Regulation Of S6 Kinase Research Articles

Mitochondrial Dysfunction Plus High-Sugar Diet Provokes a Metabolic Crisis That Inhibits Growth.

The Drosophila mutant tko25t exhibits a deficiency of mitochondrial protein synthesis, leading to a global insufficiency of respiration and oxidative phosphorylation. This entrains an organismal phenotype of developmental delay and sensitivity to seizures induced by mechanical stress. We found that the mutant phenotype is exacerbated in a dose-dependent fashion by high dietary sugar levels. tko25t larvae were found to exhibit severe metabolic abnormalities that were further accentuated by high-sugar diet. These include elevated pyruvate and lactate, decreased ATP and NADPH. Dietary pyruvate or lactate supplementation phenocopied the effects of high sugar. Based on tissue-specific rescue, the crucial tissue in which this metabolic crisis initiates is the gut. It is accompanied by down-regulation of the apparatus of cytosolic protein synthesis and secretion at both the RNA and post-translational levels, including a novel regulation of S6 kinase at the protein level.

Cross-talk between Sirtuin and Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling in the Regulation of S6 Kinase 1 (S6K1) Phosphorylation

p70 ribosomal S6 kinase (S6K1), a major substrate of the mammalian target of rapamycin (mTOR) kinase, regulates diverse cellular processes including protein synthesis, cell growth, and survival. Although it is well known that the activity of S6K1 is tightly coupled to its phosphorylation status, the regulation of S6K1 activity by other post-translational modifications such as acetylation has not been well understood. Here we show that the acetylation of the C-terminal region (CTR) of S6K1 blocks mTORC1-dependent Thr-389 phosphorylation, an essential phosphorylation site for S6K1 activity. The acetylation of the CTR of S6K1 is inhibited by the class III histone deacetylases, SIRT1 and SIRT2. An S6K1 mutant lacking acetylation sites in its CTR shows enhanced Thr-389 phosphorylation and kinase activity, whereas the acetylation-mimetic S6K1 mutant exhibits decreased Thr-389 phosphorylation and kinase activity. Interestingly, relative to the acetylation-mimetic S6K1 mutant, the acetylation-defective mutant displays higher affinity toward Raptor, an essential scaffolding component of mTORC1 that recruits mTORC1 substrates. These observations indicate that sirtuin-mediated regulation of S6K1 acetylation is an additional important regulatory modification that impinges on the mechanisms underlying mTORC1-dependent S6K1 activation.

Regulation and function of ribosomal protein S6 kinase (S6K) within mTOR signalling networks

The ribosomal protein S6K (S6 kinase) represents an extensively studied effector of the TORC1 [TOR (target of rapamycin) complex 1], which possesses important yet incompletely defined roles in cellular and organismal physiology. TORC1 functions as an environmental sensor by integrating signals derived from diverse environmental cues to promote anabolic and inhibit catabolic cellular functions. mTORC1 (mammalian TORC1) phosphorylates and activates S6K1 and S6K2, whose first identified substrate was rpS6 (ribosomal protein S6), a component of the 40S ribosome. Studies over the past decade have uncovered a number of additional S6K1 substrates, revealing multiple levels at which the mTORC1-S6K1 axis regulates cell physiology. The results thus far indicate that the mTORC1-S6K1 axis controls fundamental cellular processes, including transcription, translation, protein and lipid synthesis, cell growth/size and cell metabolism. In the present review we summarize the regulation of S6Ks, their cellular substrates and functions, and their integration within rapidly expanding mTOR (mammalian TOR) signalling networks. Although our understanding of the role of mTORC1-S6K1 signalling in physiology remains in its infancy, evidence indicates that this signalling axis controls, at least in part, glucose homoeostasis, insulin sensitivity, adipocyte metabolism, body mass and energy balance, tissue and organ size, learning, memory and aging. As dysregulation of this signalling axis contributes to diverse disease states, improved understanding of S6K regulation and function within mTOR signalling networks may enable the development of novel therapeutics.

Neutral Sphingomyelinase-2 Mediates Growth Arrest by Retinoic Acid through Modulation of Ribosomal S6 Kinase

All-trans-retinoic acid (ATRA) induces growth arrest of many cell types. Previous studies have reported that ATRA can modulate cellular sphingolipids, but the role of sphingolipids in the ATRA response is not clear. Using MCF-7 cells as a model system, we show that ATRA stimulates an increase in ceramide levels followed by G(0)/G(1) growth arrest. Notably, induction of nSMase2 was the primary effect of ATRA on the sphingolipid network and was both time- and dose-dependent. Importantly, pretreatment with nSMase2 siRNA significantly inhibited ATRA effects on ceramide levels and growth arrest. In contrast, nSMase2 overexpression was sufficient to increase ceramide levels and induce G(0)/G(1) growth arrest of asynchronous MCF-7 cells. Surprisingly, neither ATRA stimulation nor nSMase2 overexpression had significant effects on classical cell cycle regulators such as p21/WAF1 or retinoblastoma. In contrast, ATRA suppressed phosphorylation of ribosomal S6 kinase (S6K) and its downstream targets S6 and eIF4B. Importantly, these effects were significantly inhibited by nSMase2 siRNA. Reciprocally, nSMase2 overexpression was sufficient to suppress S6K phosphorylation and signaling. Notably, neither ATRA effects nor nSMase2 effects on S6K phosphorylation required the ceramide-activated protein phosphatase PP2A, previously identified as important for S6K regulation. Finally, nSMase2 overexpression was sufficient to decrease translation as measured by methionine incorporation and analysis of polyribosome profiles. Taken together, these results implicate nSMase2 as a major component of ATRA-induced growth arrest of MCF-7 cells and identify S6K as a novel downstream target of nSMase2.

Abstract 3167: Estrogenic regulation of S6 kinase 1 expression creates a positive feed-forward loop in control of breast cancer cell proliferation

Abstract The 40S ribosomal S6 kinase 1 (S6K1) acts downstream of the mammalian Target of Rapamycin (mTOR), and is sensitive to inhibition by rapamycin. Chromosomal region 17q23 containing the RPS6KB1 gene is frequently amplified in breast cancer cells, and S6K1 is overexpressed in about 30% of cases. Overexpression of S6K1 correlates with increased rapamycin sensitivity. The role of S6K1 in disease development and progression is supported by the observation that S6K1 overexpression is associated with poor prognosis in breast cancer patients. However, the reason for high-level amplification of the RPS6KB1 gene specifically in breast cancer, and not other cancer types is not well understood. We have previously reported that S6K1 regulates growth of breast cancer cells by phosphorylating Estrogen Receptor α (ERα), and co-expression of S6K1 and ERα in breast cancer cells renders them sensitive to combination therapy with rapamycin and tamoxifen. In this study, we demonstrate that in response to estrogen activation, ERα activates expression of S6K1 by upregulating transcription of the RPS6KB1 gene. Increased levels of S6K1 promote further phosphorylation and activation of ERα, creating a positive feed-forward loop. We hypothesize that this relationship promotes breast cancer cell growth, thus providing a selective advantage to S6K1- and ERα-overexpressing cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3167.

Regulation of S6 Kinase 1 Activation by Phosphorylation at Ser-411

Ribosomal S6 kinase 1 (S6K1), as a key regulator of mRNA translation, plays an important role in cell cycle progression through the G(1) phase of proliferating cells and in the synaptic plasticity of terminally differentiated neurons. Activation of S6K1 involves the phosphorylation of its multiple Ser/Thr residues, including the proline-directed sites (Ser-411, Ser-418, Thr-421, and Ser-424) in the autoinhibitory domain near the C terminus. Phosphorylation at Thr-389 is also a crucial event in S6K1 activation. Here, we report that S6K1 phosphorylation at Ser-411 is required for the rapamycin-sensitive phosphorylation of Thr-389 and the subsequent activation of S6K1. Mutation of Ser-411 to Ala ablated insulin-induced Thr-389 phosphorylation and S6K1 activation, whereas mutation mimicking Ser-411 phosphorylation did not show any effect. Furthermore, phosphomimetic mutation of Thr-389 overcame the inhibitory effect of the mutation S411A. Thus, Ser-411 phosphorylation regulates S6K1 activation via the control of Thr-389 phosphorylation. In nervous system neurons, Cdk5-p35 kinase associates with S6K1 via the direct interaction between p35 and S6K1 and catalyzes S6K1 phosphorylation specifically at Ser-411. Inhibition of the Cdk5 activity or suppression of Cdk5 expression blocked S6K1 phosphorylation at Ser-411 and Thr-389, resulting in S6K1 inactivation. Similar results were obtained by treating asynchronous populations of proliferating cells with the CDK inhibitor compound roscovitine. Altogether, our findings suggest a novel mechanism by which the CDK-mediated phosphorylation regulates the activation of S6K1.

Arabidopsis TARGET OF RAPAMYCIN Interacts with RAPTOR, Which Regulates the Activity of S6 Kinase in Response to Osmotic Stress Signals

TARGET OF RAPAMYCIN (TOR) kinase controls many cellular functions in eukaryotic cells in response to stress and nutrient availability and was shown to be essential for embryonic development in Arabidopsis thaliana. We demonstrated that Arabidopsis RAPTOR1 (a TOR regulatory protein) interacts with the HEAT repeats of TOR and that RAPTOR1 regulates the activity of S6 kinase (S6K) in response to osmotic stress. RAPTOR1 also interacts in vivo with Arabidopsis S6K1, a putative substrate for TOR. S6K1 fused to green fluorescent protein and immunoprecipitated from tobacco (Nicotiana tabacum) leaves after transient expression was active in phosphorylating the Arabidopsis ribosomal S6 protein. The catalytic domain of S6K1 could be phosphorylated by Arabidopsis 3-phosphoinositide-dependent protein kinase-1 (PDK1), indicating the involvement of PDK1 in the regulation of S6K. The S6K1 activity was sensitive to osmotic stress, while PDK1 activity was not affected. However, S6K1 sensitivity to osmotic stress was relieved by co-overexpression of RAPTOR1. Overall, these observations demonstrated the existence of a functional TOR kinase pathway in plants. However, Arabidopsis seedlings do not respond to normal physiological levels of rapamycin, which appears to be due its inability to bind to the Arabidopsis homolog of FKBP12, a protein that is essential for the binding of rapamycin with TOR. Replacement of the Arabidopsis FKBP12 with the human FKBP12 allowed rapamycin-dependent interaction with TOR. Since homozygous mutation in TOR is lethal, it suggests that this pathway is essential for integrating the stress signals into the growth regulation.

The Stress-inducted Proteins RTP801 and RTP801L Are Negative Regulators of the Mammalian Target of Rapamycin Pathway

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that plays an essential role in cell growth control. mTOR stimulates cell growth by phosphorylating p70 ribosomal S6 kinase (S6K) and eukaryote initiation factor 4E-binding protein 1 (4EBP1). The mTOR pathway is regulated by a wide variety of cellular signals, including mitogenic growth factors, nutrients, cellular energy levels, and stress conditions. Recent studies have proposed several mechanisms to explain how mTOR is regulated by growth factors and cellular energy levels. However, little is known as to how mTOR is regulated by stress conditions. We observed that two stress-induced proteins, RTP801/Redd1 and RTP801L/Redd2, potently inhibit signaling through mTOR. Our data support that RTP801 and RTP801L work downstream of AKT and upstream of TSC2 to inhibit mTOR functions. These results add a new dimension to mTOR pathway regulation and provide a possible molecular mechanism of how cellular stress conditions may regulate mTOR function.

Toxin-induced tail phosphorylation of hepatocellular S6 kinase: evidence for a dual involvement of the AMP-activated protein kinase in S6 kinase regulation.

Several protein phosphatase-inhibitory toxins (okadaic acid, microcystin, calyculin A, cantharidin, tautomycin) administered to isolated rat hepatocytes were found to induce phosphorylation in the tail region of S6 kinase (S6K; p70S6K1) as detected with a phosphospecific antibody against doubly phosphorylated Thr-421/Ser424. 5-Aminoimidazole-4-carboxamide riboside (AICAR), an adenosine analogue that elicits activation of the hepatocellular AMP-activated protein kinase (AMPK), similarly stimulated S6K tail phosphorylation. The flavonoid naringin prevented the effects of AICAR, okadaic acid, and microcystin on AMPK activation as well as on S6K tail phosphorylation, suggesting AMPK as a mediator of the latter. The effects of AICAR and the toxins were rapamycin resistant; in contrast, amino acids induced an S6K tail phosphorylation that was rapamycin sensitive, suggesting mediation by the protein kinase mammalian target of rapamycin (mTOR). Amino acids activated S6K by phosphorylation at Thr-389, but the toxins did not, and AICAR in fact suppressed the activating phosphorylation induced by the amino acids. The possibility thus must be considered that the phosphorylated S6K tail may transmit a toxin-induced signal independently of S6K enzymatic activity. Despite their inability to activate S6K, the toxins (but not AICAR) stimulated phosphorylation of the ribosomal protein S6, presumably by activating some other S6-phosphorylating protein kinase.

Critical Role of T-Loop and H-Motif Phosphorylation in the Regulation of S6 Kinase 1 by the Tuberous Sclerosis Complex

The tuberous sclerosis gene products Tsc1 and Tsc2 behave as tumor suppressors by restricting cell growth, a function conserved among metazoans. Recent evidence has indicated that hyperactivation of S6 kinase 1 (S6K1) may represent an important biochemical change in the development of tuberous sclerosis-associated lesions. We show here that deletion of either Tsc1 or Tsc2 or expression of the Rheb (Ras homolog enriched in brain) GTPase leads to hyperphosphorylation of S6K1 at a subset of regulatory sites, particularly those of two essential residues functionally conserved among AGC superfamily serine/threonine kinases, i.e. the activation loop (T-loop; Thr-229) and the hydrophobic motif (H-motif; Thr-389). These sites are reciprocally and dose-dependently regulated when S6K1 is coexpressed with the Tsc1-Tsc2 complex. Mutations that render S6K1 mTOR (mammalian target of rapamycin)-resistant also protect S6K1 activity and phosphorylation from down-regulation by Tsc1/2. We demonstrate that two disease-associated mutations in Tsc2 fail to negatively regulate S6K1 activity concomitant with a failure to modify T-loop and H-motif phosphorylation. Finally, we identify one pathological Tsc2 mutation that retains its ability to negatively regulate S6K1, suggesting that, in some cases, tuberous sclerosis may develop independently of S6K1 hyperactivation. These results also highlight the importance of dual control of T-loop and H-motif phosphorylation of S6K1 by the Tsc1-Tsc2 complex.

Osmotic Stress Inhibits p70/85 S6 Kinase through Activation of a Protein Phosphatase

While studying the stress regulation of p70/85 S6 kinase (S6K), we observed that anisomycin and UV light stimulated S6K activity, but that sorbitol inactivated S6K. Pretreatment with hyperosmotic stress also prevented the activation of S6K by both 12-O-tetradecanoylphorbol-13-acetate and anisomycin. Comparison of sorbitol and rapamycin revealed that both agents inactivated S6K and caused dephosphorylation of Ser/Thr-Pro sites in the COOH terminus of S6K, including Thr(412), a residue essential to S6K regulation, as determined by phospho-specific antibodies. Rapamycin-resistant S6K truncation mutants were similarly resistant to deactivation by sorbitol. Additionally, the PHAS-1 mobility shift, which is sensitive to rapamycin, was also found to be sensitive to osmotic stress. Experiments using the p38 inhibitor SB203580 and dominant negative mutants involving both stress-activated protein kinase/c-Jun NH(2)-terminal kinase and p38 stress pathways indicated that these pathways are probably not involved in osmotic stress inhibition of S6K. Examining the potential involvement of a phosphatase, we found that sodium pyrophosphate, sodium vanadate, cyclosporin A, tautomycin, and okadaic acid had no effect on osmotic stress inhibition of S6K. However, calyculin A prevented both rapamycin- and sorbitol-mediated deactivation of S6K. Our results suggest that osmotic stress and rapamycin act through a calyculin A-sensitive phosphatase to cause dephosphorylation and deactivation of S6K.