TOR and PKA signaling pathways converge on the protein kinase Rim15 to control entry into G0

The highly conserved Tor kinases (TOR) and the protein kinase A (PKA) pathway regulate cell proliferation in response to growth factors and/or nutrients. In Saccharomyces cerevisiae, loss of either TOR or PKA causes cells to arrest growth early in G(1) and to enter G(0) by mechanisms that are poorly understood. Here we demonstrate that the protein kinase Rim15 is required for entry into G(0) following inactivation of TOR and/or PKA. Induction of Rim15-dependent G(0) traits requires two discrete processes, i.e., nuclear accumulation of Rim15, which is negatively regulated both by a Sit4-independent TOR effector branch and the protein kinase B (PKB/Akt) homolog Sch9, and release from PKA-mediated inhibition of its protein kinase activity. Thus, Rim15 integrates signals from at least three nutrient-sensory kinases (TOR, PKA, and Sch9) to properly control entry into G(0), a key developmental process in eukaryotic cells.     

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.     

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.     

SOD2 functions downstream of Sch9 to extend longevity in yeast

Signal transduction pathways inactivated during periods of starvation are implicated in the regulation of longevity in organisms ranging from yeast to mammals, but the mechanisms responsible for life-span extension are poorly understood. Chronological life-span extension in S. cerevisiae cyr1 and sch9 mutants is mediated by the stress-resistance proteins Msn2/Msn4 and Rim15. Here we show that mitochondrial superoxide dismutase (Sod2) is required for survival extension in yeast. Deletion of SOD2 abolishes life-span extension in sch9Delta mutants and decreases survival in cyr1:mTn mutants. The overexpression of Sods--mitochondrial Sod2 and cytosolic CuZnSod (Sod1)--delays the age-dependent reversible inactivation of mitochondrial aconitase, a superoxide-sensitive enzyme, and extends survival by 30%. Deletion of the RAS2 gene, which functions upstream of CYR1, also doubles the mean life span by a mechanism that requires Msn2/4 and Sod2. These findings link mutations that extend chronological life span in S. cerevisiae to superoxide dismutases and suggest that the induction of other stress-resistance genes regulated by Msn2/4 and Rim15 is required for maximum longevity extension.     

Glucose Activates TORC2-Gad8 Protein via Positive Regulation of the cAMP/cAMP-dependent Protein Kinase A (PKA) Pathway and Negative Regulation of the Pmk1 Protein-Mitogen-activated Protein Kinase Pathway

The target of rapamycin (TOR) kinase belongs to the highly conserved eukaryotic family of phosphatidylinositol 3-kinase-related kinases. TOR proteins are found at the core of two evolutionary conserved complexes, known as TORC1 and TORC2. In fission yeast, TORC2 is dispensable for proliferation under optimal growth conditions but is required for starvation and stress responses. TORC2 has been implicated in a wide variety of functions; however, the signals that regulate TORC2 activity have so far remained obscure. TORC2 has one known direct substrate, the AGC kinase Gad8, which is related to AKT in human cells. Gad8 is phosphorylated by TORC2 at Ser-546 (equivalent to AKT Ser-473), leading to its activation. Here, we show that glucose is necessary and sufficient to induce Gad8 Ser-546 phosphorylation in vivo and Gad8 kinase activity in vitro. The glucose signal that activates TORC2-Gad8 is mediated via the cAMP/PKA pathway, a major glucose-sensing pathway. By contrast, Pmk1, similar to human extracellular signal-regulated kinases and a major stress-induced mitogen activated protein kinase (MAPK) in fission yeast, inhibits TORC2-dependent Gad8 phosphorylation and activation. Inhibition of TORC2-Gad8 also occurs in response to ionic or osmotic stress, in a manner dependent on the cAMP/PKA and Pmk1-MAPK signaling pathways. Our findings highlight the significance of glucose availability in regulation of TORC2-Gad8 and indicate a novel link between the cAMP/PKA, Pmk1/MAPK, and TORC2-Gad8 signaling.