mTOR Signalling, Nutrients and Disease 223 Activation of mTORC1 in two steps: Rheb-GTP activation of catalytic function and increased binding of substrates to raptor 1 Joseph Avruch* 2 , Xiaomeng Long*, Yenshou Lin*, Sara Ortiz-Vega*, Joseph Rapley*, Angela Papageorgiou*, Noriko Oshiro*† and Ushio Kikkawa† *Diabetes Research Laboratory, Department of Molecular Biology, Diabetes Unit, Medical Services, Massachusetts General Hospital, Simches Research Building, 6408, 185 Cambridge Street, Boston, MA 02114, U.S.A., and †Biosignal Research Center, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan Abstract The signalling function of mTOR complex 1 is activated by Rheb-GTP, which controls the catalytic competence of the mTOR (mammalian target of rapamycin) kinase domain by an incompletely understood mechanism. Rheb can bind directly to the mTOR kinase domain, and association with inactive nucleotide-deficient Rheb mutants traps mTOR in a catalytically inactive state. Nevertheless, Rheb-GTP targets other than mTOR, such as FKBP38 (FK506-binding protein 38) and/or PLD1 (phospholipase D 1 ), may also contribute to mTOR act- ivation. Once activated, the mTOR catalytic domain phosphorylates substrates only when they are bound to raptor (regulatory associated protein of mTOR), a separate polypeptide within the complex. The mechanism of insulin/nutrient stimulation of mTOR complex 1 signalling, in addition to Rheb-GTP activation of the mTOR catalytic function, also involves a stable modification of the configuration of mTORC1 (mTOR complex 1) that increases access of substrates to their binding site on the raptor polypeptide. The mechanism underlying this second step in the activation of mTORC1 is unknown. Introduction The protein kinase TOR (target of rapamycin), is a funda- mental regulator of cellular metabolism, growth and proli- feration in all eukaryotes [1], functioning through two largely independent multiprotein complexes. TORC (TOR complex) 1 contains the polypeptides raptor (regulatory associated protein of TOR) and Lst8, and controls cell growth through substrates that regulate transcriptional, translational and post-translational processes. TORC2 contains the poly- peptides Lst8, rictor (rapamycin-insensitive companion of TOR) and sin1, and, in some organisms, other less well conserved polypeptides; TORC2 controls some aspects of cell proliferation and metabolism and is especially concerned with the co-ordination of the cytoskeleton with cell enlarg- ement and cell division. We have been particularly interested in the mechanisms that underlie the activation of mTORC1 (mammalian TORC1) [2], and, in the present paper, we summarize the status of information on this topic as well as ongoing work in this laboratory. Key words: insulin, leucine, mammalian target of rapamycin (mTOR), regulatory associated protein of mTOR (raptor), Rheb, tuberous sclerosis complex (TSC). Abbreviations used: 4E-BP, eukaryotic initiation factor 4E-binding protein; FKBP, FK506-binding protein; GAP, GTPase-activating protein; IGF-I, insulin-like growth factor 1; mTOR, mammalian target of rapamycin; mTORC1, mTOR complex 1; PI3K, phosphoinositide 3-kinase; PRAS40, proline- rich Akt substrate of 40 kDa; raptor, regulatory associated protein of target of rapamycin; rictor, rapamycin-insensitive companion of target of rapamycin; SpRheb, Schizosaccharomyces pombe Rheb; SpTOR, Schizosaccharomyces pombe target of rapamycin; S6K1, S6 kinase 1; TOR, target of rapamycin; TORC, TOR complex; TSC, tuberous sclerosis complex. 1 Dedicated to our deceased colleagues K. Yonezawa and J.C. Lawrence, Jr. 2 To whom correspondence should be addressed (email avruch@molbio.mgh.harvard.edu). Insulin and growth factors stimulate signalling by mTORC1, whereas various forms of stress, especially hy- poxia and energy depletion, are inhibitory. Genetic evidence from Drosophila established unequivocally that the Ras family GTPase Rheb is an indispensable activator of TORC1 in the insulin/IGF-I (insulin-like growth factor 1) receptor pathway. Moreover, Rheb acts epistatically to the inhibitory action of the TSC (tuberous sclerosis complex) 1–TSC2 heterodimer on TORC1 signalling, a relationship explained by the finding that TSC is an activator of the Rheb GTPase activity, i.e. a Rheb-GAP (GTPase-activating protein) [3]. Studies in mammalian cells rapidly demonstrated the conservation of these relationships, and identified the TSC as a major site of regulation of TORC1 activity [4]. Insulin/IGF- I receptor/PI3K (phosphoinositide 3-kinase) activation of Akt inhibits TSC Rheb-GAP function, whereas hypoxia and energy depletion, through the transcriptional up-regulation of REDD (regulated in development and DNA damage responses) and activation of AMPK (AMP-activated protein kinase)/GSK3 (glycogen synthase kinase 3), disinhibit the TSC–GAP activity to lower Rheb-GTP. Cells lacking a functional TSC–Rheb-GAP exhibit constitutive activation of TORC1 signalling which is not increased further by insulin, or effectively inhibited by hypoxia or metabolic stress, but is inhibited by depletion of Rheb. TORC1 signalling is also regulated by amino acids; removal of extracellular amino acids, most ubiquitously and potently leucine, inactivates TORC1 signalling [5]. The site of action of leucine appears to be distal to the TSC; TORC1 signalling in TSC-deficient cells, Biochem. Soc. Trans. (2009) 37, 223–226; doi:10.1042/BST0370223 C  The Authors Journal compilation C  2009 Biochemical Society Biochemical Society Transactions www.biochemsoctrans.org