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.

The target of rapamycin (TOR) protein kinase is at the core of growth factor- and nutrient-dependent signaling pathways that are well-known for their regulation of metabolism, growth, and proliferation. However, TOR is also involved in the regulation of gene expression, genomic and epigenomic stability. TOR affects nuclear functions indirectly through its activity in the cytoplasm, but also directly through active nuclear TOR pools. The mechanisms by which TOR regulates its nuclear functions are less well-understood compared with its cytoplasmic activities. TOR is an important pharmacological target for several diseases, including cancer, metabolic and neurological disorders. Thus, studies of the nuclear functions of TOR are important for our understanding of basic biological processes, as well as for clinical implications.

The inhibition of the central growth regulatory kinase TOR, which participates in two complexes, TORC1 and TORC2, has been a focus of metabolic and cancer studies for many years. Most studies have dealt with TORC1, the canonical target of rapamycin, and the role of this complex in autophagy, protein synthesis, and cell growth control. Recent work on TORC2 in budding and fission yeast species points to a conserved role of this lesser‐known TOR complex in the survival of DNA damage. In budding yeast, TORC2 controls lipid biosynthesis and actin cytoskeleton through downstream AGC kinases, which are now, surprisingly, implicated in the survival of oxidative DNA damage. Preliminary data from mTORC2 modulation in cancer cells suggest that an extension to human chemotherapy is worth exploring.

The target of rapamycin (TOR) kinase is found at the core of two evolutionarily conserved complexes known as TOR complexes 1 and 2 (TORC1 and TORC2). In fission yeast, TORC2 is dispensable for proliferation under optimal growth conditions but is required for starvation and stress responses. We have previously reported that loss of function of TORC2 renders cells highly sensitive to DNA replication stress; however, the mechanism underlying this sensitivity is unknown. TORC2 has one known direct substrate, the kinase Gad8, which is related to AKT in human cells. Here we show that both TORC2 and its substrate Gad8 are found in the nucleus and are bound to the chromatin. We also demonstrate that Gad8 physically interacts with the MluI cell cycle box-binding factor (MBF) transcription complex that regulates the G₁/S progression and the response to DNA stress. In mutant cells lacking TORC2 or Gad8, the binding of the MBF complex to its cognate promoters is compromised, and the induction of MBF target genes in response to DNA replication stress is reduced. Consistently, the protein levels of Cdt2 and Cig2, two MBF target genes, are reduced in the absence of TORC2-Gad8 signaling. Taken together, our findings highlight critical functions of TORC2 in the nucleus and suggest a role in surviving DNA replication stress via transcriptional regulation of MBF target genes.

The conserved serine/threonine protein kinase target of rapamycin (TOR) is a major regulator of eukaryotic cellular and organismal growth and a valuable target for drug therapy. TOR forms the core of two evolutionary conserved complexes, TOR complex 1 (TORC1) and TORC2. In the fission yeast Schizosaccharomyces pombe, TORC2 responds to glucose levels and, by activating the protein kinase Gad8 (an orthologue of human AKT), is required for well-regulated cell cycle progression, starvation responses, and cell survival. Here, we report that TORC2–Gad8 is also required for gene silencing and the formation of heterochromatin at the S. pombe mating-type locus and at subtelomeric regions. Deletion of TORC2–Gad8 resulted in loss of the heterochromatic modification of histone 3 lysine 9 dimethylation (H3K9me2) and an increase in euchromatic modifications, including histone 3 lysine 4 trimethylation (H3K4me3) and histone 4 lysine 16 acetylation (H4K16Ac). Accumulation of RNA polymerase II (Pol II) at subtelomeric genes in TORC2–Gad8 mutant cells indicated a defect in silencing at the transcriptional level. Moreover, a concurrent decrease in histone 4 lysine 20 dimethylation (H4K20me2) suggested elevated histone turnover. Loss of gene silencing in cells lacking TORC2–Gad8 is partially suppressed by loss of the anti-silencer Epe1 and fully suppressed by loss of the Pol II–associated Paf1 complex, two chromatin regulators that have been implicated in heterochromatin stability and spreading. Taken together, our findings suggest that TORC2–Gad8 signaling contributes to epigenetic stability at subtelomeric regions and the mating-type locus in S. pombe.

The evolutionarily conserved TOR complex 1 (TORC1) activates cell growth and proliferation in response to nutritional signals. In the fission yeast Schizosaccharomyces pombe, TORC1 is essential for vegetative growth, and its activity is regulated in response to nitrogen quantity and quality. Yet, how TORC1 senses nitrogen is poorly understood. Rapamycin, a specific TOR inhibitor, inhibits growth in S. pombe only under conditions in which the activity of TORC1 is compromised. In a genetic screen for rapamycin-sensitive mutations, we isolated caa1-1, a loss-of-function mutation of the cytosolic form of aspartate aminotransferase (Caa1). We demonstrate that loss of caa1⁺ partially mimics loss of TORC1 activity and that Caa1 is required for full TORC1 activity. Disruption of caa1⁺ resulted in aspartate auxotrophy, a finding that prompted us to assess the role of aspartate in TORC1 activation. We found that the amino acids glutamine, asparagine, arginine, aspartate, and serine activate TORC1 most efficiently following nitrogen starvation. The glutamine synthetase inhibitor l-methionine sulfoximine abolished the ability of asparagine, arginine, aspartate, or serine, but not that of glutamine, to induce TORC1 activity, consistent with a central role for glutamine in activating TORC1. Neither addition of aspartate nor addition of glutamine restored TORC1 activity in caa1-deleted cells or in cells carrying a Caa1 variant with a catalytic site substitution, suggesting that the catalytic activity of Caa1 is required for TORC1 activation. Taken together, our results reveal the contribution of the key metabolic enzyme Caa1 to TORC1 activity in S. pombe.