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DOI: 1.138/nc286 Figure S1 e f Medium DMSO AktVIII PP242 Rp S6K1-I Gr1 + + + + + + Strvtion + + + + + IB: Akt-pT38 IB: Akt K-pT389 K IB: Rptor Gr1 shs6k1-a shs6k1-b shs6k1-c shrictor shrptor Gr1 c IB: Akt-pT38 K-pT389 K IB: Rptor TSC2 +/+ Gr1 IB: Akt totl IB: Rptor Stimuli dose IB: Akt-pT38 IB: Gr1 Stimuli dose IB: Akt-pT38 IB: d K-pT389 K j HeL TSC2 K-pT389 g h + + + S6K1-I Gr1 IRS-2 Gr1/ IB: IRS1 75 IB: Akt-pT38 IB: Akt-totl K-pT389 K K IB: Akt totl IB: Rptor k i Gr1 Gr1/ Gr1-1 Gr1-2 IB: Akt-pT38 IB: Akt-totl K-pT389 K Stimuli dose IB: IB: Gr1 IB: ppras4 IB: PRAS4 IB: pfoxo IB: Foxo3 + + + Rp IB: Gr1 l Gr1/ + + S6K1-I K-pT389 K IB: IB: Gr1 m + + Rp Gr1/ K-pT389 K IB: IB: Gr1 n Sin1 +/+ Sin1 -/- IB: Akt-pT38 IB: SGK-pS422 IB: R IB: R o 1 2 3 shsin1 1 2 3 min (short exp) (long exp) WWW.NATURE.COM/NATURECELLBIOLOGY 1 213 Mcmilln Pulishers Limited. All rights reserved.

Figure S1 Sin1 plys criticl role in negtive regultion of Akt-Ser473 phosphoryltion in response to or stimultion independent of Gr1 or.. Inhiiting C1/S6K y vrious inhiitors led to elevted Akt-Ser473 phosphoryltion. Immunolot (IB) nlysis of whole cell lystes () derived from HeL cells tht were serum-strved for 24 hours nd then collected fter insulin stimultion for 3 minutes. Where indicted, the kinse inhiitors (AktVIII: 1 mm, PP242: 1 mm, Rpmycin: 2 nm, S6K1-I: 1 mm) were dded together with insulin (1 nm). DMSO ws used s negtive control.. Depletion of endogenous S6K1 or endogenous Rptor led to elevted Akt-Ser473 phosphoryltion. IB nlysis of derived from HeL cells infected with the indicted lentivirl constructs. 24 hours post-infection, cells were selected with 1 mg/ml puromycin for 72 hours to eliminte non-infected cells. c-d. Depletion of endogenous TSC2, which ctivtes S6K, led to reduction in Akt-Ser473 phosphoryltion. IB nlysis of derived from or (c) or HeL cells depleted of endogenous TSC2 vi lentivirl infections (d). e. Depletion of endogenous Gr1 did not significntly ffect Akt-Ser473 phosphoryltion in response to or. were infected with the shgr1 (with s negtive control) lentivirl construct nd selected with 1 mg/ml puromycin for 72 hours to eliminte non-infected cells. Afterwrds, the generted vrious were serum-strved for 24 hours nd then collected fter stimultion with incresing dose of the indicted stimuli for 3 minutes for IB nlysis. f-g. Depletion of endogenous or /Gr1 doule knockdown did not significntly ffect Akt-Ser473 phosphoryltion in response to or. were infected with the sh (with s negtive control) lentivirl construct nd selected with 1 mg/ ml puromycin for 72 hours to eliminte non-infected cells (f). The - depleted were susequently infected with the shgr1 (with s negtive control) lentivirl construct to generte /Gr1 doule-depletion cell lines (g). Afterwrds, the generted vrious were serum-strved for 24 hours, stimulted with incresing doses of the indicted stimuli (insulin: 1 nm; : 1 ng/ml nd : 1 ng/ml for 3 minutes; or : 1 ng/ml for 1 minutes) then lysed for IB nlysis. h-i. IB nlysis to demonstrte the (h) or Gr1 (i) depletion efficiency. were infected with either sh or shgr1 (with s negtive control) lentivirl constructs nd selected with 1 mg/ml puromycin for 72 hours to eliminte non-infected cells. Afterwrds, the whole cell lystes were collected for IB nlysis. j-m. Akt-Ser473 phosphoryltion could still e ugmented upon S6K1-I (j) or rpmycin (k) inhiitor tretment in cells depleted of oth endogenous nd Gr1. were infected with sh or shgr1 (with s negtive control) lentivirl constructs nd selected with 1 mg/ml puromycin for 72 hours to eliminte non-infected cells. The -depleted were susequently infected with the shgr1 (with s negtive control) lentivirl construct to generte the /Gr1 doule-depletion cell lines (l-m). Afterwrds, the generted vrious were treted with 1 mm S6K inhiitor (j, l) or 2 nm rpmycin (k, m) for 12 hours efore collecting the whole cell lystes for IB nlysis. n. or Sin1 -/- were serum-strved for 24 hours nd then collected fter stimultion with the indicted stimuli for 3 minutes for IB nlysis. o. Depletion of endogenous Sin1 in led to n olishment of Akt-Ser473 phosphoryltion. IB of derived from - or shsin1- MFEs tht were serum strved for 12 hours nd then treted with the indicted stimuli for the indicted time periods efore hrvesting for IB nlysis. Specificlly, the doses of stimuli used re listed elow: insulin (1 nm), (1 ng/ ml), (1 ng/ml) nd (1 ng/ml). Other thn the indicted time points for, cells were treted for 3 min with insulin, or efore hrvesting. 2 WWW.NATURE.COM/NATURECELLBIOLOGY 213 Mcmilln Pulishers Limited. All rights reserved.

Figure S2 IP: Myc Myr-Akt1 Myr-Akt2 S6K R3A SGK1 6 Flg-SIn1 HA-AGCs + + + + + Myc-Rictor + + + + + + + + insulin IB: RxRxxpS/T IB: RxRxxpS/T IB: RxxpS/T IB: Myc -Sin1 IB: Myc HA-S6K HA-S6K R3A HA-S6K K/R c d Mss spectrometry identifiction of Sin1-T86 phosphoryltion in cells post insulin stimultion P 81 RRR.SNTAQRL.ERLR 94 e Mss spectrometry identifiction of Sin1-T398 phosphoryltion in vitro y ctive recominnt S6K1 P 393 RLR.FTTDVQLGISGDK. 48 f T86A T398A T86A/T398A + + + + + HA-S6K IB: RxxpS/T -pt86 -pt86 g h T398A T86A/T398A T86A + + + + + HA-S6K -pt398 T398A + + + insulin -pt398 i shrptor shs6k1 + + + + + + + + + -pt86 -pt398 IB: Rptor K1 j o IB: Akt ps473 IB: Akt ps473 (long expo) IB: Ak1 -pt86 K-pT389 K TSC2 +/+ +S6K1-I TSC2 +/+ +Rp p GST + + + + S6K1-I + + + + + + + + + -pt86 k + T86A/T398A strved insulin+rp + S6K GST-Sin1 - pt86 GST- Sin1 IB: GST GST -pt86 Foreskin Firolsts l HA-S6K1 + + + Rp + + + + + + q DMSO pp242 CA 3T3-L1 - pt86 AktVIII Rp S6K1-I m 118 IB: pt86-sin1 IB: pt398-sin1 IB: ps473-akt IB: pt38-akt IB: pt389-s6k K + 1%FBS Medium DMSO Rp S6K1-I r AktVIII pp242 HeL DMSO AktVIII S6K1-I Rp pp242 -pt86 n IB: pt86-sin1 IB: pt398-sin1 IB: ps473-akt IB: pt38-akt IB: pt389-s6k K Akt1 Akt2 Akt1/2 Akt1/S6K1 + + + + + -pt86 -pt398 IB: Akt2 K1 WWW.NATURE.COM/NATURECELLBIOLOGY 3 213 Mcmilln Pulishers Limited. All rights reserved.

Figure S2 S6K1 is the mjor physiologicl kinse responsile for Sin1 phosphoryltion t oth T86 nd T398 sites in tissue-specific or contextdependent mnner.. AGC kinses in Fig. 1 were ctive, s illustrted y their comprle cpility to phosphorylte Rictor in cells. Immunolot (IB) nlysis of whole cell lystes () nd immunoprecipittes derived from 293T cells trnsfected with Myc-Rictor nd indicted HA-tgged constitutive ctive AGC fmily kinses.. A dominnt negtive form of S6K (K/R) mutnt could lock Sin1 phosphoryltion in 293T cells. IB nlysis of nd immunoprecipittes derived from 293T cells trnsfected with nd HA-S6K1- or constitutive ctive form of S6K1 in the presence or sence of 2 nm rpmycin. c. Depletion of endogenous TSC2, which led to elevted S6K kinse ctivity, resulted in incresed Sin1 phosphoryltion in cells. IB of nd Flg immunoprecipittes derived from - or -HeL cells tht were trnsfected with the construct (pcdna3 plsmid ws used s negtive control). 3 hours post-trnsfection, cells were serum strved for 24 hours nd then treted with insulin for 3 minutes efore hrvesting for IB nlysis. d. Schemtic representtion of the Sin1 peptide where the T86 site ws identified to e phosphorylted in vivo y the mss spectrometry nlysis. Plese refer to the methods section Mss Spectrometry Anlysis for experimentl detils. e. Schemtic representtion of the Sin1 peptide where the T398 site ws identified to e phosphorylted in vitro y S6K1 using the mss spectrometry nlysis. Plese refer to the methods section Mss Spectrometry Anlysis for experimentl detils. f. Chrcteriztion of the generted Sin1-pT86 ntiody. IB nlysis of nd Flg immunoprecipittes (IP) derived from 293T cells trnsfected with HA-S6K1 nd the indicted constructs, y lotting with the generted phospho-sin1-t86 ntiody (Sin1-pT86) used in the rest of the studies in this pper. g. Chrcteriztion of the generted Sin1-pT398 ntiody. IB nlysis of Flg-IP derived from 293T cells trnsfected with HA-S6K1 nd the indicted constructs, y lotting with the generted phospho-sin1-t398 ntiody (Sin1-pT398) used in the rest of the studies in this pper. h. Sin1-pT398 signls could e induced y insulin in vivo. IB nlysis of Flg-IP derived from HeL cells trnsfected with the construct y the generted Sin1-pT398 ntiody. Where indicted, 1 nm insulin ws dded for 3 min prior to cell collection fter overnight serum strvtion. i. Depletion of endogenous Rptor or endogenous S6K1 resulted in n decrese in Sin1 phosphoryltion in vivo. IB nlysis of nd Flg-IP derived from -trnsfected HeL cells tht were previously infected with the indicted lentivirl to deplete endogenous Rptor or S6K1. 24 hours post trnsfection, cells were serum strved for nother 24 hours followed y stimultion (1 ng/ml) for 1 minutes efore hrvesting for IB nlysis. j. The S6K1 inhiitor, S6K1-I, could prtilly lock Sin1 phosphoryltion induced y insulin,, or. IB nlysis of nd Flg-IP derived from HeL cells trnsfected with Flg- Sin1. 24 hours post trnsfection, cells were serum strved for nother 12 hours followed y tretment with different stimuli (insulin: 1nM, : 1 ng/ml, : 1 ng/ml for 3 minutes or : 1 ng/ml for 1 min) in the presence or sence of 1 mm S6K1 inhiitor (S6K1-I) efore hrvesting for IB nlysis. k. S6K phosphoryltes Sin1 on T86 in vitro. GST- Sin1 ws incuted with recominnt ctive S6K protein s descried in the in vitro kinse ssy section. Aout 2 ng of the GST-Sin1 ws resolved on SDS-PAGE nd lotted with the Sin1-pT86 ntiody. l. Rpmycin could lock -S6K, ut not the rpmycin resistnt form of S6K (CA), in phosphorylting Sin1 in vivo. IB nlysis of nd Flg-IP derived from 293T cells trnsfected with nd HA-S6K1- () or S6K1 (CA) in the presence or sence of 2 nm rpmycin. m. At endogenous levels, Sin1 phosphoryltion ws ttenuted upon C1 or S6K inhiition. IB nlysis of derived from foreskin firolsts tht were serum-strved for 24 hours nd then collected fter stimultion with 1% FBS for 3 minutes. Where indicted, the indicted kinse inhiitors (AktVIII: 1 mm, PP242: 1 mm, Rpmycin: 2 nm, S6K1-I: 1 mm) were dded together with insulin (1 nm). DMSO ws used s negtive control. n. Sin1 phosphoryltion ws mrkedly decresed when endogenous Akt1 nd S6K1 were depleted simultneously. IB nlysis of nd Flg-IP derived from - trnsfected HeL cells depleted of endogenous Akt1, Akt2 or together with S6K1. o. Rpmycin could prtilly restore Akt-pSer473 in in prt vi reducing Sin1 phosphoryltion in vivo. IB nlysis of derived from or. Where indicted, cells were treted with 1 mm S6K inhiitor (S6K1-I) or 2 nm rpmycin efore collecting for IB nlysis. p. Rpmycin could prtilly lock in vivo Sin1 phosphoryltion induced y insulin in primry foreskin humn firolsts. IB nlysis of derived from primry foreskin firolsts tht were serum strved for 24 hours followed y insulin stimultion (1 nm) for 3 minutes with or without rpmycin (2 nm) efore hrvesting for IB nlysis. q. In 3T3-L1 dipocytes, Akt is the mjor kinse for Sin1-T86 phosphoryltion, while oth Akt nd S6K re mediting Sin1-T398 phosphoryltion. IB nlysis of derived from 3T3-L1 cells tht were serum strved for 24 hours efore ddition of 1 nm insulin for 3 min together with the indicted inhiitors (AktVIII: 1 mm, PP242: 1 mm, rpmycin: 2 nm, S6K1-I: 1 mm). r. In HeL cells, oth S6K1 nd Akt re involved in phosphorylting Sin1-T86, wheres S6K1 is the mjor kinse for Sin1-T398 phosphoryltion. IB nlysis of derived from HeL cells tht were serum strved for 24 hours efore ddition of 1 nm insulin for 3 min together with the indicted inhiitors (AktVIII: 1 mm, PP242: 1 mm, rpmycin: 2 nm, S6K1-I: 1 mm). 4 WWW.NATURE.COM/NATURECELLBIOLOGY 213 Mcmilln Pulishers Limited. All rights reserved.

IP: Myc IP: HA Figure S3 e + + + + Myc-Rictor AA 86E 398E IB: Myc IB: Myc IB: Myc IP: HA + + + HA-Rictor f IP: Myc c + + + + Myc- AA IB: Myc IB: Myc d T86E T398E K 669 44 8 75 43 1 11 12 13 14 16 17 18 19 2 21 22 23 24 25 27 28 29 3 31 32 33 + + + + + HA-RIctor -pt86 - pt86 IB: Rptor g Reltive percentge (%) Sin1-ssocited C2 complex h IgG TSC2 +/+ Sin1 IgG TSC2 Sin1 -/- IgG Sin1 +S6K1-I free Sin1 i IP (longer expo) TSC2 +/+ + S6K1-I IB: Akt-pT38 IB: Akt totl Figure S3 Sin1 phosphoryltion led to its dissocition from the C2 complex. -c. Under ectopic overexpression conditions, Sin1 phosphomimetic mutnt (Sin1-) exhiited reduced interction with Rictor (-) or (c), indicting tht Sin1 phosphoryltion led to the dissocition of Sin1 from Rictor or. Immunolot (IB) nlysis of whole cell lystes () nd immunoprecipittes (IP) derived from 293T cells trnsfected with the indicted constructs nd Myc-Rictor (), HA-Rictor () or Myc (c). d. Sin1 phosphoryltion on oth T86 nd T398 re necessry to dissocite Sin1 from other C2 components. IB nlysis of nd IP derived from 293T cells trnsfected with the indicted constructs (: empty vector control; : Sin1-; : Sin1-T86E/T398E). e. Rictor could not form complex with Sin1 phosphorylted on oth T86 nd T398. Indicted constructs were trnsfected together with HA-Rictor into Sin1 -/- nd 48 hours post trnsfection, HA-Rictor precipittion ws performed nd sujected to immunoloting with the indicted ntiodies. f. Gel filtrtion experiments to illustrte tht depletion of endogenous TSC2 resulted in disruption of Sin1 ssocition with C2 in vivo tht is ssocited with elevted Sin1 phosphoryltion. IB nlysis of the indicted frctiontions derived from the gel filtrtion experiment with - trnsfected HeL- or HeL- hrvested in EBC uffer. g. Quntifiction of totl Sin1 undnce illustrted in (f). h-i. Deletion of endogenous TSC2, which led to incresed S6K kinse ctivity, resulted in reduction of Rictor ssocition with Sin1, which could e prtilly converted y inhiiting S6K ctivity y S6K1-I. IB nlysis of nd nti-sin1 IP derived from or. Where indicted, S6K1-I (1 mm) ws dded 12 hours prior to cell collection. WWW.NATURE.COM/NATURECELLBIOLOGY 5 213 Mcmilln Pulishers Limited. All rights reserved.

8 8 8 SUPPLEMENTARY INFORMATION Figure S4 Reltive undnce 1.2 1.1.8.6.4 e Quntifiction curve for Fig. 3 Reltive undnce 1.2 1.1.8.6.4 Quntifiction curve for Fig. 3.2.2 R i c t o r R i c t o r pt86-sin1 pt86-sin1 5 1 2 25 3 35 4 45 5 1 2 25 3 tretment time (min) insulin tretment time (min) Fig2d (+ Rpmycin) Fig 2d (- Rpmycin) Quntifiction curve for Figure S4d -Rp f Quntifiction curve for Figure S4d +Rp 1.2 1.2 1.2 1 1.1 1. c -Rp +Rp 3 6 9 12 3 6 9 12 insulin (min) - pt86 d -Rp +Rp 8 16 24 32 4 8 16 24 32 4 (min) -pt86 -pt398 K-pT389 K 1. 4 1. 2 Reltive undnce.8.6.4 1. 4 1. 2 Reltive undnce.8.8.6 Rictor (- R pm ycin).6 psin1 (- R pm ycin).4 Rictor (+ R pm ycin) psin1 (+ R pm ycin) 1. 8.2 1. 8 R i c t o r pt86-sin1.2 R i c t o r pt86-sin1. 6. 6 5 1 25 2 3 35 4 45 3 4 1 2 5. 4. 4 tretment time (min) tretment time (min). 2. 2 g h i 1 2 3 4 5 1 2 3 4 5 shrptor +DMSO +Rp +S6K1-I shrptor j 16 24 32 16 24 32 16 24 32 min 3 6 12 24 3 6 12 24 insulin (min) 1 2 4 6 1 2 4 6 1 2 4 6 (min) 3 6 12 24 3 6 12 24 insulin (min) IB: Akt-pT38 IB: Akt-pT38 -pt86 IB: pfoxo KpT389 K -pt86 IB: Rptor -pt86 K-pT389 IB: FOXO3 K-pT389 K IB: Rptor K k l 3 6 12 24 3 6 12 24 insulin (min) IB: Akt-pT38 KpT389 K TSC2 +/+ insulin 1 2 3 insulin TSC2 +/+ TSC2-/- 1 2 3 min m 3 6 9 12 24 48 3 6 9 12 24 48 3 6 9 12 24 48 3 6 9 12 24 48 - - CHX (min) CHX (min) (shorter exp) IB: Mdm2 n (shorter exp) IB: Mdm2 IB: R IB: Vincullin Figure S4 Impirment of Sin1 phosphoryltion led to stilized C2 integrity nd susequently sustined Akt ctivtion under vrious stimultion conditions. -. Quntifiction curves for the reltive pt86-sin1 intensity nd Rictor undnce s function to the tretment time s presented in Figure 3,. c-d. Rpmycin tretment ttenuted in vivo Sin1 phosphoryltion nd susequent dissocition of Sin1 from Rictor/ under insulin stimultion condition (c) or stimultion condition (d). Immunolot nlysis (IB) of whole cell lystes () nd Flg immunoprecipittes (IP) derived from HeL cells trnsfected with tht were serum strved for 12 hours nd then treted with insulin (1 nm) (c) or (1ng/ ml) (d) for the indicted time periods efore hrvesting for IB nlysis. e-f. Quntifiction curves for the reltive pt86-sin1 intensity nd Rictor undnce s function to the tretment time s presented in Figure S4c,d. g. Rpmycin or S6K1-I tretment led to reltively sustined Akt ctivtion upon stimultion. IB nlysis of derived from Sin1 stimulted with with indicted inhiitors. were serum strved for 12 hours nd stimulted y 1 ng/ml. Where indicted, cells were treted with 2 nm rpmycin or 1 mm S6K inhiitor (S6K1-I) for 2 hours efore dding nd collecting the t the indicted time points. h-i. Upon insulin or stimultion, depletion of endogenous Rptor led to n elevted Akt-Ser473 phosphoryltion while depletion of endogenous TSC2 cused reduction in Akt-Ser473 phosphoryltion. IB nlysis of derived from - or shrptor- (h,i) or (i,j). HeL cells tht were serum strved for 24 hours nd then treted with insulin (h nd j) or (i) for the indicted time periods efore hrvesting for IB nlysis. k. Deletion of endogenous TSC2 led to ttenuted Akt-Ser473 phosphoryltion induced y insulin. IB nlysis of derived from TSC2 +/+ or MFEs tht were serum strved for 18 hours nd then treted with insulin for the indicted time periods efore hrvesting for IB nlysis. l. Deletion of endogenous TSC2 led to ttenuted Akt-Ser473 phosphoryltion in response to insulin,, or stimultion. IB nlysis of derived from TSC2 +/+ or MFEs tht were serum strved for 12 hours nd then treted with the indicted stimuli for the indicted time periods efore hrvesting for IB nlysis. Specificlly, the doses of stimuli used re listed elow: insulin (1 nm), (1 ng/ml), (1 ng/ml) nd (1 ng/ml). Other thn the indicted time points for, cells were treted for 3 min with other three stimuli efore hrvesting. m. Sin1 phospho-mimetic mutnt (Sin1-) exhiited similr hlf-life with Sin1-. IB nlysis of derived from HeL cells trnsfected with the indicted constructs nd hrvested t the indicted time periods of CHX tretments (1 µg/ml). Mdm2 lots were provided s control to indicte tht CHX tretment ws working. n. Depletion of endogenous TSC2, which resulted in elevted S6K kinse ctivity nd susequently incresed Sin1 phosphoryltion, did not led to noticele chnges on the stility of endogenous Sin1. IB nlysis of derived from HeL cells depleted of TSC2 vi lentivirl infections (with s negtive control). Cells were hrvested t the indicted time periods of CHX tretments (1 µg/ml). Mdm2 lots were provided s control to indicte tht CHX tretment ws working. 6 WWW.NATURE.COM/NATURECELLBIOLOGY 213 Mcmilln Pulishers Limited. All rights reserved.

Figure S5 3T3-L1 HeL OVCAR5.5 1 5 1 2 4 6 (min).5 1 5 1 2 4 6 (min).5 1 5 1 2 4 6 (min) -pt86 -pt86 c -pt86 -pt398 -pt398 -pt398 IB: ps473-akt IB: ps473-akt IB: ps473-akt IB: pt38-akt IB: pt38-akt IB: pt38-akt IB: pt389-s6k K IB: pt389-s6k K IB: pt389-s6k K d /// Stimuli 1 C2 2 Step1: Step2: C2 ctivtion C2 phosphoryltes Akt on S473 nd PDK1 phosphoryltes Akt on T38 to ctivte Akt Erly time points Erly time points pakt 3 C1 4 ps6k psin1 5 Step3: Step4: Step5: Akt phosphoryltes TSC2 nd PRAS4 to ctivte C1 C1 phosphoryltes S6K on T389 S6K phosphoryltes Sin1 to terminte C2 ctivtion Middle time points Middle time points Lte time points Figure S5 Sin1 phosphoryltion could e tissue specific nd might contriute to the timely shutting down of Akt phosphoryltion triggered y insulin. -c. time course experiments performed in 3T3-L1 (), HeL () nd OVCAR5 (c) cells indicte n oscilltion pttern of Akt1-Ser473 nd potentil kinses responsile for oth Sin1-T86 nd Sin1-T398 phosphoryltion. Immunolot (IB) nlysis of whole cell lystes () derived from the indicted cells tht were serum strved for 24 hours prior to insulin (1 nm) stimultion t the indicted time periods. Plese note tht the Sin1-pT398 signls were otined y immunolotting of the immunoprecipitted endogenous Sin1. d. A proposed model to illustrte possile role of Sin1 phosphoryltion in contriuting to the oscilltion of Akt ctivity upon the insulin stimultion. Specificlly, t erly time points, insulin initilly triggers C2 ctivtion towrds phosphorylting Akt, which susequently ctivtes C1 to ctivte S6K. Afterwrds, elevted S6K could directly phosphorylte Sin1 to disssemle C2 to inctivte Akt in epithelil cells or firolsts. In dipocytes, Akt might e the mjor kinse phosphorylting Sin1. Thus, tissue or cell context-dependent phosphoryltion of Sin1 might ply criticl role in negtively regulting Akt phosphoryltion in timely fshion. WWW.NATURE.COM/NATURECELLBIOLOGY 7 213 Mcmilln Pulishers Limited. All rights reserved.

Figure S6 Sin1 +/+ Sin1 -/- c Medium FBS Medium FBS IB: Akt-pT38 IB: Akt totl Reltive cell viility (%) 1 8 6 4 2 1 3 Reltive cell viility (%) 1 8 6 4 2 -FOXO3 T32A -FOXO3 5 1 25 5 5 1 25 5 d shsin1 + + + + Etoposide (1 µm) IB: cleved PARP 19 IB: cleved Cspse 3 e 19 1 3 1 3 shsin1 1 3 1 3 IB: cleged Cspse 3 IB: cleved PARP f - etoposide + etoposide 2.4 2.2 g 1.3 1.1 shsin1 + 2.1 14.2 1.7 18.1 h i shsin1 + Sin1- shsin1 + Sin1-2. 1.2 1.9 1.3 2.3 1.3 11.3 18.4 Figure S6 Sin1 phosphoryltion resulted in reduced Akt ctivity, susequently sensitizing cells to etoposide-induced poptosis.. Deletion of endogenous Sin1 led to drmticlly reduced Akt-Ser473, ut not Akt- Thr38, phosphoryltion. or Sin1 -/- were serum-strved for 24 hours nd then collected fter stimultion with the indicted stimuli for 3 minutes for immunolot (IB) nlysis. -c. Cell viility ssys with etoposide tretments to illustrte tht etoposide triggers cellulr poptosis in prt vi the Akt/FOXO signling pthwy. HeL cells depleted of endogenous Akt1 () or trnsfected with the indicted Flg-FOXO3 constructs (either Flg-FOXO3- or Flg-FOXO3-T32A, with empty vector s negtive control) (c) were cultured in 1% FBS-contining medium with the indicted concentrtions of etoposide for 48 hours efore performing the cell viility ssys. Dt ws shown s men + SD from n=3 independent experiments d-e. Sin1-, ut not Sin1-, could rescue the poptotic deficiency oserved in Sin1-depleted cells. IB nlysis of derived from endogenous Sin1-depleted OVCAR5 cells trnsfected with the indicted constructs. Where indicted, vrious concentrtions of etoposide were dded 36 hours prior to cell collection. f-i. FACS nlyses were performed to indicte tht compred to -infected OVCAR cells (f), in endogenous Sin1-depleted OVCAR5 cells, stle expression of Sin1- (h), ut not empty vector control (g) or Sin1- (i), could efficiently rescue the cellulr poptosis response triggered y etoposide (5 µm) for 24 hours. Dt were presented s stining s function to AnnexinV-PE stining. Cell popultions were gted to illustrte the chnges of the poptotic cell popultion. Q1: ded cells; Q2: lte poptotic cells; Q3: non-poptotic cells; Q4: erly poptotic cells. P1: poptotic cells, equls to Q2+Q4. 8 WWW.NATURE.COM/NATURECELLBIOLOGY 213 Mcmilln Pulishers Limited. All rights reserved.

Figure S7 IP: HA IP: GST S84L T86A S84L HA-Sin1 -pt86 -pt86 GST-Sin1 -pt86 IB: GST -pt86 IB: GST c 3 12 24 (min) + + + + - IB: GßL IB: GßL d Reltive quntifiction Reltive quntifiction 2 5 Rictor psin1-pt86 2 1 5 Sin1-1 5 1 2 3 4 (min) 1. 4 1. 2 1. 8. 6 Sin1-. 4. 2 1 2 3 4 (min) e g Reltive quntifiction of Akt-pS473 3 25 2 1 5 W T 1 2 3 4 (min) Sin1 f 3 6 12 3 6 12 (min) IB: Myc-Sin1 IB: pfoxo IB: FOXO3 Oncogenic Sin1 mutnt C2 dissocition pt398 pt86 Sin1 T86 nd T398 phosphpryltion Akt dectivtion C2 intct C2 Sin1 T86 cnnot e phosphorylted pt398 Hyper-Akt ctivtion C1 C1 Figure S7 Cncer ptient-derived Sin1 muttions led to n elevted nd sustined Akt phosphoryltion. -. Like the Sin1- muttion, Sin1- S84L led to reduced Sin1-pT86 phosphoryltion in vivo. Immunolot (IB) nlysis of whole cell lystes () nd immunoprecipittes (IP) derived from HeL cells trnsfected with indicted HA-Sin1 constructs () or CMV- GST-Sin1-N-terminl-2 constructs (). c. The Sin1- muttion stilized Sin1 interction with Rictor/ upon insulin stimultion. IB nlysis of derived from --trnsfected HeL cells tht were serum-strved for 24 hours nd then collected fter insulin stimultion for the indicted time periods for Flg-IP. d. Quntifiction curves for the reltive pt86-sin1 intensity nd Rictor undnce s function to the tretment time s presented in Figure 7e. e. Quntifiction curves for the reltive ps473-akt intensity in Sin1- or Sin1- expressing OVCAR5 cells depleted of endogenous Sin1, s function to the tretment time s presented in Figure 7f. f. The Sin1- muttion led to reltively sustined Akt ctivtion under stimultion. OVCAR5 ovrin cncer cells were depleted of endogenous Sin1 y constructs followed y re-introduction of Sin1- or the Sin1- mutnt vi infection with the MSCV-retrovirl vectors. The resulting cells were serum strved overnight followed y the ddition of 1 ng/ml. Cells were hrvested for IB nlysis t the indicted time periods. g. A proposed model to descrie how the ovrin cncer-derived Sin1- muttion might protect the C2 kinse from the negtive regultion y phosphoryltion of Sin1 on oth the T86 nd T398 sites. Specificlly, the Sin1 muttion led to elevted Akt S473 phosphoryltion nd susequent Akt ctivtion y ypssing the Sin1 phosphoryltion medited negtive regultion of C2/Akt signling. WWW.NATURE.COM/NATURECELLBIOLOGY 9 213 Mcmilln Pulishers Limited. All rights reserved.

1 2 3 5 SUPPLEMENTARY INFORMATION Figure S8 Sin1 -/- Sin1 -/- Sin1 +/+ Sin1 +/+ IB: Akt-pT38 IB: pfoxo IB: FOXO3 shsin1 shsin1 IB: Akt-pT38 IB: pfoxo IB: FOXO3 c 95 72 17 72 95 1 3 1 3 shsin1 1 3 1 3 IB: cleved PARP IB: cleved Cspse 3 d 95 72 17 56 1 µm 25 µm shsin1 shsin1 IB: cleved PARP IB: cleved Cspse 3 e f 12 g Reltive cell viility (%) 12 1 8 6 4 2 OVCAR5 5 1 Reltive cell viility (%) 1 8 6 4 2 5 1 1 2 2 5 86E 86A 398E 398A Reltive cell viility (%) 12 1 8 6 4 2 1 2 3 5 h i k shakt1 Repet #1 Repet #2 Reltive Reltive colony colony numer (%) OVCAR5-12 12 1 1 Akt1 Akt2 8 8 6 6 55 shakt1 4 4 2 2 Akt VIII shakt1 (um) IB: Myc-Sin1 Reltive cell viility (%) 1 8 6 4 2 2 4 8 16 32 128 256 AktVIII (µm) j 2 4 16 AktVIII (µm) 12 l m Reltive colony numer (%) 1 8 6 4 2 2 4 16 16 AktVIII (µm) B cell purity IP: Sin1 1 2 3 experiment + + nti-igm + rpmycin -pt86 IB: ERK2 n Reltive Sin1-pT86 (%) 1 9 8 7 6 5 4 3 2 1 1 2 3 Experiment # o Reltive Akt-pS473 (%) 2 1 1 2 3 Experiment # p DND41 TOPTK Lousy Molt4 SupT3 PF3 K-pT389 -pt86 IB: Akt totl K q ps473-akt Sin1-pT86 Akt-pS473-Low Sin1-pT86-Low Akt-pS473-High Sin1-pT86-High r Percentge of Ptient Smples Percentge of ptient smples (%) Correltion etween psin1 nd pakt in ptient smples 1% 9% 8% 7% 6% 5% 4% 3% 2% 1% % 6 7 11 14 2 4 4 Low Medium High Levels of psin1 4 7 pakt high pakt medium pakt low s IGF1 Cse3 Cse4 Gr1 p85 PI 3K p11 PIP2 38 473 PIP2 p11 p85 PI 3K dipocytes Rs T86 P C1 C2 P T398 epithelil cells 1 WWW.NATURE.COM/NATURECELLBIOLOGY 213 Mcmilln Pulishers Limited. All rights reserved.

Figure S8 The Sin1- muttion led to cellulr resistnce to chemotherpeutic drugs nd fcilitted tumorigenesis.. Sin1- led to elevted Akt Ser473 phosphoryltion nd susequently, n increse in FOXO phosphoryltion. Sin1 -/- were trnsfected with the indicted constructs (with empty vector s negtive control). 24 hours post-trnsfection, the resulting cells were serum strved nd hrvested fter stimultion with 1 nm insulin for 3 minutes for immunolot (IB) nlysis. Sin1- led to elevted Akt Ser473 phosphoryltion nd susequently, n increse in FOXO phosphoryltion. OVCAR5 ovrin cncer cells were infected with the shsin1 (with s negtive control) lentivirl construct nd selected with 25 mg/ml hygromycin for 72 hours to eliminte non-infected cells. Afterwrds, the resulting cell lines were trnsfected with the indicted constructs. 24 hours post-trnsfection, cells were serum-strved for 24 hours nd then collected fter stimultion with insulin for 3 minutes for IB nlysis. c-d. Sin1 restored cellulr poptotic deficiency in Sin1-deficient cells. IB nlysis of whole cell lystes () derived from endogenous Sin1 depleted OVCAR5 cells trnsfected with the indicted constructs. Where indicted, vrious concentrtions of etoposide were dded 36 hours prior to cell collection. e. Expression of Sin1- conferred cellulr resistnce to etoposide. OVCAR5 ovrin cncer cells were infected with the shsin1 (with s negtive control) lentivirl construct nd selected with 25 mg/ml hygromycin for 72 hours to eliminte non-infected cells. Afterwrds, the resulting cells were infected with indicted MSCV-Sin1-Myc retrovirl constructs (with empty vector s negtive control) nd selected with 1 mg/ml puromycin for 72 hours to eliminte non-infected cells. Otined cell lines were cultured in 1% FBS-contining medium with indicted concentrtions of etoposide for 48 hours efore performing the cell viility ssys. Dt ws shown s men + SD from n=3 independent experiments. f-g. Sin1- conferred cellulr resistnce to etoposide. Sin1 -/- were trnsfected with the indicted constructs nd cultured in 1% FBS-contining medium with the indicted concentrtions of etoposide for 24 hours (f) or 48 hours (g) efore performing the cell viility ssys. Dt ws shown s men + SD from n=3 independent experiments. h. Representtive imges of tumors in vivo from the xenogrft experiments presented in Figures 8e-g. i. Soft gr ssys to determine the criticl role of the Akt oncogenic signling in cellulr trnsformtion. Su-confluent OVCAR5 cell lines were hrvested for IB nlysis to detect Akt expression. The ove cell lines were lso pplied to soft gr ssys with the presence of 4 µm AktVIII. Briefly, 1x1 5 cells were plted in the top lyer contining.4% gr nd 4 µm AktVIII. 3 weeks lter cells were stined with iodonitrotetrzolium chloride for colony visuliztion nd counting. n=3 independent experiments were performed to generte the error r, nd dt were presented s Men + SD. The scle r represents 1 mm. j. Soft gr ssys with the indicted OVCAR5 cell lines stly expressing Sin1- or Sin1- mutnt to determine their differentil sensitivities to the Akt inhiitor, AktVIII tretment. The ove cell lines were then pplied to soft gr ssy. Briefly, 3x1 5 cells were plted in the top lyer contining.4% gr nd the indicted mount of AktVIII. 3 weeks lter cells were stined with iodonitrotetrzolium chloride for colony visuliztion nd counting. n=3 independent experiments were performed to generte the error r, nd dt were presented s men + SD. The scle r represents 1 mm. k. Cell viility ssys to illustrte tht the Sin1- muttion led to cquired cellulr resistnce to AktVIII tretment. OVCAR5 cell lines stly expressing Sin1- or Sin1- mutnt were cultured in 1% FBS-contining medium with the indicted concentrtions of AktVIII for 48 hours efore performing the cell viility ssys. Dt ws shown s men + SD from n=3 independent experiments. l-o. Splenic B cells were enriched y negtive selection from wild type B6 mice (l). Purified B cells were pretreted for minutes with rpmycin (2 nm) then stimulted with nti-igm ntiody for n dditionl 3 minutes (m). Cells were lysed in CHAPS uffer, sujected to endogenous Sin1 immunoprecipittion nd lotted with the indicted ntiodies (m). Intensities of Sin1-pT86 nds (n) nd Akt-pS473 nds (o) were quntified nd normlized from two independent experiments. p. IB nlysis of from pnel of T-ALL cell lines. q. Two representtive imges of IHC results with indicted Sin1 nd Akt phosphoryltion sttus out of 58 ovrin ptient smples under 4x mgnifiction. The scle r represents 1 mm. r. Br grph of IHC sttisticl nlysis using percentge of smples s function to the reltive levels of Sin1-T86 phosphoryltion. 58 ovrin ptient smples were nlyzed y stining with either Akt-pS473 or Sin1-pT86 ntiodies. p vlue <.3 with Fisher s exct nlysis. s. A proposed model showing how phosphoryltion of Sin1 y C1/S6K or Akt in epithelil cells or dipocytes, respectively, ctively suppresses the C2/Akt ctivtion in prt y dissociting the phosphorylted form of Sin1 from the functionl C2 complex. Importntly, distinct from the C1-medited negtive feedck loops involving either or Gr1 to suppress the PI3K/Akt pthwy tht only responds to insulin or stimultion, S6Kmedited phosphoryltion of Sin1 in firolsts or epithelil cells, or Aktmedited phosphoryltion of Sin1 in dipocytes, cn occur in response to multiple other stimuli including nd. Thus our work discovers n independent mode of negtive regultion of C2, ensuring no hyperctivtion of the Akt oncoprotein tht might fcilitte tumorigenesis. WWW.NATURE.COM/NATURECELLBIOLOGY 11 213 Mcmilln Pulishers Limited. All rights reserved.