Tuberous Sclerosis Complex Proteins 1 and 2 Control Serum-Dependent Translation in a TOP-Dependent and -Independent Manner

Protein translation is controlled by multiple signaling pathways which can affect the rate of either global protein synthesis or a small subset of transcripts (16). Different mRNAs are translated at different rates depending on the activation of signal transduction pathways in response to changes in the extracellular environment. The phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway integrates signals from nutrients, energy status, and growth factors to regulate many processes, including cell growth and division, autophagy, protein translation, and metabolism. mTOR is a conserved Ser/Thr kinase first identified as a target of the immunosuppressant rapamycin (77). It is now known that mTOR forms two functionally distinct complexes: a rapamycin-sensitive mTORC1, composed of mTOR, raptor and GβL and a rapamycin-resistant mTORC2, composed of mTOR, mSIN1, Rictor, and GβL (13, 20, 28, 35, 61). mTORC1 is activated by growth factors in part through class Ia PI3K, and the importance of this pathway in protein translation has been shown through the use of inhibitors of PI3K (50). Deregulated protein translation through mTORC1 has been implicated in many human diseases, including tuberous sclerosis, the Peutz-Jeghers and Cowdens syndromes, and cancer (18). Important effectors of PI3K mediating its effects on protein translation include protein kinase B (PKB/Akt) and the tuberous sclerosis complex protein 1 (TSC1)/TSC2 complex (also referred to as hamartin and tuberin, respectively). TSC1 and TSC2 form a complex that inhibits mTORC1 activity via inhibition of the small GTPase Rheb, a positive regulator of mTORC1. The TSC complex inhibits Rheb by decreasing its GTP levels via the GTPase-activating protein (GAP) activity of TSC2. Upon growth factor stimulation, TSC2 is phosphorylated by activated PKB/Akt at several sites which inhibit the ability of TSC2 to act as a Rheb GAP (reviewed in reference 44). PKB/Akt may also regulate mTORC1 activity by regulating AMPK phosphorylation of TSC2 (19). Moreover, mTORC1 activity is regulated by extracellular nutrients, although the signaling pathways involved and how they are coordinated with growth factors are just beginning to be unraveled (11). Activated mTORC1 and mTORC2 have distinct downstream effectors (reviewed in reference 57). mTORC2 phosphorylates PKB/Akt on Ser473 to determine PKB/Akt substrate selectivity and seems to have a role in regulating the actin cytoskeleton and cell survival (28, 29, 67). In contrast, mTORC1 regulates growth through downstream effectors such as eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP1) and the ribosomal S6 kinases (S6K1 and S6K2). mTORC1-dependent phosphorylation of 4E-BP1 results in its dissociation from the initiation factor eIF4E that binds to the 5′-end cap of the mRNAs, thereby allowing the formation of translation initiation complexes crucial for protein synthesis. mTORC1-dependent phosphorylation of S6K1 at Thr389 is essential for S6K1 activation by creating a docking site for PDK1 (14). S6K1 phosphorylates the 40S ribosomal protein (RP) S6, the RNA processing protein SKAR, the initiation factor eIF4B, and elongation factor kinase eEF2K (71). Recently, Holz et al. identified direct interactions between mTORC1, S6K1, and its substrates and components of the translation preinitiation complex, thus providing new insights into how mTORC1 is connected to components of preinitiation apparatus (24). In mammalian cells, mRNAs encoding for components of translational apparatus (RPs and initiation and elongation factors) are regulated at the translational level by mitogenic or nutritional stimuli. A structural feature common to such mRNAs is the presence of a 5′-terminal oligopyrimidine tract (5′TOP) within their 5′ untranslated region (5′UTR). Interestingly, inhibition of mTORC1 by the macrolide drug rapamycin leads to inactivation of its downstream effectors and selectively suppresses mitogen-induced translation of 5′TOP containing mRNAs, such as eEF1A, eEF2, RpS6, and Rpl32. These mRNAs are redistributed from actively translated complexes (found in polysomes) into poorly translated complexes (found in small ribonucleoprotein particles) after rapamycin treatment (7, 32). The exact mechanism whereby mTORC1 regulates the translation of 5′TOP-containing mRNAs is still unclear as is the number and identity of regulated targets (51, 56, 69). However, in many cell types, rapamycin has only minor effects on the overall rate of protein synthesis (3, 23, 73, 74), suggesting additional mTORC1-independent pathways regulating translation. Several studies demonstrate that pathways from multiple growth factors inhibit TSC1/TSC2 to regulate mTORC1 (66). Moreover, mammalian cells lacking Tsc1 or Tsc2 fail to downregulate mTORC1 function in response to growth factor deprivation, suggesting that growth factors control mTORC1 activation in a TSC1- and TSC2-dependent manner (26, 38, 79). To address whether mitogenic signals regulate translation in a TSC1/TSC2-dependent manner, we analyzed the distribution of mRNAs on polysomes/subpolysomes in wild-type (WT) and Tsc-deficient mouse embryo fibroblasts (MEFs). Using microarray analysis, we identify novel serum- and rapamycin-sensitive mRNAs translationally regulated in WT MEFs, as well as in Tsc1−/− and Tsc2−/− MEFs. This global analysis revealed three groups of mRNAs: those regulated by serum but not by rapamycin (which are mainly TSC dependent), those regulated by rapamycin but not by serum, and those regulated by both. This latter group is enriched for 5′TOP-containing mRNAs, which also possessed short 5′- and 3′UTRs. Since TSC is a disease caused (in the most part) by deregulated protein translation, identifying which subsets of mRNAs are translationally controlled by TSC1 or TSC2 signaling pathways is crucial for the discovery of new therapeutic targets.