EUKARYOTIC CELL, Nov. 2006, p. 1831–1837
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 5, No. 11
Nitrogen Availability and TOR Regulate the Snf1 Protein Kinase in
Marianna Orlova,1Ellen Kanter,2† David Krakovich,1and Sergei Kuchin1*
Department of Biological Sciences, University of Wisconsin—Milwaukee, Milwaukee, Wisconsin 53211,1and
Department of Genetics and Development, Columbia University, New York, New York 100322
Received 17 April 2006/Accepted 1 September 2006
In the yeast Saccharomyces cerevisiae, the Snf1 protein kinase of the Snf1/AMP-activated protein kinase
(AMPK) family regulates a wide range of responses to stress caused by glucose deprivation. The stress signal
is relayed via upregulation of Snf1, which depends on phosphorylation of its activation loop Thr210 residue by
upstream kinases. Although Snf1 is also required for coping with various stresses unrelated to glucose
deprivation, some evidence suggests a role for low-level basal activity of unphosphorylated Snf1, rather than
a specific signaling function. We previously found that Snf1 is required for diploid pseudohyphal differenti-
ation, a developmental response to nitrogen limitation. Here, we present evidence that Snf1 is directly involved
in nitrogen signaling. First, genetic analyses suggest that pseudohyphal differentiation depends on the stim-
ulatory phosphorylation of Snf1 at Thr210. Second, immunochemical data indicate that nitrogen limitation
improves Thr210 phosphorylation. Analyses of pseudohyphal differentiation in cells with catalytically inactive
and hyperactive Snf1 support the role of Snf1 activity. Finally, we show that Snf1 is negatively regulated by the
rapamycin-sensitive TOR kinase which plays essential roles in signaling nitrogen and amino acid availability.
This and other evidence implicate Snf1 in the integration of signals regarding nitrogen and carbon stress. TOR
and Snf1/AMPK are highly conserved in evolution, and their novel functional interaction in yeast suggests
similar mechanisms in other eukaryotes.
The Snf1/AMP-activated protein kinase (AMPK) family is
highly conserved in eukaryotes, and its members are involved
in effecting responses to cellular stress. In mammalian cells,
AMPK is activated by increased AMP:ATP ratios and controls
responses to stimuli that affect the cellular energy supply. Ev-
idence implicates the AMPK pathway in type 2 diabetes, obe-
sity, cardiac disorders, and tumorigenesis (for reviews, see ref-
erences 6, 18, 19, and 32). In the yeast Saccharomyces
cerevisiae, the Snf1 protein kinase is required for multiple as-
pects of transcriptional and metabolic adaptation to reduced
levels of available glucose, the preferred source of carbon and
energy (7, 15). Snf1 is not simply required for growth on al-
ternative carbon sources but plays a direct role in glucose
signaling, as its function is regulated by glucose availability.
Maximal catalytic activation of Snf1 requires phosphorylation
of its conserved activation loop Thr210 residue (14) by up-
stream kinases, and cellular levels of phospho-Thr210-Snf1
increase dramatically upon glucose deprivation (44). Three
Snf1 protein kinase kinases, Sak1 (Pak1), Tos3, and Elm1,
have been identified and are related to the mammalian tumor
suppressor kinase LKB1 and Ca2?/calmodulin-dependent pro-
tein kinase kinases, which activate AMPK by phosphorylation
of the cognate Thr172 residue (21, 22, 25, 26, 28, 45, 46, 64, 74).
Dephosphorylation and downregulation of Snf1 depend on
type 1 protein phosphatase Glc7 in association with its specific
targeting protein, Reg1 (44, 66, 67). The exact mechanisms by
which glucose modulates the levels of Thr210 phosphorylation
The Snf1 protein kinase functions as a heterotrimeric com-
plex containing the catalytic ? subunit Snf1, the stimulatory ?
subunit Snf4, and one of three alternative ? subunits, Sip1,
Sip2, or Gal83, which define three forms of the Snf1 complex
(30, 75). All three forms of the complex are catalytically acti-
vated on limiting glucose and perform overlapping and distinct
functions (1, 23, 36, 43, 58, 68, 70, 71).
Although Snf1 is also required for coping with a number of
stresses unrelated to glucose limitation, its involvement does
not automatically indicate the existence of a specific Snf1 sig-
naling cascade. As with glucose limitation, activation of Snf1 by
Thr210 phosphorylation was observed under conditions of so-
dium stress, suggesting a signaling mechanism (44). By con-
trast, evidence suggests a role for basal activity of unphospho-
rylated Snf1 in providing resistance to hydroxyurea and
hygromycin B (13, 50). We previously found that Snf1 is re-
quired for diploid pseudohyphal (PH) differentiation (34), a
filamentous-growth response to nitrogen limitation (16). The
requirement of Snf1 for a nitrogen-regulated phenotype sug-
gested a role in nitrogen signaling, but it remained possible
that Snf1 contributes at a basal level of activity.
Signaling mechanisms that regulate PH differentiation have
been extensively studied and involve the function of several
protein kinase pathways. The cyclic AMP-dependent protein
kinase (PKA) and mitogen-activated protein kinase (MAPK)
pathways are required for PH differentiation under conditions
of limiting nitrogen (40, 49, 53), and the Srb10-Srb11 (Cdk8-
cyclin C) kinase functions to inhibit PH differentiation under
nitrogen-rich conditions (47). Important roles are played by
* Corresponding author. Mailing address: Department of Biological
Sciences, University of Wisconsin—Milwaukee, 3209 N. Maryland
Ave., Milwaukee, WI 53211. Phone: (414) 229-3135. Fax: (414) 229-
3926. E-mail: email@example.com.
† Present address: Department of Neurology, Columbia University,
New York, NY 10032.
?Published ahead of print on 15 September 2006.
the rapamycin-sensitive TOR protein kinase pathway. The
TOR protein kinases are highly conserved in evolution and
function to signal nutrient availability and promote growth (for
reviews, see references 41 and 56). S. cerevisiae has two ho-
mologs, Tor1 and Tor2 (collectively referred to as TOR), ei-
ther of which can be employed by the rapamycin-sensitive
TOR complex called TORC1 (37). Inhibition of yeast TOR
with rapamycin broadly mimics the effects of nitrogen and
amino acid deprivation, including inhibition of translation and
ribosome biogenesis as well as greatly increased transcription
of genes regulated by nitrogen catabolite repression (for re-
views, see references 17, 51, and 54). Some of the regulators
negatively controlled by TOR in nitrogen-rich conditions, such
as the transcriptional activator Gln3, protein kinase Npr1, and
Mep2 ammonium permease/sensor, are known to play critical
positive roles in PH differentiation (2, 5, 20, 39, 57).
Here, we have further examined the role of Snf1 in the
regulation of PH differentiation and present evidence that Snf1
is directly involved in nitrogen signaling controlled by Thr210
phosphorylation. We also show that Thr210 phosphorylation is
negatively regulated by TOR.
MATERIALS AND METHODS
Strains and genetic methods. The S. cerevisiae strains used in this study are
listed in Table 1. The strains were in the ?1278b genetic background and were
descendants of wild-type strains MY1401 (MAT? ura3? leu2? his3?) and
MY1402 (MATa ura3? leu2? trp1?) of the Sigma2000 series (Microbia, Cam-
bridge, Mass.). Derivatives carrying snf1::LEU2, sip1?::KanMX6, and reg1?::
URA3 have been described previously (34, 71). To generate snf1?::KanMX6 and
reg1?::KanMX6, the KanMX6 sequence (38) was amplified by PCR with primers
flanking the corresponding open reading frames (72). The alleles sip2?3::LEU2
(75), gal83?::TRP1 (68), and snf1?::KanMX6 were introduced into wild-type
haploids by transformation; all yeast transformations were performed using stan-
dard methods (55). The reg1?::KanMX6 allele was first introduced into a wild-
type diploid, with subsequent recovery of haploid segregants by tetrad analysis.
Deletions were confirmed by PCR analysis of genomic DNA and by mutant
phenotypes. Strains carrying deletion combinations were constructed by genetic
crossing, and homozygous mutant diploids were obtained by mating appropriate
mutant haploids (55).
Construction of the chromosomal TOR1-S1972R allele. First, a 430-bp DNA
fragment corresponding to nucleotides 5515 to 5944 of the TOR1 open reading
frame was generated by PCR with Platinum Taq DNA Polymerase High Fidelity
(Invitrogen), wild-type yeast genomic DNA as a template, and primers F (5?-G
ACACGTTGAGGTTATTGACTC-3?) and M (5?-CTATGTTATGTTCAACG
in primer M, the mismatching nucleotides (lowercase type) encode the Ser1972-
to-Arg substitution and create a silent diagnostic BssHII site (underlined). The
fragment was then gel purified and used as a primer in the second PCR with the
second primer, R (5?-GGCCTTTGCTTCGAAGAGATCAC-3?), and wild-type
yeast genomic DNA as a template, to generate a more extended DNA fragment
corresponding to nucleotides 5515 to 6317 of the TOR1 open reading frame. The
obtained 803-bp DNA fragment was gel purified, confirmed to contain the
BssHII site, and used to transform a wild-type haploid, with selection on yeast
extract-peptone-dextrose agar plates (55) containing 200 ng/ml rapamycin (Sig-
ma-Aldrich). Two independent rapamycin-resistant isolates were colony purified.
The presence of the TOR1-S1972R allele at the correct chromosomal location
was confirmed by the presence of the diagnostic BssHII site following PCR
amplification of the corresponding chromosomal region. To confirm the domi-
nance of the introduced mutation, the rapamycin-resistant isolates were crossed
to a wild-type strain. In contrast to the control wild-type diploid (TOR1/TOR1),
the obtained diploids displayed resistance to 200 ng/ml rapamycin, as anticipated.
Strain KY65 (snf1?::KanMX6 TOR1-S1972R) is a segregant from a cross be-
tween one of the rapamycin-resistant isolates and a snf1?::KanMX6 (TOR1)
strain. Rapamycin hyperresistance segregated in a 2:2 manner in all nine tetrads
from this cross, and two of the tetrads were used to confirm its cosegregation with
the diagnostic BssHII site.
Plasmids. Vector pSK134HA is a derivative of pSK134 (69) and contains the
triple hemagglutinin (HA) epitope tag-encoding sequence and polycloning site
from pWS93 (63). pMO18 expresses N-terminal triple HA-tagged Snf1 (HA-
Snf1) from the yeast ADH1 promoter of vector pSK134HA; the SNF1 sequence
was from pSK119 (65). pMO19 expresses HA-Snf1-T210A (Thr210 to Ala) and
was constructed using pSK134HA by inserting the snf1-T210A sequence from
pRJ217 (35). pSK119, pSK120, and pIT517 express HA-Snf1, HA-Snf1-K84R,
and HA-Snf1-G53R, respectively, from the ADH1 promoter of vector pWS93
PH differentiation assays. Solid synthetic low-ammonia plus 2% dextrose
(SLAD) medium containing 50 ?M ammonium sulfate as the sole nitrogen
source was used for standard PH differentiation assays (16). SMAD medium was
identical to SLAD except that it contained 500 ?M ammonium sulfate as the sole
nitrogen source (48). Diploids were streaked to single cells, and the plates were
incubated at 30°C for 3 to 4 days. Colonies were photographed using a Nikon
Eclipse 50i microscope (10? objective), a CoolSnap ES camera (Photometrics),
and MetaMorph software (Universal Imaging Corporation).
Immunoblot assays of Thr210 phosphorylation. For nitrogen-rich conditions,
cells were grown at 30°C to mid-log phase (optical density at 600 nm of 0.5 to 0.7)
in synthetic complete medium with plasmid selection (55). For nitrogen-limiting
conditions, the cells were shifted to liquid SLAD medium containing 50 ?M
ammonium sulfate as the sole nitrogen source (16) for 60 min; abundant (2%)
glucose was present under both conditions. For rapamycin treatment experi-
ments, cells were grown in nitrogen-rich conditions, and rapamycin (Sigma-
Aldrich) was added to a final concentration of 200 ng/ml; a 1 mg/ml stock
solution of rapamycin was prepared in the drug vehicle, 90% ethanol plus 10%
Tween 20 (10); control cultures were treated with the drug vehicle alone. Protein
extracts were prepared by vortexing with glass beads essentially as described
previously (33), except that to arrest Snf1 in its corresponding Thr210 phospho-
rylation state (73), the cultures were placed in a boiling water bath for 3 min,
followed by cooling and harvesting. Proteins were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis, and Thr210-phosphorylated HA-Snf1
was detected by immunoblotting with anti-phospho-Thr172-AMPK (Cell Signal-
TABLE 1. S. cerevisiae strains
Strain Genotype Source
MATa/MAT? ura3?/ura3? leu2?/leu2? HIS3/his3? TRP1/trp1?
MATa/MAT? ura3?/ura3? leu2?/leu2? HIS3/his3? TRP1/trp1? snf1::LEU2/snf1::LEU2
MATa ura3? leu2? his3? snf1?::KanMX6
MATa/MAT? ura3?/ura3? leu2?/leu2? HIS3/his3? TRP1/trp1? snf1?::KanMX6/snf1?::KanMX6
MATa/MAT? ura3?/ura3? leu2?/leu? HIS3/his3? TRP1/trp1? reg1?::KanMX6/reg1?::URA3
MATa/MAT? ura3?/ura3? leu2?/leu2? HIS3/his3? TRP1/trp1? sip1?::KanMX6/sip1?::KanMX6
MATa/MAT? ura3?/ura3? leu2?/leu2? HIS3/his3? trp1?/trp1? sip1?::KanMX6/sip1?::KanMX6
MATa/MAT? ura3?/ura3? leu2?/leu2? HIS3/his3? trp1?/trp1? sip2?3::LEU2/sip2?3::LEU2
MATa/MAT? ura3?/ura3? leu2?/leu2? HIS3/his3? trp1?/trp1? sip1?::KanMX6/sip1?::KanMX6
MATa ura3? leu2? his3? snf1?::KanMX6 TOR1-S1972R
KY58 This study
1832 ORLOVA ET AL.EUKARYOT. CELL
ing Technologies). Total levels of HA-Snf1 proteins were determined by reprob-
ing the blots with monoclonal HA antibody 12CA5. Antibodies were detected by
enhanced chemiluminescence using ECL and ECLPlus (Amersham Bio-
Nonphosphorylable Snf1 does not support PH differentia-
tion. We first examined the ability of a nonphosphorylable
mutant kinase (HA-Snf1-T210A), which contains a Thr210-to-
Ala substitution previously used for studies of basal Snf1 func-
tion (13, 50), to complement the PH differentiation defect of a
diploid deleted for both genomic copies of the SNF1 gene. A
snf11/snf1 diploid was transformed with plasmids expressing
HA-Snf1 or HA-Snf1-T210A or with the parent vector, and
immunoblot analyses indicated that both Snf1 proteins are
expressed (see below). The resulting strains were tested on
SLAD plates for PH differentiation. The expression of HA-
Snf1 restored PH development in the snf1/snf1 recipient (Fig.
1A and B). By contrast, HA-Snf1-T210A was ineffective and
conferred no phenotypic improvement over the vector control
(Fig. 1C and D). Thus, these results indicate that PH differ-
entiation requires Thr210, suggesting that basal activity of un-
phosphorylated Snf1 is not sufficient.
Nitrogen limitation improves Thr210 phosphorylation. We
therefore tested whether nitrogen availability regulates Thr210
phosphorylation. The above-described snf1/snf1 cells, express-
ing HA-Snf1, expressing HA-Snf1-T210A, or carrying the
empty vector, were cultured under nitrogen-rich or nitrogen-
limiting conditions in the presence of abundant glucose (2%).
Thr210 phosphorylation was assayed by immunoblot analysis
with an anti-phospho-Thr172-AMPK antibody (Thr172 of
AMPK corresponds to Thr210 of Snf1), an approach used
previously for analyses of yeast Snf1 (64). Thr210 phosphory-
lation of HA-Snf1 under nitrogen-limiting conditions was con-
siderably increased relative to the nitrogen-rich conditions
(Fig. 2A, lanes 2 and 3). As expected, anti-phospho-Thr172-
AMPK did not detect HA-Snf1-T210A, regardless of growth
conditions (Fig. 2A, lanes 4 and 5). Reprobing the blot with
anti-HA indicated that the levels of the HA-tagged proteins
were comparable to one another (Fig. 2B). Thus, nitrogen
limitation improves Thr210 phosphorylation.
Effects of inactivation and hyperactivation of Snf1. To ad-
dress the role of Snf1 kinase activity, we first tested PH differ-
entiation in cells (snf1/snf1) expressing HA-Snf1-K84R, which
is catalytically inactive because the conserved lysine in the
ATP-binding site is replaced with arginine (14). PH differen-
tiation was defective, indicating that Snf1 catalytic activity is
required (Fig. 3A). To examine the effects of increased Snf1
activity, we similarly expressed HA-Snf1-G53R, a catalytically
hyperactive Gly53-to-Arg mutant identified by a function-
based screening (14). In the presence of nitrogen levels 10-fold
elevated relative to the optimal PH differentiation-inducing
conditions (500 ?M instead of 50 ?M ammonium sulfate as the
sole nitrogen source), wild-type HA-Snf1 was insufficient, as
anticipated (48), whereas HA-Snf1-G53R still supported PH
differentiation (Fig. 3A). In another approach, we examined
cells lacking Reg1, in which Snf1 is constitutively activated (26,
44). Unlike the wild-type diploid, which displayed PH differ-
entiation on 50 ?M but not 500 ?M ammonium sulfate me-
dium, the reg1/reg1 diploid formed filaments even under the
nonpermissive conditions (Fig. 3B). These findings indicate
that upregulation of Snf1 results in improved PH differentia-
Collectively, our immunochemical and genetic analyses
strongly suggest that the regulation of Snf1 is a major mecha-
nism by which nitrogen levels control PH differentiation and
strongly implicate Snf1 in nitrogen signaling.
FIG. 1. HA-Snf1-T210A does not support PH differentiation. Strain KY40 (snf1?/snf1?) was transformed with pMO18 expressing HA-Snf1,
pMO19 expressing HA-Snf1-T210A, or with the parent vector pSK134HA (Vector). The wild-type control strain MCY4472 (WT) carried the
empty vector pSK134HA. All transformants also carried pRS316 (62), a centromeric vector with URA3, to provide prototrophy. The strains were
examined on SLAD plates for PH differentiation. Colonies were photographed after 4 days at 30°C.
FIG. 2. Nitrogen levels regulate Thr210 phosphorylation. The
transformants of KY40 (snf1?/snf1?) analyzed in Fig. 1 expressing
T210A), or carrying the empty vector pSK134HA (Vector) were grown
in nutrient-rich synthetic medium to mid-log phase (high nitrogen, H)
and shifted for 1 h to SLAD medium (low nitrogen, L). All transfor-
mants carried pRS316 to provide prototrophy. (A) Thr210 phosphor-
ylation of HA-Snf1 was assessed by immunoblotting with anti-phos-
pho-Thr172-AMPK (?-Phospho). (B) Total levels of HA-Snf1 proteins
were determined by reprobing the blot with anti-HA (?-HA).
VOL. 5, 2006NITROGEN AND TOR REGULATE Snf11833
Requirement for the ? subunits. There are three distinct
forms of the Snf1 complex, as defined by the associated alter-
nate ? subunit. Given a new signaling role of Snf1, it was of
interest to assess the individual roles of these complexes, which
are known to have not only overlapping but also distinct func-
tions (1, 23, 36, 43, 58, 68, 70, 71). An examination of diploids
expressing individual ? subunits indicated that Sip1, Sip2, and
Gal83 can each support PH differentiation, and only the loss of
all three resulted in a strong phenotypic defect (Fig. 4). These
results suggest that all three forms of the Snf1 complex can
respond to signaling and make functionally equivalent contri-
butions to PH differentiation. However, we cannot rule out the
possibility that the different complexes also have distinct func-
tions in this and other nitrogen-regulated processes.
We also note that these findings distinguish diploid PH dif-
ferentiation from two related processes in haploids, surface
adhesion and filamentation, which are activated by glucose
limitation (11, 52, 71): only Gal83 can support haploid surface
adhesion, and haploid filamentation can be provided by Gal83
or Sip2 but not by Sip1 (71).
Snf1 is negatively regulated by TOR. The involvement of
Snf1 in nitrogen signaling suggested that Snf1 might function
downstream of the rapamycin-sensitive TOR kinase. First, un-
der nitrogen-rich conditions, TOR negatively regulates numer-
ous functions required for coping with nitrogen limitation,
some of which are known to be essential for PH differentiation.
Second, previous evidence implicated Snf1 as an effector of
rapamycin toxicity (3). We therefore examined whether treat-
ing the cells with rapamycin would affect Thr210 phosphory-
lation. Diploid cells expressing HA-Snf1 were grown in nutri-
ent-rich conditions and treated with rapamycin or with the
drug vehicle alone as a control. Rapamycin treatment resulted
in a significant improvement of Thr210 phosphorylation (Fig.
5A). A similar result was obtained for haploid cells (Fig. 5B),
suggesting that this regulatory effect is not ploidy specific.
Rapamycin treatment did not improve Thr210 phosphoryla-
FIG. 3. Effects of inactive and hyperactive Snf1 on PH differentiation. (A) A snf1/snf1 diploid, MCY4473, was transformed with plasmids
pSK119, pSK120, or pIT517 expressing HA-Snf1, catalytically inactive HA-Snf1-K84R (HA-K84R), and hyperactive HA-Snf1-G53R (HA-G53R),
respectively. Immunoblot analyses indicated that all HA-tagged proteins are expressed (panel C). Strains were examined for PH differentiation on
SLAD and SMAD media containing 50 ?M and 500 mM ammonium sulfate as the sole nitrogen source, respectively; colonies were photographed
after 3 days at 30°C. Cells expressing no Snf1 protein (carrying the corresponding empty vector pWS93) failed to undergo PH differentiation under
either condition (not shown). (B) Wild-type diploid MCY4472 (WT) and KY43 (reg1/reg1) were examined for PH differentiation as described
above. Both strains carried pLCLG-Staf (31), a centromeric plasmid with LEU2 and URA3 to confer prototrophy. (C) Cells analyzed in panel A
were grown to mid-log phase in nutrient-rich synthetic medium with plasmid selection and shifted to liquid SLAD medium for 2 h, and levels of
the HA-Snf1 proteins were compared by immunoblotting with anti-HA. V, vector pWS93; WT, HA-Snf1; KR, HA-Snf1-K84R; GR, HA-Snf1-
FIG. 4. Requirement for the ? subunits. Diploids with the indicated genotypes were grown on SLAD plates for 4 days at 30°C, and colonies
were photographed. WT, wild type. All strains carried pLCLG-Staf to confer prototrophy (see legend to Fig. 3).
1834 ORLOVA ET AL.EUKARYOT. CELL
tion in cells carrying a TOR1-S1972R allele (Fig. 5C), which
encodes a rapamycin-resistant Ser1972-to-Arg Tor1 protein (4,
24), confirming that rapamycin elicits its effect on Snf1 via
inhibition of TOR. Finally, time-course experiments showed
that a detectable response develops within 15 to 30 min of
rapamycin addition (3, 42), arguing against grossly indirect
effects (Fig. 5D). Thus, we conclude that the rapamy-
cin-sensitive TOR negatively regulates Snf1.
We have investigated the requirement of the Snf1 protein
kinase for diploid PH differentiation in S. cerevisiae and
present evidence that Snf1 is directly involved in nitrogen sig-
naling. First, nonphosphorylable Snf1-T210A does not support
PH differentiation, suggesting a requirement for maximal ac-
tivation. Second, nitrogen limitation leads to improved Thr210
phosphorylation, indicating that Snf1 responds to a nitrogen
signal. Furthermore, we show that Thr210 phosphorylation is
negatively regulated by the rapamycin-sensitive TOR kinase,
which plays essential roles in signaling nitrogen and amino acid
Evidence that the three alternative ? subunits can each sup-
port PH differentiation suggests that all three forms of the Snf1
complex are responsive to regulation by nitrogen. All three
forms are also controlled by glucose (43, 58) and, collectively,
these findings implicate Snf1 in broad integration of signals
regarding nitrogen and carbon stress. Further studies are re-
quired to decipher how such integration occurs and whether it
relies on differential roles of the known regulators, such as the
three partially redundant Snf1-activating kinases (Sak1, Tos3,
Elm1) or involves other mechanisms. In this regard, an inter-
esting possibility raised by our findings is that at least some
signal integration upstream of Snf1 in fact occurs at TOR.
Indeed, besides its broad involvement in nitrogen signaling,
TOR has also been implicated in responses to carbon and salt
stress (2, 9, 10, 27, 59).
Snf1 extends the list of known positive regulators of PH
differentiation that are negatively regulated by TOR (2, 5, 20,
39, 57), further supporting the involvement of TOR in the
negative control of this developmental process. Interestingly,
however, the net effect of rapamycin on PH differentiation is
inhibitory rather than stimulatory (12), and our experiments
confirm this conclusion (data not shown). On the one hand,
this result indicates that TOR also has a positive role, likely
reflecting the fact that PH differentiation still requires a sig-
nal(s) confirming the presence of nitrogen, albeit in limiting
amounts or in a nonpreferred form (12). On the other hand, its
involvement in both positive and negative control of PH dif-
ferentiation suggests that TOR can distinguish between differ-
ent physiological (non-rapamycin) situations to allow the acti-
vation of one set of responses but not the other. This possibility
is in line with evidence that TOR can function as a “multichan-
nel processor” that couples specific upstream inputs to specific
downstream events (10, 59).
The functional interaction between AMPK and mTOR in
mammalian cells has been intensively studied in connection
with its biomedical importance. Interestingly, it is AMPK that
has been reported to negatively control mTOR, by phosphor-
ylating and stimulating the tumor suppressor protein TSC2
which functions in mTOR inhibition (29). Upregulation of
mTOR caused by defects in the LKB1-AMPK-TSC pathway
has been implicated as an underlying molecular cause of in-
herited hamartomatous tumor syndromes, such as tuberous
sclerosis complex and Peutz-Jeghers syndrome (8, 29, 60, 61).
Our findings for S. cerevisiae suggest that other eukaryotes
may have mechanisms for the negative regulation of Snf1/
AMPK by TOR. The existence of such a mechanism in mam-
mals would suggest further clues to the molecular etiology and
therapy of tumorigenesis and other diseases associated with
deregulated energy metabolism. Regardless of whether the
specific mechanisms are conserved, however, a form of func-
tional antagonism between Snf1/AMPK and TOR clearly ap-
pears to be a common regulatory feature in eukaryotes from
yeast to humans.
This work was supported by the UWM College of Letters and
Science and a Graduate School Faculty Research Award.
We thank Mark McBride, Tom Schuck, Heather Owen, and Ray-
mond Hovey for advice and technical assistance.
This work was initiated in the laboratory of M. Carlson and sup-
ported by NIH grant GM34095.
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FIG. 5. Rapamycin-sensitive TOR negatively regulates Snf1. Cells
(snf1?/snf1? or snf1?) expressing HA-Snf1 from pMO18, expressing
HA-Snf1-T210A (HA-T210A) from pMO19, or carrying the empty
vector pSK134HA (Vector) were grown in nutrient-rich synthetic com-
plete medium lacking Leu to mid-log phase and treated with 200 ng/ml
rapamycin (?) or with the corresponding amount (1 ?l per 5-ml
culture) of drug vehicle (?) for 90 min (A to C) or for the indicated
times (D). Thr210 phosphorylation was analyzed as described for Fig.
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allele (RR), respectively, expressing HA-Snf1. (D) Cells of KY40 ex-
pressing HA-Snf1 were treated with rapamycin (?) or with the drug
vehicle (?) for the indicated times (min).
VOL. 5, 2006 NITROGEN AND TOR REGULATE Snf11835
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