RNAs, we have detected a 77S translation complex that contains the 80S ribosome, mRNA, and components of the closed-loop structure, eIF4E, eIF4G, and PAB1. This 77S structure, not readily observed previously, is consistent with the monosomal translation complex. The 77S complex abundance decreased with translational defects and following the stress of glucose deprivation that causes translational stoppage. By quantitating the abundance of the 77S complex in response to different stress conditions that block translation initiation, we observed that the stress of glucose deprivation affected translation initiation primarily by operating through a pathway involving the mRNA cap binding protein eIF4E whereas amino acid deprivation, as previously known, acted through the 43S complex. High salt conditions (1M KCl) and robust heat shock acted at other steps. The presumed sites of translational blockage caused by these stresses coincided with the types of stress granules, if any, which are subsequently formed.