Here, we conducted a search for new mutants with possible defects in the Snf1 pathway. We used the fact that the reg1
Δ mutation, which confers increased Snf1 activation, also confers very slow growth. We isolated “fast” mutations that, like snf1
, relieve the slow-growth phenotype caused by reg1
Δ. We recovered recessive mutations, not only in SNF1
, but also in SAK1
, which encodes one of three partially redundant Snf1-activating kinases. Inactivation of SAK1
alone does not cause growth defects on alternative carbon sources (30
), and identification of sak1
in our mutant search suggested that suppression of reg1
Δ is a sensitive genetic approach for identifying new components of the Snf1 regulatory network.
We also recovered multiple isolates with mutations in the RasGap genes IRA1
, the yeast orthologs of the mammalian NF1
tumor suppressor gene (79
). Inactivation of IRA1
, and BCY1
conferred a reduction in the activation of the Snf1 pathway in response to glucose limitation. These results provided evidence that activation of Ras affects Snf1 activation and that this effect is largely mediated by PKA, although additional contributions by PKA-independent mechanisms cannot be excluded.
The existence of an antagonistic relationship between Ras-PKA and Snf1 was proposed a long time ago, based on the finding that snf1
confers some of the same phenotypes as activation of Ras-PKA (70
), but whether Ras-PKA and Snf1 can engage in a hierarchical regulatory relationship remained unclear. More recent evidence indicated that PKA affects the subcellular localization of Sip1 (28
), one of three alternate β subunits of the Snf1 complex (Sip1, Sip2, and Gal83). However, Sip1 was reported to be the least abundant of the β subunits (49
), and the physiological significance of its PKA-regulated localization is not fully understood. Thus, the present study extends previous findings by implicating PKA in the control of a major aspect of Snf1 function in yeast.
We note that strain-dependent variation in the apparent strength of this PKA-Snf1 connection may be one of the factors that precluded its identification in earlier studies. For example, in our experiments with the dimorphic Σ1278b background, SUC2-lacZ
reporter expression was significantly reduced in the ira
mutants, whereas expression of constitutive Ras2Val19
had no pronounced effect on SUC2
gene expression in the more commonly used W303 genetic background (81
). Σ1278b strains have an unusually high basal level of cAMP signaling (64
), and their Snf1 pathway could therefore be more responsive to further increases in cAMP-PKA signaling. Another potential variable to consider is the rate of dephosphorylation of the relevant PKA sites by the cognate phosphatase(s), possibly including Reg1-Glc7 itself.
Our results indicate that the Snf1-activating kinase Sak1 is phosphorylated in a PKA-stimulated manner on Ser1074 within an ideal PKA motif of its C-terminal domain. The C-terminal domain of Sak1 is dispensable for its catalytic activity in vitro
). A recent study shows that this domain mediates physical interaction with Snf1 and that the segment encompassing amino acids 501 to 740 is required for full Snf1 activation in vivo
). Ser1074 is located further C-terminal of this segment, in the region that is missing in the product encoded by the inactive sak1-11
allele recovered from our screen (codons 736 to 1142). Ser1074 phosphorylation could make a fine-tuning contribution to modulating the Sak1-Snf1 interaction but could also affect an as-yet-unidentified aspect of Sak1 function. In either case, Ser1074 phosphorylation alone appears to make only a modest contribution to regulation. The underlying mechanism could, however, confer a distinct evolutionary advantage. For example, the Sak1 homolog of the pathogenic fungus C. glabrata
, whose Snf1 undergoes regulated T-loop phosphorylation (51
), has an ideal PKA recognition motif that directly corresponds to the motif addressed in this work. Moreover, this motif is embedded in a 22-amino-acid region that stands out among the surrounding sequence in sharing more than 80% identity with the region encompassing Ser1074 of S. cerevisiae
Sak1, presumably defining a conserved regulatory box. Similar boxes are also found in the Sak1 homologs of fungi representing several genera. Our results also raise the possibility that S. cerevisiae
Sak1 is additionally phosphorylated on the nearby Ser1139 residue within another putative PKA recognition motif and that the Ser1074 and Ser1139 phosphorylation events cooperate to negatively control Sak1.
Sak1, however, is not the only relevant target of the PKA pathway. Sequence analysis indicates that another Snf1-activating kinase, Tos3, has an ideal PKA recognition consensus, making it a possible substrate. The third Snf1-activating kinase, Elm1, does not have any immediately compelling PKA motifs but could be regulated by PKA either indirectly or directly by phosphorylation on a near-consensus or nonconsensus site(s). Interestingly, recent evidence indicates that mammalian AMPK is phosphorylated by PKA on a nonconsensus serine that is immediately adjacent to its critical T-loop threonine, and this phosphorylation antagonizes AMPK activation (13
). A serine residue is present at the equivalent position in S. cerevisiae
Snf1, raising the possibility that PKA could similarly antagonize Snf1 directly by inhibitory T-loop phosphorylation. It is also possible that PKA regulates Thr210 dephosphorylation by Reg1-Glc7, although such a mechanism would not have been detected in our mutant search. In addition, PKA could regulate the Sit4 protein phosphatase, which has been recently reported to contribute to Thr210 dephosphorylation (58
). Finally, we cannot exclude the possibility that PKA, whose catalytic isoforms can have opposing physiological functions, plays not only negative but also positive roles in Snf1 regulation. Further experiments will be required to address these and other possibilities in order to fully reconstruct the potentially very complex mechanistic picture.
The molecular mechanisms by which nutrient availability modulates Snf1 activity are not completely understood. PKA figures prominently in glucose signaling and collects its regulatory inputs from multiple effectors, including the Ras, RasGAP, and RasGEF proteins; the Gpr1/Gpa2 G-protein-coupled receptor system; and others (for reviews, see references 59
). The involvement of PKA could therefore account for a portion of the glucose signal affecting Snf1. In addition to glucose signaling, Snf1 participates in responses to other stress conditions, such as nitrogen limitation and exposure to rapamycin (41
). PKA is involved in nitrogen and TOR signaling (see reference 82
for a review) and could mediate a relevant signal(s) impinging on Snf1. Consistent with this possibility, we found here that, like snf1
, the ira
mutations suppress reg1
for rapamycin hypersensitivity.
Our findings lend further support to the value of the yeast Snf1 pathway as a model system for studying cancer-related signaling. The link between AMPK and cancer first transpired from the identification of tumor suppressor LKB1 as an AMPK-activating kinase (30
) and tumor suppressor TSC2 as an AMPK target (33
). Activated AMPK functions to downregulate mTOR and to stimulate p53-mediated cell cycle arrest (7
). AMPK has therefore been proposed to act as a metabolic checkpoint that coordinates cell growth and proliferation with energy availability (35
). Our results extend the idea that Snf1 has a similar function in yeast (83
). We find it fascinating that the slow-growth phenotype caused by hyperactivation of the Snf1 “tumor suppressor” pathway (reg1
Δ) can be so prominently reversed by activation of the Ras “oncogene” pathway in this simple eukaryote. Interestingly, a report using a mouse melanoma model showed that growth factor-activated Ras and oncogenic BRAFV600E
downregulate the LKB1-AMPK cascade by a mechanism involving p90RSK
-dependent phosphorylation of LKB1 (14
); LKB1 is also phosphorylated by PKA (2
). We conclude that the Snf1-dependent slow growth of the yeast reg1
Δ mutant is not only a useful genetic tool, but also an important phenotype in its own right, reflecting an energy-saving function of Snf1. The exact mechanisms by which activation of Snf1 decelerates growth in yeast are not fully understood but likely involve negative effects on the cell cycle, since the slow growth caused by reg1
Δ can be partially rescued by overexpression of the cell cycle progression kinase Cdc28 (our unpublished results). Further analysis of reg1
Δ suppressors in yeast could provide new clues to the mechanisms that couple cell growth and proliferation with nutrient availability in eukaryotes.