Molecular Cell, Vol. 10, 151–162, July, 2002, Copyright 2002 by Cell PressIdentification of the Tuberous Sclerosis Complex-2Tumor Suppressor Gene Product Tuberin as a Targetof the Phosphoinositide 3-Kinase/Akt Pathwayof these kinases is blocked by PI3K-specific inhibitors(Burgering and Coffer, 1995; Chung et al., 1994; Frankeet al., 1995). Akt contains a PH domain that is specificto PtdIns-3,4P2and PtdIns-3,4,5P3(Franke et al., 1997).Akt is thereby recruited to these PI3K-generated secondBrendan D. Manning,1,2Andrew R. Tee,1M. Nicole Logsdon,2John Blenis,1and Lewis C. Cantley1,2,31Department of Cell BiologyHarvard Medical Schoolmessengers and to the PDK1 protein kinase, which also2Division of Signal Transductionspecifically binds to these lipids (Stokoe et al., 1997).Beth Israel Deaconess Medical CenterPDK1 then phosphorylates and activates Akt (Alessi etHarvard Institutes of Medicineal., 1997).Room 1028, 4 Blackfan CircleThe regulation of S6K1 is much more complex, withBoston, Massachusetts 02115both PI3K-dependent and -independent signaling path-ways involved in its activation (Chung et al., 1994; Wenget al., 1995). Several PI3K-regulated effectors are knownSummaryto participate in the activation of S6K1 including PDK1,PKC␨/␭, Cdc42, Rac1, and Akt (Burgering and Coffer,The S/T-protein kinases activated by phosphoinositide1995; Chou and Blenis, 1996; Kohn et al., 1998; Pullen et3-kinase (PI3K) regulate a myriad of cellular processes.al., 1998; Romanelli et al., 1999). However, the molecularHere, we show that an approach using a combinationmechanism of how these contribute to S6K1 activationof biochemistry and bioinformatics can identify sub-remains unclear (reviewed by Martin and Blenis, 2002).strates of these kinases. This approach identifies theIn addition to mitogen-regulated signaling to S6K1, thetuberous sclerosis complex-2 gene product, tuberin,metabolic state of the cell and the availability of nutrientsas a potential target of Akt/PKB. We demonstrate that,control S6K1 activation through the mammalian targetupon activation of PI3K, tuberin is phosphorylated onof rapamycin (mTOR, also known as FRAP, RAFT, andconsensus recognition sites for PI3K-dependent S/TRAPT; Dennis et al., 2001; Hara et al., 1998). Recentkinases. Moreover, Akt/PKB can phosphorylate tu-studies suggest that mTOR is also regulated by mito-berin in vitro and in vivo. We also show that S939 andgenic signals (Fang et al., 2001). Interestingly, it hasT1462 of tuberin are PI3K-regulated phosphorylationbeen suggested that the point of convergence of thesites and that T1462 is constitutively phosphorylatedmitogenic and nutrient-sensing signals in the regulationin PTEN⫺/⫺tumor-derived cell lines. Finally, we findof S6K1 may be at the level of Akt directly phosphorylat-that a tuberin mutant lacking the major PI3K-depen-ing mTOR (Nave et al., 1999; Scott et al., 1998). However,dent phosphorylation sites can block the activationthis phosphorylation does not appear to affect mTORof S6K1, suggesting a means by which the PI3K-Aktactivity or S6K1 activation (Sekulic et al., 2000). Thus,pathway regulates S6K1 activity.of the PI3K-regulated effectors thought to participate inS6K1 activation, the molecular basis of how Akt regu-Introductionlates S6K1 remains the least well understood.Akt itself has been implicated in many of the PI3K-Class I phosphoinositide 3-kinases (PI3Ks) are activatedregulated cellular events, and several substrates haveby many extracellular growth and survival stimuli. Thesebeen shown to be phosphorylated in vitro and/or in vivolipid kinases catalyze the production of the second mes-by Akt (recently reviewed by Brazil and Hemmings, 2001;sengers phosphatidylinositol-3,4-bisphosphate (PtdIns-3,Vanhaesebroeck and Alessi, 2000). Therefore, the total4P2) and phosphatidylinositol-3,4,5-trisphosphate (PtdIns-cellular effect of PI3K activation and subsequent activa-3,4,5P3; reviewed by Katso et al., 2001; Rameh and Cant-tion of Akt is mediated through a variety of differentley, 1999). Downstream targets containing specializedtargets. However, it seems unlikely that the large arraydomains, such as pleckstrin-homology (PH) domains,of processes controlled by the PI3K-Akt pathway can bethat specifically bind to these lipid products of PI3K areaccounted for by our current knowledge of downstreamthen activated. These activated proteins control a widetargets.array of cellular processes, including survival, prolifera-Here, we have developed an approach to screen fortion, protein synthesis, growth, metabolism, cytoskele-substrates of PI3K-dependent S/T kinases, such as Akt.tal rearrangements, and differentiation. However, thereThis approach uses phospho-specific antibodies gener-is still much we do not know about the signaling eventsated against a phosphorylated protein kinase consen-leading from activation of PI3K effectors to downstreamsus recognition motif in combination with a protein data-changes in cell physiology.base motif scanning program called Scansite (http://Serine/threonine (S/T) protein kinases can accountscansite.mit.edu; Yaffe et al., 2001). Scansite is a web-for much of the functional diversity of PI3K signalingbased program that searches protein databases for opti-(reviewed by Toker, 2000; Vanhaesebroeck and Alessi,mal substrates of specific protein kinases and for opti-2000). Akt/protein kinase B and the 70 kDa-S6 kinase 1mal binding motifs for specific protein domains with(S6K1) are the best characterized of the PI3K-regulateddata generated by peptide library screens (e.g., ObataS/T kinases. The mitogen-stimulated activation of bothet al., 2000; Songyang and Cantley, 1998; Yaffe andCantley, 2000; Yaffe et al., 2001). The phospho-motifantibody is used to recognize proteins phosphorylated3Correspondence: [email protected] Cell152specifically under conditions in which the kinase of inter-est is active. Scansite is then used to identify candidatesubstrates of this protein kinase that have the predictedmolecular mass of the proteins recognized by the phos-pho-motif antibody. We show that this approach suc-cessfully identifies known substrates of Akt. We alsoidentify and characterize the tuberous sclerosis com-plex-2 (TSC2) tumor suppressor gene product, tuberin,as an Akt substrate. Furthermore, we find that overex-pression of a tuberin mutant lacking the major Akt phos-phorylation sites can inhibit growth factor-induced acti-vation of S6K1. These results provide a