Structure of S6 Kinase 1 Determines whether Raptor-mTOR or Rictor-mTOR Phosphorylates Its Hydrophobic Motif Site
Whitehead Institute for Biomedical Research and Massachusetts Institute of Technology, Department of Biology, Cambridge, 02142, USA. Journal of Biological Chemistry
(Impact Factor: 4.57).
06/2005; 280(20):19445-8. DOI: 10.1074/jbc.C500125200
The mTOR protein kinase is the target of the immunosuppressive and anti-cancer drug rapamycin and is increasingly recognized as a key regulator of cell growth in mammals. S6 kinase 1 (S6K1) is the best characterized effector of mTOR, and its regulation serves as a model for mTOR signaling. Nutrients and growth factors activate S6K1 by inducing the phosphorylation of threonine 389 in the hydrophobic motif of S6K1. As phosphorylation of Thr(389) is rapamycin sensitive and mTOR can phosphorylate the same site in vitro, it has been suggested that mTOR is the physiological Thr(389) kinase. This proposal is not supported, however, by the existence of mutants of S6K1 that are phosphorylated in vivo on Thr(389) in a rapamycin-resistant fashion. Here, we demonstrate that the raptor-mTOR complex phosphorylates the rapamycin-sensitive forms of S6K1, while the distinct rictor-mTOR complex phosphorylates the rapamycin-resistant mutants of S6K1. Phosphorylation of Thr(389) by rictor-mTOR is independent of the TOR signaling motif and depends on removal of the carboxyl terminal domain of S6K1. Because many members of the AGC family of kinases lack an analogous domain, rictor-mTOR may phosphorylate the hydrophobic motifs of other kinases.
Available from: Xiaolei Xu
- "In this murine HF model, the downstream mTOR signaling proteins S6K1 and S6 were both increased in TAC-HF, although the abundance of Thr389 phosphorylated S6K1 and Ser235/236 phosphorylated S6 were not increased in HF (Fig. 6). The reduction in the abundance of total and phosphorylated S6K1 in normal mice with rapamycin confirms mTOR inhibition with treatment (Fig. 2) and the significant decline in phosphorylated S6K1 in TAC-HF mice treated with rapamycin suggests that mTOR signaling through this pathway remains active in HF and is susceptible to regulation . In normal mice, rapamycin administration was associated with marked decreases in total S6 and phosphorylated S6 abundance, as expected due to reduced S6K1 activation. "
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ABSTRACT: While neurohumoral antagonists improve outcomes in heart failure (HF), cardiac remodeling and dysfunction progress and outcomes remain poor. Therapies superior or additive to standard HF therapy are needed. Pharmacologic mTOR inhibition by rapamycin attenuated adverse cardiac remodeling and dysfunction in experimental heart failure (HF). However, these studies used rapamycin doses that produced blood drug levels targeted for primary immunosuppression in human transplantation and therefore the immunosuppressive effects may limit clinical translation. Further, the relative or incremental effect of rapamycin combined with standard HF therapies targeting upstream regulators of cardiac remodeling (neurohumoral antagonists) has not been defined. Our objectives were to determine if anti-remodeling effects of rapamycin were preserved at lower doses and whether rapamycin effects were similar or additive to a standard HF therapy (angiotensin receptor blocker (losartan)). Experimental murine HF was produced by transverse aortic constriction (TAC). At three weeks post-TAC, male mice with established HF were treated with placebo, rapamycin at a dose producing immunosuppressive drug levels (target dose), low dose (50% target dose) rapamycin, losartan or rapamycin + losartan for six weeks. Cardiac structure and function (echocardiography, catheterization, pathology, hypertrophic and fibrotic gene expression profiles) were assessed. Downstream mTOR signaling pathways regulating protein synthesis (S6K1 and S6) and autophagy (LC3B-II) were characterized. TAC-HF mice displayed eccentric hypertrophy, systolic dysfunction and pulmonary congestion. These perturbations were attenuated to a similar degree by oral rapamycin doses achieving target (13.3±2.1 ng/dL) or low (6.7±2.5 ng/dL) blood levels. Rapamycin treatment decreased mTOR mediated regulators of protein synthesis and increased mTOR mediated regulators of autophagy. Losartan monotherapy did not attenuate remodeling, whereas Losartan added to rapamycin provided no incremental benefit over rapamycin alone. These data lend support to investigation of low dose rapamycin as a novel therapy in human HF.
PLoS ONE 12/2013; 8(12):e81325. DOI:10.1371/journal.pone.0081325 · 3.23 Impact Factor
Available from: Sang-Hyun Min
- "Mammalian TORC1 (mTORC1) has a primary function in autophagy regulation and contains the regulatory-associated protein of mTOR (raptor), GβL and PRAS40. Raptor is a 150 kDa mTOR-binding protein that also binds S6K1, serves as a scaffold protein of mTOR and facilitates mTOR phosphorylation of S6K1171819. It is well known that TOR has a central role in autophagy regulation, and p70 S6 kinase 1 (S6K1) is a direct substrate of TOR202122. "
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ABSTRACT: There is growing interest in identifying regulators of autophagy. The molecular mechanism underlying transforming growth factor-β activated kinase 1 (TAK1)-induced autophagy is poorly understood. We found that TAK1 inhibits p70 S6 kinase1 (S6K1) phosphorylation by interfering interaction of raptor with S6K1, thus inducing autophagy. The factors that determine whether autophagy is cytoprotective or cytotoxic have not been fully elucidated. In Drosophila, TAK1 overexpression leads to an impaired eye phenotype despite inhibition of apoptosis, indicating that the phenotype was mainly due to autophagy. Also, TAK1 overexpression increases lactate dehydrogenase (LDH) level in mammalian cells. When treated with autophagy inhibitors, the level of TAK1-induced cytotoxicity or cell death was significantly attenuated, indicating that TAK1 induces cytotoxic autophagic cell death. This study provides the first in vitro and in vivo evidence of TAK1-induced autophagy and we believe that our findings significantly contribute to the understanding of the mechanisms underlying the induction of autophagy.
Scientific Reports 03/2013; 3:1561. DOI:10.1038/srep01561 · 5.58 Impact Factor
Available from: Rotem Karni
- "Here, we report that in human cells S6K1 has two alternatively spliced short isoforms that are overproduced in breast cancer cell lines and tumors. Furthermore, all of S6K1 short splicing variants lack an autoinhibitory C terminus domain (Ali and Sabatini, 2005) and half of the kinase domain and do not exhibit kinase activity, at least on the known S6K1 substrate rpS6 (Figures 2A and S2E–S2G). Overexpression of mouse or human S6K1 short isoforms enhanced transformation, anchorage-independent growth, cell motility, and growth-factorindependent 3D acinus formation of human breast epithelial cells (Figures 2, 3A–3C, S2C, and S3A). "
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ABSTRACT: Ribosomal S6 kinase 1 (S6K1) is a major mTOR downstream signaling molecule that regulates cell size and translation efficiency. Here, we report that short isoforms of S6K1 are overproduced in breast cancer cell lines and tumors. Overexpression of S6K1 short isoforms induces transformation of human breast epithelial cells. The long S6K1 variant (Iso-1) induced opposite effects. It inhibits Ras-induced transformation and tumor formation, while its knockdown or knockout induces transformation, suggesting that Iso-1 has a tumor-suppressor activity. Furthermore, we found that S6K1 short isoforms bind and activate mTORC1, elevating 4E-BP1 phosphorylation, cap-dependent translation, and Mcl-1 protein levels. Both a phosphorylation-defective 4E-BP1 mutant and the mTORC1 inhibitor rapamycin partially blocked the oncogenic effects of S6K1 short isoforms, suggesting that these are mediated by mTORC1 and 4E-BP1. Thus, alternative splicing of S6K1 acts as a molecular switch in breast cancer cells, elevating oncogenic isoforms that activate mTORC1.
Cell Reports 12/2012; 3(1). DOI:10.1016/j.celrep.2012.11.020 · 8.36 Impact Factor
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