Tumor-promoting phorbol esters and activated Ras inactivate the tuberous sclerosis tumor suppressor complex via p90 ribosomal S6 kinase

Abstract

Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in either of the two tumor suppressor genes TSC1 or TSC2, which encode hamartin and tuberin, respectively. Tuberin and hamartin form a complex that inhibits signaling by the mammalian target of rapamycin (mTOR), a critical nutrient sensor and regulator of cell growth and proliferation. Phosphatidylinositol 3-kinase (PI3K) inactivates the tumor suppressor complex and enhances mTOR signaling by means of phosphorylation of tuberin by Akt. Importantly, cellular transformation mediated by phorbol esters and Ras isoforms that poorly activate PI3K promote tumorigenesis in the absence of Akt activation. In this study, we show that phorbol esters and activated Ras also induce the phosphorylation of tuberin and collaborates with the nutrient-sensing pathway to regulate mTOR effectors, such as p70 ribosomal S6 kinase 1 (S6K1). The mitogen-activated protein kinase (MAPK)-activated kinase, p90 ribosomal S6 kinase (RSK) 1, was found to interact with and phosphorylate tuberin at a regulatory site, Ser-1798, located at the evolutionarily conserved C terminus of tuberin. RSK1 phosphorylation of Ser-1798 inhibits the tumor suppressor function of the tuberin͞hamartin complex, resulting in increased mTOR signaling to S6K1. Together, our data unveil a regulatory mechanism by which the Ras͞MAPK and PI3K pathways converge on the tumor suppressor tuberin to inhibit its function.

Oncogenic tyrosine kinase NPM/ALK induces activation of the rapamycin-sensitive mTOR signaling pathway

“…Whereas the first is provided by the cell membrane receptors for growth factors, such as insulin, the second is generated by nutrients (Takano et al, 2001;Peng et al, 2002). The receptors activate cell-signaling pathways PI3K/Akt (Brunn et al, 1996;Sekulic et al, 2000) and ERK/ MEK (Tee et al, 2003;Roux et al, 2004;Ma et al, 2005) that indirectly activate mTOR by suppressing activity of tuberous sclerosis complex proteins TSC1 and TSC2 that, in turn, inhibit activity of mTOR through inactivating the G protein Rheb. mTOR can be functionally impaired by inhibitors from the rapamycin family.…”

Ras mediates cell survival by regulating tuberin

“…Ras regulates Raf upstream of MEK/ERK/RSK. Both, ERK-mediated phosphorylation of tuberin and RSK-dependent phosphorylation of tuberin, inhibit its potential to block mTOR/p70S6K (Dan et al, 2002;Inoki et al, 2002;Manning et al, 2002;Potter et al, 2002;Roux et al, 2004;Ballif et al, 2005;Ma et al, 2005). Since Ras regulates different pathways, it is likely that the selection of distinct effectors can lead to different outcomes.…”

“…This mutant was used to prove that RSK-dependent phosphorylation of tuberin on Ser1798 inhibits its potential to turn off Rheb and to downregulate the activities of mTOR and p70S6K. Mutation of Ser1798 inhibited most of tuberin phosphorylation and inactivation by RSK (Roux et al, 2004). Investigating PARP cleavage, caspase 3 cleavage, Figure 4 Ras-mediated cell survival depends on its potential to regulate the MEK/ERK/RSK pathway and mTOR.…”

“…For transfections the following plasmids were used: empty pCMV; pCMV-RasV12 (mutationally activated); pCMVRasN17 (dominant negative) (Clontech Laboratories Inc, Saint-Germain-en-Laye, France); the pRK7 vector, empty or harboring p70S6K ; pcDNA3 empty or harboring full-length human wild-type TSC2, or the TSC2 SATA mutant (mutations of the AKT phosphorylation sites Ser939A, Thr1462A) ; pRK7 harboring wild-type TSC2; pRK7 harboring TSC2 with the S1798A mutation of the preferred RSK1 phosphorylation site (Roux et al, 2004); the GFP-spectrin expression vector described in Kalejta et al, (1997). Since the pRK7 plasmid is not selectable, in case of selection experiments cotransfections with pCMV have been performed.…”

“…In addition, ERK-mediated phosphorylation of tuberin on Ser664 negatively regulates tuberin's function by blocking its interaction with hamartin. RSK-dependent phosphorylation of tuberin on Ser1798 inhibits its potential to turn off Rheb (Roux et al, 2004;Ballif et al, 2005;Ma et al, 2005). The role of phosphorylation by AKT, ERK or RSK for tuberin's other known functions has not been investigated in detail, so far.…”