ta. 01 eesne l,19;Wimne l,19) In 1997). al., et that Weisman ATP-analogues 1997; with cells al., mammalian et of treatment Pedersen contrast, 2001; al., et acetrM39T UK. 9PT, M13 Manchester nttt o acrRsac,Uiest fMnhse,Wlso Road, Wilmslow UK. Manchester, 4BX, of M20 University Manchester, Research, Cancer for Institute dacsmttccmimn nfsinyatadHL cells HeLa and yeast Atkin Jane fission in and commitment levels mitotic advances reduces inhibition TOR Torin1-mediated ARTICLE RESEARCH ß 1346 2013 December 13 Accepted 2013; November 15 attributed. properly Received is work original the Attribution that Commons provided Creative medium the any of distribution in terms use, reproduction the unrestricted and under permits distributed which article (http://creativecommons.org/licenses/by/3.0), Access License Open an is This 1 TORC1 the show in not does either activities it in however, effect 1996); TOR same al., et that of (Barbet arrest established growth is subset It In 2007). a complexes. al., TORC2 2006; inhibits et Matsuo Moreno, and rapamycin 2007; and al., Tor2) et (Alvarez distinct Hayashi containing Tor1) two containing least (mainly (predominantly at TORC1 in participate yeast Choder, complexes: and (Weisman Fission TOR Tor2 essential 2001). signalling. the and kinase Tor1 through non-essential (TOR) proliferation pombe and rapamycin Schizosaccharomyces of growth, target regulate Cells INTRODUCTION HeLa, WORDS: KEY cell couple to by controls. helps Wee1 growth that inhibitor and mechanism mitotic cycle conserved the a of is regulation signalling the TOR by Thus, inhibited cells. HeLa advanced was was in commitment mitotic (mTOR) and declined these levels TOR recapitulated Wee1 Torin1, mammalian lines cell observations: human yeast in in drop Experiments kinase-controlled levels. Cdr2 and Wee1 Polo by a regulation promoted Torin1 advanced rapamycin, Torin1-induced . this for with growth sufficient was inhibition inducing or TOR Wee1 before either to onset contrast mitotic In we arrest. advanced mutation, Torin1 this resistance, Using that Torin1 TOR. show for Torin1 provides of specificity to tryptophan the adjacent confirming Torin-interacting lies that key (G2040D) residue the glycine Tor2 the of mutation in completely death inhibitor cell Torin1 without the growth with division, arrests TOR cell on of rapamycin site of catalytic impact the mild and blocking the growth to we contrast Here cell in activities. that TOR regulates show of subset kinase a inhibits (TOR) only Rapamycin rapamycin division. of target The ABSTRACT Ato o orsodne([email protected]) correspondence for *Author UK. 1QN, CB2 Cambridge Road, Court Tennis oahnPines Jonathon aut fLf cecs nvriyo acetr ihe mt Building, Smith Michael Manchester, of University Sciences, Life of Faculty 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,14–36doi:10.1242/jcs.146373 1346–1356 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 1 ek Halova Lenka , acaoye cerevisiae Saccharomyces 3 lui Wellbrock Claudia , .pombe S. .pombe S. 2 acrRsac KDu icvr nt Paterson Unit, Discovery Drug UK Research Cancer 3 h udnIsiueadDprmn fZoology, of Department and Institute Gurdon The O,Trn,Wee1 Torin1, TOR, , rsm amla el (Neshat cells mammalian some or otistoTRknss the kinases: TOR two contains 1 enfrFerguson Jennifer , cioacaoye pombe Schizosaccharomyces aayi promotes rapamycin 1 n an Petersen Janni and 1 ae .Hitchin R. James , .A 1, neat ihtytpa-29i h aayi,atv ieof 3- phosphoinositide (PI3Ks). for site mTOR-related absent kinases is the active residue including Torin1 this kinases, roles Crucially, catalytic, other 2013). in of al., the et rapamycin-resistant (Yang Torin1 in effects kinase proliferation. mTOR tryptophan-2239 and These are growth with for 2009). interacts essential there are al., that mTORC1 reduced that et a protein in with (Thoreen reduce G1 treatment established in autophagy, size rapamycin progression induce cycle of cell cell they (Thoreen arrest impact that Torin1 and synthesis the as in mimics such yeast, mTOR, budding 2009), of al., domain et kinase the target ehnssb hc niiino O ciiyipcsupon impacts activity division. was TOR cell of mTOR and the mitosis inhibition into HeLa lines which insight novel by when provide cell mechanisms findings These decreased advanced Torin1. human by levels commitment inhibited Wee1 in mitotic observations: Experiments and yeast the these Wee1. advanced of recapitulated inhibition levels inhibitor the TORC1 reduced mitotic treatment complete Torin1 that commitment. Torin1-resistant mitotic show this exploited to of have We specificity mutation kinases. the TOR validated for functionally Torin1 and conferred Torin1 mutation to This of resistance Torin. (W2239 with tryptophan interacts key directly the that to mTOR) next located glycine conserved a nt CU Fg D,idctn htclsrsmdgrowth inhibition resumed Torin1 cells words, other that In indicating withdrawal. Torin1 forming 1D), colony (Fig. following vehicle- of medium (CFU) numbers and rich similar Torin1-treated units containing gave viability. plates cultures control on assess treated spread to and Torin1 whether Torin1 with hours address treated without 24 were To or As cells 9 1997). death, for al., on 1C). cell (Fig. promoting impact et was marginal (Weisman Torin1 cultures a 1A) only (Fig. control had growth vehicle-treated rapamycin reported, of less previously to proliferation 10% reduced hours than 24 for drug the with Incubation the As 2013). al., et (Ma observed is cells (wt) type tclswsihbtdb 5 by inhibited of in growth was or the (YES), media media cells solid rich minimal On wt 1A–C). on (Fig. cells halt cultures wild-type to liquid of expected (25 growth analogue be ATP inhibit (Weisman would The essential proliferation. inhibition and is TOR growth yeast 2001), Torin1 fission Choder, of of and concentration complex) low (TORC1 further a to that Torin1 shown by has (0.2 inhibition study model the TOR recent in signalling exploit TOR to characterise wanted We inhibition TOR Torin1-induced following of Growth RESULTS * ee edsrb h slto fa of isolation the describe we Here, m )ihbt OC;hwvr ogot reto wild- of arrest growth no however, TORC1; inhibits M) 2 .pombe S. gt Lichawska-Cieslar Agata , sihbtdwtotcl et rG arrest G1 or death cell without inhibited is m oi1(aantshown). not (data Torin1 M tor2 3 la .Jordan M. Allan , uainta asto maps that mutation m )ddindeed did M) .pombe S. tor2 .A 2 + ,

Journal of Cell Science EERHARTICLE RESEARCH nuigete eldaho elccears nG phase. G1 1E). in (Fig. was arrest cycle size hours cell 24 or cell death arrest, cell to inhibited data either These G1 inducing Torin1 4A). up after Fig. a that 1F; G1 of (Fig. demonstrated for inhibition in lack TOR following drug arrest this reduced Flow not despite the did 2006). Importantly, cells with al., yeast et incubation mammalian fission to Uritani wild-type contrast Tor2 in 2007; cells, following that, yeast al., demonstrated mammalian fission analysis in et cytometric in seen and (Matsuo as 2009) G1, inhibition al., in et arrest growth (Thoreen cycle the cells cell whether asked from therefore arose We arrest death. cell induce not did 25 containing plates EMMG Torin1. by on signalling TOR of inhibition following of Growth 1. Fig. eH ehnl BF iudclue eetetdwt 25 with treated were cultures Liquid (B-F) methanol. MeOH, E N otn a nlsdb lwctmty F elsz was size Cell (F) cytometry. cytometry. flow flow forward-scatter and by by plates analysed cultures. determined YES was vehicle-treated on to content spread relative DNA were shown (E) cells and vehicle 500 counted to (D) units relative (C). colony-forming proliferation hours and 24 measured after was calculated number Cell (B) DMSO. .pombe S. sihbtdwtotcl et rG arrest G1 or death cell without inhibited is m oi1 0 gm aayi rsolvent. or rapamycin ng/ml 300 Torin1, M .pombe S. A idtp el grown cells Wild-type (A) rwhwithout growth m oi1or Torin1 M oiin24 ihnteAPbnigpce ftekinase the of confirm pocket To 3C). ATP-binding (Fig. throughout the conserved is within domain 2040 a position the and with from wild-type segregated arose between resistance mating following Torin1 tor2-G2040D 2:2, 3A,B). (Fig. ratio, Mendelian drug the of complex), (TORC1 hw ht ncnrs orpmcn oi1ihbtdTRin Only have complexes. TOR inhibited TORC2 we Gad8 Torin1 and summary rapamycin, TORC1 upon to unlike the In contrast both that, in impact TORC2. that, suggested inhibited shown no This Torin1 Torin1 2D). had (Fig. rapamycin, after status rapamycin seen was whereas dephosphorylation addition, Gad8 Maf1–pk. for aigi ieyta h rwhars a osqec of consequence a was arrest growth alone. the TORC1 that of inhibition likely 2001), it Choder, and (Weisman making growth cell for essential is complex) u a nymrial eue yrpmcntreatment rapamycin by reduced marginally 2013), al., only et (Ma treatment was Torin1 of minutes but phosphorylation 30 Rps6 within cells, lost wild-type 2012; was In al., 2010). et activity. Nakashima al., 2012; et al., TORC2 Nakashima et (Du TORC2 and by regulated and is TORC1 TORC1 (Rps6) both S6 protein both ribosomal the of Phosphorylation of read-outs G1 for biochemical required is TORC2. kinases and activity TOR TORC1 the TORC2 inhibited both Torin1 in and that suggested 1E) data (Fig. these arrest, Torin1 marginal arrest inducing because without G1 a Thus, TORC1-dependent) a only is 2A). rapamycin (which (Fig. had growth contrast, proficiency inhibited TORC1, completely mating In inhibits Torin1 on cells. impact that only wild-type found which We of treatment, arrest. this mating G1 regulate is prevented the activity TORC2 TORC2 for importantly, and and, essential TORC1 exit cycle Both cell and physiological 2003). differentiation arrest sexual (Egel, Cells undergo to 2007). mating G1 al., in et progression cycle 2006; Weisman cell al., 2001; et Choder, Murai Uritani Petersen 2012; and 2012; 2007; Maeda, al., Weisman and al., et Takahara Nakashima et 2007; 2013; Nurse, (Matsuo al., and et nitrogen Nakase 2009; of regulated al., is starved et signalling are TORC2 cells and TORC1 when both in yeast, TORC2 fission and In TORC1 both inhibits Torin1 outailtlgt el eepae nomdu containing medium onto the plated in were grow cells 25 to light, exposure by cells ultraviolet mutagenesis allowed random to Following that drug. the mutations of presence isolated next Torin1 provides We Tor2 of pocket ATP-binding resistance the in mutation A concentration protein with linear is Torin1. R than staining TORC1 S of inhibitor Ponceau suggesting potent Because extent, less lesser a was a rapamycin to that but also faster- phosphorylation, Rapamycin single Maf1 TORC1. a reduced inhibited into Torin1 2012) three Thus, the al., band). of et migrating collapse (Du the note forms 2C: Maf1 (Fig. phosphorylated 2010). minutes treatment 30 al., following for et Torin1 reduced with Tatebe severely 2003; was al., phosphorylation et Maf1 whereas (Matsuo 2010), uniquely TORC2 is al., -546 on at et dependent Gad8 Michels kinase AGC 2012; the al., of phosphorylation et on dependent (Du solely is activity III, polymerase TORC1 RNA of repressor a Maf1, of next Phosphorylation substrates. We TORC2-specific phosphorylation and TOR. the TORC1- upon of targeting status addition Torin1 is of Torin1 impact the that monitored indicated This 2B). (Fig. 2 5 ocnimta oi1tresbt O iae,w used we kinases, TOR both targets Torin1 that confirm To m .9(oacy ta. 03,i a sda odn control loading as used was it 2013), al., et (Kowalczyk 0.99 oi1 on uaini h essential the in mutation point A Torin1. M ora fCl cec 21)17 3615 doi:10.1242/jcs.146373 1346–1356 127, (2014) Science Cell of Journal el Fg B,idctn htTrn resistance Torin1 that indicating 3B), (Fig. cells tor2-G2040D tor2-G2040D uainaoe h lcn at glycine The alone. mutation nbe rwhi h presence the in growth enabled , .pombe S. tor2 tor2 + (TORC1 + 1347 gene

Journal of Cell Science lsisepesn wild-type expressing plasmids EERHARTICLE RESEARCH 1348 in mutation this that agl nfetdb xouet oi1(i.3,) Together 3E,F). (Fig. Torin1 to exposure of growth by rate this unaffected growth from largely The cells 3D). protected (Fig. allele arrest mutant the of expression h tanta xrse h wild-type the expressed that strain the a into transformed tor2 tor2 eeinsri.Trn retdgot of growth arrested Torin1 strain. deletion + i nedcne Torin1-resistance, confer indeed did tor2 + tor2-G2040D and tor2 tor2-G2040D + ee whereas gene, utrswas cultures were eitnet Torin1. to resistance hs eut niae htthe that indicated results these usrtsfloigTrn treatment Torin1 following substrates The elsz tdvso rvddfrhreiec fdu resistance drug of evidence further the provided in division at size Cell tor2-G2040D tor2-G2040D ora fCl cec 21)17 3615 doi:10.1242/jcs.146373 1346–1356 127, (2014) Science Cell of Journal uainatr h ehshrlto fTORC1 of dephosphorylation the alters mutation uat h euto ncl ienormally size cell in reduction The mutant. euae ybt OC n TORC2. and TORC1 both by is regulated phosphorylation Rsp6 and specific TORC2 is phosphorylation Gad8.S546 specific, TORC1 is Maf1 phosphorylation antibodies. indicated blot the western using by analysed were and Samples harvested minutes. 30 for solvent treated were 25 (C) with cells maf1–pk Wild-type and (B-D) (B,D) indicated as incubated plates and SPA 1:1 on mixed were opposite type of mating Cells treatment. drug following and TORC1 in both TORC2 inhibits Torin1 2. Fig. acltdatr2 or (F). hours 24 after vehicle to calculated relative proliferation counted and was (E) number cell in 1B,C, as Fig. treated were Cells (E,F) Torin1. of Expression tor2.G2040D (D) red. in highlighted idn ieaeidctdb .Glycine to /. correspending by indicated are ATP- site the binding forming residues and in bold highlighted are residues kinases; conserved TOR of spores Alignment four (C) the of genotype illustrates the diagram a right the Torin1 to + plates, YES and YES onto replicated were tetrads Four Torin1- resistance. of segregation 2:2 (B) Torin1. MGpae otiig25 containing plates on EMMG grown were strains indicated Torin1. to provides resistance Tor2 ATP- of the pocket in binding mutation A 3. Fig. tor2-G2040D m oi1 0 gm aayi or rapamycin ng/ml 300 Torin1, M .pombe S. rvdsrssac to resistance provides . uainprovides mutation A aigefficiency Mating (A) tor2.G2040 AB The (A,B) m is M

Journal of Cell Science uat oehrteedt ugs htTrn a reduced the a in had activity Torin1 that TORC1 suggest on data effect the these in Together Maf1 mutant. hypo-phosphorylated less in resulting EERHARTICLE RESEARCH es rwhoc rtcllnt sahee.Cl eghat length Cell achieved. is length critical a and mitosis once to growth commit Cells cease diameter. cell constant a maintaining pombe through S. mainly mitosis into inhibition advance TORC1 to cells induces Torin1 a as use in Torin1 function validates TOR Torin1 also studying of It for specificity yeast. tool fission the in confirms kinases This in TOR drug. proliferation for the to cell of allows resistance presence thereby provides to to a and the Tor2 Torin1 residue ATP, of analogue glycine strike ATP site related a the ATP-binding highly to of the analogue mutation in the likely summary, aspartate ATP of In the binding therefore viability. support of allowing is binding and the mutant (Torin1) blocking killing between resistant thereby also compromise kinase, likely The essential most this would cell. to it ATP-binding binding, pocket block ATP-binding Torin1 the in blocked mutation an completely the inhibiting If is Tor2. that kinase, ATP-analogue essential an is Torin1 because expected Torin1-treated the in cells wild-type were to Maf1 observed of Torin1 forms nM hyper-phosphorylated still 750 contrast, within of In compromised 4D). addition was (Fig. the the activity) of in TORC1 minutes TORC2 of 10 measure inhibit (a to Maf1 able still was tor2-G2040D was Torin1 serine-546 of that at confirming treatment phosphorylation from Gad8 drug contrast, lost In after 4B). (Fig. persisted minutes phosphorylation 30 Rps6 resistance. 2B), revealed for (Fig. also cells activity wild-type Unlike TOR of read-outs in biochemical observed 25 not with treatment by induced sardsae elta rw ytpetninwhilst extension tip by grows that cell rod-shaped a is tor2-G2040D TR1cmlx uat hshrlto of Phosphorylation mutant. complex) (TORC1 tor2-G2040D el fe oi1tetet(i.4C), (Fig. treatment Torin1 after cells m oi1(i.1;Fg A was 4A) Fig. 1F; (Fig. Torin1 M el Fg A.Furthermore, 4A). (Fig. cells tor2-G2040D .pombe S. tor2-G2040D . tor2-G2040D uat hsis This mutant. tor2-G2040D mutant, cells ovrey uat nTR2( TORC1. TORC2 of in inhibition Torin1 mitotic through that mutants the suggesting mitosis of 5B), Conversely, to response (Fig. advances Torin1 impaired TORC2 primarily severely The of was strain. cells contribution this TORC1 in the expressing advance 5B). cells (Fig. of in drug assessment Torin1 the by inhibited to G2040D not exposed was were function in complex mutations To harbouring was cells again each 5A). TORC2, mitosis (Fig. or of rapamycin TORC1 inhibition acceleration through TOR elicited to Torin1-based of the than mode whether determine efficient treatment more Torin1 more a The implies to 5A,C). size (Fig. response division a the cell to rapid and leading lowered mitosis 4A), of Torin1 Fig. Consistently, burst 1F; by transient 2007). (Fig. commitment cells Nurse, mitotic wild-type for and threshold in either (Petersen inhibition cells length with TOR mitosis wild-type reduced into quality, inhibited advance a and nutrient division at is for in threshold and decrease size activity their a decrease (Petersen TOR by activity or When rapamycin TOR the 2007). with by determined Nurse, correlates is and length cells and cell Nurse which environment threshold 1977; at Nurse, This time and 1977). the (Fantes Thuriaux, of transition read-out G2-M the direct execute a therefore is division cnrsm)t rge ciaino d1cci- through Cdk1–-B of activation body trigger pole spindle with to the advance to Phosphorylation recruitment (centrosome) inhibition this Plo1. Plo1 kinase that promotes TOR Polo Plo1.S402 and kinase of mild mitotic onset, the that mitotic upon relied promotes shown also previously rapamycin have of regulators We the through factor mitosis promoting into maturation advance to cells causes Torin1 rqec fdvdn el Fg B.W ocueta the that TORC1. the conclude of inhibition in We onset the mitotic 5B). through peak advanced (Fig. was Torin1 a which cells by mechanism in dividing predominant resulting of commitment, frequency mitotic accelerated ora fCl cec 21)17 3615 doi:10.1242/jcs.146373 1346–1356 127, (2014) Science Cell of Journal TR1mtn)(is3 ) hc nbe the enabled which 4), 3, (Figs mutant) (TORC1 i.4 The 4. Fig. rae ih70n oi1fr1 minutes. 10 for Torin1 were nM Cells 750 (D) with (*). treated asterisk is an band by non-specific 25 indicated A with minutes. treated 10 were for Cells Torin1 (C) as 2. treated Fig. were in using Cells blot antibodies. Western indicated (B-D) cells the 5A). wt Fig. of (see 10% divide treatment, Torin1 of hours ( 2.5 strains indicated of division treatment. Torin1 following substrates TORC1 of dephosphorylation tor2.G2040D tor1- D uainatr the alters mutation , n A ellnt at length Cell (A) 5 sin1- 0) After 200). tor2-G2040D D both ) m M 1349 tor2-

Journal of Cell Science EERHARTICLE RESEARCH 1350 on and placed Cdc2 phosphate 1989), of inhibitory Nurse, the site and removes active Gould phosphatase 1991; the Cdc25 Russell, within and 15 (Featherstone through tyrosine Cdk1–cyclin-B of inhibits and kinase Petersen phosphorylation Wee1 2013; The al., et 2005). (Grallert Hagan, Cdc25 and Wee1 of regulation two least at of representative cells are mitotic Graphs or experiments. calculated. (A,B,D) independent were cells the (C) dividing for of DMSO percentages in or the rapamycin and Torin1, times mitosis. with indicated into treated advance were to strain cells indicated induces Torin1 5. Fig. AD el fthe of Cells (A-D) oasfiin eret dac h iigo mitotic the of because timing 15, mitotic tyrosine the Cdc2 Torin1-induced advance through acute regulated to was the degree advance sufficient Importantly, a either commitment. Cdk1–cyclin- and activates to Wee1 Wee1, of B inhibits inhibition Torin1 or and Cdc25 that of Cdc25 indicate activation (Petersen activates findings 5D) efficiently these (Fig. Together, very Torin1 2007). Nurse, of and addition following neither commitment where rapamycin, with contrast nor inhibition In TOR mutant. this mild in control to Wee1 upon activity dependent Cdk1–cyclin-B mainly hence is and Cdc25, to unresponsive largely d2 nTr5 i o fiinl dac ioi nresponse in 5C). mitosis (Fig. advance treatment efficiently Torin1 not to did Tyr15, on Cdc25 cdc2.F15 smil euae yCc5fnto.I contrast, In activity function. Cdk1–cyclin-B Cdc25 that by such regulated mainly Wee1, mutants is to Cdc2 insensitive the on largely treatment Torin1 and assessed of therefore We impact onset. mitotic the either advance to sufficient inhibition, is alone TORC1 Torin1-induced S402A phosphorylation- stronger the in the persists treatment is refractory that Torin1 mitosis by of acceleration triggered rapamycin-induced the Interestingly, weak 2007). Nurse, (Petersen this activation and for Cdk1–cyclin-B promote essential to both inhibition of is TORC1 regulation Wee1 Combined 1991). and al., Cdc25 et (Millar Wee1 by Cdc2 08.Tedciei e1lvl a eae,bti a still was it (Hagan, but Cdc25 delayed, of was levels lack Wee1 the in overcome body decline to pole The spindle sufficient 2008). the activity, is to Wee1 Cut12 recruitment regulates by Plo1 Plo1 enhanced that levels the shown Wee1 because been in previously decline has Cdr2-controlled It and Plo1- a induces Torin1 of regulation very the upon is attention inhibition our the Wee1. focused TOR of we controls to here – multiple response 4A; interesting these (Fig. in for size complex basis cell Cdk1–cyclin-B molecular reduced The a 6D) 5C). at (Fig. Fig. commitment form mitotic dephosphorylated promote active Cdk1– to the its traps seen in cells that complex wild-type was suggest in cyclin-B We Torin1 1990) 6B). by TORC1 al., (Fig. of treatment et inhibition Torin1 of Moreno minutes 30 1996; activation within its Russell, accompanies function and that SDS-PAGE (Kovelman TOR in Cdc25 observed of of not mobility was inhibition Wee1 in the the decline had in this TOR by turnover Importantly, mild protein 6A,C). Wee1 promoted (Fig. of global that actively that indicating of been and rapamycin, inhibition with treatment upon inhibition cycloheximide seen Wee1 by reduced that synthesis addition Torin1 exceeded (data which treatment to levels Torin1 degree to The following response shown). wild-type N-terminus, not a index Wee1 the controlled. has at in GFP strain mitotic decline with this are tagged a and is the with Wee1 activities 6A,D,E). coincided in (Fig. culture levels Cdc25 the rise to it and addition sharp However, Torin1 Wee1 Cdc25 2005). the Hagan, and how Interestingly, and Wee1 unclear Petersen through 2013; is al., regulated et of is (Grallert activation trigger to This to leads bodies pole turn Cdk1–cyclin-B. spindle in the which to recruitment activation, Plo1 Sty1 promotes Cdc25 reduced inhibition a TOR and levels Wee1 in migration decline a induces Torin1 cdc2.3w cdc2-3w idpnetatvto fCc5o niiino Wee1 of inhibition or Cdc25 of activation -independent tor2.G2040D ora fCl cec 21)17 3615 doi:10.1242/jcs.146373 1346–1356 127, (2014) Science Cell of Journal uat hc antb euae yete e1or Wee1 either by regulated be cannot which mutant, plo1.S402A dacdmtss ohmtnsavne mitotic advanced mutants both mitosis, advanced Fne,18;Tuiu ta. 1978). al., et Thuriaux 1981; (Fantes, uat(i.6) nadto,tedciein decline the addition, In 6E). (Fig. mutant lee(i.5) niaigta,following that, indicating 5C), (Fig. allele cdc2-1w cdc2-3w cdc2-1w cdc2.1w plo1. is is

Journal of Cell Science EERHARTICLE RESEARCH MOfridctdtms AB etr lto d2 rWe.()Ihbto fTR1b oi1i idtp el rp h d1cci- ope ni the in fr complex levels Cdk1–cyclin-B Wee1 in the of traps quantification and cells reduced levels wild-type Wee1 in comparison. of Torin1 blots for by Western twice TORC1 (C,E) shown of modified. are is Inhibition cells Cdc25 (D) and wt Wee1. reduced Torin1-treated or are experiements. Cdc25 levels individual levels. Wee1 of Wee1 as blot in form Western dephosphorylated decline (A,B) active Cdr2-controlled times. and indicated Plo1- for a DMSO induces Torin1 6. Fig. Fg E.Ti ugse htWe ol lob downregulated be also a could Wee1 through that suggested This 6E). (Fig. ioi hti rgee yTrn esse nthe in of persisted acceleration Torin1 The by triggered 6E). cdr2 (Fig. is that independently mitosis but by regulated kinases was turnover severely both Wee1 of function, was control Plo1 TORC1 and that Wee1 Cdr2 indicating combined of of absence Torin1 the decline by from compromised arise Torin1-induced that The levels Wee1 inhibition. were in Plo1 changes with the along levels for kinase) responsible (SAD zWee1 Cdr2 yeast regulate fission the also whether to defective) shown amphids been (Mu of have kinases (synapses family SAD inhibition. TOR induced le ta. 00 on n ats 97.W hrfr asked therefore We 1987). Fantes, and Young 2010; al., et ¨ller D obemtn Fg C,wihaandmntae that demonstrated again which 5C), (Fig. mutant double plo.S402A plo1.S402A idpnetmcaimfloigTorin1- following mechanism -independent uat fe oi1treatment Torin1 after mutants plo1.S402A ABCE el eetetdwt oi1 ylhxmd CX,rpmcnor rapamycin (CHX), cycloheximide Torin1, with treated were Cells (A,B,C,E) rti ytei n ret el nG tardcdcl size cell reduced a at G1 in limits cells cells arrests mammalian and in synthesis mTOR protein of inhibition levels Torin1-induced Wee1 reduces inhibition mTORC1 hshrlto ocnrlpoenlevels. protein Wee1 Russell, control modulate directly to and phosphorylation could (Kanoh kinase either Wee1 that of suggesting been 1998), phosphorylation previously control has to Cdr2 I. shown and and its Tay communication), D. (Y. personal resembles phosphorylated heavily Hagan, very Sakchaisri Wee1 being 2005; in al., However, 2004) al., et et (Harvey present. Swe1p counterpart at yeast budding unclear Wee1 Torin1-induced is novel Plo1 this turnover How control inhibition. independently onset TOR Cdr2 mitotic and Torin1-induced advance strong to sufficient the was following only Cdc25 of regulation ora fCl cec 21)17 3615 doi:10.1242/jcs.146373 1346–1356 127, (2014) Science Cell of Journal 1351 m3 om ts

Journal of Cell Science motnl ylhxmd ramn i o rmt h same the promote 7A,B). not (Fig. did cells melanoma treatment A375 cycloheximide in and was Importantly cells levels HeLa Wee1 both in of in reduction hours seen This 2 Torin1. after with in reduced inhibition levels markedly mTOR Wee1 were cultures that cell noticeable unperturbed was it populations; asynchronous e1lvl dt o hw) oee,i spsil that possible is it However, in shown). cycle decrease not small cell a (data manipulate revealed levels to cells, Wee1 used mammalian Wee1 widely in controls yeast synchronization progression are using similarly fission experiments which mTOR Initial in approaches, cells. whether control human ask in Wee1 levels to and us link The signalling prompted also arrest. cells TOR growth whether this unclear between before is commitment it mitotic However, advance 2009). al., et (Thoreen ARTICLE RESEARCH 1352 analys was blot. content western DNA by and Torin1 analysed nM and 250 Torin1 with nM treated 250 were cells with HeLa treated (E) cyclin-B1–Venus were C. Kyoto and cells A HeLa A375 of (F) (B) Quantification cytometry. (D) antibodies. flow cells. indicated HeLa by the of treatment using (CHX) blot lost. Cycloheximide western (C) is by Wee1 as analysed mitosis, and advances points inhibition mTORC1 7. Fig. treatment drug to prior that signalling (Herna mTOR signalling on impacted MAPK have could stress-induced invoked synchronization CAfc om e ie r envle ffu needn xeiet;rddt ersn el htwr nG hs ni h n fexperiment. of end the until phase G2 in the were when that point cells time represent the dots from red measured experiments; was independent cells four individual of in values length mean G2 are marker. lines mRuby–PCNA Red using form. microscopy foci time-lapse PCNA by analysed was progression ´ dze l,21;M ta. 05.W hrfr undto turned therefore We 2005). al., et Ma 2011; al., et ndez 2 /+ mRuby–PCNA A eaclswr rae ih20n oi1adsmlshretda h niae time indicated the at harvested samples and Torin1 nM 250 with treated were cells HeLa (A) 2 /+ 33CPclswr rae ih20n oi1o ovn DS) elcycle Cell (DMSO). solvent or Torin1 nM 250 with treated were cells H3.3–CFP nraei h 1pplto tteeerytm points cells time inhibition mammalian mTOR early that levels. in in Wee1 these yeast reduced experiments fission to at an led from these results for population the evidence mirrored Thus, G1 no 7A,E). the provided (Fig. Cdt1 in marker increase G1-specific level the profile the and cycle analysis cell cytometry of the flow (Watanabe However, in cycle population. change the rapid Wee1 cell of a in the reflect decline of simply Torin1-induced could rapid phase levels been this G1 therefore have the 2005), levels in al., et Wee1 low 7C,D). be (Fig. to instability reported Wee1 of degree sa omntrteipc fTrn niiino mTOR of inhibition Torin1 live-imaging of non-invasive impact a the used We monitor cycle. to G2 cell Wee1 the assay the of shortening control by of commitment mTOR phase mitotic that advance possibility would the levels addressed next onset We mitotic advances inhibition mTOR ora fCl cec 21)17 3615 doi:10.1242/jcs.146373 1346–1356 127, (2014) Science Cell of Journal ed

Journal of Cell Science uaino 2paecnrce rm27mntsto minutes 247 G2 from of duration contracted the in mitotic decrease phase 20% the This the that 7F). G2 finding (Fig. the minutes accelerated 200 by of indicated treatment as duration cells, HeLa Torin1 of commitment throughout cytoplasm. nuclear dispersed as phase PCNA G2 proliferating the nuclear of the marker when end as replication breakdown, the phase DNA and envelope G2 the (PCNA), antigen of of nuclear foci start cell last the the defined of We loss cells. HeLa on activity ARTICLE RESEARCH ie h rsneo oeueXi mle yavne ioi ntorin1-treated at in mitosis mitosis into advanced entry by driving conformation, implied resistant active Torin1 is its The X in cyan. Cdc2–cyclin-B molecule traps in of This shown presence decline. is levels The Wee1 tryptophan size. TORC1, Torin-interacting activity. inhibits Torin1 key Cdc2–cyclin-B when The control that bold. to suggesting in pathways highlighted multiple are of residues convergence The 8. Fig. putative Trp2239 a and Ile2237 through places the occur with inhibitor the complex Torin2 in to Furthermore, mTOR second-generation of Val2240. proposed structure and crystal identified is Torin1 recently mTOR between 2012) of bond this pocket al., hydrogen of ATP-binding et the site mTOR in in The (Liu docking conserved 3,4). Torin1 is (Figs 3C). pocket eukaryote (Fig. ATP-binding any the within in mutation kinases TOR for first the providing essential mammals. we in architecture signalling in the humans, mTOR of of Torin1 and understanding control of of and our yeast to use upstream contribution this the significant pathways between that signalling conserved anticipate the the are of by proliferation Because TORC1 cell function blocks yeast. TORC1 Torin1 analogue of fission ATP inhibition the of complete addition that show We DISCUSSION cells. mammalian of in inhibition by in mitosis yeast, fission advance phase previous in inhibition can like mTOR G2 just of TOR that, provide the demonstration findings these first which Together, reminiscent the reduced 2007). Nurse, cultures in and highly (Petersen 25% yeast yeast, was fission fission cells synchronous in HeLa observations in phase euaino ihrWe 1 rCc5()i ufcett dac ioi ne olwn h togTrn-nue O inhibition. TOR Torin1-induced strong the following onset mitotic advance to sufficient is (2) Cdc25 or (1) Wee1 either of regulation igegyiet-satt uaina oiin24 fthe of 2040 position at mutation glycine-to-aspartate single A .pombe S. nvivo in iaeTr ofre oi1resistance, Torin1 conferred Tor2 kinase aiaino h pcfct fTorin1 of specificity the of validation .pombe S. ilmk a make will needn ahasta euaeWe n d2 ocontrol to multiple Cdc25 of and convergence Wee1 the regulate by that achieved pathways be independent can division at size off- possible the eliminating in cells, useful mammalian effects. potency be in target will drug this studies mutant reduced of for this identification the implications where The has explain kinases. site to related conserved likely closely is these of this towards instead the – acid of 8A) site glutamic the (Fig. charged at a found having glycine yeast the in resistant here fission Torin1 isolated the the have 8A). mimic and (Fig. residue they ATR 10-fold tryptophan key However, a the ATM, contain (Liu has all Interestingly, ATR Rad3 Torin2 and homologue 2013). ATM that kinases al., shown PI3K-like (Yang two et recently the 8A) was for (Fig. affinity It lower tryptophan 2013). not Torin-interacting al., do PI3Ks et PI3Ks key Importantly, over 2009). the specificity al., et contain greater Thoreen 2013; 800-fold al., both et an Torin2 (Liu and of with Torin1 site 8A)]. mTOR the (Fig. to inhibit residue, Tor2 [equivalent yeast glycine fission Trp2239 the and in to Torin2 mutation the of adjacent moiety directly through ring found the interaction from is is pivotal atoms ten the mTOR with interacts fact, which and In 2013). Torin2 al., between et (Yang Torin1) with A 4 within s fTrn i o eyuo ulcnrlo oho these of both of control dual upon In rely 8B). not (Fig. did the Wee1 Torin1 from arising and of inhibition TORC1 required use Cdc25 of level commitment both enhanced the mitotic contrast, of inhibition of TOR control advance mild simultaneous Under the inhibition. rapamycin, TOR with of degree depending the varied phosphorylation. activation upon Cdc2–cyclin-B regulated Cdc2.Y15 of control level is the to However, Cdc25 activation and Wee1 Cdc2–cyclin-B rapamycin- through both Torin1-stimulated Importantly, and 8B). (Fig. activity Cdc2–cyclin-B l1S0Acdr2::ura4 plo1.S402A eew idta h OC oto fmttcetyadcell and entry mitotic of control TORC1 the that find we Here A lgmn fTRadTRrltdknss Conserved kinases. TOR-related and TOR of Alignment (A) ora fCl cec 21)17 3615 doi:10.1242/jcs.146373 1346–1356 127, (2014) Science Cell of Journal ˚ ftetiylcbnoahhrdnn ig(shared ring benzonaphthyridinone tricyclic the of + obemtns net ncnrs orapamycin, to contrast in Insert: mutants. double tor2-G2040D ssoni e.()Amodel A (B) red. in shown is tor2 uaini O kinases TOR in mutation tor2 uatta we that mutant eue cell reduced a 1353

Journal of Cell Science CAGTCTAAAAGGCCTGAATCCGGTG-3 eacls(i.7)i ossetwt h ointa h mTOR the that notion the with consistent Torin1- is in 7F) the observed (Fig. we cells Furthermore, that HeLa commitment signalling. mitotic of TOR novel, advancement a induced of revealing 7), aspect 6, (Figs universal cells in human levels in commitment Wee1 inhibition inhibition in mTORC1 mitotic TORC1 reduction the acute advance that find upon to we Wee1 Importantly, sufficient or 8B). Cdc25 (Fig. was either upon alone impact singular activity a rather – regulators ARTICLE RESEARCH pcrmtyadpoo M.Trn a trda oi nthe in solid 1354 a as 50 stored at was dissolved Torin1 and NMR. dark proton Institute a and yielding Research 2010), spectrometry al., Manchester et CRUK (Liu protocol with the published the at to according synthesised was Torin1 assays Growth ACTGGGTTTTGGATAGCGATAC-3 generate containing to plasmid used was the mutagenesis site-directed (Stratagene) QuikChange biology Molecular 28 at grown were cells stated, otherwise Unless S1. Table material reagents The and culture cell Yeast tumour METHODS AND particular MATERIALS TOR in of benefit inhibition therapeutic Torin1-mediated offer a contexts. which provide could was therefore by integrity activity lethal We DNA means 2012). a whose al., novel promoted et cells pre- inhibition (Kreahling cancer in compromised Wee1 inhibitor in Wee1 which catastrophe a mitotic in of success models the an clinical by it provoked made excitement been of has levels have proliferation Similar and candidate. drug growth reduced anti-cancer cell lines attractive ability reduce cell The to Torin1. A375 Torin1 with and of inhibition HeLa environmental mTOR both following to levels as commitment Wee1 couple yeast, mitotic fission that of in timing cues activity the Cdk1–cyclin-B in changes of lines. the control cell for two TOR-mediated account the to in likely inhibition is Torin1 difference of likely This impacts is lines different different. cell very HeLa 2010), and be RPE (Feng, to in inhibitors activity TOR AMP- TOR steady-state and (AMPK) the (TSC) kinase sclerosis protein tuberous on TOR activated impacts the also increased through signalling have because TOR addition, therefore In and They 2010). in (Feng, deficient. levels, case signalling p53 the PTEN are not cells reduced is HeLa have this Furthermore, whereas lines. RPE 2013), cell because al., other in for et differently required (Parrales However, absolutely function activity specific. is its to AKT1 context of appears activation and is TORC2-dependent signalling line commitment TOR cell shown). cells, be mitotic not to epithelium (data advanced likely pigment treatment Torin1-induced Torin1 retinal following in Therefore, lines observed cell was (RPE) mitosis of timing commitment. mitotic controls kinase ih2 Mguaae(MG.Clswr rw xoetal at exponentially grown were Cells supplemented (EMMG). ForMedium) glutamate 28 (EMM2-N mM 20 2 with medium minimal Edinburgh npae otiigYSo EMMG. or YES containing plates on itfo .Hgn.Pamd eetasomdinto transformed were Plasmids (Ba acetate Hagan). lithium I. from gift G2040D ˚ nsmay amla el a xli h aetpsof types same the exploit can cells mammalian summary, In the in advancement no experiments, pilot in Interestingly, o 8hus o rwho oi ei,clswr rw t30 at grown were cells media, solid on growth For hours. 48 for C .pombe S. . 5 uiya eemndb iudcrmtgah mass chromatography liquid by determined as purity 95% eesbeunl lndit h RP2NTvco (kind vector pREP42–NAT the into cloned subsequently were tan sdi hssuyaelse nsupplementary in listed are study this in used strains he ta. 1998). al., et ¨hler tor2-G2040D ˚ nDS SgaAdih immediately (Sigma-Aldrich) DMSO in C fradpie:5 primer: (forward 9 ees rmr 5 primer: reverse , .pombe S. 9 .Wild-type ). sas enupon seen also is .pombe S. 9 tor2 -CCTTTTAG- 9 -CAAAACC- + and using ˚ tor2- Cin ˚ C al MMgCl mM 5 NaCl, .pombe S. cytometry Flow 2.5 to grown were cells Wild-type screen Torin1 el eegonepnnilyfr2 or nYSa 28 at YES in hours 24 for exponentially grown were Cells assays Mating of blotting western blotting For (Beckman western and software extraction 4.3 Protein Summit using analysed processed events were HeLa 5000 Coulter). and Data bandpass. ethanol nm above. 70% 530/40 with as and drop-wise filter fixed were detection instrument cells excitation ADP Cyan nm Coulter 488 and Beckman sonicated with a were using Samples processed 1986). events al., 30,000 et (Costello described previously el eegonepnnilyfr4 or o2.5 to hours assays, 48 growth cell for For exponentially mM. grown 7.5 were of concentration cells stock a with use, before ramn ihTrn tafnlcnetaino 25 of 28 concentration at final Growth control. a vehicle at DMSO Torin1 with treatment ocnrto f2.4 of concentration uat eesrae osnl ooiso E ltsadreplica- and 25 plates containing YES plates on colonies EMMG single to to plated streaked were mutants nioyt eetCt 110;NwEgadBiolabs). anti-Cdt1 England rabbit New Biolabs), (1:1000; England Cdt1 New detect to (1:500; antibody Wee1 antibody human anti-Wee1 rabbit detect anti- Roche), to (1:100; anti-GFP mouse Wee1–GFP mouse 2012), detect Millipore), to (1:2000; National al., antibody Cdc25–myc Scottish et detect by to (Du anti- antibody raised Midlothian) sheep myc (1:200; 2012), Service, Gad8 al., Transfusion total et Blood detect phospho- (Du to Eurogentec) detect antibody by to (1:2000; Gad8 raised antibody (1:500; Rps6 AbD S546 S546 (1:1000; total Gad8 anti-phospho-Gad8 Maf1–pk detect detect rabbit to to Serotec), antibody anti-V5-Tag antibody mouse Akt anti-S6 Abcam), Signaling anti-phospho-(Ser/Thr) Cell mouse (1:2000; mouse phospho-Rps6 Technology), detect study: to this antibody (PAS) in used were antibodies oi1(25 Torin1 olwn e lcrpoei,poen eetaserdot a TBS in antibodies onto 4 primary at with overnight incubated transferred milk in and 3% milk blocked plus were dried (Millipore), 3% plus membrane TBS (PVDF) electrophoresis, difluoroide 10% 6.8), polyvinylidene (pH gel A375 Tris-HCl mM and 2% Following [60 HeLa SDS, buffer 2000). sample 1% al., SDS glycerol, in et lysed (Caspari acid were trichloroacetic described cells with previously precipitation as by (TCA) prepared were extracts protein o005Jo VirdainprpaeuigaU apcbnt(Uvitec, cabinet 30 lamp UV at a incubated using plate were per Plates irradiation UV Cambridge). of J 0.015 25 containing to plates EMMG 10 onto plated ubro idtp el (h cells wild-type of number oi1o MOa bv,50clswr pedo E ltsin plates YES on with spread treatment were 30 following cells at incubation assays, 500 after counted survival above, were Colonies as cell duplicate. DMSO For or nm. Torin1 595 at (OD) oi ei 2.5 media solid eedvlpdatrwsiga bv yadto fsbtae nitro- substrate: secondary of International)/5-bromo-4-chloro- addition with VWR 3 by (NBT; above chloride probed as tetrazolium Membranes blue washing were (Sigma-Aldrich). after phosphatase developed membranes alkaline were to Tween20), linked antibodies 0.05% plus nosouainaa SA ltso P ltssplmne with supplemented plates SPA or plates (SPA) agar sporulation onto i)adsqecdb hl eoesqecn GT Biotech, (GATC sequencing genome whole by Germany). sequenced Yeast/Bact. Konstanz, Puregene and Gentra times (Qiagen three isolated Kit) of was minimum DNA a genomic backcrossed before were candidates Three positives. MOMO 01%fnlvv.Frpoentroe sas 100 1:1 assays, or turnover Sigma-Aldrich) protein stock; For v/v). mg/ml final 2 (0.15% from DMSO:MeOH final ng/ml (300 rapamycin ylhxmd a used. was cycloheximide 9 idllhsht -oudn BI;Mlkl)i Pbfe 10mM (100 buffer AP in Molekula) (BCIP; p-toluidine -indolylphosphate ora fCl cec 21)17 3615 doi:10.1242/jcs.146373 1346–1356 127, (2014) Science Cell of Journal N otn n elsz eemaue yfo yoer as cytometry flow by measured were size cell and content DNA m ia)o MO(.3 ia /) rEM + EMMG or v/v), final (0.33% DMSO or final) M 6 10 6 2 6 0 MTi-C H95.Tefloigprimary following The 9.5). pH Tris-HCl mM 100 , el/lwr pte npae otiigEM + EMMG containing plates on spotted were cells/ml 10 .pombe S. 6 b el/l 4 cells/ml. mratehnl .1 rmpeo Blue]. Bromophenol 0.01% -mercaptoethanol, ˚ .Atrwsig(4 washing After C. + /h 2 6 ,wse nseiewtradspotted and water sterile in washed ), el,6 cells, 10 6 ˚ a oioe yotcldensity optical by monitored was C 10 6 el/l 0,0 el/lt were cells/plate 100,000 cells/ml. 6 6 el eemxdwt nequal an with mixed were cells m 10 ˚ m o o5dy.Isolated days. 5 to 3 for C oi1t eoefalse remove to Torin1 M oi1adwr exposed were and Torin1 M 7 el eefxdadtotal and fixed were cells 6 0mntswt TBS with minutes 30 6 10 m ˚ .Frgot on growth For C. 6 requivalent or M el/lbefore cells/ml ˚ Ctoa m g/ml

Journal of Cell Science oi1o .3 MO ltswr nuae t30 at incubated were Plates DMSO. 0.33% or Torin1 counted. 35clswr rw nDE Gbo upeetdwt 0 FBS 10% 50 with assays, supplemented turnover (Gibco) protein DMEM For in (PAA). (Gibco). grown FBS were 2% cells with supplemented A375 (Gibco) DMEM Advanced in grown el eegonepnnilyfr2 or nyi E t28 at YES in only hours 24 for exponentially grown were Cells tests Spot (25 Torin1 ARTICLE RESEARCH Ba S. Moreno, and B. Alvarez, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.146373/-/DC1 online available material Supplementary material Supplementary for PMC UK in Research Deposited Cancer J.P.]. a release. to and C10888/A11178 immediate Trust; number Wellcome [grant the Fellowship by Senior supported was work This authors. all Funding from the input designed J. with Petersen and manuscript J. A.L. the and by wrote J.A. Torin1. cultures done and synthesized cell was experiments A.J. all microscopy and provided live C.W. J.H. single-cell and Pines. the J.F. 7F, experiments, Fig the Except did extracts. L.H. and J.A. contributions Author interests. competing no declare authors The interests Competing on comments valuable for Hagan I. and reagents; manuscript. and the strains Paul for and UK) University) (LRI, Aviv Nurse (Tel Mohan Weisman NH), Ronit Singapore), School, (NAIST (MBI Medical Balasubramanian Shiozaki (Dartmouth Kaz Tokyo), Moseley CRUK of James (Manchester (University Japan), Hagan Yamamoto Iain Gray Torin1; Masayuki Nathanael of UK), aliquot and Institute, an MA) for Cambridge, School) cell (MIT Medical CFP Sabatini (Harvard H3.3 David mRuby-PCNA-/+ thank B1-Venus-/+ We Cyclin line. Kyoto HeLa Jo the to generating grateful very are We Acknowledgements cyclin-B1–Venus Kyoto HeLa microscopy live cell Single cyclin-B1–Venus Kyoto HeLa were cells culture Cell non-septating fluorescence bi-nucleate cells. only anaphase a determine as to population; counted under used anaphase was staining observed the white calcoflour/DAPI calcofluor Combined were with time-point. stained 100 cells at were microscope with every Septa and fixed microscopy hours. samples fluorescence 3 (Sigma-Aldrich) and for for assays v/v) minutes growth final 15–60 liquid (10% for formaldehyde as 30% treated were Cells microscopy Fluorescence 2.4 oioe al o days. 4 for daily monitored pte noYSpae rEM ltssplmne ih25 with supplemented plates EMMG or plates YES onto spotted iews20mlieod o h Rb filter. mRuby the for a milliseconds with 200 hours was 18–24 time of duration an total Cells the using for 6 minutes Devices). performed 15 was (Molecular every filmed imaging were microscope Live-cell high-throughput filming. to ImageXpress with prior supplemented (Invitrogen) FBS medium 10% ( L-15 plate Leibovitz’s with 96-well replaced a on grown were nuae t30 at incubated he,J,W,J . ogie .S,Sa,N . ceze . r,Steever, 3rd, A., McKenzie, G., N. Shah, S., M. Longtine, Q., J. Wu, J., ¨hler, .B,Wc,A,Piipe,P n rnl,J R. J. Pringle, and P. Philippsen, A., Wach, B., A. differentiation. cell represses 0ojcie ort i oiin eeaqie e el Exposure well. per acquired were positions six to Four objective. 20 6 10 6 el/l efl iuinsre trigwt 1 with starting series dilution tenfold A cells/ml. m ia)o aayi 30n/lfnl.Pae were Plates final). ng/ml (300 rapamycin or final) M ˚ o 8husbfr 0–0 el rzgtswere zygotes or cells 200–500 before hours 28 for C 6 anfcto.A es 0 el eecutdper counted were cells 200 least At magnification. r asedad atclry oSme esrfor Weiser Samuel to particularly, and, Mansfeld ¨rg 20) iso es o2pooe elgot and growth cell promotes Tor2 yeast Fission (2006). .Cl Sci. Cell J. 2 /+ 2 m mRuby–PCNA /+ la,Genr.Cluemdu was medium Culture Greiner). Clear, mRuby–PCNA 119 m ylhxmd a used. was cycloheximide M 4475-4485. , 2 /+ 2 33CPclswere cells H3.3–CFP /+ 19) Heterologous (1998). ˚ n rwhwas growth and C 33CPcells H3.3–CFP 6 10 6 el was cells ˚ Cto m M aso . tuo . rn,J,Tmni .adYmmt,M. Yamamoto, and F. Tamanoi, J., Urano, Y., Otsubo, T., Matsuo, aso . uo . aaae .adYmmt,M. Yamamoto, and Y. Y. Watanabe, Ma, Y., Kubo, and T., P. Matsuo, E. Henske, L., Zhang, Q., Liu, N., Ma, P. P. Pandolfi, and P. Tempst, H., Erdjument-Bromage, Z., Chen, L., Ma, P. Russell, and O. Mondesert, B., Furnari, A., Lopez-Girona, i,Q,X,C,Krbkrn . hn,X,Hr . i,Y,Kitosi .P., N. Kwiatkowski, Y., Liu, W., Hur, X., Zhang, S., Kirubakaran, C., Xu, C., Q., C. Liu, Thoreen, K., Westover, M., Niepel, W., Hur, S., Kirubakaran, Q., Liu, Hur, A., Markhard, C., C. Thoreen, A., S. Kang, S. J., Altiok, Wang, W., and J. M. Chang, Q., Bui, Liu, D., Letson, D., Reed, Y., J. Gemmer, M., J. Kreahling, oacy,K . atuh . eea . tnfed .adPetersen, and P. Stansfield, D., Perera, S., Hartmuth, M., K. Kowalczyk, P. Russell, and R. Kovelman, P. Russell, and J. Kanoh, otlo . ogr,L n ec,D. Beach, and L. Rodgers, K., G., Hofmann, Costello, D., H. Lindsay, G., Kanter-Smoler, M., Dahlen, T., Caspari, kd,K,Mrgsk,S,Ttb,H,Tmni .adSizk,K. Shiozaki, and F. Tamanoi, H., Tatebe, S., Morigasaki, K., Ikeda, avy .L,Calt . as . yi .P n elg,D R. D. Kellogg, and P. S. Gygi, W., Haas, A., Charlet, L., S. A., Harvey, Krapp, S., Bagley, Y., K. M. Chan, I. Hagan, A., V. Tallada, A., Patel, A., Grallert, P. Nurse, and L. K. Gould, Z. Feng, P. Russell, and C. Featherstone, P. Nurse, and P. Fantes, Ha W., Du, Bimbo Herna A., Kokubu, J., Kanoh, Y., Nakaseko, K., Nagao, M., Hatanaka, T., Hayashi, ats .A. P. Fantes, R. Egel, fteTRkns o2mmc irgnsavto n ciae h sexual the activates and starvation nitrogen yeast. fission mimics in Tor2 pathway kinase development TOR the of ob G aiykns a8 om osre inln ouewt O and TOR with module signaling conserved a kinases. forms PDK1-like Gad8p kinase family AGC yeast. pombe fission in regulators for negative implications two by Erk governed by TSC2 pathogenesis. cancer of and inactivation sclerosis tuberous functional and Phosphorylation protein. 14-3-3 a and damage DNA by 397 regulated is Cdc25 of localization T-opttv niio fmO,AM n ATR. and al. ATM, mTOR, et P. of Gao, inhibitor ATP-competitive D., K. Westover, J., Wang, inhibitors kinetics. (mTOR) binding rapamycin their of of characterization target and mammalian al. ATP-competitive et K. of Vogel, P., profiling M. Patricelli, J., of Ni, J., Wang, target cancer. of mammalian treatment selective the potent, for 7146-7155. inhibitor highly al. (mTOR) et a rapamycin M. as D. naphthyridin-2(1H)-one Sabatini, T., Sim, antitumor propionylpiperazin-1-yl)-3-(trifluoromethyl)phenyl)-9-(quinolin-3-yl)benzo[h][1,6]- J., single-agent Zhang, shows W., inhibitor, Wee1 cells. sarcoma selective against a activity MK1775, (2012). phosphatase. J. pombe. Schizosaccharomyces in 16 arrest checkpoint replication yeast. fission in mitosis of onset 3321-3334. the regulates inducer, mitotic fShzschrmcspmeHs:aPN-eae rti htassociates that Rad9. protein and PCNA-related Rad1 a with Hus1: M. pombe A. Carr, Schizosaccharomyces and of P. Sunnerhagen, K., Papadimitriou, iso es O ope ciae h G-aiyGd iaeessential control. kinase cycle Gad8 cell AGC-family and the resistance activates stress 2 for complex TOR yeast Fission omteti h iso es cioacaoye pombe. Schizosaccharomyces yeast Trans. fission the in commitment yeast. fission of switches M. morphogenetic I. Hagan, and V. Simanis, mitosis. into entry regulates kinase protein cdc2+ compartment. endosomal kinase. tyrosine/serine a is division. nuclear over control size growth-modulated g2. or g1 mitotic either from state stationary kinases. protein al. yeast et fission K. of M. analysis Balasubramanian, T., deletion E. Liu, M., O’Connell, L., eedn euaino h ioi niio Wee1. inhibitor mitotic the of regulation dependent elRes. Cell highly M. C. Pombo, two of organization subunits. common and and 12 specific mutant by M. complexes TOR tor2 phosphorylated Yanagida, pombe and Schizosaccharomyces M. Ebe, eetv ehd hrceiaino uat n mlctosfrdivision for implications and mutants of mechanisms. characterization control method: selective Beyond in and S6 Genomics protein ribosomal the of phosphorylation yeast. regulate fission Gad8 kinase AGC the in targeting gene PCR-based versatile and pombe. efficient Schizosaccharomyces for modules 21) oto fSy AKatvt hog tblsto ftePp MAPK Pyp2 the of stabilisation through activity MAPK Sty1 of Control (2013). 86-93. , 1357-1370. , ne,G,Ll . iag,M,Gerr,A,Zlie . oc,T and T. Force, J., Zalvide, A., Guerrero, M., Fidalgo, H., Lal, G., ´ndez, . i,Y,Ph .L,Krtr,R . e le,N,Pn,X,Zheng, X., Peng, N., Elzen, den K., R. Karuturi, L., S. 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