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RESEARCH ARTICLE

Torin1-mediated TOR kinase inhibition reduces Wee1 levels andadvances 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

The target of rapamycin (TOR) kinase regulates cell growth and

division. Rapamycin only inhibits a subset of TOR activities. Here we

show that in contrast to the mild impact of rapamycin on cell division,

blocking the catalytic site of TOR with the Torin1 inhibitor completely

arrests growth without cell death in Schizosaccharomyces pombe. A

mutation of the Tor2 glycine residue (G2040D) that lies adjacent to

the key Torin-interacting tryptophan provides Torin1 resistance,

confirming the specificity of Torin1 for TOR. Using this mutation, we

show that Torin1 advanced mitotic onset before inducing growth

arrest. In contrast to TOR inhibition with rapamycin, regulation by

eitherWee1 or Cdc25 was sufficient for this Torin1-induced advanced

mitosis. Torin1 promoted a Polo and Cdr2 kinase-controlled drop in

Wee1 levels. Experiments in human cell lines recapitulated these

yeast observations: mammalian TOR (mTOR) was inhibited by

Torin1, Wee1 levels declined and mitotic commitment was advanced

in HeLa cells. Thus, the regulation of the mitotic inhibitor Wee1 by

TOR signalling is a conserved mechanism that helps to couple cell

cycle and growth controls.

KEY WORDS: HeLa, S. pombe, TOR, Torin1, Wee1

INTRODUCTIONCells regulate growth, metabolism and proliferation through

target of rapamycin (TOR) kinase signalling. Fission yeast

Schizosaccharomyces pombe contains two TOR kinases: the

non-essential Tor1 and the essential Tor2 (Weisman and Choder,

2001). TOR kinases participate in at least two distinct protein

complexes: TORC1 (mainly containing Tor2) and TORC2

(predominantly containing Tor1) (Alvarez and Moreno, 2006;

Hayashi et al., 2007; Matsuo et al., 2007). It is established that

rapamycin inhibits a subset of TOR activities in TORC1

complexes. In Saccharomyces cerevisiae rapamycin promotes

growth arrest (Barbet et al., 1996); however, it does not show the

same effect in either S. pombe or some mammalian cells (Neshat

et al., 2001; Pedersen et al., 1997; Weisman et al., 1997). In

contrast, treatment of mammalian cells with ATP-analogues that

target the kinase domain of mTOR, such as Torin1 (Thoreen

et al., 2009), mimics the impact of rapamycin treatment in

budding yeast, in that they induce autophagy, reduce protein

synthesis and arrest cell cycle progression in G1 with a reduced

cell size (Thoreen et al., 2009). These effects of Torin1

established that there are rapamycin-resistant roles for

mTORC1 that are essential for growth and proliferation. Torin1

interacts with tryptophan-2239 in the catalytic, active site of

mTOR kinase (Yang et al., 2013). Crucially, this residue is absent

in other kinases, including the mTOR-related phosphoinositide 3-

kinases (PI3Ks).

Here, we describe the isolation of a tor2 mutation that maps to

a conserved glycine located next to the key tryptophan (W2239 of

mTOR) that directly interacts with Torin. This mutation conferred

resistance to Torin1 and functionally validated the specificity of

Torin1 for TOR kinases. We have exploited this Torin1-resistant

mutation to show that complete TORC1 inhibition advanced

mitotic commitment. Torin1 treatment reduced the levels of the

mitotic inhibitor Wee1. Experiments in human cell lines

recapitulated these yeast observations: Wee1 levels decreased

and mitotic commitment advanced when HeLa mTOR was

inhibited by Torin1. These findings provide novel insight into the

mechanisms by which inhibition of TOR activity impacts upon

mitosis and cell division.

RESULTSGrowth of S. pombe is inhibited without cell death or G1 arrestfollowing Torin1-induced TOR inhibition

We wanted to exploit TOR inhibition by Torin1 to further

characterise TOR signalling in the model eukaryote S. pombe. A

recent study has shown that a low concentration of Torin1

(0.2 mM) inhibits TORC1; however, no growth arrest of wild-

type (wt) cells is observed (Ma et al., 2013). As the tor2+

(TORC1 complex) gene of fission yeast is essential (Weisman

and Choder, 2001), TOR inhibition would be expected to halt

growth and proliferation. The ATP analogue (25 mM) did indeed

inhibit growth of wild-type cells on minimal solid media or in

liquid cultures (Fig. 1A–C). On rich media (YES), the growth of

wt cells was inhibited by 5 mM Torin1 (data not shown).

Incubation with the drug for 24 hours reduced proliferation to less

than 10% of vehicle-treated control cultures (Fig. 1C). As

previously reported, rapamycin had only a marginal impact on

growth (Fig. 1A) (Weisman et al., 1997). To address whether

Torin1 was promoting cell death, cells were treated with Torin1

for 9 or 24 hours and spread on plates containing rich medium

without Torin1 to assess viability. Torin1-treated and vehicle-

treated control cultures gave similar numbers of colony forming

units (CFU) (Fig. 1D), indicating that cells resumed growth

following Torin1 withdrawal. In other words, Torin1 inhibition

1Faculty of Life Sciences, University of Manchester, Michael Smith Building,Manchester M13 9PT, UK. 2Cancer Research UK Drug Discovery Unit, PatersonInstitute for Cancer Research, University of Manchester, Wilmslow Road,Manchester, M20 4BX, UK. 3The Gurdon Institute and Department of Zoology,Tennis Court Road, Cambridge CB2 1QN, UK.

*Author for correspondence (Janni.Petersen@manchester.ac.uk)

This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distributionand reproduction in any medium provided that the original work is properly attributed.

Received 15 November 2013; Accepted 13 December 2013

� 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 1346–1356 doi:10.1242/jcs.146373

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did not induce cell death. We therefore asked whether the growth

arrest arose from cell cycle arrest in G1, as seen in mammaliancells (Thoreen et al., 2009) and in fission yeast following Tor2inhibition (Matsuo et al., 2007; Uritani et al., 2006). Flow

cytometric analysis demonstrated that, in contrast to mammaliancells, wild-type fission yeast cells did not arrest in G1 afterincubation with the drug for up to 24 hours (Fig. 1E).

Importantly, despite this lack of a G1 arrest, cell size wasreduced following TOR inhibition (Fig. 1F; Fig. 4A). These datademonstrated that Torin1 inhibited S. pombe growth withoutinducing either cell death or cell cycle arrest in G1 phase.

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; Muraiet al., 2009; Nakase et al., 2013; Nakashima et al., 2012; Petersenand 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 andmating (Egel, 2003). Both TORC1 and TORC2 regulate thisphysiological cell cycle exit and, importantly, TORC2 activity is

essential for the G1 arrest. We found that Torin1 completelyprevented mating of wild-type cells. In contrast, rapamycintreatment, which only inhibits TORC1, had only a marginal

impact on mating proficiency (Fig. 2A). Thus, because Torin1inhibited growth (which is TORC1-dependent) without inducinga G1 arrest (Fig. 1E) and TORC2 activity is required for G1

arrest, these data suggested that Torin1 inhibited the TOR kinasesin both TORC1 and TORC2.

To confirm that Torin1 targets both TOR kinases, we usedbiochemical read-outs of both TORC1 and TORC2 activity.

Phosphorylation of the ribosomal protein S6 (Rps6) is regulated byboth 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 phosphorylationstatus of TORC1- and TORC2-specific substrates. Phosphorylationof Maf1, a repressor of RNA polymerase III, is solely dependent on

TORC1 activity (Du et al., 2012; Michels et al., 2010), whereasphosphorylation of the AGC kinase Gad8 at serine-546 is uniquelydependent 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 threephosphorylated 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, suggestingthat 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 controlfor Maf1–pk. Gad8 dephosphorylation was seen after Torin1addition, whereas rapamycin had no impact upon Gad8phosphorylation status (Fig. 2D). This suggested that, unlike

rapamycin, Torin1 inhibited TORC2. In summary we haveshown that, in contrast to rapamycin, Torin1 inhibited TOR inboth the TORC1 and TORC2 complexes. Only tor2+ (TORC1

complex) is essential for cell growth (Weisman and Choder, 2001),making it likely that the growth arrest was a consequence ofinhibition of TORC1 alone.

A mutation in the ATP-binding pocket of Tor2 provides Torin1resistanceWe next isolated mutations that allowed cells to grow in thepresence of the drug. Following random mutagenesis by exposureto ultraviolet light, cells were plated onto medium containing25 mM Torin1. A point mutation in the essential tor2+ gene

(TORC1 complex), tor2-G2040D, enabled growth in the presenceof the drug (Fig. 3A,B). Torin1 resistance segregated with aMendelian ratio, 2:2, following mating between wild-type and

tor2-G2040D cells (Fig. 3B), indicating that Torin1 resistancearose from the tor2-G2040D mutation alone. The glycine atposition 2040 within the ATP-binding pocket of the kinase

domain is conserved throughout eukaryotes (Fig. 3C). To confirm

Fig. 1. Growth of S. pombe is inhibited without cell death or G1 arrestfollowing inhibition of TOR signalling by Torin1. (A) Wild-type cells grownon EMMG plates containing 25 mM Torin1, 300 ng/ml rapamycin or solvent.MeOH, methanol. (B-F) Liquid cultures were treated with 25 mM Torin1 orDMSO. (B) Cell number was measured and proliferation relative to vehiclecalculated after 24 hours (C). (D) 500 cells were spread on YES plates andcolony-forming units counted and shown relative to vehicle-treated cultures.(E) DNA content was analysed by flow cytometry. (F) Cell size wasdetermined by forward-scatter flow cytometry.

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that this mutation in tor2+ did indeed confer Torin1-resistance,

plasmids expressing wild-type tor2+ and tor2-G2040D weretransformed into a tor2 deletion strain. Torin1 arrested growth ofthe strain that expressed the wild-type tor2+ gene, whereas

expression of the mutant allele protected cells from this growtharrest (Fig. 3D). The growth rate of tor2-G2040D cultures waslargely unaffected by exposure to Torin1 (Fig. 3E,F). Together

these results indicated that the tor2-G2040D mutation provides

resistance to Torin1.

The tor2-G2040D mutation alters the dephosphorylation of TORC1substrates following Torin1 treatmentCell size at division provided further evidence of drug resistancein the tor2-G2040D mutant. The reduction in cell size normally

Fig. 2. Torin1 inhibits both TORC1 andTORC2 in S. pombe. (A) Mating efficiencyfollowing drug treatment. Cells of oppositemating type were mixed 1:1 and incubatedon SPA plates as indicated (B-D) Wild-type(B,D) and maf1–pk cells (C) were treatedwith 25 mM Torin1, 300 ng/ml rapamycin orsolvent for 30 minutes. Samples wereharvested and analysed by western blotusing the indicated antibodies. Maf1phosphorylation is TORC1 specific,Gad8.S546 phosphorylation is TORC2specific and Rsp6 phosphorylation isregulated by both TORC1 and TORC2.

Fig. 3. A mutation in the ATP-binding pocket of Tor2 providesresistance to Torin1. (A,B) Theindicated strains were grown onEMMG plates containing 25 mMTorin1. (B) 2:2 segregation of Torin1-resistance. Four tetrads werereplicated onto YES and YES + Torin1plates, to the right a diagram illustratesthe genotype of the four spores(C) Alignment of TOR kinases;conserved residues are highlighted inbold and residues forming the ATP-binding site are indicated by /. Glycinecorrespending to tor2.G2040 ishighlighted in red. (D) Expression oftor2.G2040D provides resistance toTorin1. (E,F) Cells were treated as inFig. 1B,C, cell number was counted(E) and proliferation relative to vehiclecalculated after 24 hours (F).

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induced by treatment with 25 mM Torin1 (Fig. 1F; Fig. 4A) wasnot observed in tor2-G2040D cells (Fig. 4A). Furthermore,biochemical read-outs of TOR activity also revealed resistance.

Unlike wild-type cells (Fig. 2B), Rps6 phosphorylation persistedfor 30 minutes after drug treatment of tor2-G2040D cells(Fig. 4B). In contrast, Gad8 phosphorylation at serine-546 was

lost from tor2-G2040D cells after Torin1 treatment (Fig. 4C),confirming that Torin1 was still able to inhibit TORC2 in thetor2-G2040D (TORC1 complex) mutant. Phosphorylation of

Maf1 (a measure of TORC1 activity) was compromised within10 minutes of the addition of 750 nM Torin1 to wild-type cells(Fig. 4D). In contrast, hyper-phosphorylated forms of Maf1 were

still observed in the Torin1-treated tor2-G2040D mutant,resulting in less hypo-phosphorylated Maf1 in the tor2-G2040D

mutant. Together these data suggest that Torin1 had a reducedeffect on TORC1 activity in the tor2-G2040D mutant. This is

expected because Torin1 is an ATP-analogue that is inhibiting anessential kinase, Tor2. If the mutation in the ATP-binding pocketcompletely blocked Torin1 binding, it would most likely also

block ATP-binding to this essential kinase, thereby killing thecell. The resistant mutant is therefore likely to strike acompromise between blocking binding of the ATP analogue

(Torin1) and allowing binding of the highly related ATP, tosupport viability. In summary, mutation of a glycine residue toaspartate in the ATP-binding site of Tor2 provides resistance to

the ATP analogue Torin1 and thereby allows cell proliferation inthe presence of the drug. This confirms the specificity of Torin1for TOR kinases in fission yeast. It also validates Torin1 use as atool for studying TOR function in S. pombe.

Torin1 induces cells to advance into mitosis mainly throughTORC1 inhibitionS. pombe is a rod-shaped cell that grows by tip extension whilstmaintaining a constant cell diameter. Cells commit to mitosis andcease growth once a critical length is achieved. Cell length at

division is therefore a direct read-out of the time at which cellsexecute the G2-M transition (Fantes and Nurse, 1977; Nurse andThuriaux, 1977). This threshold cell length is determined by the

environment and correlates with TOR activity (Petersen andNurse, 2007). When TOR activity is inhibited with eitherrapamycin or by a decrease in nutrient quality, wild-type cells

decrease their size threshold for division and advance into mitosisat a reduced length (Petersen and Nurse, 2007). Consistently,TOR inhibition in wild-type cells by Torin1 lowered the size

threshold for mitotic commitment (Fig. 1F; Fig. 4A), leading to atransient burst of mitosis and cell division (Fig. 5A,C). The morerapid response to Torin1 treatment than to rapamycin again

implies a more efficient mode of TOR inhibition (Fig. 5A). Todetermine whether the Torin1-based acceleration of mitosis waselicited through TORC1 or TORC2, cells harbouring mutations ineach complex were exposed to the drug (Fig. 5B). TORC1

function was not inhibited by Torin1 in cells expressing tor2-

G2040D (TORC1 mutant) (Figs 3, 4), which enabled theassessment of the contribution of TORC2 to the mitotic

advance in this strain. The Torin1 response of tor2-G2040D

cells was severely impaired (Fig. 5B), suggesting that Torin1primarily advances mitosis through inhibition of TORC1.

Conversely, mutants in TORC2 (tor1-D, sin1-D) bothaccelerated mitotic commitment, resulting in a peak in thefrequency of dividing cells (Fig. 5B). We conclude that the

predominant mechanism by which Torin1 advanced mitotic onsetwas through the inhibition of TORC1.

Torin1 causes cells to advance into mitosis through the regulators ofmaturation promoting factorWe have previously shown that mild TOR inhibition withrapamycin also promotes mitotic onset, and that this advance

relied upon the mitotic kinase Polo kinase Plo1. Phosphorylationof Plo1.S402 promotes Plo1 recruitment to the spindle pole body(centrosome) to trigger activation of Cdk1–cyclin-B through

Fig. 4. The tor2.G2040D mutation alters thedephosphorylation of TORC1 substratesfollowing Torin1 treatment. (A) Cell length atdivision of indicated strains (n5200). After2.5 hours of Torin1 treatment, 10% of wt cellsdivide (see Fig. 5A). (B-D) Western blot usingthe indicated antibodies. Cells were treated asin Fig. 2. (C) Cells were treated with 25 mMTorin1 for 10 minutes. A non-specific band isindicated by an asterisk (*). (D) Cells weretreated with 750 nM Torin1 for 10 minutes.

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regulation of Wee1 and Cdc25 (Grallert et al., 2013; Petersen and

Hagan, 2005). The Wee1 kinase inhibits Cdk1–cyclin-B throughphosphorylation of tyrosine 15 within the active site of Cdc2(Featherstone and Russell, 1991; Gould and Nurse, 1989), andCdc25 phosphatase removes the inhibitory phosphate placed on

Cdc2 by Wee1 (Millar et al., 1991). Combined regulation of bothCdc25 and Wee1 is essential for this weak rapamycin-induced

TORC1 inhibition to promote Cdk1–cyclin-B activation (Petersenand Nurse, 2007). Interestingly, the acceleration of mitosis that istriggered by Torin1 treatment persists in the phosphorylation-refractory plo1.S402A allele (Fig. 5C), indicating that, following

the stronger Torin1-induced TORC1 inhibition, either plo1.

S402A-independent activation of Cdc25 or inhibition of Wee1alone is sufficient to advance mitotic onset. We therefore assessed

the impact of Torin1 treatment on the Cdc2 mutants cdc2-1w

and cdc2-3w (Fantes, 1981; Thuriaux et al., 1978). cdc2-1w islargely insensitive to Wee1, such that Cdk1–cyclin-B activity

is mainly regulated by Cdc25 function. In contrast, cdc2-3w islargely unresponsive to Cdc25, and hence Cdk1–cyclin-B activityis mainly dependent upon Wee1 control in this mutant. In contrast

to mild TOR inhibition with rapamycin, where neither cdc2.1w

nor cdc2.3w advanced mitosis, both mutants advanced mitoticcommitment following addition of Torin1 (Fig. 5D) (Petersenand Nurse, 2007). Together, these findings indicate that Torin1

very efficiently activates Cdc25 and inhibits Wee1, and eitheractivation of Cdc25 or inhibition of Wee1 activates Cdk1–cyclin-B to a sufficient degree to advance the timing of mitotic

commitment. Importantly, the acute Torin1-induced mitoticadvance was regulated through Cdc2 tyrosine 15, because thecdc2.F15 mutant, which cannot be regulated by either Wee1 or

Cdc25 on Tyr15, did not efficiently advance mitosis in responseto Torin1 treatment (Fig. 5C).

Torin1 induces a decline in Wee1 levels and a reduced Cdc25migrationTOR inhibition promotes Sty1 activation, which in turn leads toPlo1 recruitment to the spindle pole bodies to trigger activation of

Cdk1–cyclin-B. This is regulated through Wee1 and Cdc25(Grallert et al., 2013; Petersen and Hagan, 2005). However, itis unclear how Wee1 and Cdc25 activities are controlled.

Interestingly, the sharp rise in the mitotic index followingTorin1 addition to the culture coincided with a decline in Wee1levels (Fig. 6A,D,E). Wee1 is tagged with GFP at the N-terminus,

and this strain has a wild-type response to Torin1 treatment (datanot shown). The degree to which Torin1 addition reduced Wee1levels exceeded that seen upon inhibition of global proteinsynthesis by cycloheximide treatment and that of mild TOR

inhibition with rapamycin, indicating that Wee1 turnover hadbeen actively promoted by the inhibition of TOR function(Fig. 6A,C). Importantly, this decline in Wee1 was not observed

in the tor2.G2040D mutant (Fig. 6E). In addition, the decline inmobility of Cdc25 in SDS-PAGE that accompanies its activation(Kovelman and Russell, 1996; Moreno et al., 1990) was seen

within 30 minutes of Torin1 treatment (Fig. 6B). We suggest thatinhibition of TORC1 by Torin1 in wild-type cells traps the Cdk1–cyclin-B complex in its active dephosphorylated form (Fig. 6D)

to promote mitotic commitment at a reduced cell size (Fig. 4A;Fig. 5C). The molecular basis for these multiple controls of theCdk1–cyclin-B complex in response to TOR inhibition is veryinteresting – here we focused our attention upon the regulation of

Wee1.

Torin1 induces a Plo1- and Cdr2-controlled decline in Wee1 levelsIt has previously been shown that Plo1 regulates Wee1 activity,because the enhanced Plo1 recruitment to the spindle pole bodyby Cut12 is sufficient to overcome the lack of Cdc25 (Hagan,

2008). The decline in Wee1 levels was delayed, but it was still

Fig. 5. Torin1 induces cells to advance into mitosis. (A-D) Cells of theindicated strain were treated with Torin1, rapamycin or DMSO for theindicated times and the percentages of dividing cells (A,B,D) or mitotic cellsin anaphase (C) were calculated. Graphs are representative of at least twoindependent experiments.

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reduced in the plo1.S402A mutants after Torin1 treatment

(Fig. 6E). This suggested that Wee1 could also be downregulatedthrough a plo.S402A-independent mechanism following Torin1-induced TOR inhibition. SAD (synapses of amphids defective)

family kinases have been shown to also regulate zWee1 levels(Muller et al., 2010; Young and Fantes, 1987). We therefore askedwhether the fission yeast Cdr2 (SAD kinase) along with Plo1 were

responsible for the changes in Wee1 levels that arise from Torin1inhibition. The Torin1-induced decline of Wee1 was severelycompromised by the absence of combined Cdr2 and Plo1 function,

indicating that TORC1 control of Wee1 turnover was regulated byboth kinases but independently (Fig. 6E). The acceleration ofmitosis that is triggered by Torin1 persisted in the plo1.S402A

cdr2D double mutant (Fig. 5C), which again demonstrated that

regulation of Cdc25 only was sufficient to advance mitotic onset

following the strong Torin1-induced TOR inhibition. How Plo1and Cdr2 independently control this novel Torin1-induced Wee1turnover is unclear at present. However, Wee1 resembles its

budding yeast counterpart Swe1p (Harvey et al., 2005; Sakchaisriet al., 2004) in being very heavily phosphorylated (Y. D. Tay and I.Hagan, personal communication), and Cdr2 has previously been

shown to control phosphorylation of Wee1 (Kanoh and Russell,1998), suggesting that either kinase could directly modulate Wee1phosphorylation to control protein levels.

mTORC1 inhibition reduces Wee1 levelsTorin1-induced inhibition of mTOR in mammalian cells limitsprotein synthesis and arrests cells in G1 at a reduced cell size

Fig. 6. Torin1 induces a Plo1- and Cdr2-controlled decline in Wee1 levels. (A,B,C,E) Cells were treated with Torin1, cycloheximide (CHX), rapamycin orDMSO for indicated times. (A,B) Western blot of Cdc25 or Wee1. (D) Inhibition of TORC1 by Torin1 in wild-type cells traps the Cdk1–cyclin-B complex in itsactive dephosphorylated form as Wee1 levels are reduced and Cdc25 is modified. (C,E) Western blots of Wee1 levels and quantification of Wee1 levels from 3individual experiements. Torin1-treated wt cells are shown twice for comparison.

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(Thoreen et al., 2009). However, it is unclear whether cells alsoadvance mitotic commitment before this growth arrest. The link

between TOR signalling and Wee1 control in fission yeastprompted us to ask whether mTOR similarly controls Wee1levels in human cells. Initial experiments using synchronizationapproaches, which are widely used to manipulate cell cycle

progression in mammalian cells, revealed a small decrease inWee1 levels (data not shown). However, it is possible thatsynchronization invoked stress-induced MAPK signalling that

could have impacted on mTOR signalling prior to drug treatment(Hernandez et al., 2011; Ma et al., 2005). We therefore turned toasynchronous populations; it was noticeable that Wee1 levels in

unperturbed cell cultures were markedly reduced after 2 hours ofmTOR inhibition with Torin1. This reduction in Wee1 levels wasseen in both HeLa cells and in A375 melanoma cells (Fig. 7A,B).

Importantly cycloheximide treatment did not promote the same

degree of Wee1 instability (Fig. 7C,D). Wee1 levels have beenreported to be low in the G1 phase of the cell cycle (Watanabe

et al., 2005), therefore this rapid Torin1-induced decline in Wee1levels could simply reflect a rapid change in the cell cycle profileof the population. However, flow cytometry analysis and the levelof the G1-specific marker Cdt1 provided no evidence for an

increase in the G1 population at these early time points(Fig. 7A,E). Thus, these experiments in mammalian cellsmirrored the results from fission yeast in that mTOR inhibition

led to reduced Wee1 levels.

mTOR inhibition advances mitotic onsetWe next addressed the possibility that mTOR control of Wee1levels would advance mitotic commitment by shortening the G2phase of the cell cycle. We used a non-invasive live-imaging

assay to monitor the impact of Torin1 inhibition of mTOR

Fig. 7. mTORC1 inhibition advances mitosis, as Wee1 is lost. (A) HeLa cells were treated with 250 nM Torin1 and samples harvested at the indicated timepoints and analysed by western blot using the indicated antibodies. (B) A375 cells were treated with 250 nM Torin1 and analysed by western blot.(C) Cycloheximide (CHX) treatment of HeLa cells. (D) Quantification of A and C. (E) HeLa cells were treated with 250 nM Torin1 and DNA content was analysedby flow cytometry. (F) HeLa Kyoto cyclin-B1–Venus2/+ mRuby–PCNA2/+ H3.3–CFP cells were treated with 250 nM Torin1 or solvent (DMSO). Cell cycleprogression was analysed by time-lapse microscopy using mRuby–PCNA marker. G2 length in individual cells was measured from the time point when thePCNA foci form. Red lines are mean values of four independent experiments; red dots represent cells that were in G2 phase until the end of experiment.

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activity on HeLa cells. We defined the start of G2 phase as theloss of the last foci of the DNA replication marker proliferating

cell nuclear antigen (PCNA), and the end of G2 phase as nuclearenvelope breakdown, when nuclear PCNA dispersed throughoutthe cytoplasm. Torin1 treatment accelerated the mitoticcommitment of HeLa cells, as indicated by the finding that the

duration of G2 phase contracted from 247 minutes to200 minutes (Fig. 7F). This 20% decrease in the duration of G2phase in HeLa cells was highly reminiscent of previous

observations in fission yeast, in which TOR inhibition insynchronous fission yeast cultures reduced the G2 phase by25% (Petersen and Nurse, 2007). Together, these findings provide

the first demonstration that, just like in fission yeast, inhibition ofmTOR can advance mitosis in mammalian cells.

DISCUSSIONWe show that complete inhibition of TORC1 function by theaddition of the ATP analogue Torin1 blocks cell proliferation offission yeast. Because the signalling pathways upstream of

TORC1 are conserved between this yeast and humans, weanticipate that the use of Torin1 in S. pombe will make asignificant contribution to our understanding of the architecture

and control of mTOR signalling in mammals.A single glycine-to-aspartate mutation at position 2040 of the

essential S. pombe kinase Tor2 conferred Torin1 resistance,

providing the first in vivo validation of the specificity of Torin1for TOR kinases in any eukaryote (Figs 3,4). The site of thismutation within the ATP-binding pocket is conserved in mTOR

(Fig. 3C). Torin1 docking in the ATP-binding pocket of mTOR(Liu et al., 2012) is proposed to occur through a putativehydrogen bond between Torin1 and Val2240. Furthermore, therecently identified crystal structure of mTOR in complex with the

second-generation Torin2 inhibitor places Ile2237 and Trp2239

within 4 A of the tricyclic benzonaphthyridinone ring (sharedwith Torin1) (Yang et al., 2013). In fact, the pivotal interaction

between Torin2 and mTOR is through the Trp2239 residue,which interacts with ten atoms from the ring moiety of Torin2 andis found directly adjacent to the glycine [equivalent to the site ofmutation in fission yeast Tor2 (Fig. 8A)]. Torin1 and Torin2 both

inhibit mTOR with an 800-fold greater specificity over PI3Ks(Liu et al., 2013; Thoreen et al., 2009). Importantly, PI3Ks do notcontain the key Torin-interacting tryptophan (Fig. 8A) (Yang

et al., 2013). It was recently shown that Torin2 has a 10-foldlower affinity for the two PI3K-like kinases ATM and ATR (Liuet al., 2013). Interestingly, ATM, ATR and the fission yeast

homologue Rad3 all contain the key tryptophan residue (Fig. 8A).However, they mimic the Torin1 resistant tor2 mutant that wehave isolated here in having a charged glutamic acid instead of

the glycine found at the site of the tor2 mutation in TOR kinases(Fig. 8A) – this is likely to explain the reduced drug potencytowards these closely related kinases. The identification of thisconserved site has implications for studies in mammalian cells,

where this mutant will be useful in eliminating the possible off-target effects.

Here we find that the TORC1 control of mitotic entry and cell

size at division can be achieved by the convergence of multipleindependent pathways that regulate Wee1 and Cdc25 to controlCdc2–cyclin-B activity (Fig. 8B). Importantly, both rapamycin-

and Torin1-stimulated Cdc2–cyclin-B activation is regulatedthrough Wee1 and Cdc25 to control Cdc2.Y15 phosphorylation.However, the level of Cdc2–cyclin-B activation varied depending

upon the degree of TOR inhibition. Under mild TOR inhibitionwith rapamycin, the advance of mitotic commitment requiredsimultaneous control of both Cdc25 and Wee1 (Fig. 8B). Incontrast, the enhanced level of TORC1 inhibition arising from the

use of Torin1 did not rely upon dual control of both of these

Fig. 8. The convergence of multiple pathways to control Cdc2–cyclin-B activity. (A) Alignment of TOR and TOR-related kinases. Conservedresidues are highlighted in bold. The key Torin-interacting tryptophan is shown in cyan. The Torin1 resistant tor2-G2040D is shown in red. (B) A modelsuggesting that when Torin1 inhibits TORC1, Wee1 levels decline. This traps Cdc2–cyclin-B in its active conformation, driving entry into mitosis at a reduced cellsize. The presence of molecule X is implied by advanced mitosis in torin1-treated plo1.S402A cdr2::ura4+ double mutants. Insert: in contrast to rapamycin,regulation of either Wee1 (1) or Cdc25 (2) is sufficient to advance mitotic onset following the strong Torin1-induced TOR inhibition.

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regulators – rather a singular impact upon either Cdc25 or Wee1activity alone was sufficient to advance mitotic commitment

(Fig. 8B). Importantly, we find that the reduction in Wee1 levelsupon acute TORC1 inhibition in S. pombe is also seen uponmTORC1 inhibition in human cells (Figs 6, 7), revealing a novel,universal aspect of TOR signalling. Furthermore, the Torin1-

induced advancement of mitotic commitment that we observed inHeLa cells (Fig. 7F) is consistent with the notion that the mTORkinase controls mitotic commitment.

Interestingly, in pilot experiments, no advancement in thetiming of mitosis was observed in retinal pigment epithelium(RPE) cell lines following Torin1 treatment (data not shown).

Therefore, Torin1-induced advanced mitotic commitment islikely to be cell line and context specific. However, in RPEcells, TOR signalling appears to function differently because

TORC2-dependent activation of AKT1 is absolutely required forits activity (Parrales et al., 2013), whereas this is not the case inother cell lines. Furthermore, HeLa cells are p53 deficient. Theyhave reduced PTEN levels, and therefore have increased TOR

signalling (Feng, 2010). In addition, because p53 also impacts onTOR signalling through the tuberous sclerosis (TSC) and AMP-activated protein kinase (AMPK) TOR inhibitors (Feng, 2010),

the steady-state TOR activity in RPE and HeLa cell lines is likelyto be very different. This difference is likely to account for thedifferent impacts of Torin1 inhibition in the two cell lines.

In summary, mammalian cells can exploit the same types ofTOR-mediated control of Cdk1–cyclin-B activity that couplechanges in the timing of mitotic commitment to environmental

cues in fission yeast, as both HeLa and A375 cell lines reducedWee1 levels following mTOR inhibition with Torin1. The abilityof Torin1 to reduce cell growth and proliferation has made it anattractive anti-cancer drug candidate. Similar levels of excitement

have been provoked by the success of a Wee1 inhibitor in pre-clinical models in which Wee1 inhibition promoted a lethalmitotic catastrophe in cancer cells whose DNA integrity was

compromised (Kreahling et al., 2012). We therefore provide anovel means by which Torin1-mediated inhibition of TORactivity could offer therapeutic benefit in particular tumour

contexts.

MATERIALS AND METHODSYeast cell culture and reagentsThe S. pombe strains used in this study are listed in supplementary

material Table S1. Unless otherwise stated, cells were grown at 28 C in

Edinburgh minimal medium 2 (EMM2-N ForMedium) supplemented

with 20 mM glutamate (EMMG). Cells were grown exponentially at

28 C for 48 hours. For growth on solid media, cells were grown at 30 C

on plates containing YES or EMMG.

Molecular biologyQuikChange (Stratagene) site-directed mutagenesis was used to generate

the plasmid containing tor2-G2040D (forward primer: 59-CCTTTTAG-

ACTGGGTTTTGGATAGCGATAC-39, reverse primer: 59-CAAAACC-

CAGTCTAAAAGGCCTGAATCCGGTG-39). Wild-type tor2+ and tor2-

G2040D were subsequently cloned into the pREP42–NAT vector (kind

gift from I. Hagan). Plasmids were transformed into S. pombe using

lithium acetate (Bahler et al., 1998).

Growth assaysTorin1 was synthesised at the CRUK Manchester Research Institute

according to the published protocol (Liu et al., 2010), yielding a product

with .95% purity as determined by liquid chromatography mass

spectrometry and proton NMR. Torin1 was stored as a solid in the

dark and dissolved at 50 C in DMSO (Sigma-Aldrich) immediately

before use, with a stock concentration of 7.5 mM. For cell growth assays,

cells were grown exponentially for 48 hours to 2.56106 cells/ml before

treatment with Torin1 at a final concentration of 25 mM or equivalent

DMSO vehicle control. Growth at 28 C was monitored by optical density

(OD) at 595 nm. For cell survival assays, following treatment with

Torin1 or DMSO as above, 500 cells were spread on YES plates in

duplicate. Colonies were counted after incubation at 30 C. For growth on

solid media 2.56106 cells/ml were spotted on plates containing EMMG +

Torin1 (25 mM final) or DMSO (0.33% final v/v), or EMMG +

rapamycin (300 ng/ml final from 2 mg/ml stock; Sigma-Aldrich) or 1:1

DMSO:MeOH (0.15% final v/v). For protein turnover assays, 100 mg/ml

cycloheximide was used.

Torin1 screenWild-type cells were grown to 2.56106 cells/ml. 100,000 cells/plate were

plated onto 10 EMMG plates containing 25 mM Torin1 and were exposed

to 0.015 J of UV irradiation per plate using a UV lamp cabinet (Uvitec,

Cambridge). Plates were incubated at 30 C for 3 to 5 days. Isolated

mutants were streaked to single colonies on YES plates and replica-

plated to EMMG plates containing 25 mM Torin1 to remove false

positives. Three candidates were backcrossed a minimum of three times

before genomic DNA was isolated (Qiagen Gentra Puregene Yeast/Bact.

Kit) and sequenced by whole genome sequencing (GATC Biotech,

Konstanz, Germany).

Flow cytometryS. pombe DNA content and cell size were measured by flow cytometry as

previously described (Costello et al., 1986). Samples were sonicated and

30,000 events processed using a Beckman Coulter Cyan ADP instrument

with 488 nm excitation detection filter and 530/40 nm bandpass. HeLa

cells were fixed drop-wise with 70% ethanol and 5000 events processed

as above. Data were analysed using Summit 4.3 software (Beckman

Coulter).

Protein extraction and western blottingFor western blotting of S. pombe cells, 66107 cells were fixed and total

protein extracts were prepared by precipitation with trichloroacetic acid

(TCA) as previously described (Caspari et al., 2000). HeLa and A375

cells were lysed in SDS sample buffer [60 mM Tris-HCl (pH 6.8), 10%

glycerol, 1% SDS, 2% b-mercaptoethanol, 0.01% Bromophenol Blue].

Following gel electrophoresis, proteins were transferred onto a

polyvinylidene difluoroide (PVDF) membrane (Millipore), blocked in

TBS plus 3% dried milk and incubated with primary antibodies in TBS

plus 3% milk overnight at 4 C. After washing (4630 minutes with TBS

plus 0.05% Tween20), membranes were probed with secondary

antibodies linked to alkaline phosphatase (Sigma-Aldrich). Membranes

were developed after washing as above by addition of substrate: nitro-

blue tetrazolium chloride (NBT; VWR International)/5-bromo-4-chloro-

39-indolylphosphate p-toluidine (BCIP; Molekula) in AP buffer (100 mM

NaCl, 5 mM MgCl2, 100 mM Tris-HCl pH 9.5). The following primary

antibodies were used in this study: mouse anti-phospho-(Ser/Thr) Akt

substrate (PAS) antibody to detect phospho-Rps6 (1:2000; Cell Signaling

Technology), mouse anti-S6 antibody to detect total Rps6 (1:2000;

Abcam), mouse anti-V5-Tag antibody to detect Maf1–pk (1:1000; AbD

Serotec), rabbit anti-phospho-Gad8 S546 antibody to detect phospho-

Gad8 S546 (1:500; raised by Eurogentec) (Du et al., 2012), sheep anti-

Gad8 antibody to detect total Gad8 (1:200; raised by Scottish National

Blood Transfusion Service, Midlothian) (Du et al., 2012), mouse anti-

myc antibody to detect Cdc25–myc (1:2000; Millipore), mouse anti-GFP

antibody to detect Wee1–GFP (1:100; Roche), rabbit anti-Wee1 antibody

to detect human Wee1 (1:500; New England Biolabs), rabbit anti-Cdt1

antibody to detect Cdt1 (1:1000; New England Biolabs).

Mating assaysCells were grown exponentially for 24 hours in YES at 28 C to a

concentration of 2.46106 cells/ml. 46106 cells were mixed with an equal

number of wild-type cells (h+/h2), washed in sterile water and spotted

onto sporulation agar (SPA) plates or SPA plates supplemented with

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Torin1 (25 mM final) or rapamycin (300 ng/ml final). Plates were

incubated at 30 C for 28 hours before 200–500 cells or zygotes were

counted.

Spot testsCells were grown exponentially for 24 hours only in YES at 28 C to

2.46106 cells/ml. A tenfold dilution series starting with 16106 cells was

spotted onto YES plates or EMMG plates supplemented with 25 mM

Torin1 or 0.33% DMSO. Plates were incubated at 30 C and growth was

monitored daily for 4 days.

Fluorescence microscopyCells were treated as for liquid growth assays and samples fixed with

30% formaldehyde (10% final v/v) for fluorescence microscopy every

15–60 minutes for 3 hours. Septa were stained with calcofluor white

(Sigma-Aldrich) and cells were observed under a fluorescence

microscope at 1006 magnification. At least 200 cells were counted per

time-point. Combined calcoflour/DAPI staining was used to determine

the anaphase population; only bi-nucleate non-septating cells were

counted as anaphase cells.

Cell cultureHeLa Kyoto cyclin-B1–Venus2/+ mRuby–PCNA2/+ H3.3–CFP cells were

grown in Advanced DMEM (Gibco) supplemented with 2% FBS (Gibco).

A375 cells were grown in DMEM (Gibco) supplemented with 10% FBS

(PAA). For protein turnover assays, 50 mM cycloheximide was used.

Single cell live microscopyHeLa Kyoto cyclin-B1–Venus2/+ mRuby–PCNA2/+ H3.3–CFP cells

were grown on a 96-well plate (mClear, Greiner). Culture medium was

replaced with Leibovitz’s L-15 medium (Invitrogen) supplemented with

10% FBS prior to filming. Live-cell imaging was performed using an

ImageXpress high-throughput microscope (Molecular Devices). Cells

were filmed every 15 minutes for the total duration of 18–24 hours with a

620 objective. Four to six positions were acquired per well. Exposure

time was 200 milliseconds for the mRuby filter.

AcknowledgementsWe are very grateful to Jorg Mansfeld and, particularly, to Samuel Weiser forgenerating the HeLa Kyoto Cyclin B1-Venus-/+ mRuby-PCNA-/+ H3.3 CFP cellline. We thank David Sabatini (MIT Cambridge, MA) and Nathanael Gray(Harvard Medical School) for an aliquot of Torin1; Iain Hagan (Manchester CRUKInstitute, UK), Masayuki Yamamoto (University of Tokyo), Kaz Shiozaki (NAISTJapan), James Moseley (Dartmouth Medical School, NH), MohanBalasubramanian (MBI Singapore), Ronit Weisman (Tel Aviv University) and PaulNurse (LRI, UK) for strains and reagents; and I. Hagan for valuable comments onthe manuscript.

Competing interestsThe authors declare no competing interests.

Author contributionsJ.A. and L.H. did the experiments, J.F. and C.W. provided all cell culturesextracts. Except Fig 7F, the single-cell live microscopy was done by A.L. and J.Pines. J.H. and A.J. synthesized Torin1. J.A. and J. Petersen designed theexperiments and wrote the manuscript with input from all authors.

FundingThis work was supported by the Wellcome Trust; and a Cancer Research UKSenior Fellowship [grant number C10888/A11178 to J.P.]. Deposited in PMC forimmediate release.

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.146373/-/DC1

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