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Supplement

Amino Acid Regulation of Gene Expression^{1 ,2}

Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, PA 17033

³To whom correspondence should be addressed. E-mail: jjefferson{at}psu.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION Regulation of guanine nucleotide... Regulation involving signaling... Regulation involving signaling... Regulation involving mTOR... CONCLUSIONS LITERATURE CITED

 
Regulation of gene expression by amino acids is mediated through a number of mechanisms affecting both the transcription of DNA and the translation of mRNA. This report reviews recent findings demonstrating a role for amino acids in regulating the initiation phase of mRNA translation. The report focuses on key regulatory events in translation initiation and discusses some of the signaling pathways through which amino acid sufficiency or the lack thereof is communicated within the cell. It concludes with a consideration of some of the important unanswered questions in this rapidly advancing area of research.

KEY WORDS: • leucine • translation initiation • signal transduction

	INTRODUCTION

TOP ABSTRACT INTRODUCTION Regulation of guanine nucleotide... Regulation involving signaling... Regulation involving signaling... Regulation involving mTOR... CONCLUSIONS LITERATURE CITED

 
Recent advances in biomedical research reveal a key role for amino acids as nutritional signals in the regulation of a number of cellular processes. Studies employing a variety of cell types and different tissues demonstrate that one such process affected is the regulation of gene expression through modulation of the translation of messenger RNA (mRNA). The studies show that cells recognize alterations in amino acid sufficiency and respond by either upregulating or downregulating translation initiation, i.e., the process during which initiator methionyl-tRNA (met-tRNA_i)⁴ and mRNA bind to a 40S ribosomal subunit followed by the joining of a 60S ribosomal subunit to form a translationally competent 80S ribosome (Fig. 1 ). Translation initiation is mediated by over a dozen proteins referred to as eukaryotic initiation factors (eIF). Binding of met-tRNA_i is mediated by eIF2, the met-tRNA_i binding protein, and eIF2B, a GDP/GTP exchange factor for eIF2 that is subject to regulation by the phosphorylation status of the -subunit of eIF2 and the -subunit of eIF2B. Binding of mRNA is mediated by eIF4F, a complex consisting of eIF4E, the mRNA cap-binding protein, eIF4A, an RNA helicase, eIF4B, a stimulator of eIF4A helicase activity, and eIF4G, a scaffolding protein that binds eIF4E, eIF4A, eIF3 and the poly(A) binding protein, PABP. This step is regulated by the phosphorylation status of eIF4E, eIF4B and eIF4G, as well as by binding of eIF4E to a family of binding proteins (4E-BP1, -2 and -3) that prevents its association with eIF4G. The response of translation initiation to a change in amino acid availability can be general, i.e., affecting the translation of most if not all mRNAs, and/or specific, i.e., affecting the translation of a single class or subset of mRNAs. The general response is mediated through regulation of both the met-tRNA_i and mRNA binding steps, whereas the specific response involves the mRNA binding step and an additional regulatory site, i.e., the phosphorylation of S6, one of the proteins comprising the 40S ribosomal subunit. Sufficient availability of amino acids for optimal rates of protein synthesis is characterized by hypophosphorylation of eIF2 allowing for unimpeded eIF2B activity, hyperphosphorylation of 4E-BP1, resulting in its dissociation from eIF4E and thus allowing association of eIF4E with eIF4G to form the active eIF4F complex, and hyperphosphorylation of S6. The increased availability of eIF4E caused by 4E-BP1 phosphorylation results in preferential translation of mRNAs containing highly structured 5'-untranslated regions. Similarly, the hyperphosphorylation status of S6 favors translation of mRNAs containing a 5'-terminal oligopyrimidine (TOP) tract. The Ser⁵¹ residue of the -subunit of eIF2 is a substrate for four different protein kinases; however, the identity of the one involved in mediating the amino acid response is presently not established, and the mode through which the cell recognizes amino acid sufficiency is presently unknown. Both 4E-BP1 and the protein kinase that phosphorylates S6, i.e., S6K1, are downstream in a signal transduction pathway involving a protein kinase referred to as the mammalian target of rapamycin (mTOR), which appears to be a point of convergence of signals generated by the action of hormones such as insulin and those generated by the cell’s recognition of a sufficiency of amino acids. Learning how the cell recognizes a sufficiency of amino acids is presently the object of intense research. Present evidence, however, suggests multiple recognition sites and multiple signaling pathways. In this review, we describe mechanisms by which alterations in amino acid sufficiency mediate control of translation initiation in mammalian cells. We also provide examples of different modes of amino acid–induced regulation and discuss potential signaling pathways through which each mode of regulation is mediated.

View larger version (24K): [in this window] [in a new window]   Figure 1. Translation initiation pathway in mammalian cells. The figure depicts the methionyl-tRNA (met-tRNAi) binding step (left side) and the mRNA binding step (right side) in translation initiation. The various eukaryotic translation initiation factors (eIF) and their roles in mRNA translation are described in the text. In this scheme, mRNA translation begins with the binding of met-tRNAi to the 40S ribosomal subunit, a reaction mediated by eIF2. The binding of mRNA to the 40S ribosomal subunit occurs through the interaction of the eIF4E component of the eIF4F complex with the m7GTP cap structure at the 5'-end of the mRNA and the association of eIF4G with the eIF3 already bound to the 40S ribosomal subunit. Initiation ends when the initiation complex scans to the initiator AUG start codon and assembles with the 60S ribosomal subunit. At this point, initiation factors are thought to be released from the complex.

	Regulation of guanine nucleotide exchange activity of eIF2B

TOP ABSTRACT INTRODUCTION Regulation of guanine nucleotide... Regulation involving signaling... Regulation involving signaling... Regulation involving mTOR... CONCLUSIONS LITERATURE CITED

View larger version (9K): [in this window] [in a new window]   Figure 2. Regulation of eukaryotic initiation factor (eIF)2B activity through phosphorylation of the -subunit of eIF2. Under amino acid sufficient conditions, eIF2B catalyzes the exchange of GDP bound to eIF2 for free GTP, allowing formation of the GTP · eIF2 · methionyl-tRNA (Met-tRNAi) complex. Deprivation of essential amino acids activates the eIF2 kinase GCN2, at least in Saccharomyces cerevisiae. Phosphorylation of eIF2 on Ser51 increases the affinity of the protein for eIF2B, resulting in formation of an inactive complex. Sequestration of eIF2B into the inactive complex decreases the rate of guanine nucleotide exchange on the unsequestered eIF2, leading to a fall in ternary complex formation and ultimately to a reduction in protein synthesis.

The guanine nucleotide exchange activity of eIF2B is also regulated indirectly through allosteric mechanisms. Examples of such regulation include the inhibition of activity by ATP (Kimball and Jefferson 1995 ) and oxidized pyridine dinucleotides (Dholakia et al. 1986 ), and its reversal by reduced pyridine dinucleotides (Dholakia et al. 1986 , Kimball and Jefferson 1995 ). Sugar phosphates, such as fructose-1,6-bisphosphate, and polyamines also allosterically activate eIF2B (Gross et al. 1991 and 1988 , Singh and Wahba 1995 ). Although allosteric regulation of eIF2B activity has been demonstrated in vitro, it is important to note that such mechanisms have not been shown to be involved in mediating effects induced by alterations in amino acid sufficiency.

In addition to the indirect mechanisms described above, the guanine nucleotide exchange activity of eIF2B may also be regulated directly through phosphorylation of the -subunit of the protein. The -subunit is phosphorylated by at least three different protein kinases, i.e., casein kinases (CK)-I and -II, and glycogen synthase kinase (GSK)-3. Phosphorylation by either CK-I (Singh et al. 1996 ) or CK-II (Dholakia and Wahba 1988 , Singh et al. 1994 ) stimulates the guanine nucleotide exchange activity of eIF2B. In contrast, phosphorylation by GSK-3 is inhibitory (Welsh et al. 1998 ). A precedent for this mode of regulation is suggested by studies in CHO.T cells in which phosphorylation of eIF2B by GSK-3 is concluded to be an important mechanism for mediating the action of insulin on the guanine nucleotide exchange activity of eIF2B (Welsh et al. 1997 and 1998 ). In CHO.T cells, insulin regulates GSK-3 through a PI 3-kinase–dependent signaling pathway. In contrast, this pathway does not appear to be modulated by amino acid sufficiency in FAO (Patti et al. 1998 ) or Jurkat T-lymphoblastoid (Iiboshi et al. 1999 ) cells. Moreover, in L6 myoblasts, deprivation of single essential amino acids results in a decrease in the activity of an uncharacterized eIF2B kinase but has no effect on GSK-3 activity (Kimball et al. 1998 ). The direction of change in the eIF2B kinase activity observed in L6 myoblasts deprived of single essential amino acids is opposite to what would be expected if the eIF2B kinase were GSK-3. Thus, because phosphorylation by GSK-3 is inhibitory, a decrease in eIF2B activity should be associated with an increase in GSK-3 activity if GSK-3 is causative in the effect. Thus, it is unlikely that GSK-3 mediates regulation of eIF2B activity by amino acid sufficiency. However, because the activity of an as yet to be characterized eIF2B kinase decreases in response to deprivation of single essential amino acids, a role for other kinases in the regulation of eIF2B must be considered.

In vivo, feeding a diet lacking a single essential amino acid results in disaggregation of hepatic polyribosomes and repression of protein synthesis (Sidransky et al. 1967 , Wunner et al. 1966 ). The reduction in protein synthesis is associated with a decrease in the guanine nucleotide exchange activity of eIF2B, with only minor changes in eIF2 phosphorylation (Anthony et al. 2001 ), suggesting that eIF2B is regulated through mechanisms distinct from eIF2 phosphorylation under these conditions. An important distinction between in vitro studies using cells in culture and in vivo studies in which diets lacking single essential amino acids are fed to animals is in the amount of the deprived amino acid that is available to the cells or tissue. In in vitro studies, the concentration of the deprived amino acid in cell culture medium is essentially zero. In contrast, in food-deprived rats fed a diet lacking a single essential amino acid, plasma concentrations of the missing amino acid fall only slightly below the value observed in a food-deprived animal. Thus, the continued availability in the plasma of the amino acid missing from the diet, albeit at fasting rather than fed concentrations, likely accounts for the lack of effect on eIF2 phosphorylation. Furthermore, in response to provision of an imbalanced mixture of amino acids, eIF2B is regulated through mechanisms other than phosphorylation of the -subunit of eIF2.

	Regulation involving signaling through mTOR to modulate the phosphorylation status of 4E-BP1

TOP ABSTRACT INTRODUCTION Regulation of guanine nucleotide... Regulation involving signaling... Regulation involving signaling... Regulation involving mTOR... CONCLUSIONS LITERATURE CITED

 
In addition to the changes in eIF2B activity noted above, in rats fed a diet lacking a single essential amino acid, eIF4F assembly and S6 phosphorylation, i.e., the steps involved in regulating the binding of mRNA to the 40S ribosomal subunit, are repressed (Anthony et al. 2001 ). Assembly of the active eIF4F complex is regulated in part through reversible association of eIF4E with a family of translation repressors that currently consists of three members, eIF4E binding protein (4E-BP)1, 4E-BP2 and 4E-BP3 [reviewed in Raught and Gingras (1999) ]. Both eIF4G and the eIF4E binding proteins contain a common structural domain that is involved in recognition of eIF4E; consequently, binding of eIF4G and the binding proteins to eIF4E is mutually exclusive. Thus, association of eIF4E with an eIF4E binding protein prevents its interaction with eIF4G and precludes assembly of the active eIF4F complex. The dynamics of eIF4E interaction with the eIF4E binding proteins is modulated by phosphorylation of the binding proteins, whereby phosphorylation causes dissociation of eIF4E from the inactive 4E-BP · eIF4E complex.

Although a number of studies have shown changes in phosphorylation of 4E-BP1 in response to amino acid sufficiency (Fox et al. 1998 , Hara et al. 1998 , Patti et al. 1998 , Wang et al. 1998 , Xu et al. 1998 ), few have examined its consequences on the interaction of 4E-BP1 or eIF4G with eIF4E. An exception is a recent study showing that in L6 myoblasts in culture, replacement of the essential amino acid leucine to leucine-deprived myoblasts resulted in a concomitant decrease in 4E-BP1 binding to eIF4E and an increase in eIF4G binding to eIF4E (Kimball et al. 1998 ). A similar effect was observed in skeletal muscle of food-deprived rats in which oral administration of leucine both promoted binding of eIF4G to eIF4E and repressed 4E-BP1 association with eIF4E (Anthony et al. 2000 ). In each of these studies, alterations in 4E-BP1 binding to eIF4E were associated with hyperphosphorylation of the binding protein.

The kinase(s) that phosphorylates 4E-BP1 in vivo remains elusive. For example, inhibition of the extracellular-signal regulated kinase (ERK) protein kinase, MEK, by PD98059 prevents 4E-BP1 phosphorylation induced by physiologic, but not supraphysiologic concentrations of insulin in CHO or 3T3-L1 cells (Scott and Lawrence 1997 ), suggesting that ERK phosphorylates 4E-BP1 in vivo. Similarly, PD98059 attenuates prostaglandin F₂-induced phosphorylation of 4E-BP1 in rat vascular smooth muscle cells (Rao et al. 1999 ). However, inhibition of the mammalian homolog of the yeast target of rapamycin protein kinase (mTOR), a protein kinase on a separate intracellular signal transduction pathway, also prevents the insulin- or prostaglandin F₂-induced stimulation of 4E-BP1 phosphorylation. Inhibition of mTOR also represses 4E-BP1 phosphorylation induced by amino acids or by hormones, such as insulin-like growth factor-I (Graves et al. 1995 ). Thus, it may be that multiple protein kinases mediate phosphorylation of 4E-BP1. In this regard, six 4E-BP1 phosphorylation sites have been identified in cells treated with insulin or serum (Fadden et al. 1997 , Gingras et al. 1999 , Heesom et al. 1998 , Mothe-Satney et al. 2000 ). Two sites, Thr³⁶ and Thr⁴⁵, reside N-terminal to the eIF4E binding domain and the remaining sites, Ser⁶⁴, Thr⁶⁹, Ser⁸² and Ser¹¹¹, are C-terminal to the binding domain. In in vitro studies, both ERK2 and mTOR phosphorylate each of the sites with the exception of Ser¹¹¹, although not all sites are phosphorylated by the two kinases with equal efficiency (Brunn et al. 1997 , Gingras et al. 1999 , Haystead et al. 1994 ). In addition, dephosphorylation of Thr³⁶ and Thr⁴⁵ is refractory to inhibition of mTOR by rapamycin (Mothe-Satney et al. 2000 ). In contrast, phosphorylation of Ser⁶⁴ and Thr⁶⁹ in quiescent cells is acutely blocked by inhibitors or mTOR (Mothe-Satney et al. 2000 ). Thus, it may be that mTOR, or an mTOR-regulated protein kinase, phosphorylates Ser⁶⁴ and Thr⁶⁹, whereas a different kinase phosphorylates Thr³⁶ and Thr⁴⁵.

Of the five sites that are phosphorylated by ERK2 and mTOR, phosphorylation at Thr⁴⁵ and Ser⁶⁴, i.e., those sites that flank the eIF4E binding domain, appear to be dominant in attenuating binding to eIF4E (Mothe-Satney et al. 2000 ). Thus, phosphorylation of Thr⁴⁵ and Ser⁶⁴ abolishes eIF4E binding in vitro. If phosphorylation at only one or two sites can prevent eIF4E binding, then what is the function of the remaining sites? One possibility is that phosphorylation at certain sites acts to "prime" 4E-BP1 for phosphorylation at other sites. In this regard, it has been suggested that 4E-BP1 phosphorylation occurs in an ordered process, with phosphorylation at Thr³⁶ and Thr⁴⁵ occurring before, and being required for phosphorylation at Ser⁶⁴ and Thr⁶⁹ [reviewed in Raught et al. (2000b) ]. This suggestion is consistent with results from a study showing that mutation of Thr³⁶ and Thr⁴⁵ to nonphosphorylatable Ala residues reduces serum-stimulated phosphorylation of the remaining sites (Gingras et al. 1999 ).

In addition to phosphorylating 4E-BP1, mTOR also phosphorylates S6K1 in vitro (Isotani et al. 1999 ). However, the sites in 4E-BP1 phosphorylated by mTOR are flanked by proline residues, whereas those sites on S6K1 (Thr³⁸⁹) have neighboring large, bulky residues. Moreover, mTOR exhibits a strong (>10-fold) preference for S6K1 over 4E-BP1 (Burnett et al. 1998 ). Therefore, mTOR may not act as a 4E-BP1 kinase in vivo. Instead, it may act by inhibiting the activity of a protein phosphatase that dephosphorylates 4E-BP1 (Fig. 3 ). In Saccharomyces cerevisiae, two TOR isoforms, Tor1p and Tor2p, control translation initiation in response to nutrient availability (Barbet et al. 1996 ). In this system, TOR is linked to PPH21 and PPH22, two type 2A protein phosphatase catalytic subunits (PP2A_C) (Jiang and Broach 1999 ), and SIT4, a homolog of the mammalian type 6 protein phosphatase (Bastians and Ponstingl 1996 ). Pph21p, Pph22p and Sit4p all interact with the essential protein, Tap42p (Di Como and Arndt 1996 ). The interaction between Tap42p and the protein phosphatase catalytic subunits is observed in growing cells, but not in cells in stationary phase. Furthermore, the association is prevented by rapamycin, and mutations in the TAP42 gene can result in rapamycin resistance, implicating Tor in the effect. Further evidence that Tor is upstream of, and regulates Tap42p interaction with protein phosphatase catalytic subunits is that at the nonpermissive temperature, a yeast strain expressing a temperature-sensitive variant of Tap42p exhibits a defect in translation initiation (Di Como and Arndt 1996 ) similar to wild-type cells treated with rapamycin (Barbet et al. 1996 ). Moreover, Tap42p is phosphorylated in growing cells, but not in cells treated with rapamycin (Jiang and Broach 1999 ). Finally, expression of a rapamycin-insensitive Tor variant confers rapamycin resistance to Tap42p phosphorylation. Overall, results obtained in yeast suggest that Tor phosphorylates and thereby promotes association of Tap42p with the catalytic subunit of PP2A and PP6 and that such an association is a requisite step in Tor-mediated signaling.

View larger version (16K): [in this window] [in a new window]   Figure 3. Regulation of translation initiation by mammalian target of rapamycin (mTOR)-mediated alterations in phosphorylation of 4E-BP1 and S6K1. Hormones, such as insulin and insulin-like growth factor-I, cause activation of mTOR and subsequently, hyperphosphorylation of 4E-BP1 and S6K1. Although still not completely defined in mammalian cells, in yeast, mTOR functions through inhibition of a protein phosphastase rather than through activation of a protein kinase. Although hyperphosphorylation of 4E-BP1 and S6K1 in response to regulatory amino acids requires mTOR to be active, there is no evidence that amino acids actually act directly through mTOR. In the figure, the alternative possibility that amino acids regulate a protein kinase(s) distinct from mTOR is presented. In this model, mTOR activity would be required to repress the phosphatase that dephosphorylates 4E-BP1 and S6K1, whereas amino acids signal to 4E-BP1 and S6 protein kinases. Regardless of the mechanism involved in hyperphosphorylation, it is clear that phosphorylation of 4E-BP1 stimulates eukaryotic initiation factor (eIF)4F assembly, leading to enhanced translation of mRNAs having high secondary structure at their 5'-termini. Increased eIF4F assembly would also be expected to increase global rates of protein synthesis. Activation of S6K1 by hyperphosphorylation leads to a preferential increase in translation of mRNAs with a terminal oligopyrimidine (TOP) sequence adjacent to their 5'-termini.

Regardless of the mechanism, it is clear that essential amino acids, and in particular leucine, regulate eIF4F assembly. This effect is observed in cells in culture and in liver and skeletal muscle of food-deprived animals. Thus, in contrast to phosphorylation of eIF2, which manifests only during severe amino acid deprivation, modulation of the steps involved in mRNA binding to the 40S ribosomal subunit is observed under conditions of both amino acid deprivation and administration of the essential amino acid leucine.

	Regulation involving signaling through mTOR to modulate the phosphorylation status of S6K1

TOP ABSTRACT INTRODUCTION Regulation of guanine nucleotide... Regulation involving signaling... Regulation involving signaling... Regulation involving mTOR... CONCLUSIONS LITERATURE CITED

 
The S6K are a family of mitogen-activated, Ser/Thr protein kinases that participate in translational and cell cycle control [reviewed in Fumagalli and Thomas (2000) ]. The S6K1 gene encodes cytoplasmic and nuclear proteins referred to as S6K1ß/p70^S6k and S6K1/p85^S6k, respectively, whereas the S6K2 gene encodes S6K2ßI and S6K2ßII. Both S6K1 and S6K2 are phosphoproteins whose enzymatic activities are regulated by phosphorylation at multiple Ser and Thr residues. With regard to S6K1, current studies suggest that activation of the enzyme involves an ordered series of phosphorylation events whereby phosphorylation of a cluster of sites located near the C-terminus permits phosphorylation of three internal sites, Thr²²⁹, Ser³⁷¹ and Thr³⁸⁹, which collectively confer maximal kinase activity. Mitogen- and amino acid–induced phosphorylation of one of the activating sites, Thr³⁸⁹, is blocked by the mTOR inhibitor, rapamycin, which prevents activation of the kinase. Thus, both 4E-BP1 and S6K1 are phosphorylated in an mTOR-dependent manner. Although phosphorylation of Thr³⁸⁹ is inhibited by rapamycin, it is unlikely that mTOR is the relevant kinase because deletion of the C-terminal region of the protein containing the cluster of phosphorylation sites abrogates the effect, suggesting that the C-terminus confers rapamycin sensitivity.

Although mitogen-stimulated phosphorylation of S6 has been known to occur for many years, its role in regulating translation initiation has been delineated only recently. In this regard, rather than stimulating global rates of protein synthesis, activation of S6K1 results in the preferential translation of mRNAs encoding proteins that play important roles in protein synthesis [reviewed in Dufner and Thomas (1999) ]. Thus, activation of S6K1 causes a preferential increase in the synthesis of proteins, such as ribosomal proteins and elongation factors eEF1A and eEF2, which are encoded by mRNAs containing 5'-terminal oligopyrimidine tracts (referred to as TOPS mRNAs). For example, in liver of food-deprived rats, oral administration of leucine promoted phosphorylation of S6K1 and ribosomal protein S6 (Anthony et al. 2001 ). Similar results were observed in rat livers perfused in situ with medium containing leucine at a concentration four times that found in plasma of food-deprived animals, with the remaining amino acids present at plasma concentrations (Shah et al. 1999 ). In both cases, unbalanced provision of leucine did not stimulate global rates of protein synthesis, but rather specifically increased translation of mRNAs containing the TOP sequence (Anthony et al. 2001 ). Thus, in the liver of food-deprived rats, mRNAs encoding ribosomal proteins S4, S8 and L26 are not associated predominantly with polysomes. Leucine administration results in a redistribution of ribosomal protein mRNAs, such that the bulk of these mRNAs exhibit a polysomal distribution. In contrast, mRNAs encoding albumin and ß-actin are predominantly polysome associated, regardless of feeding status, indicating that imbalanced provision of leucine preferentially upregulates the translation of TOP mRNAs.

	Regulation involving mTOR-independent signaling to modulate the activation state of the eIF4F complex

TOP ABSTRACT INTRODUCTION Regulation of guanine nucleotide... Regulation involving signaling... Regulation involving signaling... Regulation involving mTOR... CONCLUSIONS LITERATURE CITED

 
Although association of eIF4E with 4E-BP1 is an important mechanism for modulating assembly of the eIF4F complex, it is not the sole means of regulating the interaction of eIF4E and eIF4G. For example, in skeletal muscle of food-deprived rats, oral administration of leucine enhanced 4E-BP1 phosphorylation concomitant with dissociation of the 4E-BP1 · eIF4E complex and increased association of eIF4G with eIF4E (Anthony et al. 2000 ). Treatment with rapamycin before leucine administration prevented completely leucine-induced changes in 4E-BP1 phosphorylation and dissociation of the 4E-BP1 · eIF4E complex. However, binding of eIF4G to eIF4E was nonetheless significantly enhanced by leucine administration in rapamycin-treated rats. A similar phenomenon was observed in neonatal pigs, in which rapamycin treatment abrogated the feeding-induced dissociation of the 4E-BP1 · eIF4E complex, but only partially reduced the increase in eIF4G binding to eIF4E (Kimball et al. 2000 ).

A possible mechanism for TOR-independent signaling to eIF4F is suggested by a recent study showing that stimulation of eIF4G binding to eIF4E by epidermal and nerve growth factors (EGF and NGF, respectively) was blocked by the MEK inhibitor, PD98059 (Kleijn and Proud 2000 ). In that study, both EGF and NGF also promoted dissociation of the 4E-BP1 · eIF4E complex. However, PD98059 had no effect on 4E-BP1 binding to eIF4E, indicating that signaling through the mitogen-activated protein (MAP) kinase pathway can modulate eIF4F assembly in a 4E-BP1-independent manner.

How signaling through the MAP kinase pathway promotes eIF4G binding to eIF4E remains an unanswered question. On the basis of observations that eIF4G is a phosphoprotein, and that its phosphorylation is enhanced by mitogen- and serum-stimulation of cells in culture [reviewed in Raught et al. (2000b) ], it is tempting to speculate that phosphorylation of eIF4G might stimulate eIF4F assembly. However, phosphorylation of the three serum-stimulated phosphorylation sites in the C-terminal one third of the protein is inhibited by rapamycin (Raught et al. 2000a ), suggesting that signaling through mTOR is important in regulating eIF4G phosphorylation. Interestingly, similar to the rapamycin-sensitive phosphorylation of S6K1 described above, deletion of the N-terminus of eIF4G abolishes the inhibition of phosphorylation of the C-terminal sites caused by rapamycin. Moreover, in eIF4G variants lacking the N-terminus, phosphorylation of the C-terminal sites is constitutively elevated, even in the absence of serum. Thus, although mTOR activation enhances phosphorylation of the serum-sensitive sites in the C-terminus of eIF4G, it is unlikely that mTOR phosphorylates those sites directly.

Whether phosphorylation of eIF4G is regulated by alterations in amino acid sufficiency is unknown. In unpublished studies, we have found that at least two of the three rapamycin-sensitive phosphorylation sites in eIF4G are phosphorylated in response to feeding a complete meal to food-deprived rats. However, in that study, an increase in plasma insulin concentration may have contributed to the increased phosphorylation. Further studies are required to establish what, if any role, changes in amino acid sufficiency have in regulating mTOR-independent signaling to eIF4F assembly.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION Regulation of guanine nucleotide... Regulation involving signaling... Regulation involving signaling... Regulation involving mTOR... CONCLUSIONS LITERATURE CITED

 
The recent identification of amino acids as signaling molecules that regulate translation initiation has provided the incentive to gain a better understanding of the mechanisms through which they operate. A plethora of studies have demonstrated that essential amino acids, and in particular leucine, stimulate phosphorylation of 4E-BP1 and S6K1. In contrast, relatively few studies have examined their effects on eIF4F assembly or eIF2B activity. On the basis of information presently available, a number of questions remain unanswered. One key question concerns the specificity of the response. In particular, why do certain amino acids stimulate 4E-BP1 and/or S6K1 phosphorylation in some cells, but not others? Another important question is how amino acid sufficiency is sensed by the cell. It would appear that at least two mechanisms exist through which amino acid sufficiency is detected, although their identities remain elusive. One mechanism by which amino acid sufficiency is detected appears to involve a recognition molecule whose signaling to 4E-BP1 and S6K1 is dependent upon mTOR (Lynch et al. 2000 ) (Fig. 4 ). Although incompletely characterized, the recognition molecule preferentially responds to leucine sufficiency, although other essential amino acids appear to be recognized to lesser extents. In addition to modulating eIF4F complex assembly indirectly through phosphorylation of 4E-BP, the recognition molecule may also regulate eIF4F complex assembly directly through the MAP kinase signaling pathway. A second mechanism by which amino acid sufficiency is detected involves an eIF2 kinase, most likely mGCN2. By modulating the phosphorylation state of eIF2, the kinase indirectly regulates the guanine nucleotide exchange activity of eIF2B. In contrast to the specificity demonstrated by the putative leucine recognition molecule, activation of the eIF2 kinase is responsive to insufficiency of any essential amino acid. The activity of eIF2B may also be regulated through modulation of an unidentified eIF2B kinase. A third unanswered question concerns the exact role of mTOR in amino acid signaling to translation initiation. It is clear that such signaling requires mTOR to be active, but whether changes in amino acid sufficiency modulate mTOR activity is unclear. Finally, a topic not discussed in this report, but of considerable interest, involves the possible cooperativity between amino acids and growth-promoting hormones in the regulation of translation initiation.

View larger version (18K): [in this window] [in a new window]   Figure 4. Potential signaling pathways involved in the response of translation initiation to amino acid sufficiency. At least two mechanisms exist that detect the sufficiency of essential amino acids. As discussed in the text, a recognition molecule that preferentially, but not exclusively, senses leucine sufficiency signals to S6K1 and eukaryotic initiation factor (eIF)4F complex assembly, the latter through both direct and indirect (i.e., through phosphorylation of 4E-BP1) mechanisms. The leucine recognition molecule may also signal to eIF2B. A second mechanism for detecting amino acid sufficiency involves regulation of eIF2B through changes in eIF2 phosphorylation and possibly through changes in eIF2B phosphorylation.

	FOOTNOTES

 
¹ Presented at the International Symposium on Glutamine, October 2–3, 2000, Sonesta Beach, Bermuda. The symposium was sponsored by Ajinomoto USA, Incorporated. The proceedings are published as a supplement to The Journal of Nutrition. Editors for the symposium publication were Douglas W. Wilmore, the Department of Surgery, Brigham and Women’s Hospital, Harvard Medical School and John L. Rombeau, the Department of Surgery, the University of Pennsylvania School of Medicine.

² Supported by research grants DK13499 and DK15658 from the National Institutes of Health.

⁴ Abbreviations used: BP, binding proteins; CK, casein kinase; EGF, epidermal growth factor; eIF, eukaryotic initiation factor; ER, endoplasmic reticulum; ERK, extracellular-signal regulated kinase; GSK, glycogen synthase kinase; HRI, heme-regulated translational inhibitor; MAP, mitogen-activated protein; met-tRNA_i, methionyl-tRNA; mGCN2, mammalian homolog of yeast GCN2; mTOR, mammalian target of rapamycin; NGF, nerve growth factor; PEK/PERK, pancreatic eIF2 kinase/PKR-like ER kinase; PKR, RNA-dependent protein kinase; TOP, terminal oligopyrimidine.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION Regulation of guanine nucleotide... Regulation involving signaling... Regulation involving signaling... Regulation involving mTOR... CONCLUSIONS LITERATURE CITED

 

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