Regulation of G0 entry by the Pho80-Pho85 cyclin-CDK complex

Eukaryotic cell proliferation is controlled by growth factors and essential nutrients. In their absence, cells may enter into a quiescent state (G0). In Saccharomyces cerevisiae, the conserved protein kinase A (PKA) and rapamycin-sensitive TOR (TORC1) pathways antagonize G0 entry in response to carbon and/or nitrogen availability primarily by inhibiting the PAS kinase Rim15 function. Here, we show that the phosphate-sensing Pho80-Pho85 cyclin-cyclin-dependent kinase (CDK) complex also participates in Rim15 inhibition through direct phosphorylation, thereby effectively sequestering Rim15 in the cytoplasm via its association with 14-3-3 proteins. Inactivation of either Pho80-Pho85 or TORC1 causes dephosphorylation of the 14-3-3-binding site in Rim15, thus enabling nuclear import of Rim15 and induction of the Rim15-controlled G0 program. Importantly, we also show that Pho80-Pho85 and TORC1 converge on a single amino acid in Rim15. Thus, Rim15 plays a key role in G0 entry through its ability to integrate signaling from the PKA, TORC1, and Pho80-Pho85 pathways.     

Caffeine extends yeast lifespan by targeting TORC1

Dietary nutrient limitation (dietary restriction) is known to increase lifespan in a variety of organisms. Although the molecular events that couple dietary restriction to increased lifespan are not clear, studies of the model eukaryote Saccharomyces cerevisiae have implicated several nutrient-sensitive kinases, including the t arget o f r apamycin c omplex 1 (TORC1), Sch9, protein kinase A (PKA) and Rim15. We have recently demonstrated that TORC1 activates Sch9 by direct phosphorylation. We now show that Sch9 inhibits Rim15 also by direct phosphorylation. Treatment of yeast cells with the specific TORC1 inhibitor rapamycin or caffeine releases Rim15 from TORC1-Sch9-mediated inhibition and consequently increases lifespan. This kinase cascade appears to have been evolutionarily conserved, suggesting that caffeine may extend lifespan in other eukaryotes, including man.     

PKA and Sch9 control a molecular switch important for the proper adaptation to nutrient availability

In the yeast Saccharomyces cerevisiae, PKA and Sch9 exert similar physiological roles in response to nutrient availability. However, their functional redundancy complicates to distinguish properly the target genes for both kinases. In this article, we analysed different phenotypic read-outs. The data unequivocally showed that both kinases act through separate signalling cascades. In addition, genome-wide expression analysis under conditions and with strains in which either PKA and/or Sch9 signalling was specifically affected, demonstrated that both kinases synergistically or oppositely regulate given gene targets. Unlike PKA, which negatively regulates stress-responsive element (STRE)- and post-diauxic shift (PDS)-driven gene expression, Sch9 appears to exert additional positive control on the Rim15-effector Gis1 to regulate PDS-driven gene expression. The data presented are consistent with a cyclic AMP (cAMP)-gating phenomenon recognized in higher eukaryotes consisting of a main gatekeeper, the protein kinase PKA, switching on or off the activities and signals transmitted through primary pathways such as, in case of yeast, the Sch9-controlled signalling route. This mechanism allows fine-tuning various nutritional responses in yeast cells, allowing them to adapt metabolism and growth appropriately.     

Sch9 is a major target of TORC1 in Saccharomyces cerevisiae

The Target of Rapamycin (TOR) protein is a Ser/Thr kinase that functions in two distinct multiprotein complexes: TORC1 and TORC2. These conserved complexes regulate many different aspects of cell growth in response to intracellular and extracellular cues. Here we report that the AGC kinase Sch9 is a substrate of yeast TORC1. Six amino acids in the C terminus of Sch9 are directly phosphorylated by TORC1. Phosphorylation of these residues is lost upon rapamycin treatment as well as carbon or nitrogen starvation and transiently reduced following application of osmotic, oxidative, or thermal stress. TORC1-dependent phosphorylation is required for Sch9 activity, and replacement of residues phosphorylated by TORC1 with Asp/Glu renders Sch9 activity TORC1 independent. Sch9 is required for TORC1 to properly regulate ribosome biogenesis, translation initiation, and entry into G0 phase, but not expression of Gln3-dependent genes. Our results suggest that Sch9 functions analogously to the mammalian TORC1 substrate S6K1 rather than the mTORC2 substrate PKB/Akt.     

TOR regulation of AGC kinases in yeast and mammals

The TOR (target of rapamycin), an atypical protein kinase, is evolutionarily conserved from yeast to man. Pharmacological studies using rapamycin to inhibit TOR and yeast genetic studies have provided key insights on the function of TOR in growth regulation. One of the first bona fide cellular targets of TOR was the mammalian protein kinase p70 S6K (p70 S6 kinase), a member of a family of kinases called AGC (protein kinase A/protein kinase G/protein kinase C-family) kinases, which include PKA (cAMP-dependent protein kinase A), PKG (cGMP-dependent kinase) and PKC (protein kinase C). AGC kinases are also highly conserved and play a myriad of roles in cellular growth, proliferation and survival. The AGC kinases are regulated by a common scheme that involves phosphorylation of the kinase activation loop by PDK1 (phosphoinositide-dependent kinase 1), and phosphorylation at one or more sites at the C-terminal tail. The identification of two distinct TOR protein complexes, TORC1 (TOR complex 1) and TORC2, with different sensitivities to rapamycin, revealed that TOR, as part of either complex, can mediate phosphorylation at the C-terminal tail for optimal activation of a number of AGC kinases. Together, these studies elucidated that a fundamental function of TOR conserved throughout evolution may be to balance growth versus survival signals by regulating AGC kinases in response to nutrients and environmental conditions. This present review highlights this emerging function of TOR that is conserved from budding and fission yeast to mammals.     

Low temperature highlights the functional role of the cell wall integrity pathway in the regulation of growth in Saccharomyces cerevisiae

Unlike other stresses, the physiological significance and molecular mechanisms involved in the yeast cold response are largely unknown. In the present study, we show that the CWI (cell wall integrity) pathway plays an important role in the growth of Saccharomyces cerevisiae at low temperatures. Cells lacking the Wsc1p (wall integrity and stress response component 1) membrane sensor or the MAPKs (mitogen-activated protein kinases) Bck1p (bypass of C kinase 1), Mkk (Mapk kinase) 1p/Mkk2p or Slt2p (suppressor of lyt2) exhibited cold sensitivity. However, there was no evidence of either a cold-provoked perturbation of the cell wall or a differential cold expression program mediated by Slt2p. The results of the present study suggest that Slt2p is activated by different inputs in response to nutrient signals and mediates growth control through TORC1 (target of rapamycin 1 complex)-Sch9p (suppressor of cdc25) and PKA (protein kinase A) at low temperatures. We found that absence of TOR1 (target of rapamycin 1) causes cold sensitivity, whereas a ras2Δ mutant shows increased cold growth. Lack of Sch9p alleviates the phenotype of slt2Δ and bck1Δ mutant cells, as well as attenuation of PKA activity by overexpression of BCY1 (bypass of cyclase mutations 1). Interestingly, swi4Δ mutant cells display cold sensitivity, but the phenotype is neither mediated by the Slt2p-regulated induction of Swi4p (switching deficient 4)-responsive promoters nor influenced by osmotic stabilization. Hence, cold signalling through the CWI pathway has distinct features and might mediate still unknown effectors and targets.     

cAMP signaling regulates histone H3 phosphorylation and mitotic entry through a disruption of G2 progression

cAMP signaling is known to have significant effects on cell growth, either inhibitory or stimulatory depending on the cell type. Study of cAMP-induced growth inhibition in mammalian somatic cells has focused mainly on the combined role of protein kinase A (PKA) and mitogen-activated protein (MAP) kinases in regulation of progression through the G1 phase of the cell cycle. Here we show that cAMP signaling regulates histone H3 phosphorylation in a cell cycle-dependent fashion, increasing it in quiescent cells but dramatically reducing it in cycling cells. The latter is due to a rapid and dramatic loss of mitotic histone H3 phosphorylation caused by a disruption in G2 progression, as evidenced by the inhibition of mitotic entry and decreased activity of the CyclinB/Cdk1 kinase. The inhibition of G2 progression induced through cAMP signaling is dependent on expression of the catalytic subunit of PKA and is highly sensitive to intracellular cAMP concentration. The mechanism by which G2 progression is inhibited is independent of both DNA damage and MAP kinase signaling. Our results suggest that cAMP signaling activates a G2 checkpoint by a unique mechanism and provide new insight into normal cellular regulation of G2 progression.     

Nutrient sensing kinases PKA and Sch9 phosphorylate the catalytic domain of the ubiquitin-conjugating enzyme Cdc34

Cell division is controlled in part by the timely activation of the CDK, Cdc28, through its association with G1 and G2 cyclins. Cdc28 complexes are regulated in turn by the ubiquitin conjugating enzyme Cdc34 and SCF ubiquitin ligase complexes of the ubiquitin-proteasome system (UPS) to control the initiation of DNA replication. Here we demonstrate that the nutrient sensing kinases PKA and Sch9 phosphorylate S97 of Cdc34. S97 is conserved across species and restricted to the catalytic domain of Cdc34/Ubc7-like E2s. Cdc34-S97 phosphorylation is cell cycle regulated, elevated during active cell growth and division and decreased during cell cycle arrest. Cell growth and cell division are orchestrated to maintain cell size homeostasis over a wide range of nutrient conditions. Cells monitor changes in their environment through nutrient sensing protein kinases. Thus Cdc34 phosphorylation by PKA and Sch9 provides a direct tether between G1 cell division events and cell growth.     

Down-regulating sphingolipid synthesis increases yeast lifespan

Knowledge of the mechanisms for regulating lifespan is advancing rapidly, but lifespan is a complex phenotype and new features are likely to be identified. Here we reveal a novel approach for regulating lifespan. Using a genetic or a pharmacological strategy to lower the rate of sphingolipid synthesis, we show that Saccharomyces cerevisiae cells live longer. The longer lifespan is due in part to a reduction in Sch9 protein kinase activity and a consequent reduction in chromosomal mutations and rearrangements and increased stress resistance. Longer lifespan also arises in ways that are independent of Sch9 or caloric restriction, and we speculate on ways that sphingolipids might mediate these aspects of increased lifespan. Sch9 and its mammalian homolog S6 kinase work downstream of the target of rapamycin, TOR1, protein kinase, and play evolutionarily conserved roles in regulating lifespan. Our data establish Sch9 as a focal point for regulating lifespan by integrating nutrient signals from TOR1 with growth and stress signals from sphingolipids. Sphingolipids are found in all eukaryotes and our results suggest that pharmacological down-regulation of one or more sphingolipids may provide a means to reduce age-related diseases and increase lifespan in other eukaryotes.     

Regulation of the cell integrity pathway by rapamycin-sensitive TOR function in budding yeast

The TOR (target of rapamycin) pathway controls cell growth in response to nutrient availability in eukaryotic cells. Inactivation of TOR function by rapamycin or nutrient exhaustion is accompanied by triggering various cellular mechanisms aimed at overcoming the nutrient stress. Here we report that in Saccharomyces cerevisiae the protein kinase C (PKC)-mediated mitogen-activated protein kinase pathway is regulated by TOR function because upon specific Tor1 and Tor2 inhibition by rapamycin, Mpk1 is activated rapidly in a process mediated by Sit4 and Tap42. Osmotic stabilization of the plasma membrane prevents both Mpk1 activation by rapamycin and the growth defect that occurs upon the simultaneous absence of Tor1 and Mpk1 function, suggesting that, at least partially, TOR inhibition is sensed by the PKC pathway at the cell envelope. This process involves activation of cell surface sensors, Rom2, and downstream elements of the mitogen-activated protein kinase cascade. Rapamycin also induces depolarization of the actin cytoskeleton through the TOR proteins, Sit4 and Tap42, in an osmotically suppressible manner. Finally, we show that entry into stationary phase, a physiological situation of nutrient depletion, also leads to the activation of the PKC pathway, and we provide further evidence demonstrating that Mpk1 is essential for viability once cells enter G(0).