2nd Amino Acid Workshop

Amino Acids as Regulators of Gene Expression at the Level

of mRNA Translation1,2
Leonard S. Jefferson3 and Scot R. Kimball
Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine,
Hershey, PA 17033

KEY WORDS:  mRNA leucine  upstream open reading frame  internal ribosome entry site  eukaryotic
initiation factor

g-subunits)4 on Ser-51 has been recognized for a number of

years as a mechanism for suppressing global protein synthesis
[reviewed in (1)]. The phosphorylation event is mediated by at
least four different protein kinases, all of which are activated in
response to some specific type of cellular stress. For example,
the protein kinase known as PERK [double-stranded RNAactivated protein kinase (PKR)-like endoplasmic reticulumassociated kinase] is activated in response to any number of
conditions, and all of them result in malfunctioning of the
process of protein folding in the endoplasmic reticulum (2,3).
PKR and heme-regulated inhibitor are activated by doublestranded RNA during viral infection and hemin deprivation,
respectively (4,5). Finally, general control non-derepressing
kinase-2 (GCN2), which is an eIF2a kinase that is conserved
from yeast to mammals, is activated in response to amino acid
starvation, purine limitation or DNA damage (610). Acting
alone or in combination, the eIF2a kinases mediate hyperphosphorylation of Ser-51 in response to a variety of stress
conditions including suboptimal levels of amino acids, glucose
or serum; exposure to heat, heavy metals or arsenite; formation
of free oxygen radicals or hypoxic or hyperosmotic conditions
[c.f. (10)]. Hyperphosphorylation of eIF2a leads to suppression
of global protein synthesis through a mechanism whereby
binding of the initiator met-tRNAi to the 40S ribosomal
subunit is inhibited [reviewed in (1)]. Initiator met-tRNAi
joins the 40S ribosomal subunit as a ternary complex with eIF2

A growing number of examples illustrate that many and

perhaps all genes are regulated at multiple steps that include
transcription, posttranscriptional processing, nuclear export,
stability and translation of mature mRNA molecules.
Translation itself is regulated by a diverse array of mechanisms
that act not only at the initiation step but also during
elongation and termination. This review focuses on the initiation step and some of the regulatory mechanisms involved in
the discrimination as to which mRNA are selected for
translation and to what extent they are translated into protein.
Moreover, it focuses specifically on amino acids as nutrient
regulators of initiation and the signaling pathways through
which amino acids exert their effects on translation.

Phosphorylation of eukaryotic initiation factor-2 as

a regulatory mechanism for the translational control
of gene expression
Phosphorylation of the a-subunit of eukaryotic initiation
factor-2 (eIF2, a trimeric complex composed of a-, b- and

1
Presented at the conference The Second Workshop on the Assessment of
Adequate Intake of Dietary Amino Acids held October 31November 1, 2002, in
Honolulu, Hawaii. The conference was sponsored by the International Council on
Amino Acid Science. The Workshop Organizing Committee included Vernon R.
Young, Yuzo Hayashi, Luc Cynober and Motoni Kadowaki. Conference proceedings were published in a supplement to The Journal of Nutrition. Guest editors for
the supplement publication were Dennis M. Bier, Luc Cynober, Yuzo Hayashi and
Motoni Kadowaki.
2
The studies described in this article that were performed in the laboratories of
the authors were supported by research grants DK-13499 and DK-15658 from the
National Institutes of Health.
3
To whom correspondence should be addressed. E-mail: jjefferson@psu.edu.

4
Abbreviations used: 4E-BP1, eIF4E binding protein-1; eIF, eukaryotic
initiation factor; GCN, general control non-derepressing; FAT, FRAP-ATM-TRRAP;
IRES, internal ribosome entry site; mTOR, mammalian target of rapamycin; PI3K,
phosphatidylinositol 3-kinase; PKB, protein kinase B; raptor, regulatory associated
protein of mTOR; rpS6, ribosomal protein S6; S6K1, 70-kDa ribosomal protein S6
kinase; SIN1, stress-associated protein kinase interacting protein; TOP, terminal
oligopyrimidine tract; TSC, tuberous sclerosis complex; uORF, upstream open
reading frame.

0022-3166/03 $3.00 2003 American Society for Nutritional Sciences.

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ABSTRACT Amino acids act through a number of signaling pathways and mechanisms to mediate control of gene
expression at the level of mRNA translation. This report reviews recent ndings that illustrate the manner through
which amino acids act to regulate the initiation phase of mRNA translation. The report focuses on signaling pathways
that involve the eukaryotic initiation factor-2 (eIF2) protein kinase, general control non-derepressing kinase-2 and the
mammalian target of rapamycin (mTOR) protein kinase. It also describes the mechanisms through which amino
acidinduced modulation of eIF2 phosphorylation and mTOR-mediated signaling cause derepression of translation of
specic mRNAs and result in an overall change in the pattern of gene expression. Finally, it provides examples of
mRNAs whose translation is modulated through these mechanisms. J. Nutr. 133: 2046S2051S, 2003.

AMINO ACID REGULATION OF GENE EXPRESSION

basic fibroblast growth factor (27), c-myc (28), X-linked

inhibitor of apoptosis (29), p97 [also known as death-associated
protein or N-acetyltransferase-1 (30)] and ornithine decarboxylase (31). Although the advantages for viruses to contain
an IRES are quite clear (i.e., 59 cap-independent initiation), the
advantages for a cellular mRNA IRES were not immediately
understood. However, it is now becoming recognized that the
59 untranslated regions of many cellular mRNA that serve in
critical biological functions are long and structured and contain
upstream AUG codons, so that scanning ribosomes are unlikely
to efficiently initiate translation (32). Efficient initiation of the
translation of these mRNA in many examples including some of
those listed above has been shown to be mediated by an IRES.
Recent evidence (33,34) suggests that phosphorylation of
eIF2a is required for activation of IRES-mediated translation
initiation. Whereas production of poorly translated mRNA
seems inefficient, evolution has clearly tolerated and apparently
exploited uORF and IRES for regulatory purposes. As future
work unfolds, it will certainly be exciting to identify the genes
regulated by amino acid availability through these novel
regulatory elements.
Phosphorylation of eIF4E binding protein-1, ribosomal
protein S6 and eIF4G as regulatory mechanisms
for translational control of gene expression
Three proteins involved in the joining of mRNA with the
40S ribosomal subunit, i.e., eIF4E-binding protein-1 (4E-BP1),
ribosomal protein S6 (rpS6) and eIF4G, are downstream targets
of a signaling pathway that includes the protein kinase referred
to as the target of rapamycin (TOR) [reviewed in (35)]. TOR
was first identified in S. cerevisiae as two genes (TOR-1 and -2)
that permit growth in the presence of the immunosuppressant
rapamycin [reviewed in (35)]. Subsequent studies revealed that
TOR orthologs are present in flies, worms and mammals. The
TOR proteins are members of a family of proteins that have in
common a C-terminal protein kinase domain with homology to
the catalytic domain of phosphatidylinositol 3-kinase (PI3K).
Members of the PI3K-related family of kinases also contain
a FAT (FRAP, ATM, TRRAP) domain that is speculated to
serve as a site that might be involved in protein-protein
interaction. In addition to the catalytic and FAT domains,
TOR contains multiple HEAT (Huntingtin eIF3, A-subunit of
protein phosphatase 2A, TOR) motifs which, like the FAT
domain, are thought to be involved in protein-protein
interaction. Finally, the TOR protein also has an FK506
binding protein (FKB)-rapamycin binding domain that binds
the FKB protein-12drapamycin complex and mediates inhibition of TOR activity by rapamcyin.
In yeasts and mammals, one of the major functions of TOR
is to coordinate nutrient availability with cell growth and
proliferation [reviewed in (36,37)]. In mammalian cells,
mammalian TOR (mTOR) is involved in regulating the
binding of mRNA to the 40S ribosomal subunit [reviewed in
(35)]. The mRNA binding step in translation initiation is
mediated by a complex of translation initiation factors that is
referred to as eIF4F and is comprised of eIF4A (an RNA
helicase), eIF4E (the protein that binds to the m7GTP cap
structure at the 59 end of mammalian cytoplasmic mRNA) and
eIF4G, a scaffolding protein that in addition to binding to
eIF4A and eIF4E binds to the poly(A) binding protein (PABP)
and eIF3 [reviewed in (38)]. The binding of eIF4G to eIF3 is of
particular importance because it is through this interaction that
the eIF4FdmRNA complex binds to the 40S ribosomal subunit.
Assembly of the eIF4F complex is regulated in part through the
association of eIF4E with the so-called eIF4E binding proteins

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and GTP. After each cycle of initiation, the eIF2 is released as

a binary complex with GDP, and for eIF2 to function in another
round of initiation, the GDP must be exchanged for GTP. This
guanine nucleotide exchange reaction is catalyzed by another
initiation factor, eIF2B. When eIF2a becomes phosphorylated,
it forms an unproductive complex with eIF2B, the binding of
initiator met-tRNAi to the 40S ribosomal subunit becomes
restricted and global protein synthesis is suppressed.
Although phosphorylation of eIF2a leads to a suppression of
global protein synthesis, accumulating evidence demonstrates
that it also results in the derepression of translation of a number
of specific mRNA. The first evidence in support of this type of
role for phosphorylated eIF2a was provided by studies in the
yeast Saccharomyces cerevisiae in which it was shown that
GCN2 mediates activation of the general amino acid control
pathway when one or more amino acids becomes limiting for
growth [reviewed in (11)]. It does so by mediating the derepression of translation of the mRNA for GCN4, which is a
transcriptional activator of genes that are involved in amino
acid biosynthesis and related metabolic pathways. GCN4
mRNA contains a unique cluster of upstream open reading
frames (uORF) that mediate translational derepression of the
GCN4 coding sequence only when a certain threshold level of
eIF2a phosphorylation is achieved as a result of activation of
GCN2. Limitation of amino acids is thought to activate GCN2
through a mechanism that involves accumulation of deacylated
tRNA.
The first evidence that the mammalian ortholog of GCN2
is also activated by deacylated tRNA is provided by a recent
report (12) in which liver from wild-type (GCN21/1) mice or
mice with a chromosomal disruption of the GCN2 gene
(GCN22/2) were perfused in situ with medium that contained
or lacked the histidinyltRNA synthetase inhibitor, histidinol.
In liver from GCN21/1 mice, histidinol treatment resulted in
enhanced phosphorylation of eIF2a as well as inhibition of
eIF2B activity. In contrast, in liver from GCN22/2 mice,
histidinol had no effect on either eIF2a phosphorylation or
eIF2B activity. It is noteworthy that in the absence of
histidinol, eIF2a phosphorylation was lower and eIF2B activity
was higher in liver from GCN22/2 compared with GCN21/1
mice, which suggests that under these conditions, GCN2 may
be the predominant eIF2a kinase activity in mouse liver. The
same report (12) showed that phosphorylation of eIF2a in
response to leucine deprivation of mouse embryonic stem cells
was also dependent on GCN2.
Besides the yeast GCN4, there is a growing list of examples
of genes that are regulated by uORF. Some well-characterized
transcription factor examples include activating transcription
factor 4 (7), CCAAT/enhancer binding proteins-a and -b (13);
the oncoprotein mouse double-minute 2, which is a part of
a negative-feedback loop that regulates the activity of the
tumor suppressor p53 (14) and the human epidermal growth
factor receptor-2 (15); S-adenosylmethionine decarboxylase,
which is a key enzyme in the biosynthetic pathway for the
polyamines spermidine and spermine (16,17); and b2-adrenergic (18), retinoic acid (19), glucocorticoid (20) and estrogen
(21) receptors. Thus, uORF appear frequently in genes with
critical biological functions.
An alternative mode of translational control of gene
expression mediated by eIF2a phosphorylation involves recruitment of the translation initiation complex by an internal
ribosome entry site (IRES). Translation by internal ribosome
entry was first identified in picornaviruses (22,23), but a number
of cellular mRNA have subsequently been found to contain an
IRES including vascular endothelial growth factor (24),
hypoxia-inducible factor-1a (25), protein kinase C-d (26),

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rapamycin treatment prevented the leucine-induced phosphorylation of 4E-BP1 and S6K1. However, rapamycin attenuated
but did not prevent the stimulation of protein synthesis or
assembly of the eIF4F complex that was caused by leucine
administration. A similar result was reported for skeletal muscle
of perfused rat hindlimb preparations whereby increasing the
concentrations of amino acids in the perfusate from those
observed in fasted rats to 10 times those amounts stimulated
protein synthesis and the binding of eIF4G to eIF4E but had no
effects on 4E-BP1 or S6K1 phosphorylation (53).
Recent evidence suggests that insulin is required for amino
acidinduced signaling through mTOR. For example, in diabetic rats, oral leucine administration enhances protein synthesis in skeletal muscle, although the absolute rate is only
;50% of the value observed in control animals that are
administered leucine (54). Moreover, unlike in control animals,
in diabetic rats, leucine is unable to stimulate eIF4G binding to
eIF4E or phosphorylation of 4E-BP1 or S6K1. In muscle from
diabetic rats, phosphorylation of protein kinase B (PKB) is
almost undetectable. Infusion of insulin at a rate sufficient to
restore the plasma insulin concentration to a value equivalent to
that observed in plasma from a fasted control rat restores PKB
phosphorylation to levels observed in a fasted control animal.
Although leucine does not stimulate PKB phosphorylation
(even in a control animal), oral leucine administration to
diabetic rats infused with insulin enhances the binding of eIF4G
to eIF4E as well as phosphorylation of 4E-BP1 and S6K1. The
idea that both insulin and amino acids are required for activation
of the mRNA binding step is also supported by results from
a study in which diazoxide was used to acutely attenuate insulin secretion (55). In that study, protein synthesis, 4E-BP1
and S6K1 phosphorylation and eIF4G binding to eIF4E were
all enhanced by feeding fasted rats a protein-containing but
not protein-free meal. However, in fasted rats treated with
diazoxide, the protein-containing meal had no effect on any of
these variables. The result of these and similar studies suggest
that by maintaining a minimal level of activation of the PI3KPKB signaling pathway, insulin has a permissive effect on
signaling by amino acids to targets downstream of mTOR.
Mechanisms for regulation of mTOR signaling:
TOR-interacting proteins
One mechanism through which the activity of mTOR
can be modulated is through interactions with other proteins.
Two proteins recently shown to repress mTOR signaling are
tuberous sclerosis complexes (TSC)-1 and -2 (56). The genes
for TSC1 and TSC2 encode proteins (hamartin and tuberin,
respectively) that were originally identified as tumor suppressor
genes in humans, and mutations in either gene are associated
with the growth of disorganized benign tumors in a variety of
organs (57,58). Such tumors are characterized by the presence
of abnormally large cells.
TSC1 and TSC2 are also present in Drosophila melanogaster.
Mutations in the D. melanogaster TSC2 gene result in the Gigas
phenotype; like tuberous sclerosis in humans, this phenotype is
characterized by an unusually large cell size [reviewed in (59)].
Genetic analysis in D. melanogaster place the TSC1dTSC2
complex downstream of PKB and upstream of S6K1 in the
insulin signal transduction pathway (60,61). Indeed, two recent
studies (62,63) report that PKB directly phosphorylates TSC2
on at least two residues, Ser-924 and Thr-1518, in D.
melanogaster and mammalian cells. A more recent genetic
analysis places TSC1 and TSC2 upstream of TOR (56). In fact,
both proteins bind to mTOR when coexpressed in D.
melanogaster S2 cells and inhibit mTOR signaling to S6K1

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(4E-BP), of which 4E-BP1 is the prototype. The binding site on

eIF4E for 4E-BP1 overlaps with that for eIF4G, such that either
4E-BP1 or eIF4G can bind individually to eIF4E, but both
cannot bind at the same time (39). Thus, binding of eIF4E to
4E-BP1 prevents mRNA from binding to the 40S ribosomal
subunit. Formation of the 4E-BP1deIF4E complex only occurs
when 4E-BP1 is hypophosphorylated; hyperphosphorylated
forms of the protein do not bind to eIF4E. Of particular
relevance to this article is the finding that the hyperphosphorylation of 4E-BP1 that occurs in response to provision
of amino acids to deprived cells in culture, and in particular to
provision of leucine to amino aciddeprived cells, is blocked
by rapamycin (40,41). A similar effect is observed in fasted
rats, where either provision of dietary protein (42) or oral
administration of leucine (43) promotes 4E-BP1 hyperphosphorylation and assembly of the eIF4GdeIF4E complex in
skeletal muscle. A similar effect of amino acids on 4E-BP1
phosphorylation is observed in skeletal muscle of pigs (44) and
humans (45). However, administration of rapamycin before
feeding or administering oral leucine completely prevents the
increase in 4E-BP1 phosphorylation (46,47) and thereby
implicates mTOR in the response.
Another control point in the mRNA binding step in
translation initiation that is regulated by mTOR involves
changes in phosphorylation of rpS6. Phosphorylation of rpS6
occurs on multiple sites and is mediated by a 70-kDa protein
kinase termed S6K1 [reviewed in (48)]. The activity of S6K1 is
itself regulated by multisite phosphorylation, whereby phosphorylation of sites in the C-terminus of the protein permits
phosphorylation of other internal sites such as Ser-229 and
Thr-389. Phosphorylation of the internal sites is critical for
optimal activation of the kinase. Thr-389 is phosphorylated in
vitro by mTOR (49) and treatment of cells in culture [c.f. (40)]
and animals in vivo (46) with rapamycin prevents amino acid
induced phosphorylation of the residue.
Early studies suggested that phosphorylation of rpS6 might
enhance global protein synthesis. However, recent studies
suggest that rather than controlling translation of mRNA in
a global manner, phosphorylation of rpS6 enhances the
translation of a subset of mRNAs that contain a common
structural feature: an uninterrupted stretch of ;715 pyrimidine residues that are adjacent to the 59 cap structure
[terminal oligopyrimidine tract (TOP) mRNA; reviewed in
(50)]. Proteins encoded by such mRNAs include the ribosomal
proteins, eIF4G, PABP and eukaryotic elongation factor-2; i.e.,
proteins that are involved in mRNA translation. Related
studies have shown that ribosomal DNA transcription is also
inhibited by rapamycin (51). Thus, mTOR controls the
synthesis of both ribosomal proteins and ribosomal RNA; in
other words, mTOR controls ribosome biogenesis. It should be
noted that a recent report (52) questions the assumptions that
activation of S6K1 and phosphorylation of rpS6 are required for
upregulated translation of TOP mRNA. In the study reported,
amino acid stimulation of TOP mRNA translation was
dependent upon intact signaling through PI3K but was not
dependent upon either S6K1 or phosphorylation of rpS6. Thus,
there may exist multiple signaling pathways, one or more of
which may be independent of mTOR, that can regulate TOP
mRNA translation.
Evidence suggesting that amino acids may regulate a step in
translation initiation that does not depend on rapamycinsensitive signaling through mTOR is provided by a study
wherein fasted rats were treated with rapamycin before oral
administration of leucine (46). In control animals, leucine
stimulated protein synthesis, phosphorylation of 4E-BP1 and
S6K1 and binding of eIF4G to eIF4E. As noted above,

AMINO ACID REGULATION OF GENE EXPRESSION

mTOR signaling to S6K1 and 4E-BP1, whereas mutation to

acidic residues to mimic phosphorylation of the protein
represses the effectiveness of the protein in inhibiting mTOR
signaling (62,64). This demonstrates the important role played
by TSC2 phosphorylation in regulating signaling through
mTOR. Finally, rapamycin-resistant variants of S6K1 are also
resistant to overexpression of TSC1 and TSC2 (62). Overall, the results are consistent with a model whereby the
TSC1dTSC2 complex binds to mTOR and prevents PKBmediated phosphorylation and activation of mTOR. However,
this model may be insufficient to explain the observation that
TSC1 and TSC2 repress not only PKB signaling to targets
downstream of mTOR, but also suppress amino acid signaling
to such targets (56,67). Amino acids do not activate PKB,
although they do promote phosphorylation of Ser-2448 on
mTOR. Thus, amino acids may promote dissociation of the
TSC1dTC2 complex from mTOR, which would subsequently
allow PKB access to Ser-2448. Such a mechanism would not
require activation of PKB but would require release of the
TSC1dTSC2 complex.
In addition to TSC1 and TSC2, several other recently
identified proteins were shown to associate with and regulate
the activity of mTOR [reviewed in (68)]. For example, the
regulatory associated protein of mTOR (raptor) regulates both
the activity of mTOR and its sensitivity to rapamycin (69,70).
Although the mechanism(s) through which raptor modulates
mTOR activity is still incompletely defined, it is clear that it
binds to mTOR and that such an association is required for
mTOR to efficiently phosphorylate 4E-BP1 and S6K1. Thus,
suppression of raptor expression in either HeLa (69) or HEK293T (70) cells results in decreased phosphorylation of 4E-BP1
and S6K1, respectively. Such studies also revealed that raptor
has a positive role in nutrient signaling because suppression of

FIGURE 1 Regulation of gene expression through modulation of translation of mRNA that encode specic proteins by amino acids and insulin.
The diagram highlights the mechanisms through which amino acids regulate gene expression through modulation of mRNA translation. Details of the
individual steps are discussed in the text. eIF, eukaryotic initiation factor; eIF2(P), eIF2 phosphorylated on residue 51 of its a-subunit; BP1, eIF4E binding
protein-1; GCN2, general control non-derepressing kinase-2 amino acidregulated eIF2a kinase; GSK3, glycogen synthase kinase-3; PKB, protein kinase
B; mTOR, mammalian target of rapamycin protein kinase; S6K1, 70-kDa ribosomal protein S6 protein kinase; uORF, upstream open reading frame; UTR,
untranslated region.

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and 4E-BP1 (56). The mechanism by which phosphorylation of

TSC2 results in inhibition of TOR may involve dissociation
of the TSC1dTSC2 complex (62). Thus, phosphorylation of
TSC2 by PKB results in dissociation of the complex and rapid
degradation of the protein (63). Moreover, an exogenously
expressed variant of TSC2 wherein the two PKB phosphorylation sites were changed to acidic residues to mimic
phosphorylation does not bind to TSC1 and is unstable (62).
However, it should be noted that in a different study (64),
phosphorylation of TSC2 by PKB did not affect its ability to
bind to TSC1, which suggests that additional studies in this
area are warranted.
Further evidence implicating TSC1 and TSC2 in modulating signaling through mTOR is provided by studies in which
the expression of the proteins is exogenously enhanced or
suppressed. Suppressed expression of either TSC1 or TSC2
results in constitutive hyperphosphorylation and activation of
S6K1 that is sensitive to rapamycin (62,65,66), which supports
the idea that TSC1 and TSC2 act upstream of mTOR.
Furthermore, RNAi-mediated suppression of TSC2 expression
results in enhanced phosphorylation of mTOR on Ser-2448
(62). In contrast, when both proteins are simultaneously
overexpressed in human embryonic kidney (HEK)-293 cells,
phosphorylation of S6K1 and 4E-BP1 is low relative to control
cells (62,67). However, expression of either TSC1 or TSC2
alone has little if any effect on phosphorylation of S6K1 or 4EBP1, which indicates that the two proteins function as
a complex. The phosphorylation sites on S6K1 and 4E-BP1
that are affected by TSC1 and TSC2 overexpression are those
that have been demonstrated to be phosphorylated by mTOR
in vitro; e.g., Thr-389 on S6K1 and Thr-36, Thr-45, Ser-65 and
Thr-70 on 4E-BP1. Mutation of the PKB phosphorylation sites
on TSC2 to Ala enhances the ability of the protein to suppress

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raptor expression represses leucine-stimulated activation of

S6K1 (70).
In addition to regulating the activity of mTOR toward
downstream targets, raptor also may be a determinant in
controlling the sensitivity of mTOR to rapamycin. In S.
cerevisiae, five TOR-interacting proteins were recently identified: KOG1, LST8, AVO1, AVO2 and AVO3 (71). In yeasts,
the TOR2 isoform can bind to either KOG1 or the AVO
proteins, whereas LST8 is present in both the TORdKOG1 and
TORdAVO1dAVO2dAVO3 complexes. In contrast, the
TOR1 isoform only binds to KOG1 and LST8. Interestingly,
when bound to KOG1, the activities of TOR1 and TOR2 are
sensitive to inhibition by rapamycin, but when TOR2 is bound
to the AVO proteins, it is rapamycin insensitive. Thus, at least
two independent TORsignaling pathways exist in yeast: one
that is repressed by rapamycin and one that is not repressed.
Whether the rapamycin-independent signaling pathway is present in mammals is unknown. However, mammalian orthologs of the KOG1, LST8 and AVO1 proteins have been
identified: raptor, mLST8 and SAP kinase interacting protein
(SIN1), respectively, and both raptor and mLST8 coimmunoprecipitate with mTOR (71). Whether SIN1 interacts with
mTOR needs to be examined in future studies.
Although the precise mechanisms through which the TORinteracting proteins regulate the activity of the kinase have not
been determined, the available evidence supports a model
whereby TSC1 and TSC2 bind to mTOR and thereby suppress
phosphorylation of downstream targets such as 4E-BP1 and
S6K1. In this model, amino acids and insulin promote release of
the TSC1dTSC2 complex from mTOR and permit PKB to
phosphorylate the kinase on Ser-2448. In addition, release of
the TSC1dTSC2 complex allows mTOR to bind to LST1 and
raptor, which is an event that enhances the ability of the kinase
to phosphorylate 4E-BP1 and S6K1. Release of the
TSC1dTSC2 complex might also allow mTOR to bind to
SIN1 and result in activation of an undetermined rapamycininsensitive signaling pathway(s).
In summary, as is evident from the studies described, amino
acids regulate gene expression through multiple mechanisms
that include modulation of eIF2B activity, change in eIF4F
assembly,and alteration of phosphorylation of rpS6 (see Fig. 1).
In each case, the translation of mRNAs that encode individual
proteins is enhanced or repressed based upon specific structural
features present in the 59 untranslated region of the mRNA.
For example, in the case of rpS6 phosphorylation, mRNA that
contain a TOP sequence adjacent to the 59 cap structure are
preferentially translated. For those targets downstream of
mTOR such as rpS6, amino acidinduced translation of specific mRNA may also require that a basal amount of insulin (i.e.,
the concentration observed in plasma of fasted animals) be
available so that protein kinases upstream of mTOR are at least
minimally activated. The recent identification of proteins that
interact with and appear to modulate the activity and substrate
specificity of mTOR is an exciting development that in the
future may provide the answer to the as-yet unanswered
question of how amino acids regulate the function of targets
downstream of mTOR.

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