Abstract:The tuberous sclerosis proteins TSC1 and TSC2 are key integrators of growth factor signaling. They suppress cell growth and proliferation by acting in a heteromeric complex to inhibit the mammalian target of rapamycin complex 1 (mTORC1). In this study, we identify TSC1 as a component of the transforming growth factor β (TGF-β)-Smad2/3 pathway. Here, TSC1 functions independently of TSC2. TSC1 interacts with the TGF-β receptor complex and Smad2/3 and is required for their association with one another. TSC1 regul… Show more
“…In contrast to WT and Tsc2 − / − MEFs, SMAD2/3 phosphorylation was significantly reduced in Tsc1 − / − MEFs after 1 h of stimulation with TGF-β1 (Fig. 5a), showing that TSC1, but not TSC2, is required for activation of canonical TGF-β signaling, as previously observed in HeLa cells [11]. Fig.…”
Section: Resultssupporting
confidence: 79%
“…Activated SMAD2/3 then forms a heterotrimeric complex with SMAD4, which translocates to the nucleus for expression of target genes. Interestingly, several reports have indicated that GLI2 is a target gene for SMAD2/3 signaling in various human cell lines including fibroblasts [61, 62], and it has recently been shown that TSC1, independently of TSC2, is required for the association between TGF-β receptors and SMAD2/3, so that the knockdown of TSC1 causes an impaired phosphorylation and nuclear translocation of SMAD2/3 in HeLa cells [11]. We therefore speculated whether canonical TGF-β signaling is impaired in Tsc1 − / − MEFs, leading to a reduced expression of Gli2 .…”
Section: Resultsmentioning
confidence: 99%
“…For example, TSC inactivation has been shown to cooperate with non-canonical Smoothened (SMO)-independent Hedgehog (HH) signaling to drive tumor growth in murine cerebellar granule neuron precursors [10]. Furthermore, TSC1 was demonstrated to be required for the proper activation of the transforming growth factor-β (TGF-β)-SMAD2/3 pathway in HeLa cells [11]. Despite these advances, the roles of TSC1 and TSC2 in regulating signaling pathways independently of mTORC1/2 remain poorly understood.…”
Primary cilia are sensory organelles that coordinate multiple cellular signaling pathways, including Hedgehog (HH), Wingless/Int (WNT) and Transforming Growth Factor-β (TGF-β) signaling. Similarly, primary cilia have been implicated in regulation of mTOR signaling, in which Tuberous Sclerosis Complex proteins 1 and 2 (TSC1/2) negatively regulate protein synthesis by inactivating the mTOR complex 1 (mTORC1) at energy limiting states. Here we report that TSC1 and TSC2 regulate Smoothened (SMO)-dependent HH signaling in mouse embryonic fibroblasts (MEFs). Reduced SMO-dependent expression of Gli1 was demonstrated in both Tsc1−/− and Tsc2−/− cells, and we found that Tsc1 is required for TGF-β induced phosphorylation of SMAD2/3 and subsequent expression of the HH signaling effector and transcription factor GLI2. Hedgehog signaling was restored in Tsc1−/− cells after exogenous expression of Gli2, whereas rapamycin restored HH signaling in Tsc2−/− cells. Furthermore, we observed that Tsc1−/− MEFs display significantly elongated cilia, whereas cilia in Tsc2−/− MEFs were shorter than normal. The elongated cilium phenotype of Tsc1−/− MEFs is likely due to increased mTORC1-dependent autophagic flux observed in these cells, as both the autophagic flux and the cilia length phenotype was restored by rapamycin. In addition, ciliary length control in Tsc1−/− MEFs was also influenced by reduced expression of Gli2, which compromised expression of Wnt5a that normally promotes cilia disassembly. In summary, our results support distinct functions of Tsc1 and Tsc2 in cellular signaling as the two genes affect ciliary length control and HH signaling via different mechanisms.Electronic supplementary materialThe online version of this article (10.1007/s00018-018-2761-8) contains supplementary material, which is available to authorized users.
“…In contrast to WT and Tsc2 − / − MEFs, SMAD2/3 phosphorylation was significantly reduced in Tsc1 − / − MEFs after 1 h of stimulation with TGF-β1 (Fig. 5a), showing that TSC1, but not TSC2, is required for activation of canonical TGF-β signaling, as previously observed in HeLa cells [11]. Fig.…”
Section: Resultssupporting
confidence: 79%
“…Activated SMAD2/3 then forms a heterotrimeric complex with SMAD4, which translocates to the nucleus for expression of target genes. Interestingly, several reports have indicated that GLI2 is a target gene for SMAD2/3 signaling in various human cell lines including fibroblasts [61, 62], and it has recently been shown that TSC1, independently of TSC2, is required for the association between TGF-β receptors and SMAD2/3, so that the knockdown of TSC1 causes an impaired phosphorylation and nuclear translocation of SMAD2/3 in HeLa cells [11]. We therefore speculated whether canonical TGF-β signaling is impaired in Tsc1 − / − MEFs, leading to a reduced expression of Gli2 .…”
Section: Resultsmentioning
confidence: 99%
“…For example, TSC inactivation has been shown to cooperate with non-canonical Smoothened (SMO)-independent Hedgehog (HH) signaling to drive tumor growth in murine cerebellar granule neuron precursors [10]. Furthermore, TSC1 was demonstrated to be required for the proper activation of the transforming growth factor-β (TGF-β)-SMAD2/3 pathway in HeLa cells [11]. Despite these advances, the roles of TSC1 and TSC2 in regulating signaling pathways independently of mTORC1/2 remain poorly understood.…”
Primary cilia are sensory organelles that coordinate multiple cellular signaling pathways, including Hedgehog (HH), Wingless/Int (WNT) and Transforming Growth Factor-β (TGF-β) signaling. Similarly, primary cilia have been implicated in regulation of mTOR signaling, in which Tuberous Sclerosis Complex proteins 1 and 2 (TSC1/2) negatively regulate protein synthesis by inactivating the mTOR complex 1 (mTORC1) at energy limiting states. Here we report that TSC1 and TSC2 regulate Smoothened (SMO)-dependent HH signaling in mouse embryonic fibroblasts (MEFs). Reduced SMO-dependent expression of Gli1 was demonstrated in both Tsc1−/− and Tsc2−/− cells, and we found that Tsc1 is required for TGF-β induced phosphorylation of SMAD2/3 and subsequent expression of the HH signaling effector and transcription factor GLI2. Hedgehog signaling was restored in Tsc1−/− cells after exogenous expression of Gli2, whereas rapamycin restored HH signaling in Tsc2−/− cells. Furthermore, we observed that Tsc1−/− MEFs display significantly elongated cilia, whereas cilia in Tsc2−/− MEFs were shorter than normal. The elongated cilium phenotype of Tsc1−/− MEFs is likely due to increased mTORC1-dependent autophagic flux observed in these cells, as both the autophagic flux and the cilia length phenotype was restored by rapamycin. In addition, ciliary length control in Tsc1−/− MEFs was also influenced by reduced expression of Gli2, which compromised expression of Wnt5a that normally promotes cilia disassembly. In summary, our results support distinct functions of Tsc1 and Tsc2 in cellular signaling as the two genes affect ciliary length control and HH signaling via different mechanisms.Electronic supplementary materialThe online version of this article (10.1007/s00018-018-2761-8) contains supplementary material, which is available to authorized users.
“…Interestingly, the cancer-associated TSC mutations described above involve the TSC1 gene, whereas the majority of alterations that underpin tuberous sclerosis and LAM target the TSC2 subunit. It is tempting to speculate that in addition to activating mTORC1, these mutations disrupt an mTORC1-independent function(s) of TSC1, such as the regulation of transforming growth factor-β signaling (Thien et al, 2015). …”
Section: Tissue Overgrowth and Hamartoma Syndromesmentioning
Phosphoinositide 3-kinase (PI3K) activity is stimulated by diverse oncogenes and growth factor receptors, and elevated PI3K signaling is considered a hallmark of cancer. Many PI3K pathway-targeted therapies have been tested in oncology trials, resulting in regulatory approval of one isoform-selective inhibitor (idelalisib) for treatment of certain blood cancers, and a variety of other agents at different stages of development. In parallel to PI3K research by cancer biologists, investigations in other fields have uncovered exciting and often unpredicted roles for PI3K catalytic and regulatory subunits in normal cell function and in disease. Many of these functions impinge upon oncology by influencing the efficacy and toxicity of PI3K-targeted therapies. Here we provide a perspective on the roles of class I PI3Ks in the regulation of cellular metabolism and in immune system functions, two topics closely intertwined with cancer biology. We also discuss recent progress developing PI3K-targeted therapies for treatment of cancer and other diseases.
“…One possibility is that the TSC1-TBC1D7 interaction regulates pathways other than mTORC1. For example, it has recently been shown that TSC1 interacts with SMAD2/3 to activate TGF- signaling (22). Alternatively, elevated levels of FIGURE 7.…”
Mutations in TSC1 or TSC2 cause tuberous sclerosis complex (TSC), an autosomal dominant disorder characterized by the occurrence of benign tumors in various vital organs and tissues. TSC1 and TSC2, the TSC1 and TSC2 gene products, form the TSC protein complex that senses specific cellular growth conditions to control mTORC1 signaling. TBC1D7 is the third subunit of the TSC complex, and helps to stabilize the TSC1-TSC2 complex through its direct interaction with TSC1. Homozygous inactivation of TBC1D7 causes intellectual disability and megaencephaly. Here we report the crystal structure of a TSC1-TBC1D7 complex and biochemical characterization of the TSC1-TBC1D7 interaction. TBC1D7 interacts with the C-terminal region of the predicted coiled-coil domain of TSC1. The TSC1-TBC1D7 interface is largely hydrophobic, involving the ␣4 helix of TBC1D7. Each TBC1D7 molecule interacts simultaneously with two parallel TSC1 helices from two TSC1 molecules, suggesting that TBC1D7 may stabilize the TSC complex by tethering the C-terminal ends of two TSC1 coiled-coils.Tuberous sclerosis complex (TSC) 4 is a multisystem genetic disease that is characterized by the development of hamartomas or benign tumors in various organs, including skin, brain, and kidneys, and is frequently associated with epilepsy, intellectual disability, and autism (1, 2). TSC is caused by mutations to either of two genes, TSC1 and TSC2, which encode for proteins hamartin (TSC1) and tuberin (TSC2), respectively. TSC1 and TSC2 form a protein complex that acts as a GTPase-activating protein (GAP) for the small G-protein Rheb, which in turn serves as a crucial activator of the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1). The TSC complex is a tumor suppressor, inhibiting the Rheb-mTORC1-dependent stimulation of anabolic metabolism and cell growth (2-11). TSC2 contains a GAP domain and is the catalytic subunit of the complex. TSC1 is believed to enhance TSC2 function by activating TSC2 GAP activity, stabilizing TSC2, and/or maintaining the correct intracellular localization of the TSC complex (12-19). Recent work indicates that TSC1 might also have TSC2-independent activities in the cell (20 -22).Tre2-Bub2-Cdc16 domain family member 7 (TBC1D7) is a TSC1-binding protein (23-25), and has been recently identified as the third subunit of the TSC complex (25). TSC1, TSC2, and TBC1D7 form the core of the TSC protein complex in the cell, which migrates in size exclusion chromatography with a peak mass of ϳ2 MDa (26). Consistent with its important role in the TSC protein complex, knockdown of TBC1D7 results in decreased association of TSC1 and TSC2, leading to decreased Rheb-GAP activity, increased mTORC1 signaling, delayed induction of autophagy, and enhanced cell growth under poor growth conditions (25). Intriguingly, overexpression of TBC1D7 was also reported to increase mTORC1 activity (23). TBC1D7 mutations have not been found in TSC patients, but homozygous loss of TBC1D7 causes intellectual disability and megalencephaly, a developmental disorder a...
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