If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
* This work was supported by NCI, National Institutes of Health Grants CA77935 and CA89242 and Department of Defense Grants DAMD17-00-0559 and DAMD17-01-1-0394.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § These authors contributed equally to this work.
Normal cellular functions of hamartin and tuberin, encoded by the TSC1 and TSC2tumor suppressor genes, are closely related to their direct interactions. However, the regulation of the hamartin-tuberin complex in the context of the physiologic role as tumor suppressor genes has not been documented. Here we show that insulin or insulin growth factor (IGF) 1 stimulates phosphorylation of tuberin, which is inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 but not by the mitogen-activated protein kinase inhibitor PD98059. Expression of constitutively active PI3K or active Akt, including Akt1 and Akt2, induces tuberin phosphorylation. We further demonstrate that Akt/PKB associates with hamartin-tuberin complexes, promoting phosphorylation of tuberin and increased degradation of hamartin-tuberin complexes. The ability to form complexes, however, is not blocked. Akt also inhibits tuberin-mediated degradation of p27kip1, thereby promoting CDK2 activity and cellular proliferation. Our results indicate that tuberin is a direct physiological substrate of Akt and that phosphorylation of tuberin by PI3K/Akt is a major mechanism controlling hamartin-tuberin function.
). However, the regulation of hamartin and tuberin in the context of physiologic role as tumor suppressor genes has not been documented.
Among the various properties of these two proteins, the ability to interact and to form stable complex has been the most consistent finding. This led to the hypothesis that hamartin and tuberin function as a complex and that factors regulating their interaction are important in understanding physiologic roles. There is evidence to suggest that phosphorylation of tuberin may be a major mechanism of regulation of the hamartin-tuberin complex (
). However, the kinases that are responsible for phosphorylation of this complex are currently unknown. Recent Drosophila genetic studies showed thatdTsc1 and dTsc2 play an important role in the insulin/dPI3K/dakt signal transduction pathway by demonstrating that reduced cell size and cell proliferation caused by either mutations indINR and dakt or by overexpression ofdPTEN are overridden by homozygous mutants ofdTsc1 or dTsc2. This implies thatdTsc1 and dTsc2 are either direct downstream targets of dakt or on a parallel pathway of the insulin cascade downstream from dakt (
). Akt, also known as protein kinase B (PKB), represents a subfamily of the serine/threonine protein kinase. Three isoforms of Akt have been identified including Akt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ, all of which are activated by growth factors and insulin in a PI3K-dependent manner and are inhibited by PTEN tumor suppressor (
). Akt regulates a wide spectrum of cell functions, including cell survival, cell growth, differentiation, angiogenesis, and glucose metabolism, through phosphorylation of a number of proteins that contain the RXRXXS/T motif (
Here we show that Akt physically interacts with and phosphorylates tuberin, leading to degradation of the hamartin-tuberin complex and p27kip1 without interfering with hamartin-tuberin complex formation. Moreover, IGF1 and insulin induce tuberin phosphorylation, which is mediated by the PI3K/Akt pathway but not by the MAPK pathway. As a result, cyclin-dependent kinase (CDK) 2 activity, DNA synthesis, and S phase of the cell cycle are elevated. We thus have identified Akt as a major tuberin kinase to negatively regulate hamartin-tuberin tumor suppressor function by inducing degradation.
Recent studies have demonstrated that phosphorylation of hamartin and/or tuberin may play an important role in the formation of the tuberin-hamartin complex. Tuberin is phosphorylated at serine and tyrosine residues, and a disease-related TSC2 tyrosine 1571 mutation (Y1571H) nearly abolishes tuberin tyrosine phosphorylation and disrupts tuberin-hamartin binding, implying that the phosphorylation of tyrosine 1571 of TSC2 is required for tuberin-hamartin complex formation (
). Our study, however, shows that phosphorylation of tuberin by Akt and mitogenic factors (insulin and IGF1) abrogates hamartin-tuberin tumor suppressor activity without interfering with binding but by inducing degradation of both proteins through the proteosome pathway. Therefore, we provide a new paradigm for regulation of the TSC1/TSC2 tumor suppressor pathway.
In addition to the Forkhead transcription factor family (
), tuberin is the second Akt downstream target that has been uncovered by genetic studies so far. In this study, we present molecular evidence that tuberin is a direct physiological substrate of Akt by demonstrating that Akt binds to and phosphorylates tuberin. It has been documented that Akt induces cell cycle progression and cell proliferation through transcription repression and degradation of p27kip1 (
). We observed in this study that Akt attenuates the tuberin action but does not induce translocation of p27kip1 from nuclear to cytoplasm (data not shown). Previous studies have shown that three isoforms of Akt share almost the same upstream regulators and downstream targets. Similarly, we have observed that Akt1, Akt2, and Akt3 all phosphorylate and interact with tuberin, even though Akt2 displays a slightly higher binding affinity to tuberin. The model in Fig. 5illustrates the mechanism through which the PI3K/Akt pathway mediates insulin and IGF1 signals to down-regulate hamartin-tuberin function by phosphorylation of tuberin. Our results define a possible new mechanism through which Akt induces cell proliferation and transformation by inhibiting TSC1/TSC2 tumor suppressor functions.
We thank the DNA Sequence and Flow Cytometry Facilities at the H. Lee Moffitt Cancer Center.