Insulin activation of Rheb , a mediator of mTOR / S 6 K / 4 E - BP signaling

Tumor suppressor genes evolved as negative effectors of mitogen and nutrient signaling pathways, such that mutations in these genes can lead to pathological states of growth. Tuberous sclerosis (TSC) is a potentially devastating disease associated with mutations in two tumor suppressor genes, TSC1 and 2, that function as a complex to suppress signaling in the mTOR/S6K/4E-BP pathway. However, the inhibitory target of TSC1/2 and the mechanism by which it acts are unknown. Here we provide evidence that TSC1/2 is a GAP for the small GTPase Rheb and that insulin-mediated Rheb activation is PI3K dependent. Moreover, Rheb overexpression induces S6K1 phosphorylation and inhibits PKB phosphorylation, as do loss-of-function mutations in TSC1/2, but contrary to earlier reports Rheb has no effect on MAPK phosphorylation. Finally, coexpression of a human TSC2 cDNA harboring a disease-associated point mutation in the GAP domain, failed to stimulate Rheb GTPase activity or block Rheb activation of S6K1. Molecular Cell, Vol. 11, 1457–1466, June, 2003, Copyright 2003 by Cell Press Insulin Activation of Rheb, a Mediator of mTOR/S6K/4E-BP Signaling, Is Inhibited by TSC1 and 2 which in rare cases progress to renal cell carcinomas (Young and Povey, 1998). The contribution of TSC1/2 mutations to cancer progression may be much more prevalent than noted, as one in six thousand individuals is born with a mutation in either TSC1 or 2 (Montagne Attila Garami,1,4 Fried J.T. Zwartkruis,2,4 Takahiro Nobukuni,1 Manel Joaquin,1 Marta Roccio,2 Hugo Stocker,3 Sara C. Kozma,1 Ernst Hafen,3 Johannes L. Bos,2 and George Thomas1,* et al., 2001; Sparagana and Roach, 2000). Consistent Friedrich Miescher Institute with this, studies in Drosophila and in mammals demonfor Biomedical Research strate that TSC1/2 acts to constrain both mitogenand Maulbeerstrasse 66 nutrient-induced activation of the mTOR/S6K/4E-BP 4058 Basel signal transduction pathway (Marygold and Leevers, Switzerland 2002; McManus and Alessi, 2002), which has been impli2 Department of Physiological Chemistry and cated in the progression of a number of solid tumors Centre for Biomedical Genetics Universiteitsweg (Abraham, 2002; Hidalgo and Rowinsky, 2000; Sekulic 100 3584 CG Utrecht et al., 2000). At least in part, TSC1/2 repression of the The Netherlands mitogen-induced segment of the mammalian mTOR/ 3 Zoologisches Institut der Universität Zürich S6K/4E-BP signaling pathway appears to be relieved Winterthurerstrasse 190 by protein kinase B (PKB) phosphorylation of TSC2, a 8057 Zürich response mediated by insulin-induced activation of Switzerland phosphatidylinositide-3OH kinase (PI3K) (Goncharova et al., 2002; Inoki et al., 2002; Jaeschke et al., 2002; Manning et al., 2002). Moreover, hamartomatous lesions Summary are also observed in patients with one of three related autosomal-dominant disorders associated with germTumor suppressor genes evolved as negative effectors line mutations in PTEN, a negative effector of PI3K: Cowof mitogen and nutrient signaling pathways, such that den disease (CD), Lhermitte-Duclos disease (LDD), and mutations in these genes can lead to pathological states Bannayan-Zonana syndrome (BZS) (Liaw et al., 1997). of growth. Tuberous sclerosis (TSC) is a potentially devHowever, unlike the case in PTEN, the exact mechanism astating disease associated with mutations in two tuby which TSC1/2 represses mTOR/S6K/4E-BP signaling mor suppressor genes, TSC1 and 2, that function as is unknown, as is the identity of the inhibitory target. a complex to suppress signaling in the mTOR/S6K/4EThe severest forms of TSC syndrome are associated BP pathway. However, the inhibitory target of TSC1/2 with mutations that map to the GTPase activating doand the mechanism by which it acts are unknown. main of TSC2 (Maheshwar et al., 1997 and D. Franz, Here we provide evidence that TSC1/2 is a GAP for the personal communication). Consistent with TSC2 funcsmall GTPase Rheb and that insulin-mediated Rheb tioning as a GTPase activating protein (GAP), it had been activation is PI3K dependent. Moreover, Rheb overexreported that the GTPase activity of Rap1, a member of pression induces S6K1 phosphorylation and inhibits the Ras superfamily of small GTPases, is stimulated PKB phosphorylation, as do loss-of-function mutain vitro by TSC2 (Wienecke et al., 1995). However, we tions in TSC1/2, but contrary to earlier reports Rheb previously found that insulin had no effect on Rap1 activhas no effect on MAPK phosphorylation. Finally, coexity in cells overexpressing the insulin receptor (Zwartpression of a human TSC2 cDNA harboring a diseasekruis et al., 1998), while potently activating S6K1 in these associated point mutation in the GAP domain, failed same cells (Ming et al., 1994). To identify potential to stimulate Rheb GTPase activity or block Rheb actiGTPases as targets of TSC2 GAP, we set out to detervation of S6K1. mine whether the activity of any Ras superfamily member (Bos, 1997) was elevated in TSC2 / versus TSC2 / Introduction mouse embryo fibroblasts (MEFs). From such studies, Rheb (Ras homologue enriched in brain) emerged as a Tuberous sclerosis (TSC) syndrome is an autosomalpossible candidate. In parallel, a role for Rheb in the dominant genetic disorder, characterized by mutations mTOR/S6K/4E-BP signaling pathway became apparent in either the TSC1 or TSC2 gene, whose protein products from genetic studies in Drosophila (see Discussion). hamartin (TSC1) and tuberin (TSC2) form a putative tumor Rheb was first identified in a differential screen of suppressor complex (Montagne et al., 2001; Sparagana mRNAs induced in neurons by agents that provoke seiand Roach, 2000). TSC can cause severe pathological zures (Yamagata et al., 1994) and is ubiquitously exconsequences, including mental retardation, epilepsy, pressed, but particularly abundant, in muscle and brain and autism as well as leading to cardiac, pulmonary, (Yamagata et al., 1994). Moreover, Rheb was shown to and renal failure (Montagne et al., 2001; Sparagana and have intrinsic GTPase activity (Yamagata et al., 1994) Roach, 2000). These pathological states arise from haand to be as efficient as wild-type Raf-1 or H-Ras in martomas, largely manifested as benign tumors, but inducing transformation in NIH3T3 cells (Yee and Worley, 1997). Importantly, in S. pombe deletion of the Rheb ortholog, Rhb1, affects amino acid transport and nutri*Correspondence: [email protected] These authors contributed equally to this work. ent sensing (Mach et al., 2000), as has been recently