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: firstname.lastname@example.org.
† Present address: Department of Neurology, Columbia University,
New York, NY 10032.
?Published ahead of print on 15 September 2006.
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VOL. 5, 2006NITROGEN AND TOR REGULATE Snf11837