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INTRODUCTION |
Exposure of cells to UV light results in dramatic changes in the
spectrum and levels of gene expression. In prokaryotes most of the
genes induced encode DNA repair enzymes, but some encode proteins
involved in growth control (1, 2). Similarly, in eukaryotes, UV
radiation evokes expression of the DNA repair system (3-5) as well as
the cell cycle checkpoint machinery (6-8). Mammalian cells, in
addition to inducing those responses, also activate a battery of
transcription factors whose target genes are not directly involved in
DNA repair or cell cycle arrest. Many of these factors are members of
the AP-1 family, e.g. c-Jun, ATF2, and cAMP-response
element-binding protein (9-12). Their activation is obtained through
phosphorylation and is controlled by UV-responsive signal transduction
pathways (9, 10, 12-18). In addition to activation of AP-1 proteins,
UV radiation induces a dramatic increase in mRNA levels of
c-jun, c-fos, and ATF2 (9, 10, 12). Thus, both
AP-1 transcriptional activity and expression of AP-1 components are
elevated in response to UV radiation. The increase in mRNA levels
of AP-1 genes is mediated at least at two levels: through increased
transcription rate (9-12) and through an increase in mRNA
stability (19).
The biological role of UV-induced AP-1 activation is not entirely
clear. In some cell types it seems to be essential for apoptosis induction in damaged cells, whereas in other systems AP-1 activation plays a protective role (20-22). Paradoxically, AP-1 activity and expression are also induced by mitogenic signals (9, 12, 13, 15, 18,
23). Similar signal transduction components, including tyrosine kinase
receptors, Ras proteins, and mitogen-activated protein kinase cascades
activate AP-1 in response to both UV and growth signals (9, 10, 12, 13,
15, 18, 20, 23). It is still a puzzle how the same signal transduction
pathways and the AP-1 transcription activators respond to both UV and
growth factors and induce the appropriate but diverse biological responses.
Many aspects of the Ras signaling pathway are similar in yeast and
mammals. In the yeast Saccharomyces cerevisiae UV
irradiation stimulates the Ras signaling pathway and leads to increased
transcriptional activity of the yeast AP-1 factor Gcn4 (24). Gcn4 is a
functional homolog of c-jun (25). In yeast Ras proteins are
involved in regulation of intracellular cAMP, which is essential for
entering the "start" at G1 phase of cell cycle
(26-28). Glucose response in yeast is also regulated by Ras signaling.
Addition of glucose to glucose-starved cells causes a rapid and
dramatic increase in the intracellular cAMP concentration (26, 29, 30).
The increase in cAMP levels is transient and last 1-2 min. The
physiological role of this rapid and transient production of cAMP is
not fully understood because cells defective in this response are fully viable and show normal growth. Yet, a recent report suggested that the
transient induction in cAMP levels is important for efficient activation of glycolysis and reentry of cell cycle from
stationary phase (30).
Addition of glucose to glucose-starved cells has a dramatic effect on
gene expression. The most prominent effect is rapid suppression of
stress-related genes whose expression is elevated under glucose
starvation. Among these genes are some that encode stress-related
proteins, (e.g. heat shock proteins, enzymes that scavenge
free oxygen radicals, and enzymes involved in glycerol synthesis)
(31-33) and others that encode G1 cyclins (34, 35). Expression of both stress genes and G1 cyclins is elevated
under glucose starvation and rapidly suppressed upon addition of
glucose (32, 34, 35). Suppression of both stress genes and
G1 cyclins is regulated by the Ras/cAMP pathway. Another
effect of glucose is the induction of expression of ribosomal genes
(36, 37). Expression of these genes is elevated following glucose
induction and remains high. So far, genes whose pattern of expression
reflects directly the pattern of the cAMP response have not been
reported. Here we show that addition of glucose to glucose-starved
cells leads to a transient activation of the AP-1 factor Gcn4. This pattern of expression reflects faithfully the cAMP response. We show
that this transient activation is indeed dependent on the Ras/cAMP
pathway. Namely, both the UV signal (24) and the growth signal are
mediated through the same pathway. Activation of Gcn4 in response to
glucose is unexpected because Gcn4 is not known to play any role in
cell proliferation. Gcn4 is known in fact to be activated by stress
signals such as UV radiation and amino acid starvation (24, 38). Thus,
it seems that the paradox of AP-1 activation by both stress and growth
signals is evolutionarily conserved and may be addressed in yeast.
We also show that activation of Gcn4 target genes by either UV
radiation or glucose requires the GCN2 gene. GCN2
encodes a serine kinase whose sole known substrate is the translation
initiation factor eIF2
(39-41). Gcn2 phosphorylates eIF2
under
conditions of amino acid starvation (38-40). Phosphorylation of
eIF2
suppresses its activity and consequently reduces the cellular
translation activity. The resulting cease in translation is a
protective response that provides ample time for the cell to activate
endogenous biosynthesis of amino acids. Those biosynthetic pathways are
not active in media supplemented with amino acids and are
activated under amino acid starvation following induction of Gcn4
expression (38, 40). Most if not all Gcn4 target genes encode amino
acids and purine biosynthetic enzymes. Increased expression of Gcn4
during the period of amino acid starvation is achieved through an
unusual mechanism that increases translation of GCN4
mRNA when eIF2
is phosphorylated (40, 41). Thus, when
translational activity in the cell is mostly suppressed,
GCN4 translation is specifically increased. As we show in
this study however, activation of Gcn4 in response to UV radiation or
glucose is not mediated through an increase in GCN4
translation, suggesting that GCN2 functions in these
responses through another, novel mechanism.
Based on our results we suggest that cAMP coordinates activation of
carbohydrate metabolism and energy production with synthesis of amino
acids and nucleotides prior to re-start of cell proliferation.
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EXPERIMENTAL PROCEDURES |
Yeast Strains and Growth
Conditions--
Yeast strains used are
described in Table I.
YPD1 medium is composed of
2% glucose, 1% yeast extract and 2% Bacto Peptone. YNB
minimal medium is composed of 0.17% yeast nitrogen base without amino
acids and ammonium sulfate, 0.5%
NH4(SO4)2, 2% glucose, and the
required amino acids or nitrogenous bases. For glucose-response experiments cultures were grown to logarithmic phase
(A600 = 0.5) on YPD medium, collected,
and resuspended in the same volume of YPD medium containing 0.01%
glucose instead of 2% (42). Cultures were further grown for about
17 h. Then glucose (or other sugars as indicated in particular
experiments) were added from a stock solution of 20% to reach
concentration of 2%. Samples were collected by centrifugation at the
indicated time points, frozen immediately in liquid nitrogen, and
stored at
70 °C prior to RNA or lysate preparations or frozen at
20 °C prior to
-galactosidase assay. Yeast cultures were
exposed to UV radiation as previously described (24).
RNA Preparation and Analysis--
Frozen pellets were thawed on
ice, and total RNA was prepared and analyzed by primer extension as
previously described (43). Specific primers for the genes analyzed are
described in Stanhill et al. (44).
Preparation of Protein Lysates and Western Blot
Analysis--
Protein lysates were prepared using a modified
trichloroacetic acid precipitation protocol (45). Frozen pellets
were washed with 20% trichloroacetic acid and resuspended in 200 µl
of trichloroacetic acid 20%. 600 mg of glass beads were added, and the
cells were vortexed two times for 4 min. After vortexing the
supernatant was transferred to a new Eppendorf tube, and the beads were
washed twice with 200 µl of trichloroacetic acid 5%. The
supernatants were combined and centrifuged for 10 min at 3000 rpm. The
pellet was suspended in 200 µl of 2x sample buffer followed by an
addition of 100 µl of Tris base 1 M. The samples were
vortexed for 30 s, boiled for 3 min, and cleared by centrifugation
(10 min at 3000 rpm). The lysates were stored in aliquots at
20 °C
Protein concentrations were determined using a modified Bradford assay.
Briefly, protein aliquots were loaded on 3-mm Whatman filter papers in
parallel with 5-25 µg of bovine serum albumin calibration curve. The
samples were dried, stained for 30 min in Coomassie staining solution
(0.25% Coomassie Blue, 10% acetic acid, and 40% methanol), washed
with destaining solution (7% acetic acid and 20% methanol), and
dried. Color was eluted in 500 µl of 3% SDS solution and the
absorbance at A590 was determined.
For Western blot analysis protein samples were separated on 10%
SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose paper using LKB (Sweden) semi-dry blotter. Nitrocellulose papers were
incubated with the appropriate antibodies. Polyclonal anti-Gcn4 antibodies were obtained from Daniel Kornitzer (Technion, Haifa, Israel) and were used in a 1:15,000 dilution. Polyclonal anti-eIF2
antibodies were obtained from Ronald Wek (Indiana University, Indianapolis, IN) and were used in a 1:10,000 dilution.
Anti-phospho-eIF2
antibodies were purchased from Research Genetics
and used in a 1:10,000 dilution.
Plasmids and
-Galactosidase Assay--
The
GCN4-LacZ plasmid used is the p180I, which is an integrated
version of p180 (46). The construction of p180I was described previously (24). p180I was digested with SmaI prior to
transfection of yeast cells. Disruption of GCN2 was obtained
using a gcn2::LEU2 construct obtained
from A. Hinnebusch (National Institutes of Health). Disruption of
GPA2 was obtained by using a
gpa2-1::TRP1 construct obtained from
J. P. Hirsch. SP1 yeast culture was transformed with a
1.4-kilobase BamHI fragment of the
gpa2-1::TRP1 plasmid.
-Galactosidase assays were performed following lysis of cells in
SDS/chloroform as described (47). Each assay was repeated at least two
times in duplicate.
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RESULTS |
Transcription of HIS4 Is Activated by Growth Signals--
Activity
and expression of the mammalian c-Jun transcriptional activator are
induced in response to either stress signals such as UV radiation or in
response to growth stimulation (9, 15, 20). The yeast Gcn4 activity is
induced in response to stresses such as amino acid starvation or UV
radiation (24, 38). To test if, similar to c-Jun, its yeast homolog
Gcn4 is also activated in response to growth signals, we tested the
mRNA levels of HIS4, a major Gcn4 target gene (48)
following addition of glucose to glucose-starved cells. As shown in
Fig. 1A, upon addition of
glucose there is a sharp and transient increase in HIS4
expression (peaks at about 20'). Thus, HIS4, known to be induced by stresses such as amino acid limitation and UV radiation, is
induced here by a growth signal.

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Fig. 1.
Transcription of HIS4 is
transiently induced upon addition of fermentable sugars to
glucose-starved cells. A, primer extension analysis of
RNAs isolated from: (i) cells grown at logarithmic phase just before
their transfer to starvation medium (Log. phase); (ii) cells
maintained for 17 h under glucose starvation
(starvation); and (iii) same cells after glucose was added.
Specific primers for HIS4, HSP26, and ACTIN were
used. B, HIS4 transcription is not induced by
sugars that are not metabolized. Primer extension analysis of mRNA
prepared from cells treated as in A except that after
starvation the culture was divided to four and each fraction was
treated with a different sugar (glucose, fructose, xylose, or
2-deoxyglucose). C, schematic presentation of the three
patterns of gene expression observed in response to glucose
addition.
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The possibility remains, however, that a rapid increase in external
glucose concentration could be sensed in fact as a stress signal, such
as osmotic shock, rather than as a growth signal. To verify this point
we added to glucose-starved cells several sugars: glucose and fructose
that are easily metabolized and support growth and xylose and
2-deoxyglucose that enter the cell but cannot be used metabolically
(29). If HIS4 induction is a response to stress such as
osmotic shock, it should be observed in response to all sugars. Yet, as
is apparent in Fig. 1B, only the metabolizable carbon
sources, glucose and fructose, elicited HIS4 transcription suggesting that HIS4 responds to growth stimulation and not
to stress signals. This result also shows that glucose transport is not
sufficient for Gcn4 activation. Further metabolism is required.
Notably, all experiments were performed using YPD medium, and only
glucose concentrations were manipulated (for starvation 0.01% glucose
was used). As all other nutrients including amino acids are not
limited, induction of HIS4 is unexpected and intriguing (see below).
The transient mode of HIS4 expression is somewhat unusual
and reveals a novel pattern of gene expression in response to glucose. Previous studies described glucose-dependent suppression of
transcription of stress genes and G1 cyclins (26, 32-35).
We also observed rapid suppression of stress genes following addition
of glucose in our system. The level of HSP26 mRNA for
example that is increased upon glucose starvation decreases rapidly
after addition of glucose (Figs. 1 and
2). Another type of glucose response is
the slow, but continuous increase in expression of many genes. This
type of response is manifested here by the steady increase in the
levels of ACTIN mRNA (Fig. 1A and 2). Thus,
it seems that at least three responses could be measured at the level
of gene expression following addition of glucose to starved cells (Fig.
1C): (i) rapid suppression of stress genes and
G1 cyclins, (ii) slow induction of structural and
proliferative genes, and (iii) rapid and transient increase in Gcn4
target genes.

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Fig. 2.
The transient induction of HIS4
expression in response to glucose is mediated through the
RAS/cAMP pathway and the Gcn4 transcriptional activator. The
effect of glucose on HSP26 and ACTIN mRNA on
the other hand is not mediated via the Ras/Gcn4 system. Cells of the
ras2 (A), gpa2 (B)
and gcn4 (C) strains were starved to glucose
for about 17 h when glucose was added again. Experimental
procedures were identical to those performed with wild type cells (Fig.
1A). RNA was isolated at the indicated time points and
analyzed by the primer extension method.
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Glucose-dependent HIS4 Induction Is Mediated via the
Ras/Gcn4 Cascade--
Addition of glucose to glucose-starved cells is
known to induce rapid activation of the Ras/cAMP pathway that is
manifested in a transient increase in cAMP levels (29, 33). As the
kinetic pattern of HIS4 expression following addition of
glucose is strikingly similar to the pattern of changes in cAMP
concentrations, it could be that the Ras/cAMP system is involved in
glucose-induced Gcn4 activation. To test this possibility we measured
glucose-dependent induction of HIS4
transcription in isogenic strains deleted in either RAS2 or
GPA2 (Fig. 2, A and B).
GPA2 encodes the
subunit of a heterotrimeric G protein
that has been shown recently to function either upstream or parallel to
Ras2 (33). The results presented in Fig. 2, A and
B clearly show that the mutants are unable to induce
HIS4 transcription in response to addition of glucose. Thus,
induction of HIS4 expression in response to glucose is
mediated via the Ras pathway. The unexpected observation that HIS4 is activated in response to a growth stimulus in the
presence of amino acids raises the possibility that under these
conditions it is not induced by Gcn4. To test this idea we measured
HIS4 induction in gcn4
cells. In these cells
the low levels of HIS4 mRNA did not change when glucose
was added to glucose-starved cells (Fig. 2C). Notably, the
RAS2, GPA2, and GCN4 genes are also essential for
HIS4 induction in response to UV radiation (Ref. 24 and data
not shown).
In contrast to their effect on HIS4 activation, the
ras2
, gpa2
, and gcn4
mutations had no effect on the other glucose-mediated responses. The
reduction in HSP26 mRNA and elevation of
ACTIN mRNA following addition of glucose were intact in
all strains analyzed (Fig. 2, A-C), suggesting that the
ras2
, gpa2
, and gcn4
cells are specifically defective in only one of the
three measured glucose responses.
Activation of Gcn4 by a mitogenic signal through the mitogenic Ras/cAMP
cascade is unexpected because this transcriptional activator is known
to be activated by stresses such as amino acid starvation. To test
whether the Ras signaling pathway is also involved in Gcn4 activation
in response to amino acid starvation, we monitored HIS4
induction in the various mutants in response to amino acid starvation
(Fig. 3, A-D). As expected,
the gcn4
strain was not able to induce HIS4
under these conditions, similar to its inability to induce
HIS4 in response to glucose (Fig. 3D). The
response of gpa2
cells, however, was indistinguishable
from the wild type response (compare Fig. 3A to
3C). The response of ras2
cells to amino acid
starvation was somewhat delayed but reached wild type levels of
HIS4 mRNA (Fig. 3B). Thus, unlike the case of
the glucose response, amino acid starvation-mediated Gcn4
activation is independent of the Ras pathway.

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Fig. 3.
The Ras cascade is dispensable for induction
of HIS4 in response to amino acids starvation.
RNA was prepared from wild type (A), ras2
(B), gpa2 (C), and
gcn4 (D) cells. Cells were grown on rich YPD
medium to A600 = 0.5, collected, washed with
water, and split at time 0. Half of each culture was resuspended in the
same volume of rich medium (YPD) and the other half was resuspended in
amino acid starvation medium (YNB without histidine, supplemented with
20 mM 3-aminotriazole (3-AT)). Cultures were
allowed to grow, and samples were removed from both halves for RNA
preparations at the indicated time points. RNA was analyzed by the
primer extension method.
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Glucose-dependent HIS4 Induction Is Not Mediated
through GCN4 Translation--
Having verified that
glucose-dependent Gcn4 activation requires the Ras/cAMP
pathway, we sought more components of the Ras/Gcn4 pathway in
particular those that function between the Ras/cAMP cascade and Gcn4.
The main mechanism known to induce Gcn4 activation/expression is
activated by amino acid starvation and involves Gcn2-mediated increase
in translation of GCN4 mRNA (40, 41). Yet, there must be
other mechanisms because in the RAS2val19 and
bcy1
strains, the constitutive activity of Gcn4 is
explained by a cooperation of two mechanisms: 1) a moderate increase
(~2.5-fold) in GCN4 translation and 2) another, unknown
mechanism that operates posttranslationally (24). We tested if the
increase in HIS4 mRNA following addition of glucose may
be a consequence of elevated translation of GCN4. To this
end we used a strain harboring an integrated copy of the
GCN4-LacZ fusion gene (46) (provided by A. Hinnebusch, NIH).
This construct contains the promoter 5'UTR and 153 base pairs of
the GCN4 coding sequence fused to
-galactosidase. As
GCN4 transcription is constitutive,
-galactosidase
activity derived from this construct reflects GCN4
translation (41, 46). Surprisingly, the activity of this construct that
was very low on YPD not only was not reduced, as was expected from the
decrease in HIS4 mRNA (Fig. 1A), but was even
somewhat increased upon glucose starvation (Fig.
4A). An increase in
GCN4-LacZ activity under glucose starvation was also
observed recently by Yang et al. (49). Namely,
GCN4 translation increased under conditions that caused suppression of HIS4 transcription (Figs. 1 and 2).
Furthermore
-galactosidase activity of this construct did not change
when glucose was provided and Gcn4 was activated (Fig. 4A),
suggesting that GCN4 translation was not correlated to
HIS4 transcription (Fig. 1). To verify that the
GCN4-lacZ construct that was integrated in the cells is
intact and responsive, the same culture was starved not for glucose but
for amino acids. Fig. 4B shows that under these conditions
GCN4-LacZ activity, reflecting GCN4 translation, was induced ~10-fold. This increase in GCN4 translation is
well correlated with the increase in HIS4 transcription
(Fig. 3A).

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Fig. 4.
Induction of HIS4 mRNA
by glucose response is not mediated through increase in GCN4
translation nor through increase in Gcn4 levels.
A, -galactosidase activity of the GCN4-LacZ
reporter gene integrated in the genome of the wild type SP1 strain.
Samples for -galactosidase assay were collected from logarithmically
growing cells (Logarithmic Phase), from cells under glucose
starvation (Starvation), and from same cells after glucose
was added. B, GCN4 translation increases
dramatically in response to amino acid starvation. Same cells used in
A were grown on rich YPD medium to logarithmic phase,
collected, and divided to three cultures. One culture was resuspended
in YPD, one in YNB, and one in YNB without histidine supplemented with
20 mM 3-AT. Samples for -galactosidase assay were
removed at the indicated time points. C, Western blot
analysis of Gcn4 proteins. Yeast lysates were prepared from
logarithmically growing cells (of the wild type strain SP1), from
glucose-starved cells, and at the indicated time points after glucose
was added. As positive controls, lysates were prepared from cells of
the same strain treated with 3-AT (eighth lane from the
left), from cells of the GCN4c
strain (24) in which GCN4 is constitutively translated, and
from cells of the RAS2val19 strain in which
GCN4 translation is partially elevated (24). Lysate prepared
from gcn4 cells was used as a negative control (tenth
lane from the left). Lysates were separated on 10%
SDS-polyacrylamide gel electrophoresis and blotted to nitrocellulose
paper, which was reacted with polyclonal anti-Gcn4 antibodies as
described under "Experimental Procedures."
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To show unequivocally that Gcn4 expression is not increased upon
addition of glucose, we measured directly through Western blot analysis
the level of endogenous Gcn4p (antibodies were kindly provided by
Daniel Kornitzer). This analysis shows that Gcn4p levels remain very
low during glucose starvation and following addition of glucose (Fig.
4C). We found Gcn4 levels to increase only in response to
amino acid starvation (lane 8 in Fig. 4C) in the
RAS2val19 strain (lane 11 in Fig.
4C) as expected (24) and in the
GCN4c strain in which GCN4
mRNA is constitutively translated (lane 9 in Fig.
4C). The results of the Western blot agree with the results
obtained with the GCN4-LacZ constructs. Taken together, these results show that in response to the addition of glucose, Gcn4
increases HIS4 transcription via a mechanism that does not involve an increase in GCN4 translation.
Activation of Gcn4 by Glucose Is a General Phenomenon That Occurs
in All Laboratory Strains--
The results above described two
unexpected findings: (i) Gcn4 is activated by growth signals and (ii)
this activation is obtained via a mechanism that is independent of
GCN4 translation. These unexpected observations raised the
concern that the phenomenon is restricted to one genetic background
that may be utilizing a peculiar mechanism for Gcn4 activation. To test
whether activation of Gcn4 by glucose is a general phenomenon in yeast,
we tested a battery of commonly used laboratory strains for their
ability to activate HIS4 transcription in response to
glucose. Cells of the W303,
1278b, and H4 (a derivative of the S288C
strain) genetic backgrounds were grown on YPD medium, starved for
glucose for 17 h before readdition of glucose. As is shown in Fig.
5, transcription of HIS4 was
suppressed upon glucose starvation and rapidly increased when glucose
was added in all the strains. Thus, activation of Gcn4 target genes in
response to glucose was measured in all laboratory strains tested. It
is interesting to note that the pattern of HIS4 activation
is different in each genetic background. In the
1278b background
(strain
L5527LH) for example, glucose-induced increase in
HIS4 mRNA is sustained and not transient (Fig.
5B). This result is expected because in the
1278b genetic
background HIS4 mRNA levels are high during exponential
growth even on YPD medium, similar to the situation in
RAS2val19 mutants (44). This is a consequence of
the high levels of cAMP in the
1278b background (44). Another
reflection of the high cAMP levels in
L5527LH cells is suppression
of the stress response (note HSP26 levels in Fig.
5B and Ref. 44).

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Fig. 5.
Glucose-induced Gcn4
activation is a general phenomenon measured in a variety of
laboratory strains. A similar glucose response experiment that was
performed with cells of the SP1 strain (Fig. 1A) was
repeated with cells of strains W303 (A), L5527LH (of the
1278b background) (B), and H4 (a derivative of S288C)
(C). RNA was prepared at the indicated time points and
analyzed by primer extension using the specific HIS4 and
HSP26 primers.
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To test whether in all genetic background Gcn4 activation and
HIS4 transcription are not correlated with GCN4
translation, we integrated the GCN4-LacZ gene into the
genome of the strains shown above as well as into the EG328-1A (used
by Yang et al. (49)) and ras2
strains. As is
shown in Fig. 6, in all genetic backgrounds GCN4-LacZ activity increased upon glucose
starvation (when HIS4 transcription is almost totally
suppressed, Fig. 5) and did not change when glucose was added. The
observed increase in GCN4-LacZ activity upon glucose
starvation is in agreement with the observation made by Yang et
al. (49). Thus, induction of Gcn4 activity by glucose via a
mechanism independent of GCN4 translation was measured in
all yeast strains. Even the ras2
strain, which does not
induce HIS4 in response to glucose (Fig. 2A),
showed GCN4-LacZ activity similar to that of its parental wild type strain (Fig. 6), emphasizing the lack of correlation between
HIS4 induction and GCN4-LacZ activity in response
to glucose.

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Fig. 6.
In all laboratory strains tested,
glucose-dependent increase in HIS4
transcription is not mediated through GCN4
translation. -Galactosidase activity was measured in
cells of the laboratory strains SP1, W303, L5527LH, H4, and
EG328-1A harboring an integrated GCN4-LacZ reporter gene.
Cells of the ras2 strain, which is incapable of
HIS4 induction in response to glucose (Fig. 2A),
were also included in this experiment. Samples for -galactosidase
assay were collected from cells grown under the indicated growth
conditions. Minutes refer to time after glucose addition to
glucose-starved cells.
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GCN2 Is Essential for Glucose- and UV-dependent HIS4
Induction--
Unlike the situation described here, under conditions
of amino acid starvation GCN4 expression is induced at the
translational level. This induction requires Gcn2-mediated
phosphorylation and inhibition of eIF2
(39-41, 49). As we showed
previously (24), in the bcy1
strain GCN2 is
not required for the high and constitutive HIS4 expression.
To further verify that Gcn2 is not required for Ras/cAMP-dependent Gcn4 activation, we disrupted the
GCN2 gene in the RAS2val19 strain and
in wild type cells. Similar to the case of the bcy1
strain, deletion of GCN2 in the
RAS2val19 strain did not abolish constitutive
HIS4 expression, although it was somewhat reduced (Fig.
7A). Thus, in mutants
harboring a constitutively active Ras pathway, GCN2 is not
an essential mediator of the constitutive Gcn4 activity. However,
deletion of GCN2 in wild type cells destroyed their
capability to induce HIS4 in response to glucose or UV
radiation (Fig. 7, B and C). Thus, although
GCN2 is not required for HIS4 expression in
RAS2val19 and bcy1
strains, it is
required for induction of the Ras/Gcn4 pathway by external signals. The
requirement of GCN2 for the transmission of these signals is
further intriguing because GCN4 translation is not elevated
in response to glucose or UV radiation (Figs. 4 and 6 and data not
shown). It seems therefore that Gcn2 is required for the UV and glucose
responses but functions through a novel mechanism. Under amino acid
starvation Gcn2 phosphorylates and inhibits eIF2
and thereby
increases GCN4 translation (40). To verify that in response
to glucose Gcn2 functions in a different way, we tested the level of
phospho-eIF2
during the course of glucose response. Fig.
8 depicts the results of a Western blot analysis showing that eIF2
is somewhat phosphorylated upon
starvation to glucose. This observation explains the increase in
GCN4 translation under these conditions (Figs. 4A
and 6) and is in agreement with previous observations (49). Yet, it is
not correlated with HIS4 transcription that is
suppressed under glucose starvation (Fig. 1). Furthermore, no changes
in eIF2
phosphorylation were measured upon addition of glucose (Fig.
8), which caused strong induction of Gcn4-dependent
HIS4 expression (Figs. 1 and 2). Thus, Gcn2 mediates Gcn4
activation in response to UV or glucose through a mechanism that does
not involve eIF2
phosphorylation and GCN4 translation.
This is the first indication of another mechanism of action of
Gcn2.

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Fig. 7.
GCN2 is essential for glucose and UV
induction of HIS4 but not for
Ras2Val19-mediated HIS4 induction.
A, primer extension analysis of RNAs prepared from the
RAS2val19 and the
RAS2val19gcn2 strains grown on YPD
to logarithmic phase. B, analysis of HIS4 and
ACTIN mRNA levels in gcn2 cells grown in
logarithmic phase (Log. phase), exposed to glucose
starvation (starvation), and at different time points after
glucose was added. C, primer extension analysis of
RNAs prepared from cells of the SP1 and gcn2 strains at
various time points after exposure to UV radiation (40 joules/m2).
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Fig. 8.
Induction of HIS4
transcription in response to glucose is not correlated with
changes in eIF-2 phosphorylation. Shown
are Western blots in which the levels of phosphorylated and
nonphosphorylated eIF-2 were analyzed. Lysates were prepared from
cells of the SP1 strain at the indicated time points prior to and after
addition of glucose to glucose-starved cells. Cells from another
culture that was exposed to 3-AT treatment were used as a positive
control (compare YPD to 3-AT).
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DISCUSSION |
In this study we describe a novel glucose response in yeast at the
level of gene expression. We show that addition of glucose to
glucose-starved cells caused a transient increase in the level of
HIS4 mRNA, a target gene of Gcn4 transcription factor
(Fig. 1). We verified that glucose-dependent
HIS4 induction is indeed mediated through Gcn4 (Fig.
2C). This is an unexpected finding because Gcn4 is usually
activated not by growth stimuli but rather by stress conditions. Yet,
the findings described here are reminiscent of the situation in
mammalian cells where AP-1 factors are activated by either stress or
growth stimuli (9, 12, 15, 18). Thus, our results show that activation
of AP-1 factors by these contradictory signals is conserved from yeast
to mammals. This conservation points at the importance of AP-1
activation by these signals but does not explain the paradox of
induction of an identical response by mitogenic signals on one hand and
by stress signals on the other hand.
Induction of Gcn4 in response to amino acid starvation is explained
readily because it leads to activation of de novo
biosynthetic pathways of amino acids. It could be that the same
biosynthetic pathways need to be activated when glucose becomes
available after starvation to ensure availability of amino acids and
nucleotides for reinitiation of cell cycle. These biosynthetic pathways
seem to be suppressed under glucose starvation as is manifested by the
undetectable levels of HIS4 mRNA under these conditions
(Figs. 1 and 2). The question remains however, why is Gcn4 induced in medium so rich in amino acids? It could be that amino acid permeases and sensors are shut off under glucose starvation (to ensure
maintenance of G1 arrest) so that the cells sense in fact
some degree of amino acid limitation. The increase in
GCN4-LacZ activity under glucose starvation (Figs.
4A and 5B; and Ref. 49) supports this notion. Addition of glucose, which was the only limiting factor in the medium,
leads to induction of biosynthetic pathways as is manifested by the
rise in GCN4 target genes (Figs. 1 and 5), but
simultaneously it may reactivate the amino acid permeases.
Consequently, the high concentrations of nutrients in YPD medium
immediately suppress the cascades again. This model explains the
transient mode of the response. Such a pattern of transient response to
glucose has not been described previously at the transcriptional level but only at the enzymatic level showing activation of Ras and adenylyl
cyclase (26, 29, 33). As we showed here the two responses are connected
(Fig. 2; see below). In fact, the case shown here for HIS4
is the first example for a gene whose expression reflects the cAMP response.
The effect of glucose addition on Gcn4 activity points at a previously
unidentified link between glucose signaling and amino acids and purine
biosynthesis. Clearly, to resume growth after starvation cells must
produce ATP but concomitantly have to synthesize nucleotides and amino
acids. It seems that the glucose-induced cAMP burst orchestrates
coinduction of glucose metabolism for the production of energy (26, 30)
and amino acids/purine metabolism as is shown in this work. Most
interestingly however, the transient rise in cAMP is essential but not
sufficient for Gcn4 activation. Only metabolized sugars such as glucose
or fructose activate Gcn4 (Fig. 1 A and B),
whereas sugars that are transported to the cell but not metabolized,
like xylose and 2-deoxyglucose, do not activate Gcn4 (Fig.
1B). These non-metabolizable sugars were shown to cause a
transient or permanent rise in cAMP (29). Similarly, deletion of the
RAS2 gene has no effect on the transient rise in cAMP (54) but dramatically suppresses Gcn4 activation (Fig. 2). Thus sugar metabolism and intact Ras2, which are not essential for the cAMP response, are essential for Gcn4 activation.
Activation of Gcn4 in response to UV radiation (Ref. 24 and Fig. 6) may
be required more specifically for induction of nucleotide biosynthesis
(needed for DNA repair). However, also in the case of UV radiation it
could be that an immediate cellular protective response is the
suppression of amino acid transport (as part of the checkpoint/growth
arrest system that provides time for repair). Consequently, the cells
rely on de novo synthesis until permeases are activated
again. Gcn4 activation in response to UV is indeed not transient and
HIS4 mRNA levels remain high for at least 75 min after
irradiation (24). Thus, activation of Gcn4 by contradictory stimuli may
seem less paradoxical if de novo synthesis of amino acids
and purine is required for appropriate cellular response for each of
those signals. It is obvious that Gcn4 activation is just one aspect of
the complex cellular response to a given stimulus, implying that not
Gcn4 activation alone but the particular combination of responses to
each stimulus (UV, glucose, amino acid starvation) determines the
appropriate overall biological phenotype. Similar to the case described
here for Gcn4, the mammalian c-Jun is also activated by either growth
stimuli or stress signals. It is tempting to suggest that a similar
explanation would resolve the paradox in mammalian cells too. Yet, most
c-Jun target genes are currently unknown and it is difficult to predict
if c-Jun induces its various responses via the same set of genes.
Determination of the subset of c-Jun target genes induced in response
to each stimulus is required to test this idea.
As each of the different signals that activate Gcn4 is sensed and
transmitted via a specific signal transduction pathway, it seems that
Gcn4 is recognized by many cascades. Indeed, in the case of the UV- and
glucose-response, it is the Ras/cAMP pathway that activates Gcn4,
whereas in response to amino acid and purine starvation this pathway is
dispensable for Gcn4 activation. Under these conditions Gcn4 is
activated by the Gcn2/eIF2
machinery. The latter pathway also
activates GCN4 translation in response to glucose starvation
(49). Strikingly, the increased expression of Gcn4 under glucose
starvation does not result in transcription of HIS4. In
fact, HIS4 mRNA is barely measurable under glucose starvation (Figs. 1, 2, and 5A). This result suggests that
an increase in Gcn4 expression is not always correlated with Gcn4 transcriptional activity. There must be regulators that do not affect
Gcn4 expression, but rather affect Gcn4 activity. Such a
regulator may be for example the multiprotein bridging factor 1 (Mbf1).
Mbf1 functions as a mediator between Gcn4 and the basal transcriptional
machinery (50). Its expression is essential for Gcn4 transcriptional
activity and for cell growth under amino acids starvation. It is not
known how Mbf1 is regulated or to which external signal it may respond.
Another Gcn4 regulator is the Cpc2 repressor that suppresses Gcn4 under
optimal growth conditions (51). It is not known how Cpc2 is inactivated
to derepress Gcn4 activity. Analysis of Cpc2 effect on Gcn4 points at a
novel mechanism of Gcn4 regulation that is not mediated through Gcn4
translation (51). Our findings also suggest a mechanism that activates
Gcn4 not through GCN4 translation (Figs. 4 and 5). Deletion
of Cpc2 in gcn2
cells restores the expression of Gcn4
target genes and the ability to grow under amino acids starvation (51).
This result may suggest that Cpc2 is epistatic to Gcn2, but further studies are obviously required to reveal the relationships (if any)
between Gcn2, Mbf1, Cpc2, and/or yet unknown regulators of Gcn4. These
studies should also reveal which of those Gcn4 regulators is responsive
to the Ras/cAMP cascade and mediates Gcn4 activation in response to
glucose and UV radiation.
The function of Gcn2 in the glucose response is rather peculiar.
Although it is essential for transmitting the signal to Gcn4 (Fig. 6,
B and C), translation of GCN4 is not
induced (Fig. 4, A and C) and phosphorylation of
eIF2
is not changed (Fig. 7). It may be that Gcn2 phosphorylates and
modulates a yet unknown substrate, maybe one of Gcn4 coactivators or
transcriptional mediators. Alternatively, Gcn2 may function through
protein-protein interactions. It was recently suggested that the
mammalian homolog of Gcn2, PKR, may recognize more substrates in
addition to eIF2
and was shown to physically interact with but not
necessarily phosphorylate Stat1 and p53 (Ref. 52 and reviewed in Ref.
53). In any case, Gcn2 functions in the glucose- and UV-response in a
novel way that should be explored to fully understand the cellular
response to these important signals.