Oncogene (2007) 26, 5655–5661 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE Akt inhibitor A-443654 induces rapid Akt Ser-473 independent of mTORC1 inhibition

EK-H Han1, JD Leverson1, T McGonigal1, OJ Shah1,2, KW Woods1, T Hunter2, VL Giranda1 and Y Luo1

1Abbott Laboratories, Global Pharmaceutical Research Division, Research, IL, USA and 2Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA, USA

Rapamycin, a natural product inhibitor of the Raptor- Introduction mammalian target of rapamycin complex (mTORC1), is known to induce Protein B (Akt/PKB) Ser-473 The (Akt/PKB) serine/ phosphorylation in a subset of human cancer cell lines protein play major roles in , through inactivation of S6K1, stabilization of insulin recep- , proliferation and inhibition of apoptosis tor substrate (IRS)-1, and increased signaling through the (see reviews Datta et al., 1999; Thompson and insulin/insulin-like growth factor-I/phosphatidylinositol Thompson, 2004; Altomare and Testa, 2005; Barnett 3-kinase (PI3K) axis. We report that A-443654, a potent et al., 2005; Cheng et al., 2005; Testa and Tsichlis, 2005; small-molecule inhibitor of Akt serine/threonine kinases, Granville et al., 2006). There are three structurally induces Akt Ser-473 phosphorylation in all human cancer related homologues in humans designated as Akt1, Akt2 cell lines tested, including PTEN- and TSC2-deficient and Akt3. Hallmarks of the Akt family include an lines. This phenomenon is dose-dependent, manifests amino-terminal pleckstrin homology (PH) domain, a coincident with Akt inhibition and likely represents an short a-helical linker and a carboxy-terminal kinase alternative, rapid-feedback pathway that can be function- domain. The PH domain facilitates anchorage of Akt to ally dissociated from mTORC1 inhibition. Experi- membrane phospholipids produced by phosphatidyli- ments performed in TSC2À/À cells indicate that TSC2 nositol 3-kinase (PI3K). PI3K is activated by growth and IRS-1 cooperate with, but are dispensable for, A- factors, and insulin and produces phosphati- 443654-mediated Akt phosphorylation. This feedback dylinositol 3,4-phosphate (PIP2) and phosphatidylino- event does require PI3K activity, however, as it can be sitol 3,4,5-phosphate (PIP3), which interact with Akt inhibited by LY294002 or wortmannin. Small interfering and recruit it from the cytosol to cellular membranes. RNA-mediated knockdown of mTOR or Rictor, compo- Following membrane localization, Akt is phosphoryla- nents of the rapamycin-insensitive mTORC2 complex, but ted at Thr-308 in the kinase activation loop (T-loop) by not the mTORC1 component Raptor, also inhibited Akt phosphoinositide-dependent kinase 1 (PDK1), which, Ser-473 phosphorylation induced by A-443654. Our data by virtue of its own PH domain, colocalizes with Akt at thus indicate that Akt phosphorylation and activity are sites of PIP2/PIP3 production. Phosphorylation of Akt coupled in a manner not previously appreciated and within the carboxy-terminal tail (hydrophobic motif) at provide a novel mode of Akt regulation that is distinct Ser-473 is required for maximal activation (Alessi et al., from the previously described rapamycin-induced IRS-1 1997). The identity of the kinase(s) responsible for stabilization mechanism. phosphorylating Ser-473 (provisionally referred to as Oncogene (2007) 26, 5655–5661; doi:10.1038/sj.onc.1210343; ‘PDK2’) has remained enigmatic, but recent work by published online 5 March 2007 Sarbassov et al. (2005) has provided compelling genetic and biochemical evidence indicating that the rapamycin- Keywords: A-443654; Akt; phosphorylation; rapamycin; insensitive mammalian target of rapamycin complex mTOR (mTORC2) mediates this event. Other kinases have been implicated as ‘PDK2’, including PDK1 (Balendran et al., 1999), DNA-PK (Feng et al., 2004), ILK (Persad et al., 2001) and Akt itself (Toker and Newton, 2000). Akt is overexpressed or amplified in a variety of human , including prostate, lung, breast, ovar- ian, colon and head and neck. Inappropriately high Akt activity is pervasive in tumors with deregulated PIP2/ PIP3 production, namely those in which PTEN (phos- Correspondence: Dr EK-H Han, Abbott Laboratories, GPRD, Cancer phatase and tensin homolog) has been inactivated or Research, Dept. R47S, Bldg. AP9A, Abbott Park, IL 60064, USA. E-mail:[email protected] silenced or those harboring oncogenic mutations in the Received 6 October 2006; revised 4 December 2006; accepted 22 January catalytic subunit of PI3K, p110a. The preponderance of 2007; published online 5 March 2007 data implicating Akt as a valid cancer target led us to Akt inhibitor induces Akt Ser-473 phosphorylation EK-H Han et al 5656 generate small-molecule inhibitors of this kinase as rapid induction of Akt Ser-473 phosphorylation caused potential therapeutics. We have previously described a by A-443654. We thus compared the effects of pan-Akt inhibitor, A-443654, that inhibits the phos- rapamycin and A-443654 in multiple human cancer cell phorylation of Akt substrates and demonstrates toxicity- lines. A 1 h treatment with 10 nM rapamycin was not limited efficacy in human-xenografted murine tumor sufficient to induce Akt Ser-473 phosphorylation in any models (Luo et al., 2005). During our studies, we of the cell lines tested, despite complete inhibition of observed that A-443654 induces rapid and robust S6K1 Thr-389 and S6 Ser-235/236 phosphorylation phosphorylation of Akt Ser-473, indicative of a com- (Figure 1c). In contrast, Akt Ser-473 phosphorylation pensatory, but futile, upregulation of Akt activity when was induced by A-443654 in each cell line, often within the kinase is pharmacologically inhibited. In the current 5 min at a time when little or no inhibition of S6 Ser-235/ study, we have examined the mechanism(s) by which 236 phosphorylation was observed (Figure 1c). A-443654 triggers this event. Although A-443654 and rapamycin inhibited S6 Ser- Recent work has shown that S6K1 functions down- 235/236 phosphorylation to similar degrees (cf., 60 min stream of Akt and Raptor-mTOR (mTORC1) to promote time points, Figure 1c), only A-443654 induced robust the degradation of substrates (IRS-1/2), Akt Ser-473 phosphorylation in these time courses. adaptor molecules that mediate PI3K activation by A-443654 also induced an acute, although transient, insulin and IGF-I receptors. This feedback activity is increase in S6K1 Thr-389 phosphorylation in several of sufficient to dampen insulin- and IGF-I-induced Akt the cell lines tested (Figure1c, DU-145, MCF-7, HeLa signaling both in response to prolonged insulin treatment and H460; Figure 2, TSC2 þ / þ ). and in tumor models in which the mTORC1/S6K1 axis is constitutively engaged (Harrington et al., 2004; Shah et al., 2004; Um et al., 2004). Conversely, decreased Akt A-443654 induces Akt phosphorylation in the absence activity leads to a decrease in S6K1 activity, allowing IRS- of TSC2 1 to accumulate and thereby upregulate Akt Ser-473 Given the rapid kinetics of Akt Ser-473 phosphorylation phosphorylation via insulin receptor and PI3K signaling following A-443654-mediated Akt inhibition, it seemed (Hay, 2005). We thus investigated the role of this pathway unlikely to require IRS-1 stabilization downstream of in A-443654-mediated Akt phosphorylation. Although S6K1 inhibition. We therefore asked whether A-443654 Akt Ser-473 phosphorylation can be induced after could induce Akt phosphorylation in TSC2-deficient approximately 20 min of S6K1 inhibition by the mouse embryo fibroblasts (MEFs), which possess a mTORC1 inhibitor rapamycin (O’Reilly et al., 2006), constitutively active mTORC1/S6K axis and therefore the phosphorylation induced by A-443654 is extremely express little or no functional IRS-1 (Harrington et al., rapid (5 min or less) and does not appear to require S6K1 2004; Shah et al., 2004). S6K1 activity is independent of inhibition. Instead, there seems to be a novel, fast-acting Akt in this model, and Akt activation is uncoupled mechanism that responds to acute Akt inhibition by from, and thus refractory to, insulin or IGF-I stimula- upregulating its phosphorylation. tion. Notably, TSC2 þ / þ and TSC2À/À MEFs both showed an induction of Akt Ser-473 phosphorylation upon A-443654 treatment, indicating that the feedback can occur in the absence of TSC2 and IRS-1. A-443654 Results and discussion also induced Akt phosphorylation in both cell lines in the absence of serum (Figure 2), further indicating that Induction of Akt Ser-473 phosphorylation by A-443654 is this effect can occur in the absence of growth factors. time- and concentration dependent We noted that the stimulation of Akt Thr-308 and A-443654 is a potent inhibitor of all three Akt family Ser-473 phosphorylation by A-443654 was reduced in kinases (Luo et al., 2005). We have shown that A- TSC2À/À MEFs relative to their TSC2 þ / þ counterparts. 443654 inhibits the phosphorylation of the Akt sub- This could reflect the downregulation of some Akt strates GSK3a/b, mTOR and TSC2 in a dose-dependent effector pathway. Akt has been reported to phosphor- manner (Luo et al., 2005). At the same concentrations, ylate and inhibit IRS-1, thereby playing a role in its we now find that A-443654 induces phosphorylation of negative feedback regulation (Li et al., 1999). It is thus Akt Ser-473, perhaps reflecting a homeostatic feedback possible that Akt inhibition by A-443654 leads to a mechanism by which the cell attempts to maintain Akt rapid activation of IRS-1, independent of its stabiliza- activity. A-443654 induces Akt Ser-473 phosphorylation tion, and that the reduced Akt phosphorylation in a concentration- and time-dependent manner, an observed in TSC2À/À cells reflects the diminished pool effect that is stable for up to 6 h after the drug is of functional IRS-1 in these cells. removed from the culture medium (Figure 1a and b). In the presence of insulin, only TSC2 þ / þ cells showed The mTORC1 inhibitor rapamycin has been shown to a potentiation of Akt Ser-473 phosphorylation induced induce Akt Ser-473 phosphorylation concomitant with by A-443654 (Figure 2). Thus, although Akt Ser-473 inhibition of S6K1 Thr-389 phosphorylation (Sun et al., phosphorylation induced by Akt inhibition can occur 2005; O’Reilly et al., 2006). As Akt acts upstream to independent of InsR/IGF-IR/IRS1 signaling, this path- positively regulate Raptor-mTOR and S6K1, it was way can potentiate it when it is functional. The addition conceivable that S6K1 inhibition and consequent loss of of 10% fetal calf serum (FCS) was also able to S6K1-IRS-1/2 negative feedback could also explain the potentiate the induction of Akt Ser-473 phosphorylation

Oncogene Akt inhibitor induces Akt Ser-473 phosphorylation EK-H Han et al 5657 Time after a µ b A-443654 ( M) removal (h) A-443654 (min) 0 0.03 0.10.3 1.03.0 10 0.5 1.0 3.0 6.0 0 15 30 60 120 240 P-Akt Ser-473 P-Akt Ser-473

P-GSK3α/β Akt

β- β-Actin

c DU-145 HeLa Rapamycin A-443654 Rapamycin A-443654 Time (min):0 5 15 30 60 5 15 30 60 Time (min): 0 5 15 30 60 5 15 30 60 P-Akt 473 P-Akt 473 Akt Akt

P-S6K1 389 P-S6K1 389

S6K1 S6K1

P-S6 235/236 P-S6 235/236

S6 S6

MCF-7 PC3 Rapamycin A-443654 Rapamycin A-443654 Time (min):0 5 15 30 60 5 15 30 60 Time (min): 0 5 15 30 60 5 15 30 60

P-Akt 473 P-Akt 473

Akt Akt

P-S6K1 389 P-S6K1 389

S6K1 S6K1

P-S6 235/236 P-S6 235/236

S6 S6

A431 H460 Rapamycin A-443654 Rapamycin A-443654 Time (min): 0 5 15 30 60 5 15 30 60 Time (min): 0 5 15 30 60 5 15 30 60 P-Akt 473 P-Akt 473

Akt Akt

P-S6K1 389 P-S6K1 389

S6K1 S6K1

P-S6 235/236 P-S6 235/236

S6 S6

MiaPaCa A-443654 Rapamycin Time (min): 0 5 15 30 60 5 15 30 60 P-Akt 473

Akt P-S6K1 389 S6K1

P-S6 235/236

S6

Figure 1 Concentration- and time-dependent induction of Akt Ser-473 phosphorylation by the Akt-specific inhibitor, A-443654. (a) MiaPaCa cells were treated with increasing concentrations of A-443654 for 2 h. Cell lysates were analysed for Akt (Ser-473) and GSK3a/b (Ser-9/21) phosphorylation by immunoblotting. (b) MiaPaCa cells were treated with 1.0 mM A-443654 for various times, up to 2 h. Cell lysates were analysed for Akt Ser-473 phosphorylation by immunoblotting. (c) Various human tumor cell lines were treated with 1.0 mM A-443654 or 10 nM rapamycin for the times indicated. Cell lysates were analysed for Akt (Ser-473), S6K1 (Thr-389) and S6 (Ser-235/236) phosphorylation by immunoblotting.

Oncogene Akt inhibitor induces Akt Ser-473 phosphorylation EK-H Han et al 5658 TSC2+/+ TSC2-/- a 1.0 µM A -443654 µ A-443654 (1.0 M): - + - + - + - + - + - + LY294002 (µM): - 10 30 50 - 10 30 50 Insulin (200 nM): - - + + - - - - + + - - 10% Serum: - - - - + + - - - - + + P-Akt Ser-473 P-Akt Ser-473 P-Akt Thr-308

P-Akt Thr-308 P-GSK3α/β

P-S6K1 Thr-389 β-Actin

P-S6 Ser- 240/244 b Serum-starved µ α-Tubulin A-443654 (1.0 M): - - - - + + LY294002 (10 µM): - - - + - + Figure 2 Inhibition of Akt kinase activity induces Akt phosphor- 2h Serum Add-back: - - + - - - þ / þ À/À ylation in the absence of TSC function. TSC2 or TSC2 P-Akt Ser-473 MEFs were serum-starved overnight and then treated with 1.0 mM A-443654 for 1 h before stimulation with 200 nM insulin or 10% FCS for 30 min. Cell lysates were analysed for Akt (Ser-473 and P-GSK3α/β Thr-308), S6K1 (Thr-389) and S6 (Ser-240/244) phosphorylation by immunoblotting. β-Actin 12 34 56

c 5 nM rapamycin: - - - + þ / þ by A-443654 in TSC2 and, to a lesser extent, in 100 nM wortmannin: - - + - TSC2À/À MEFs (Figure 2). Presumably, it is growth µ factors other than insulin and IGF-I in FCS that act to 1.0 M A-443654: - + + + / stimulate Akt phosphorylation in TSC2À À MEFs, as has P-Akt Ser-473 been shown previously (Zhang et al., 2003; Harrington et al., 2004; Shah et al., 2004). Figure 3 PI3K activity is required for A-443654-induced Akt Ser- Interestingly, A-443654 induced S6K1 Thr-389 phos- 473 phosphorylation. (a) MiaPaCa cells were treated with 10, 30 or phorylation in TSC2 þ / þ MEFs but not in cells lacking 50 mM LY294002 for 30 min and then treated for an additional 2 h with or without 1.0 mM A-443654. Cell lysates were analysed for TSC2 (Figure 2). We were also surprised to note that Akt Ser-473 phosphorylation by immunoblotting. (b) MiaPaCa S6K1-mediated phosphorylation of the ribosomal pro- cells were serum starved for 2 days and then treated with 10 mM tein S6 decreased in the presence or absence of TSC2. LY294002 or 1 mM A-443654 for 2 h. Cells were also pretreated These data indicated that, at 1.0 mM, A-443654 may non- with LY294002 for 30 min followed by 2 h treatment with A- selectively inhibit S6K1. Indeed, we found that A- 443654. Cell lysates were analysed for Akt Ser-473 phosphorylation by immunoblotting. (c) MiaPaCa cells were pretreated with 5 nM 443654 can inhibit S6K1 in vitro, though much less rapamycin or 100 nM wortmannin for 30 min before treatment with potently than Akt (Ki ¼ 30 nM, versus Ki ¼ 160 pM 1.0 mM A-443654 for an additional 30 min. Cell lysates were against Akt). Although these findings were unexpected, analysed for Akt Ser-473 phosphorylation by immunoblotting. S6K1 inhibition does not appear sufficient for the kind of rapid Akt Ser-473 phosphorylation caused by A- 443654. Rapamycin rapidly and potently inhibits S6 phosphorylation mediated by S6K1, and yet it did not induce immediate Akt Ser-473 phosphorylation, in so we asked whether it is also involved in the response to contrast to A-443654 (Figure 1c). Rapamycin inhibits A-443654. As shown in Figure 3a, pretreatment of S6K1 indirectly, via its effect on mTORC1, and thus it MiaPaCa cells with the PI3K inhibitor LY294002 remains formally possible that direct inhibition of S6K1 inhibited A-443654-mediated Akt Ser-473 phosphoryla- by an adenosine 50-trisphosphate-competitive inhibitor, tion in a dose-dependent manner under serum-replete such as A-443654, may account for Akt Ser-473 conditions. Pretreatment with 50 mM LY294002 comple- phosphorylation. However, A-443654 is a more potent tely inhibited A-443654-mediated Akt phosphorylation at inhibitor of Akt, and we observed the induction of Akt both Ser-473 and Thr-308 (Figure 3a). LY294002 inhibits Ser-473 phosphorylation at low A-443654 concentra- PI3K and mTOR with IC50’s of 3 and 5 mM, respectively tions that are unlikely to inhibit S6K1 in vivo (Figure 1a (Brunn et al., 1996; Davies et al., 2000), and thus our and data not shown). results indicate that PI3K and/or mTOR is involved in mediating Akt phosphorylation in response to A-443654. Similar results were obtained when serum-starved cells Induction of Akt phosphorylation by A-443654 is were pretreated with LY294002 (Figure 3b). Basal Akt dependent on PI3K and/or mTORC2 phosphorylation is very low in MiaPaCa cells, whether PI3K activity is required for Akt Ser-473 phosphoryla- grownin10%serumortheabsenceofserum(Figure3b, tion in response to insulin and other growth factors, and lanes 1 and 2). The addition of 10% serum following

Oncogene Akt inhibitor induces Akt Ser-473 phosphorylation EK-H Han et al 5659 serum starvation caused a slight increase in Akt So how does A-443654 induce Akt Ser-473 phosphor- phosphorylation (Figure 3b, lane 3). As in TSC2 þ / þ ylation? Given the rapidity of A-443654-induced Akt and TSC2À/À MEFs, significant Akt phosphorylation phosphorylation, and the fact that it can be observed in occurred in response to A-443654 even in the absence of TSC2À/À MEFs, it seems unlikely to require IRS-1 serum (Figure 3b, lane 5). stabilization downstream of mTORC1 inhibition. Wortmannin is an inhibitor of PI3K family kinases, Although IRS-1 levels are not elevated after short (1 h) including PI3K p110a and mTOR. Although wortman- A-443654 treatments (data not shown), it is possible that nin inhibited A-443654-induced Akt Ser-473 phosphor- inhibiting Akt leads to rapid IRS-1 activation through a ylation, specific inhibition of mTORC1 with rapamycin distinct mechanism. As mentioned previously, Akt has had no effect (Figure 3c). Our data thus indicate that been implicated as a negative feedback regulator PI3K and/or mTORC2, but not mTORC1, is required through direct IRS-1 phosphorylation (Li et al., 1999), for Akt feedback phosphorylation induced by A- and so pools of IRS-1 activated upon A-443654 443654. Further support for the role of the mTORC2 treatment may be able to rapidly activate PI3K and complex derives from small interferance RNA (siRNA) Akt Ser-473 phosphorylation. experiments in which specific knockdown of mTOR or Another possibility is that A-443654-bound Akt is Rictor, but not Raptor, inhibited Akt Ser-473 phos- simply locked into a conformation that is not amenable phorylation following A-443654 treatment (Figure 4). to Ser-473 dephosphorylation, and thus the phosphory- mTOR and Raptor siRNAs reproducibly caused a lated form rapidly accumulates. Even after washout of decrease in S6K1 Thr-389 phosphorylation (data not A-443654, Akt Ser-473 phosphorylation was maintained shown), indicating successful knockdown of this com- for long periods of time (Figure 1a), and so it is possible plex. These data demonstrate that mTORC2 complexes that this compound continues to bind Akt avidly in the are responsible, at least in part, for the phosphorylation cell. It is also possible that Akt, once activated by PDK1 of Akt Ser-473 observed upon treatment with the Akt and ‘PDK2’, triggers its own release from PIP3-contain- inhibitor A-443654. ing membranes by phosphorylating some tethering Taken as a whole, our data point to the existence of a substrate. A similar mechanism has been described for novel feedback mechanism that quickly responds to the release of Erk1/2 following activation by mitogen- acute Akt inhibition. A-443654, a potent pharmacolo- induced extracellular kinase 1 (Xu et al., 1999). gical inhibitor of Akt, afforded us a unique means of Assuming Akt relocalization is required for its subse- probing cellular Akt function. A-443654 consistently quent dephosphorylation, A-443654 might lead to an induces dose-dependent phosphorylation of a crucial accumulation of inactive, but highly phosphorylated, activating site in Akt, Ser-473. Perhaps not surprisingly, Akt simply by preventing its release from sites of this event requires continued PI3K and mTORC2 activation. There are other examples in which treat- activity, which are known to be requisite for Akt Ser- ment with a kinase inhibitor results in increased 473 phosphorylation. Based on studies performed in activating phosphorylation of the kinase itself or an TSC2À/À cells and time courses performed with A- upstream kinase (Hall-Jackson et al., 1999; Cheung 443654 and the mTORC1 inhibitor rapamycin, we et al., 2003). conclude that this rapid feedback event does not involve Although our studies point to the existence of the mTORC1-S6K1 pathway that regulates IRS-1 previously undescribed modes of Akt regulation, it is stability. clear that further studies will be required to elucidate the mechanisms involved.

Materials and methods siRNA: SCR mTOR Raptor Rictor A-443654 (1.0 µM):-+-+-+-+ Cell culture All human cell lines and human pancreatic carcinoma MiaPaCa mTOR cells were obtained from ATCC (Manassas, VA, USA) and were maintained in 5% CO2 at 371C. MiaPaCa were grown in Raptor Dulbecco’s modified Eagle’s medium (DMEM) plus 10% FBS. TSC2 þ / þ and TSC2À/À MEFs, provided by David Kwiatkowski (Harvard University), were maintained in DMEM supplemen- Rictor ted with 10% (v/v) FBS. All medium components were obtained from Invitrogen (Carlsbad, CA, USA). P-Akt Ser-473 Antibodies and reagents Akt Antibodies for Akt, phospho-Akt Ser-473, phospho-Akt Thr- 308, phospho-GSK3a/b, mTOR, Raptor, S6K1, phospho- Figure 4 Akt feedback phosphorylation requires mTORC2, but S6K1 Thr-389 and phospho-S6 Ser-235/236 and Ser-240/244 not mTORC1. MiaPaCa cells were transfected with 50 nM scrambled, mTOR, Raptor or Rictor siRNA and grown for 72 h were obtained from Cell Signaling Technology (Beverly, MA, before treatment with or without 1.0 mM A-443654 for 1 h. Cell USA). Antibodies to Rictor and additional mTOR antibodies lysates were analysed for mTOR, Raptor, Rictor and Akt (total were obtained from Bethyl Laboratories (Montgomery, TX, protein and Ser-473-phosphorylated) by immunoblotting. USA). a-Tubulin antibodies were purchased from Sigma

Oncogene Akt inhibitor induces Akt Ser-473 phosphorylation EK-H Han et al 5660 (St Louis, MO, USA). Secondary anti-rabbit-horseradish Protein extraction and immunoblot analysis peroxidase (HRP) and anti-mouse-HRP antibodies were Cells were scraped into cold phosphate-buffered saline, purchased from Amersham Biosciences (Piscataway, NJ, pelleted, and then resuspended in 100–200 ml ice-cold insect USA). All chemicals were purchased from Sigma. cell lysis buffer supplemented with protease inhibitors (BD Biosciences Pharmingen, San Diego, CA, USA). Cells were lysed by sonication and the debris was cleared in a siRNA transfections microcentrifuge. Forty micrograms of lysate was loaded in SMARTpool siRNAs targeting mTOR and Raptor were each well of 6 or 10% Tris-Glycine polyacrylamide mini-gels obtained from Dharmacon (Lafayette, CO, USA), as was an (Invitrogen) for sodium dodecyl sulfate–polyacrylamide gel individual siRNA targeting Rictor (see Sarbassov et al., 2005, electrophoresis analysis. Proteins were transferred to PVDF for sequence). MiaPaCa cells were seeded into 10 cm dishes at membranes (Invitrogen), blocked for 1 h in TBS-T plus 5% (w/ 1.5 Â 106 per dish the day before transfections. For each v) powdered blotting grade milk (Bio-Rad Laboratories, 1 transfection 12.5 mlof20mM siRNA stock was mixed with Hercules, CA, USA), and then probed overnight at 4 C with 1.4 ml Opti-MEM (Invitrogen) for 5 min at room temperature. primary antibodies at a 1:2000 dilution in blocking solution. Lipofectamine 2000 (22.4 ml; Invitrogen) was likewise mixed Blots were developed using enhanced chemiluminescence (ECL with 1.4 ml Opti-MEM for 5 min. The two solutions were then Plus) reagents from Amersham Biosciences. mixed for 30 min before being added to cells covered by 2.2 ml medium (final siRNA concentration of 50 nM). After 4 h of Acknowledgements incubation at 371C the transfection solution was removed and replaced by fresh medium. Cell lysates were prepared 3 days TH is a Frank and Else Schilling American Cancer Society later. Research Professor.

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