ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 1346–1356 doi:10.1242/jcs.146373 RESEARCH ARTICLE Torin1-mediated TOR kinase inhibition reduces Wee1 levels and advances mitotic commitment in fission yeast and HeLa cells Jane Atkin1, Lenka Halova1, Jennifer Ferguson1, James R. Hitchin2, Agata Lichawska-Cieslar3, Allan M. Jordan2, Jonathon Pines3, Claudia Wellbrock1 and Janni Petersen1,* ABSTRACT target the kinase domain of mTOR, such as Torin1 (Thoreen et al., 2009), mimics the impact of rapamycin treatment in The target of rapamycin (TOR) kinase regulates cell growth and budding yeast, in that they induce autophagy, reduce protein division. Rapamycin only inhibits a subset of TOR activities. Here we synthesis and arrest cell cycle progression in G1 with a reduced show that in contrast to the mild impact of rapamycin on cell division, cell size (Thoreen et al., 2009). These effects of Torin1 blocking the catalytic site of TOR with the Torin1 inhibitor completely established that there are rapamycin-resistant roles for arrests growth without cell death in Schizosaccharomyces pombe.A mTORC1 that are essential for growth and proliferation. Torin1 mutation of the Tor2 glycine residue (G2040D) that lies adjacent to interacts with tryptophan-2239 in the catalytic, active site of the key Torin-interacting tryptophan provides Torin1 resistance, mTOR kinase (Yang et al., 2013). Crucially, this residue is absent confirming the specificity of Torin1 for TOR. Using this mutation, we in other kinases, including the mTOR-related phosphoinositide 3- show that Torin1 advanced mitotic onset before inducing growth kinases (PI3Ks). arrest. In contrast to TOR inhibition with rapamycin, regulation by Here, we describe the isolation of a tor2 mutation that maps to either Wee1 or Cdc25 was sufficient for this Torin1-induced advanced a conserved glycine located next to the key tryptophan (W2239 of mitosis. Torin1 promoted a Polo and Cdr2 kinase-controlled drop in mTOR) that directly interacts with Torin. This mutation conferred Wee1 levels. Experiments in human cell lines recapitulated these resistance to Torin1 and functionally validated the specificity of yeast observations: mammalian TOR (mTOR) was inhibited by Torin1 for TOR kinases. We have exploited this Torin1-resistant Torin1, Wee1 levels declined and mitotic commitment was advanced mutation to show that complete TORC1 inhibition advanced in HeLa cells. Thus, the regulation of the mitotic inhibitor Wee1 by mitotic commitment. Torin1 treatment reduced the levels of the TOR signalling is a conserved mechanism that helps to couple cell mitotic inhibitor Wee1. Experiments in human cell lines cycle and growth controls. recapitulated these yeast observations: Wee1 levels decreased and mitotic commitment advanced when HeLa mTOR was KEY WORDS: HeLa, S. pombe, TOR, Torin1, Wee1 inhibited by Torin1. These findings provide novel insight into the mechanisms by which inhibition of TOR activity impacts upon INTRODUCTION mitosis and cell division. Cells regulate growth, metabolism and proliferation through target of rapamycin (TOR) kinase signalling. Fission yeast RESULTS Schizosaccharomyces pombe contains two TOR kinases: the Growth of S. pombe is inhibited without cell death or G1 arrest non-essential Tor1 and the essential Tor2 (Weisman and Choder, following Torin1-induced TOR inhibition 2001). TOR kinases participate in at least two distinct protein We wanted to exploit TOR inhibition by Torin1 to further complexes: TORC1 (mainly containing Tor2) and TORC2 characterise TOR signalling in the model eukaryote S. pombe.A (predominantly containing Tor1) (Alvarez and Moreno, 2006; recent study has shown that a low concentration of Torin1 Hayashi et al., 2007; Matsuo et al., 2007). It is established that (0.2 mM) inhibits TORC1; however, no growth arrest of wild- rapamycin inhibits a subset of TOR activities in TORC1 type (wt) cells is observed (Ma et al., 2013). As the tor2+ complexes. In Saccharomyces cerevisiae rapamycin promotes (TORC1 complex) gene of fission yeast is essential (Weisman growth arrest (Barbet et al., 1996); however, it does not show the and Choder, 2001), TOR inhibition would be expected to halt same effect in either S. pombe or some mammalian cells (Neshat growth and proliferation. The ATP analogue (25 mM) did indeed et al., 2001; Pedersen et al., 1997; Weisman et al., 1997). In inhibit growth of wild-type cells on minimal solid media or in contrast, treatment of mammalian cells with ATP-analogues that liquid cultures (Fig. 1A–C). On rich media (YES), the growth of wt cells was inhibited by 5 mM Torin1 (data not shown). 1Faculty of Life Sciences, University of Manchester, Michael Smith Building, Incubation with the drug for 24 hours reduced proliferation to less Manchester M13 9PT, UK. 2Cancer Research UK Drug Discovery Unit, Paterson than 10% of vehicle-treated control cultures (Fig. 1C). As Institute for Cancer Research, University of Manchester, Wilmslow Road, previously reported, rapamycin had only a marginal impact on Manchester, M20 4BX, UK. 3The Gurdon Institute and Department of Zoology, Tennis Court Road, Cambridge CB2 1QN, UK. growth (Fig. 1A) (Weisman et al., 1997). To address whether Torin1 was promoting cell death, cells were treated with Torin1 *Author for correspondence ([email protected]) for 9 or 24 hours and spread on plates containing rich medium This is an Open Access article distributed under the terms of the Creative Commons Attribution without Torin1 to assess viability. Torin1-treated and vehicle- License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. treated control cultures gave similar numbers of colony forming units (CFU) (Fig. 1D), indicating that cells resumed growth Received 15 November 2013; Accepted 13 December 2013 following Torin1 withdrawal. In other words, Torin1 inhibition Journal of Cell Science 1346 RESEARCH ARTICLE Journal of Cell Science (2014) 127, 1346–1356 doi:10.1242/jcs.146373 Torin1 inhibits both TORC1 and TORC2 in S. pombe In fission yeast, both TORC1 and TORC2 signalling is regulated when cells are starved of nitrogen (Matsuo et al., 2007; Murai et al., 2009; Nakase et al., 2013; Nakashima et al., 2012; Petersen and Nurse, 2007; Takahara and Maeda, 2012; Uritani et al., 2006; Weisman and Choder, 2001; Weisman et al., 2007). Cells arrest cell cycle progression in G1 to undergo sexual differentiation and mating (Egel, 2003). Both TORC1 and TORC2 regulate this physiological cell cycle exit and, importantly, TORC2 activity is essential for the G1 arrest. We found that Torin1 completely prevented mating of wild-type cells. In contrast, rapamycin treatment, which only inhibits TORC1, had only a marginal impact on mating proficiency (Fig. 2A). Thus, because Torin1 inhibited growth (which is TORC1-dependent) without inducing a G1 arrest (Fig. 1E) and TORC2 activity is required for G1 arrest, these data suggested that Torin1 inhibited the TOR kinases in both TORC1 and TORC2. To confirm that Torin1 targets both TOR kinases, we used biochemical read-outs of both TORC1 and TORC2 activity. Phosphorylation of the ribosomal protein S6 (Rps6) is regulated by both TORC1 and TORC2 (Du et al., 2012; Nakashima et al., 2012; Nakashima et al., 2010). In wild-type cells, Rps6 phosphorylation was lost within 30 minutes of Torin1 treatment (Ma et al., 2013), but was only marginally reduced by rapamycin treatment (Fig. 2B). This indicated that Torin1 is targeting TOR. We next monitored the impact of Torin1 addition upon the phosphorylation status of TORC1- and TORC2-specific substrates. Phosphorylation of Maf1, a repressor of RNA polymerase III, is solely dependent on TORC1 activity (Du et al., 2012; Michels et al., 2010), whereas phosphorylation of the AGC kinase Gad8 at serine-546 is uniquely dependent on TORC2 (Matsuo et al., 2003; Tatebe et al., 2010). Maf1 phosphorylation was severely reduced following treatment with Torin1 for 30 minutes (Fig. 2C: note the collapse of the three phosphorylated Maf1 forms (Du et al., 2012) into a single faster- migrating band). Thus, Torin1 inhibited TORC1. Rapamycin also reduced Maf1 phosphorylation, but to a lesser extent, suggesting that rapamycin was a less potent inhibitor of TORC1 than Torin1. Because Ponceau S staining is linear with protein concentration R250.99 (Kowalczyk et al., 2013), it was used as loading control for Maf1–pk. Gad8 dephosphorylation was seen after Torin1 addition, whereas rapamycin had no impact upon Gad8 Fig. 1. Growth of S. pombe is inhibited without cell death or G1 arrest phosphorylation status (Fig. 2D). This suggested that, unlike following inhibition of TOR signalling by Torin1. (A) Wild-type cells grown on EMMG plates containing 25 mM Torin1, 300 ng/ml rapamycin or solvent. rapamycin, Torin1 inhibited TORC2. In summary we have MeOH, methanol. (B-F) Liquid cultures were treated with 25 mM Torin1 or shown that, in contrast to rapamycin, Torin1 inhibited TOR in + DMSO. (B) Cell number was measured and proliferation relative to vehicle both the TORC1 and TORC2 complexes. Only tor2 (TORC1 calculated after 24 hours (C). (D) 500 cells were spread on YES plates and complex) is essential for cell growth (Weisman and Choder, 2001), colony-forming units counted and shown relative to vehicle-treated cultures. making it likely that the growth arrest was a consequence of (E) DNA content was analysed by flow cytometry. (F) Cell size was inhibition of TORC1 alone. determined by forward-scatter flow cytometry. A mutation in the ATP-binding pocket of Tor2 provides Torin1 resistance did not induce cell death. We therefore asked whether the growth We next isolated mutations that allowed cells to grow in the arrest arose from cell cycle arrest in G1, as seen in mammalian presence of the drug. Following random mutagenesis by exposure cells (Thoreen et al., 2009) and in fission yeast following Tor2 to ultraviolet light, cells were plated onto medium containing inhibition (Matsuo et al., 2007; Uritani et al., 2006).