Tuberin Binds p27 and Negatively Regulates Its Interaction with the SCF Component Skp2

Abstract

TSC1 (tuberous sclerosis complex 1) encoding hamartin and TSC2 encoding tuberin are tumor suppressor genes responsible for the autosomal dominantly inherited disease tuberous sclerosis. These genes have been demonstrated to negatively regulate cell cycle progression, the activity of cdk2, and the degradation of the cyclin-dependent kinase inhibitor p27. To date, the underlying molecular mechanism remains elusive. Here, we show that tuberin binds to p27. Whereas tuberin also binds p27 in TSC1-negative cells, hamartin does not bind p27 without tuberin. p27 protein levels are regulated through ubiquitin-dependent degradation. Skp2 is the F-box protein, which, together with other proteins, forms an SCF (Skp1/cullin/F-box protein)-type E3 ubiquitin ligase complex whose task is to target p27 for degradation by the proteasome. We found that neither tuberin nor hamartin are in a complex with Skp2. Tuberin does not affect Skp2 protein levels, and the SCF Skp2 ubiquitin ligase does not regulate tuberin stability. But binding of tuberin to p27 sequesters p27 from Skp2 accompanied by an up-regulation of the p27 interaction with cdk2. Skp2-induced p27 degradation and cell cycle progression is abolished by tuberin s protective binding to p27. This work, the first description of the direct interaction of a tumor suppressor protein with p27, provides a molecular explanation for the effects of tuberous sclerosis complex genes on the cell cycle and demonstrates a new aspect of the SCF Skp2 -mediated regulation of p27 stability.

Tuberin activates the proapoptotic molecule BAD

“…For transfections, the following plasmids were used: the empty pcDNA3 vector; pcDNA3 harboring full-length human wildtype TSC1 or TSC2 (already used in Miloloza et al, 2000;Rosner and Hengstschla¨ger, 2004); pcDNA3 harboring fulllength BCL-2 (Borner, 1996); pcDNA3 harboring full-length BAD (Harada et al, 2001); the pSG5 vector, empty as a negative control or harboring either full-length human TSC2 cDNA or the TSC2 naturally occurring disease-causing mutant 6 2/25 (C to G at nucleotide 4947 ¼ Asn to Lys at amino acid 1643) (already used in Rosner et al, 2003); the pRK7 vector, empty or harboring full-length p70S6K ; pcDNA3 harboring the TSC2 SATA mutant (S939A/T1462A) ; the Myc-tagged activated AKT (Upstate, Lake Placid, NY, USA); the GFP-spectrin expression vector described in Kalejta et al (1997). All cell transfections were performed using the Lipofectamine reagents obtained from Invitrogen (Life Technologies, Lofer, Austria) following the transfection protocol provided by the manufacturer.…”

“…Tuberin is assumed to be the functional component of the complex (Kwiatkowski, 2003;Li et al, 2003;Pan et al, 2004;Astrinidis and Henske, 2005) and has been implicated in the regulation of different cellular functions, such as endocytosis (Xiao et al, 1997) or transcription (Henry et al, 1998). Tuberin also regulates cell cycle progression by binding to the cyclin-dependent kinase inhibitor p27 and, thereby, preventing p27 degradation via its SCFtype E3 ubiquitin ligase complex (Soucek et al, 1997(Soucek et al, , 1998Rosner and Hengstschla¨ger, 2004). Recent studies in Drosophila and mammalian cells demonstrated that the TSC1/TSC2 complex functions as GTPase-activating protein against Rheb (Ras homolog enriched in brain), which in turn regulates mTOR (mammalian target of rapamycin) signaling.…”

“…The TSC genes have been implicated in the regulation of cell cycle (Soucek et al, 1997(Soucek et al, , 1998Rosner and Hengstschla¨ger, 2004) and here, we investigated whether these genes are also involved in the regulation of cell death. The BCL-2 family of proteins constitutes a critical intracellular checkpoint in the intrinsic pathway of apoptosis.…”

Ras mediates cell survival by regulating tuberin

“…Tuberin also regulates cell cycle progression. Tuberin binds to the cyclin-dependent kinase inhibitor p27 and, thereby, prevents p27 degradation via its SCFtype E3 ubiquitin ligase complex (Soucek et al, 1997(Soucek et al, , 1998bMiloloza et al, 2000;Rosner and Hengstschla¨-ger, 2004).…”

Hamartin-Hsp70 Interaction Is Necessary for Akt-Dependent Tuberin Phosphorylation during Heat Shock

“…[3][4][5] Recent studies have shown that hamartin and tuberin interact strongly, forming a heterodimer that negatively regulates a small GTPase, Ras homolog enriched in the brain (Rheb), through the GTPase-activating (GAP) domain of tuberin. 6,7) Akt and other factors phosphorylate tuberin and inhibit its downstream signaling targets, including Rheb, the mammalian target of rapamycin (mTOR), and the ribosomal protein S6 kinase (S6K), via the hamartintuberin complex.…”