Ribosomal protein S6 kinase
The insect prothoracic glands are the source of steroidal molting hormone precursors and the glands are stimulated by a brain neuropeptide, prothoracicotropic hormone (PTTH). PTTH acts via a cascade including Ca2+/calmodulin activation of adenylate cyclase, protein kinase A, and the subsequent phosphorylation of a 34 kDa protein (p34) hypothesized, but not proven, to be the S6 protein of the 40S ribosomal subunit. The immunosuppressive macrolide, rapamycin, is a potent inhibitor of cell proliferation, a signal transduction blocker, and also prevents ribosomal S6 phosphorylation in mammalian systems. Rapamycin inhibits PTTH-stimulated ecdysteroidogenesis in vitro by the prothoracic glands of the tobacco hornworm, Manduca sexta, with half-maximal inhibition at a concentration of about 5 nM. At concentrations above 5 nM, there is a 75% inhibition of ecdysteroid biosynthesis. Similar results are observed with the calcium ionophore (A23187), a known stimulator of ecdysteroidogenesis. Most importantly, the inhibition of ecdysteroid biosynthesis is accompanied by the specific inhibition of the phosphorylation of p34, indicating that p34 indeed is ribosomal protein S6. In vivo assays reveal that injection of rapamycin into day 6 fifth instar larvae results in a decreased hemolymph ecdysteroid titer and a dose-dependent delay in molting and metamorphosis. When S6 kinase (S6K) activity is examined using rapamycin-treated prothoracic glands as the enzyme source and a synthetic peptide (S6-21) or a 40S ribosomal subunit fraction from Manduca tissues as substrate, the data reveal that rapamycin inhibits S6K activity. It is concluded that S6 kinase plays a role in prothoracicotropic hormone stimulation of insect prothoracic glands by targeting ribosomal protein S6 (Song, 1994).
Phosphorylation of ribosomal protein S6 is requisite for prothoracicotropic hormone (PTTH)-stimulated specific protein synthesis and subsequent ecdysteroidogenesis in the prothoracic glands of the tobacco hornworm, Manduca sexta. To better understand the role of S6 in regulating ecdysteroidogenesis, S6 cDNA was isolated from a Manduca prothoracic gland cDNA library and sequenced. The deduced protein is comprised of 253 amino acids, has a molecular weight of 29,038, and contains four copies of a 10-amino acid motif defining potential DNA-binding sites. This Manduca S6 possesses a consensus recognition sequence for the p70(s6k) binding domain as well as six seryl residues at the carboxyl-terminal sequence of 17 amino acids. Phosphoamino acid analysis reveals that the phosphorylation of Manduca prothoracic gland S6 is limited exclusively to serine residues. Although alterations in the quantity of S6 mRNA throughout the last larval instar and early pupal-adult development are not well correlated with the hemolymph ecdysteroid titer, developmental expression and phosphorylation of S6 are temporally correlated with PTTH release and the hemolymph ecdysteroid titer. These data provide additional evidence that S6 phosphorylation is a critical element in the transduction pathway leading to PTTH-stimulated ecdysteroidogenesis (Song, 1997).
A cDNA encoding the Drosophila melanogaster p90 ribosomal S6 kinase II (RSK) has been isolated from an eye-antennal imaginal disc library and sequenced. The conceptually translated protein is 60%-63% identical to vertebrate RSK homologs and contains a perfectly conserved mitogen-activated protein kinase phosphorylation site. The gene was mapped to the base of the X chromosome in division 20 by in situ hybridization to polytene chromosomes (Wassarman, 1994).
The Wnt signaling pathway was first discovered as a key event in embryonic development and cell polarity in Drosophila. Recently, several reports have shown that Wnt stimulates translation and cell growth by activating the mTOR pathway in mammals. Previous studies have demonstrated that the Target of Rapamycin (TOR) pathway plays an important role in mosquito vitellogenesis. However, the interactions between these two pathways are poorly understood in the mosquito. In this study, it was hypothesized that factors from the TOR and Wnt signaling pathways interacted synergistically in mosquito vitellogenesis. The results showed that silencing Aedes aegypti Frizzled 2 (AaFz2), a transmembrane receptor of the Wnt signaling pathway, decreased the fecundity of mosquitoes. AaFz2 was highly expressed at the transcriptional and translational levels in the female mosquito 6 h after a blood meal, indicating amino acid-stimulated expression of AaFz2. Notably, the phosphorylation of S6K, a downstream target of the TOR pathway, and the expression of vitellogenin were inhibited in the absence of AaFz2. A direct link was found in this study between Wnt and TOR signaling in the regulation of mosquito reproduction (Weng, 2015).
FoxA factors are critical regulators of embryonic development and postembryonic life, but little is know about the upstream pathways that modulate their activity. C. elegans pha-4 encodes a FoxA transcription factor that is required to establish the foregut in embryos and to control growth and longevity after birth. The AAA+ ATPase homolog ruvb-1 has been identified as a potent suppressor of pha-4 mutations. This study shows that ruvb-1 is a component of the Target of Rapamycin (TOR) pathway in C. elegans (CeTOR). Both ruvb-1 and let-363/TOR control nucleolar size and promote localization of box C/D snoRNPs to nucleoli, suggesting a role in rRNA maturation. Inactivation of let-363/TOR or ruvb-1 suppresses the lethality associated with reduced pha-4 activity. The CeTOR pathway controls protein homeostasis and also contributes to adult longevity. This study found that pha-4 is required to extend adult lifespan in response to reduced CeTOR signaling. Mutations in the predicted CeTOR target rsks-1/S6 kinase or in ife-2/eIF4E also reduce protein biosynthesis and extend lifespan, but only rsks-1 mutations require pha-4 for adult longevity. In addition, rsks-1, but not ife-2, can suppress the larval lethality associated with pha-4 loss-of-function mutations. In conclusion this data suggests that pha-4 and the CeTOR pathway antagonize one another to regulate postembryonic development and adult longevity. A model is suggested in which nutrients promote TOR and S6 kinase signaling, which represses pha-4/FoxA, leading to a shorter lifespan. A similar regulatory hierarchy may function in other animals to modulate metabolism, longevity, or disease (Sheaffer, 2008).
The mammalian target of rapamycin (mTOR) controls the translation machinery via activation of S6 kinases 1 and 2 (S6K1/2) and inhibition of the eukaryotic initiation factor 4E (eIF4E) binding proteins 1, 2, and 3 (4E-BP1/2/3). S6K1 and 4E-BP1 are regulated by nutrient-sensing and mitogen-activated pathways. The molecular basis of mTOR regulation of S6K1 and 4E-BP1 remains controversial. A conserved TOR signaling (TOS) motif has been identified in the N terminus of all known S6 kinases and in the C terminus of the 4E-BPs; the TOS motif is crucial for phosphorylation and regulation S6K1 and 4E-BP1 activities. Deletion or mutations within the TOS motif significantly inhibit S6K1 activation and the phosphorylation of its hydrophobic motif, Thr389. In addition, this sequence is required to suppress an inhibitory activity mediated by the S6K1 C terminus. The TOS motif is essential for S6K1 activation by mTOR, since mutations in this motif mimic the effect of rapamycin on S6K1 phosphorylation, and render S6K1 insensitive to changes in amino acids. Furthermore, overexpression of S6K1 with an intact TOS motif prevents 4E-BP1 phosphorylation by a common mTOR-regulated modulator of S6K1 and 4E-BP1. It is concluded that S6K1 and 4E-BP1 contain a conserved five amino acid sequence (TOS motif) that is crucial for their regulation by the mTOR pathway. mTOR seems to regulate S6K1 by two distinct mechanisms. The TOS motif appears to function as a docking site for either mTOR itself or a common upstream activator of S6K1 and 4E-BP1 (Schalm, 2002).
Inhibition of DAF-2 (insulin-like growth factor 1 [IGF-1] receptor) or RSKS-1 (S6K), key molecules in the insulin/IGF-1 signaling (IIS) and target of rapamycin (TOR) pathways, respectively, extend lifespan in Caenorhabditis elegans. However, it has not been clear how and in which tissues they interact with each other to modulate longevity. This study demonstrates that a combination of mutations in daf-2 and rsks-1 produces a nearly 5-fold increase in longevity that is much greater than the sum of single mutations. This synergistic lifespan extension requires positive feedback regulation of DAF-16 (FOXO) via the AMP-activated protein kinase (AMPK) complex. Furthermore, germline was identified as the key tissue for this synergistic longevity. Moreover, germline-specific inhibition of rsks-1 activates DAF-16 in the intestine. Together, these findings highlight the importance of the germline in the significantly increased longevity produced by daf-2 rsks-1, which has important implications for interactions between the two major conserved longevity pathways in more complex organisms (Chen, 2013).
IIS and TOR pathways play conserved roles in modulating lifespan in multiple species. However, it is unclear how they might interactively modulate aging. This study set out to address this question by constructing a daf-2 rsks-1 double mutant, which has reduced function of IIS and an important branch of the TOR pathway. Surprisingly, the daf-2 rsks-1 double mutant showed a nearly 5-fold lifespan extension. This phenotype is defined as a synergistic lifespan extension, based on the observation that the longevity of the daf-2 rsks-1 double mutant is beyond the combined effects of rsks-1 and daf-2 single mutants. This synergistic longevity phenotype cannot be explained by the hypothesis that daf-2 and rsks-1 function in parallel to modulate lifespan independently, since an additive effect would be expected under such an assumption (Chen, 2013).
The synergistic longevity phenotype is different from what we previously reported; i.e., that rsks-1 RNAi further extended the daf-2 lifespan by 24%. One major difference in the experimental procedures used is that in the previous study, daf-2 animals were treated with rsks-1 RNAi only during adulthood, whereas in the current work, a double mutant was made that carries the putative null allele of rsks-1 throughout life (Chen, 2013).
When daf-2 animals were treated with rsks-1 RNAi for two generations, resulting in a more complete reduction in rsks-1 mRNA levels, a 54% further lifespan extension was observed. These results suggest that inhibition of rsks-1 during development is critical for the synergistic longevity phenotype. Consistently, inhibition of the RSKS-1 upstream activator LET-363/CeTOR in daf-2 during adulthood led to a 17% additive lifespan extension. Since let-363 is an essential gene, inhibition of which during development leads to larval arrest, a pharmaceutical approach was used to inhibit let-363 by treating animals with rapamycin. Rapamycin treatment throughout life extended the lifespan of N2 and daf-2 animals by 26% and 45%, respectively. There are multiple possible reasons why rapamycin treatment could not extend the lifespan of daf-2 animals as much as the rsks-1 deletion mutant did. One possibility is that rapamycin treatment did not fully block RSKS-1, which is required for the synergistic longevity. Another possibility is that since rapamycin treatment at this dosage has been shown to inhibit both TOR complex 1 and complex 2 activities (Robida-Stubbs et al., 2012), the drug might also affect other lifespan-determinant genes. Nevertheless, these results are consistent with the idea that inhibiting rsks-1 in daf-2 during development leads to a synergistic lifespan extension (Chen, 2013).
Previous studies showed that null mutants of age-1, which encodes a catalytic subunit of the phosphatidylinositol-3-kinase (PI3K) in the IIS pathway, exhibit an exceptional lifespan extension in a DAF-16-dependent manner (Chen, 2013).
Since the daf-2 mutations that were used in this study are not null alleles, one possible explanation for the synergistic longevity produced by daf-2 rsks-1 is that the rsks-1 deletion makes daf-2 mutant phenotypes more severe. This is unlikely to be true, because many aging-related phenotypes of daf-2 are not enhanced by the rsks-1 deletion.rsks-1 does not affect daf-2-mediated dauer arrest, and rsks-1 has a minor or even opposite effect on most stress resistance. Understanding why these phenotypes are uncoupled from the synergistically prolonged longevity produced by daf-2 rsks-1 will help to elucidate the basic mechanisms of aging (Chen, 2013).
TOR plays a conserved role in dietary restriction (DR)-mediated lifespan extension. The effect of nutrients on the synergistic longevity was tested using the DR-FD regimen (FD stands for food deprivation). The rsks-1 single mutant did not show a lifespan extension under DR, which is consistent with the idea that DR and reduced TOR signaling function through overlapping mechanisms to extend lifespan. Interestingly, the synergistic longevity produced by daf-2 rsks-1 is nutrient independent, suggesting that rsks-1 functions through unidentified mechanisms to further extend the lifespan of daf-2 animals (Chen, 2013).
To better understand the molecular mechanisms of the synergistic longevity produced by daf-2 rsks-1, this study set out to identify critical mediators by testing known regulators of IIS or rsks-1. Heat-shock factor 1 (HSF-1) is critical for daf-2-mediated lifespan extension. Inhibition of hsf-1 almost completely abolished the lifespan extension produced by daf-2 rsks-1. Lifespan extension via genetic or pharmaceutical inhibition of TOR requires the IIS downstream transcription factor SKN-1. Surprisingly, inhibition of skn-1 by RNAi had little effect on the synergistic longevity produced by daf-2 rsks-1. Similarly, inhibition of PHA-4, a FOXA transcription factor that is required for the rsks-1 single mutant-mediated lifespan extension, did not affect the lifespan of daf-2 rsks-1. This is further evidence that the mechanism of the synergistic longevity in the daf-2 rsks-1 double mutant is distinct from the lifespan extension caused by the single mutants (Chen, 2013).
Microarray studies were performed and genes were identified that are differentially expressed in daf-2 rsks-1. A genetic screen using RNAi helped identify the AMPK complex (see Drosophila AMP-activated protein kinase alpha subunit) as the key mediator of the synergistic longevity produced by daf-2 rsks-1. Quantitative analysis of the lifespan data indicated that suppression of the daf-2 rsks-1 lifespan by inhibition of AMPK was not due to general sickness. Instead, inhibition of AMPK suppressed the synergy part of the lifespan extension. Further analysis identified a positive feedback regulation of DAF-16 via AMPK in the daf-2 rsks-1 mutant. AMPK plays important roles in various cellular functions. Under energy-starved conditions, AMPK is activated to promote catabolism and thus ATP production. Further characterization of the role of AMPK in metabolism will enhance understanding of the synergistic longevity produced by daf-2 rsks-1 (Chen, 2013).
Both IIS and signals from the reproductive system have endocrine functions. Modulation of these pathways in one tissue leads to nonautonomous activation of DAF-16 in the intestine. To better understand how aging is coordinately modulated across multiple tissues, the involvement of key regulators of the daf-2 rsks-1-mediated synergistic longevity were tested by tissue-specific RNAi. It was found that rsks-1, daf-16, and aak-2 function in the germline to regulate the synergistic lifespan extension, which can also be suppressed by a genetic mutation that causes germline overproliferation or by inhibition of key mediators of germline signaling. In addition, inhibiting rsks-1 in the germline leads to nonautonomous activation of DAF-16 in the intestine. Previous studies on the tissue-specific requirements of key longevity determinants, including DAF-16, mainly employed transgenic rescue approaches. However, the traditional microinjection method creates transgenic lines with a high copy number of transgenes, which will be silenced in the germline. The results indicate that the germline is an important tissue for integrating signals from the IIS pathway and S6K for lifespan determination (Chen, 2013).
Similar to the rsks-1 single mutant, daf-2 rsks-1 animals showed significantly delayed, prolonged, and overall reduced reproduction. This is consistent with a recent study showing that RSKS-1 acts in parallel with the IIS pathway to play an essential role in establishing the germline stem cell/progenitor pool. Interestingly, RSKS-1 functions cell autonomously to regulate establishment of the germline progenitor. This effect is independent of its known suppressors in the regulation of lifespan. These findings suggest that the synergistic longevity of daf-2 rsks-1 cannot simply be linked with its functions in germline development and reproduction (Chen, 2013).
In C. elegans, the intestine carries out multiple nutrient-related functions and is the site for food digestion and absorption, fat storage, and immune response. DAF-16 is one of the essential transcription factors that function in the intestine to modulate lifespan. It was found that intestinal-specific inhibition of daf-16, aak-2, or hsf-1 largely abolishes the synergistic lifespan extension of daf-2 rsks-1. However, knockdown of rsks-1 in the intestine only has an additive effect on daf-2 lifespan, suggesting that rsks-1 may function through nonautonomous mechanisms to activate DAF-16 (Chen, 2013).
The hypodermis is considered as part of the epithelial system in C. elegans. It is involved in basic body plan establishment, cell fate specification, axon migration, apoptotic cells removal, and fat storage. Hypodermis-specific knockdown of rsks-1 in daf-2 also leads to synergistic lifespan extension, and that hypodermis-specific knockdown of daf-16 significantly reduces the synergistic lifespan extension. These results provide evidence for the important role of the hypodermis in lifespan determination. In future studies, it will be interesting to examine which biological functions of the hypodermis are involved in regulating the synergistic longevity by daf-2 rsks-1 (Chen, 2013).
Previous studies showed that muscle decline is one of the major physiological causes of aging in C. elegans. Neither rsks-1 nor the downstream regulators daf-16, hsf-1, and aak-2 seem to function in the muscle to modulate the synergistic lifespan extension. However, the possibility that these regulators may function in other tissues to nonautonomously regulate muscle functions in daf-2 rsks-1 cannot be ruled out. Characterization of age-dependent muscle decline in daf-2 rsks-1 will help to elucidate whether muscle functions are important for the synergistic lifespan extension (Chen, 2013).
There are limitations to assessing tissue-specific involvement of key regulators in lifespan determination by RNAi, such as uncertainty of knockdown efficiency and potential leakiness. It has been reported that in rrf-1 mutants, RNAi can be processed in certain somatic tissues, including the intestine, at least for the genes tested. However, the critical function of rsks-1 in the germline is unlikely to be an artifact, as rsks-1 knockdown in the intestine of daf-2 animals did not lead to synergistic lifespan extension. Moreover, inhibition of certain strong suppressors of daf-2 rsks-1 (e.g., hsf-1) in the intestine, but not in the germline, significantly decreased the synergistic lifespan extension produced by daf-2 rsks-1. Further analyses with single-copied, isoform-specific transgenic rescue will help to quantitatively determine the tissue-specific involvement of key regulators in the synergistic lifespan extension produced by daf-2 rsks-1 (Chen, 2013).
It has not been clear whether DAF-16 is quantitatively more active or is uniquely activated in certain tissues, such as the germline of daf-2 rsks-1. Although the AMPK-mediated positive feedback regulation of DAF-16 was identified based on genes that are expressed to a greater extent in daf-2 rsks-1 animals, it is speculated that the double mutant has some unique properties, as shown in dauer formation and various stress-tolerance assays. The data from the phenotypic analysis of the double mutant and epistasis analysis of tissue requirement of DAF-16 suggest that with the rsks-1 deletion, DAF-16 plays a more important role in certain tissues, such as the germline, to further extend the lifespan of daf-2. Characterization of the genes that are uniquely upregulated in daf-2 rsks-1 or those that are regulated independently of DAF-16 will help distinguish these models (Chen, 2013).
In conclusion, this study found that the daf-2 rsks-1 double mutant shows a synergistic lifespan extension, which is achieved through positive feedback regulation of DAF-16 by AMPK. Tissue- specific epistasis analysis suggests that this enhanced activation of DAF-16 is initiated by signals from the germline, and that the germline tissue may play a key role in integrating the interactions between daf-2 and rsks-1 to cause a synergistic lifespan extension. Since DAF-2, RSKS-1, AMPK, and DAF-16 are highly conserved molecules, similar regulation may also exist in mammals. Further characterization of the daf-2 rsks-1-mediated synergistic longevity will contribute to a better understanding of the molecular mechanisms of aging and age-related diseases (Chen, 2013).
The p70 S6 kinase (p70s6k) gene was disrupted in murine embryonic stem cells to determine the role of this kinase in cell growth, protein synthesis, and rapamycin sensitivity. p70s6k-/- cells proliferate at a slower rate than parental cells, suggesting that p70s6k has a positive influence on cell proliferation but is not essential. In addition, rapamycin inhibits proliferation of p70s6k-/- cells, indicating that other events inhibited by the drug, independent of p70s6k, also are important for both cell proliferation and the action of rapamycin. In p70s6k-/- cells, which exhibit no ribosomal S6 phosphorylation, translation of mRNA encoding ribosomal proteins is not increased by serum nor specifically inhibited by rapamycin. In contrast, rapamycin inhibits (1) phosphorylation of initiation factor 4E-binding protein 1 (4E-BP1); (2) general mRNA translation, and (3) overall protein synthesis in p70s6k-/- cells, indicating that these events proceed independently of p70s6k activity. This study localizes the function of p70s6k to ribosomal biogenesis by regulating ribosomal protein synthesis at the level of mRNA translation (Kawasome, 1998).
The p70s6k/p85s6k signaling pathway plays a critical role in cell growth by modulating the translation of a family of mRNAs termed 5'TOPs, which encode components of the protein synthetic apparatus. Homozygous disruption of the p70s6k/p85s6k gene does not affect viability or fertility of mice, but it has a significant effect on animal growth, especially during embryogenesis. Surprisingly, S6 phosphorylation in liver or in fibroblasts from p70s6k/p85s6k-deficient mice proceeds normally in response to mitogen stimulation. Furthermore, serum-induced S6 phosphorylation and translational up-regulation of 5'TOP mRNAs are equally sensitive to the inhibitory effects of rapamycin in mouse embryo fibroblasts derived from p70s6k/p85s6k-deficient and wild-type mice. A search of public databases has identified a novel p70s6k/p85s6k homolog that contains the same regulatory motifs and phosphorylation sites known to control kinase activity. This newly identified gene product, termed S6K2, is ubiquitously expressed and displays both mitogen-dependent and rapamycin-sensitive S6 kinase activity. More striking, in p70s6k/p85s6k-deficient mice, the S6K2 gene is up-regulated in all tissues examined, especially in thymus, a main target of rapamycin action. The finding of a new S6 kinase gene, which can partly compensate for p70s6k/p85s6k function, underscores the importance of S6K function in cell growth (Shima, 1998).
Activation of 40S ribosomal protein S6 kinases (S6Ks) is mediated by anabolic signals triggered by hormones, growth factors, and nutrients. Stimulation by any of these agents is inhibited by the bacterial macrolide rapamycin, which binds to and inactivates the mammalian target of rapamycin, an S6K kinase. In mammals, two genes encoding homologous S6Ks, S6K1 and S6K2, have been identified. Mice deficient for S6K1 or S6K2 are born at the expected Mendelian ratio. Compared to wild-type mice, S6K1(-/-) mice are significantly smaller, whereas S6K2(-/-) mice tend to be slightly larger. However, mice lacking both genes showed a sharp reduction in viability due to perinatal lethality. Analysis of S6 phosphorylation in the cytoplasm and nucleoli of cells derived from the distinct S6K genotypes suggests that both kinases are required for full S6 phosphorylation but that S6K2 may be more prevalent in contributing to this response. Despite the impairment of S6 phosphorylation in cells from S6K1(-/-)/S6K2(-/-) mice, cell cycle progression and the translation of 5'-terminal oligopyrimidine mRNAs were still modulated by mitogens in a rapamycin-dependent manner. Thus, the absence of S6K1 and S6K2 profoundly impairs animal viability but does not seem to affect the proliferative responses of these cell types. Unexpectedly, in S6K1(-/-)/S6K2(-/-) cells, S6 phosphorylation persists at serines 235 and 236, the first two sites phosphorylated in response to mitogens. In these cells, as well as in rapamycin-treated wild-type, S6K1(-/-), and S6K2(-/-) cells, this step is catalyzed by a mitogen-activated protein kinase (MAPK)-dependent kinase, most likely p90rsk. These data reveal a redundancy between the S6K and the MAPK pathways in mediating early S6 phosphorylation in response to mitogens (Pende, 2004).
Protein kinase B and p70 S6 kinase are members of the cyclic AMP-dependent/cyclic GMP-dependent/protein kinase C subfamily of protein kinases and are activated by a phosphatidylinositol 3-kinase-dependent pathway when cells are stimulated with insulin or growth factors. Both of these kinases are activated in cells by phosphorylation of a conserved residue in the kinase domain [Thr-308 of protein kinase B (PKB) and Thr-252 of p70 S6 kinase] and another conserved residue located C-terminal to the kinase domain (Ser-473 of PKB and Thr-412 of p70 S6 kinase). Thr-308 of PKBalpha and Thr-252 of p70 S6 kinase are phosphorylated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) in vitro. Recent work has shown that PDK1 interacts with a region of protein kinase C-related kinase-2, termed the PDK1 interacting fragment (PIF). Interaction with PIF converts PDK1 from a form that phosphorylates PKB at Thr-308 alone to a species capable of phosphorylating Ser-473 as well as Thr-308. This suggests that PDK1 may be the enzyme that phosphorylates both residues in vivo. PDK1 is capable of phosphorylating p70 S6 kinase at Thr-412 in vitro. The effect of PIF on the ability of PDK1 to phosphorylate p70 S6 kinase has been examined. Surprisingly, PDK1 bound to PIF is no longer able to interact with or phosphorylate p70 S6 kinase in vitro at either Thr-252 or Thr-412. The expression of PIF in cells prevents insulin-like growth factor 1 from inducing the activation of the p70 S6 kinase and its phosphorylation at Thr-412. Overexpression of PDK1 in cells induces the phosphorylation of p70 S6 kinase at Thr-412 in unstimulated cells, and a catalytically inactive mutant of PDK1 prevents the phosphorylation of p70 S6K at Thr-412 in insulin-like growth factor 1-stimulated cells. These observations indicate that PDK1 regulates the activation of p70 S6 kinase and provides evidence that PDK1 mediates the phosphorylation of p70 S6 kinase at Thr-412 (Balendran, 1999).
Activation of the protein p70s6k by mitogens leads to increased translation of a family of messenger RNAs that encode essential components of the protein synthetic apparatus. Activation of the kinase requires hierarchical phosphorylation at multiple sites, culminating in the phosphorylation of the threonine in position 229 (Thr229), in the catalytic domain. The homologous site in protein kinase B (PKB), Thr308, has been shown to be phosphorylated by the phosphoinositide-dependent protein kinase PDK1. A regulatory link between p70s6k and PKB has been demonstrated, since PDK1 was found to selectively phosphorylate p70s6k at Thr229. More importantly, PDK1 activates p70s6k in vitro and in vivo, whereas the catalytically inactive PDK1 blocks insulin-induced activation of p70s6k (Pullen, 1998).
The complex of rapamycin with its intracellular receptor, FKBP12, interacts with RAFT1/FRAP/mTOR (the in vivo rapamycin-sensitive target) and a member of the ataxia telangiectasia mutated (ATM)-related family of kinases that share homology with the catalytic domain of phosphatidylinositol 3-kinase. The function of RAFT1 in the rapamycin-sensitive pathway and its connection to downstream components of the pathway, such as p70 S6 kinase and 4E-BP1, are poorly understood. RAFT1 directly phosphorylates p70S6k, 4E-BP1, and 4E-BP2. Serum stimulates RAFT1 kinase activity with kinetics similar to those of p70S6k and 4E-BP1 phosphorylation. RAFT1 phosphorylates p70S6k on Thr-389, a residue whose phosphorylation is rapamycin-sensitive in vivo and necessary for S6 kinase activity. RAFT1 phosphorylation of 4E-BP1 on Thr-36 and Thr-45 blocks its association with the cap-binding protein, eIF-4E, in vitro, and phosphorylation of Thr-45 seems to be the major regulator of the 4E-BP1-eIF-4E interaction in vivo. RAFT1 phosphorylates p70(S6k) much more effectively than 4E-BP1, and the phosphorylation sites on the two proteins show little homology. This raises the possibility that, in vivo, an unidentified kinase analogous to p70S6k is activated by RAFT1 phosphorylation and acts at the rapamycin-sensitive phosphorylation sites of 4E-BP1 (Burnett, 1998).
p70 S6 kinase alpha (p70alpha) is activated in vivo through a multisite phosphorylation in response to mitogens if a sufficient supply of amino acids is available or in response to high concentrations of amino acids per se. The immunosuppressant drug rapamycin inhibits p70alpha activation in a manner that can be overcome by coexpression of p70alpha with a rapamycin-resistant mutant of the mammalian target of rapamycin (mTOR) but only if the mTOR kinase domain is intact. A mammalian recombinant p70alpha polypeptide, extracted in an inactive form from rapamycin-treated cells, can be directly phosphorylated by the mTOR kinase in vitro predominantly at the rapamycin-sensitive site Thr-412. mTOR-catalyzed p70alpha phosphorylation in vitro is accompanied by a substantial restoration in p70alpha kinase activity toward its physiologic substrate, the 40 S ribosomal protein S6. Moreover, sequential phosphorylation of p70alpha by mTOR and 3-phosphoinositide-dependent protein kinase 1 in vitro results in a synergistic stimulation of p70alpha activity to levels similar to those attained by serum stimulation in vivo. These results indicate that mTOR is likely to function as a direct activator of p70 in vivo, although the relative contribution of mTOR-catalyzed p70 phosphorylation in each of the many circumstances that engender p70 activation remains to be defined (Isotani, 2000).
PDK1-catalyzed phosphorylation of Thr-252 on the p70alpha activation loop is a critical and probably final step in the physiologic activation of p70alpha in vivo. The ability of PDK1 to phosphorylate Thr-252 is regulated primarily by the accessibility of the p70alpha activation loop to PDK1, which in turn is controlled by a series of prior p70alpha phosphorylations. Phosphorylation of the multiple Ser/Thr-Pro motifs clustered in the pseudosubstrate autoinhibitory segment of the p70alpha carboxyl-terminal tail serves to disocclude the catalytic domain, greatly enhancing access to PDK1. A similar effect can be achieved by deletion of the p70alpha carboxyl-terminal tail (to give p70alpha-DeltaCT104). At any level of PDK1 activity, the extent of Thr-252 phosphorylation of p70alpha-DeltaCT104 is substantially greater than with a similar amount of full-length p70alpha polypeptide. In addition, the S6 kinase activity generated by any extent of PDK1-catalyzed Thr-252 phosphorylation is significantly higher for p70alpha-DeltaCT104 as compared with full-length p70alpha. Displacement of the p70alpha carboxyl-terminal tail is also necessary for the phosphorylation of Thr-412 in vivo, and modification of Thr-412 itself significantly enhances the ability of PDK1 to phosphorylate Thr-252. In addition, the simultaneous phosphorylation of Thr-412 and Thr-252 appears to generate a synergistic activation of p70alpha. Thus, the substitution of Thr-412 by Glu in p70alpha-DeltaCT104 alone gives a 6-fold increase in S6 kinase activity, and the PDK1 catalyzed phosphorylation of p70alpha-DeltaCT104 Thr-252 alone gives a 15-fold increase, but the two modifications together give at least a 240-fold increase in S6 kinase activity over the unmodified p70alpha-DeltaCT104 polypeptide. The importance of the strong positively cooperative effect of Thr-252 and Thr-412 phosphorylation for the physiologic activation of p70alpha is illustrated by the response of p70alpha to the inhibitors rapamycin and wortmannin; these agents each cause a rapid dephosphorylation of Thr-412 but a slower and lesser dephosphorylation of Thr-252. Despite the preservation of Thr-252 phosphorylation, S6 kinase activity in the presence of rapamycin or wortmannin declines in parallel to Thr-412 dephosphorylation. In view of the ability of mTOR to catalyze the in vitro phosphorylation of p70alpha Thr-412 as well as sites within the autoinhibitory segment in the p70alpha carboxyl-terminal tail and the potential effects of such phosphorylations on the response of p70alpha to PDK1, the p70alpha activation achieved in vitro by mTOR or PDK1 alone have been compared to that achieved by sequential phosphorylation by mTOR and PDK1 and to that achieved in vivo by stimulation of the cells with 10% serum. mTOR alone increases the S6 kinase activity of p70alpha in vitro by more than 10-fold, whereas PDK1 alone hardly activates p70alpha, presumably reflecting the relatively poor access of Thr-252 to PDK1 in full-length, inactive p70alpha. In contrast, phosphorylation of p70alpha by PDK1 after a prior phosphorylation by mTOR increases the p70alpha activity by 10-fold over that engendered by mTOR alone, to a level roughly 70-fold greater than that generated by PDK1 acting alone. Moreover, the S6 kinase activity generated in vitro by the sequential action of mTOR and PDK1 is indistinguishable from that achieved in vivo by stimulation of cells with 10% serum (Isotani, 2000).
mTOR and raptor are components of a signaling pathway that regulates mammalian cell growth in response to nutrients and growth factors. A member of this pathway, a protein named GbetaL, has been identified that binds to the kinase domain of mTOR and stabilizes the interaction of raptor with mTOR. Like mTOR and raptor, GbetaL participates in the control of cell size and in nutrient- and growth factor-mediated signaling to S6K1, a downstream effector of mTOR. The binding of GbetaL to mTOR strongly stimulates the kinase activity of mTOR toward S6K1 and 4E-BP1, an effect reversed by the stable interaction of raptor with mTOR. Interestingly, nutrients and rapamycin regulate the association between mTOR and raptor only in complexes that also contain GbetaL. Thus, it is proposed that the opposing effects on mTOR activity of the GbetaL- and raptor-mediated interactions regulate the mTOR pathway (Kim, 2003).
The FKBP12-rapamycin-associated protein (FRAP; also called RAFT1/mTOR) regulates translation initiation and entry into the cell cycle. Depriving cells of amino acids or treating them with the small molecule rapamycin inhibits FRAP and results in rapid dephosphorylation and inactivation of the translational regulators 4E-BP1(eukaryotic initiation factor 4E-binding protein 1) and p70s6k. Data published recently have led to the view that FRAP acts as a traditional mitogen-activated kinase, directly phosphorylating 4E-BP1 and p70s6k in response to mitogenic stimuli. Evidence indicates that FRAP controls 4E-BP1 and p70s6k phosphorylation indirectly by restraining a phosphatase. A calyculin A-sensitive phosphatase is required for the rapamycin- or amino acid deprivation-induced dephosphorylation of p70s6k, and treatment of Jurkat I cells with rapamycin increases the activity of the protein phosphatase 2A (PP2A) toward 4E-BP1. PP2A associates with p70s6k but not with a mutated p70s6k that is resistant to rapamycin- and amino acid deprivation-mediated dephosphorylation. FRAP also is shown to phosphorylate PP2A in vitro, consistent with a model in which phosphorylation of PP2A by FRAP prevents the dephosphorylation of 4E-BP1 and p70s6k, whereas amino acid deprivation or rapamycin treatment inhibits FRAP's ability to restrain the phosphatase (Peterson, 1999).
On TGF-beta binding, the TGF-beta receptor directly phosphorylates and activates the transcription factors Smad2/3, leading to G1 arrest. Evidence is presented for a second, parallel, TGF-beta-dependent pathway for cell cycle arrest, achieved via inhibition of p70s6k. TGF-beta induces association of its receptor with protein phosphatase-2A (PP2A)-Balpha. Concomitantly, three PP2A-subunits, Balpha, Abeta, and Calpha, associate with p70s6k, leading to its dephosphorylation and inactivation. Although either pathway is sufficient to induce G1 arrest, abrogation of both, the inhibition of p70s6k, and transcription through Smad proteins is required for release of epithelial cells from TGF-beta-induced G1 arrest. TGF-beta thereby modulates the translational and posttranscriptional control of cell cycle progression (Petritsch, 2000).
On receptor activation, PP2A-Balpha specifically binds the activated TbetaRI and is catalytically activated by TGF-beta. PP2A-Balpha then recruits PP2A-Abeta and PP2A-C to bind and dephosphorylate p70s6k. Complexes containing at the same time PP2A, TbetaRI and p70s6k, could not be detected, indicating that on activation the phosphatase is released from the receptor to bind to the target molecule. Immunolocalization of the endogenous proteins supports this model. p70s6k activity controls the translational upregulation of proteins important for G1/S progression and is itself essential for cell cycle progression. Most of the transcripts isolated to date represent ribosomal proteins and elongation factors of protein synthesis. TGF-beta-induced inactivation of p70s6k leads to the translational regulation of a group of cell cycle regulators for G1 progression. It remains unclear, however, if the repression of those cell cycle regulators result from global repression of protein translation or represent a class of specifically translationally repressed mRNAs. It is conceivable that the regulation of crucial components of the cell cycle machinery is mediated at the transcriptional, translational, and posttranslational levels (Petritsch, 2000).
Expression of the regulatory subunit PP2A-Balpha itself appears to be a prerequisite for the PP2A-mediated inhibition of p70s6k by TGF-beta. Cells with nondetectable PP2A-Balpha expression remain solely responsive to TGF-beta-mediated transcriptional responses. p70s6k is not inhibited by TGF-beta in these cells; this reflects the differential sensitivity of epithelial cells and mesenchymal cells to growth inhibitory effects of TGF-beta. The chromosomal localization of PP2A-Balpha has not been investigated; PP2A-Abeta, however, has been mapped to a human tumor suppressor locus on 11q22-24 and appears to be mutated in a subset of human lung tumors. It is tempting to speculate that mutations of the regulatory subunits of PP2A in human tumors abolish the regulation of p70s6k to TGF-beta and confer a selective advantage to growing tumors (Petritsch, 2000).
The 70 kDa ribosomol S6 kinase (pp70S6k) plays an important role in the progression of cells through G1 phase of the cell cycle. However, little is known of the signaling molecules that mediate its activation. Rho family G proteins regulate pp70S6k activity in vivo. Activated alleles of Cdc42 and Rac1, but not RhoA, stimulate pp70S6k activity in multiple cell types. Activation requires an intact effector domain and isoprenylation of Cdc42 and Rac1. Coexpression of Dbl, an exchange factor for Cdc42, also activates pp70S6k. Growth factor-induced activation of pp70S6k is abrogated by dominant negative alleles of Cdc42 and Rac1. In addition, Cdc42 and Rac1 form GTP-dependent complex with the catalytically inactive form of pp70S6k in vitro and in vivo, suggesting a mechanism by which these G proteins activate pp70S6k (Chou, 1996).
This paper reviews signaling upstream of p70S6K and the role of p70S6K in controlling translation and cell growth. The highly homologous 40S ribosomal protein S6 kinases (S6K1 and S6K2) play a key role in the regulation of cell growth by controlling the biosynthesis of translational components which make up the protein synthetic apparatus, most notably ribosomal proteins. In the case of S6K1, at least eight phosphorylation sites are believed to mediate kinase activation in a hierarchical fashion. Activation is initiated by phosphatidylinositide-3OH kinase (PI3K)-mediated phosphorylation of key residues in the carboxy-terminus of the kinase, allowing phosphorylation of a critical residue residing in the activation loop of the catalytic domain by phosphoinositide-dependent kinase 1 (PDK1). The kinases responsible for phosphorylating the carboxy-terminal sites have yet to be identified. Additionally, S6 kinases are under the control of the PI3K relative, mammalian Target Of Rapamycin (mTOR), which may serve an additional function as a checkpoint for amino acid availability (Dufner, 2000).
After an initial burst of cell proliferation, the type 1 insulin-like growth factor receptor (IGF-IR) induces granulocytic differentiation of 32D IGF-IR cells, an interleukin-3-dependent murine hemopoietic cell line devoid of insulin receptor substrate-1 (IRS-1). The combined expression of the IGF-IR and IRS-1 (32D IGF-IR/IRS-1 cells) inhibits IGF-I-mediated differentiation, and causes malignant transformation of 32D cells. Because of the role of IRS-1 in changing the fate of 32D IGF-IR cells from differentiation (and subsequent cell death) to malignant transformation, differences in IGF-IR signaling between 32D IGF-IR and 32D IGF-IR/IRS-1 cells were examined. A focus was placed on p70S6K, which is activated by the IRS-1 pathway. The ectopic expression of IRS-1 and the inhibition of differentiation correlates with a sustained activation of p70S6K and an increase in cell size. Phosphorylation in vivo of threonine 389 and, to a lesser extent, of threonine 421/serine 424 of p70S6K seems to be a requirement for inhibition of differentiation. A role for IRS-1 and p70S6K in the alternative between transformation or differentiation of 32D IGF-IR cells was confirmed by evidence that inhibition of p70S6K activation or IRS-1 signaling, by rapamycin or okadaic acid, induces differentiation of 32D IGF-IR/IRS-1 cells. Expression of myeloperoxidase mRNA (a marker of differentiation, which sharply increases in 32D IGF-IR cells), does not increase in 32D IGF-IR/IRS-1 cells, suggesting that the expression of IRS-1 in 32D IGF-IR cells causes the extinction of the differentiation program initiated by the IGF-IR, while leaving intact its proliferation program (Valentinis, 2000).
The c-Abl protein-tyrosine kinase is activated by ionizing radiation and certain other DNA-damaging agents. The rapamycin and FKBP-target 1 (RAFT1), also known as FKBP12-rapamycin-associated protein (FRAP, mTOR), regulates the p70S6 kinase (p70S6k) and the eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). The present results demonstrate that c-Abl binds directly to RAFT1 and phosphorylates RAFT1 in vitro and in vivo. c-Abl inhibits autophosphorylation of RAFT1 and RAFT1-mediated phosphorylation p70S6k. The functional significance of the c-Abl-RAFT1 interaction is further supported by the finding that eIF4E-dependent translation in mouse embryo fibroblasts from Abl(-/-) mice is significantly higher than that compared in wild-type cells. The results also demonstrate that exposure of cells to ionizing radiation is associated with c-Abl-mediated binding of 4E-BP1 to eIF4E and inhibition of translation. These findings with the c-Abl tyrosine kinase represent the first demonstration of a negative physiologic regulator of RAFT1-mediated 5' cap-dependent translation (Kumar, 2000b).
Extracellular zinc ion may play a role in the activation of p70S6k, which in turn functions in regulating the progression of cells from the G(1) to S phase of the cell cycle. Treatment of Swiss 3T3 cells with zinc sulfate leads to the activation and phosphorylation of p70S6k in a dose-dependent manner. The activation of p70S6k by zinc treatment is biphasic, the early phase being at 30 min followed by the late phase at 120 min. The zinc-induced activation of p70S6k is partially inhibited by down-regulation of phorbol 12-myristate 13-acetate-responsive protein kinase C (PKC) by chronic treatment with phorbol 12-myristate 13-acetate, but this is not significant. Moreover, Go6976, a specific calcium-dependent PKC inhibitor, does not significantly inhibit the activation of p70S6k by zinc. These results demonstrate that the zinc-induced activation of p70S6k is not related to PKC. Also, extracellular calcium is not involved in the activation of p70S6k by zinc. Further characterization of the zinc-induced activation of p70S6k using specific inhibitors of the p70S6k signaling pathway, namely rapamycin, wortmannin, and LY294002, has shown that zinc acts upstream of mTOR/FRAP/RAFT and phosphatidylinositol 3-kinase (PI3K), because these inhibitors cause the inhibition of zinc-induced p70S6k activity. In addition, Akt, the upstream component of p70S6k, is activated by zinc in a biphasic manner, as is p70S6k. Moreover, dominant interfering alleles of Akt and PDK1 block the zinc-induced activation of p70S6k, whereas the lipid kinase activity of PI3K is potently activated by zinc. Taken together, these data suggest that zinc activates p70S6k through the PI3K signaling pathway (Kim, 2000).
Expression of the insulin-like growth factor-binding protein 5 (IGFBP-5) gene in vascular smooth muscle cells is up-regulated by IGF-I through an IGF-I receptor-mediated mechanism. The possible involvement of the mitogen-activated protein kinase (MAPK) and PI 3-kinase signaling pathways in mediating IGF-I-regulated IGFBP-5 gene expression has been examined. The addition of Des(1-3)IGF-I, an IGF analog with reduced affinity to IGFBPs, results in a transient activation of p44 and p42 MAPK. Inhibition of the MAPK activation by PD98059, however, does not affect IGF-I-stimulated IGFBP-5 expression. Des(1-3)IGF-I treatment also strongly activates PI 3-kinase. This activation is probably mediated through IRS-1, because IGF-I stimulation results in a significant increase in IRS-1- but not IRS-2-associated PI 3-kinase activity. This activation occurs within 5 min and is sustained at high levels for over 6 h. Likewise, Des(1-3)IGF-I causes a long lasting activation of PKB/Akt and p70s6k. When LY294002 and wortmannin, two specific inhibitors of PI 3-kinase, are added with Des(1-3)IGF-I, the IGF-I-regulated IGFBP-5 expression is negated. The addition of rapamycin, which inhibits IGF-I-induces p70s6k activation, significantly inhibits IGF-I-regulated IGFBP-5 gene expression. These results suggest that the action of IGF-I on IGFBP-5 gene expression requires the activation of the PI 3-kinase-PKB/Akt-p70s6k pathway but not the MAPK pathway in vascular smooth muscle cells (Duan, 1999).
Ligation of the B cell antigen receptor (BCR) induces a cascade of signaling pathways that lead to clonal expansion, differentiation, or abortive activation-induced apoptosis of B lymphocytes. BCR-mediated cross-linking induces the rapid phosphorylation of protein tyrosine kinases. However, the pathways leading to the activation of downstream serine/threonine kinases such as mitogen-activated protein kinase, p90Rsk, and p70S6 kinase (p70S6k) that mediate reorganization of the actin cytoskeleton, cell cycle progression, gene transcription, and protein synthesis have not been delineated. Cross-linking of BCR leads to activation of p70S6k in B lymphocytes. Multiple protein tyrosine kinase-dependent signal transduction pathways induced by BCR lead to the activation of p70S6k. These distinct pathways exhibit different thresholds with respect to the extent of receptor cross-linking required for their activation. Activation of p70S6k by suboptimal doses of anti-Ig is Syk-dependent and is mediated by protein kinase C and phosphoinositol 3-kinase. Moreover, the activation of p70S6k results in phosphorylation of S6 protein which is important for ribosomal protein synthesis and may be coupled to BCR-induced protein and DNA synthesis in primary murine B cells (Hsiu-Ling, 1999).
The present study identifies the operation of a signal tranduction pathway in mammalian cells that provides a checkpoint control, linking amino acid sufficiency to the control of peptide chain initiation. Withdrawal of amino acids from the nutrient medium of CHO-IR cells results in a rapid deactivation of p70 S6 kinase and dephosphorylation of eIF-4E BP1, which becomes unresponsive to all agonists. Readdition of the amino acid mixture quickly restores the phosphorylation and responsiveness of p70 and eIF-4E BP1 to insulin. Increasing the ambient amino acids to twice that usually employed increases basal p70 activity to the maximal level otherwise attained in the presence of insulin and abrogates further stimulation by insulin. Withdrawal of most individual amino acids also inhibits p70, although with differing potency. Amino acid withdrawal from CHO-IR cells does not significantly alter insulin stimulation of tyrosine phosphorylation, phosphotyrosine-associated phosphatidylinositol 3-kinase activity, c-Akt/protein kinase B activity, or mitogen-activated protein kinase activity. The selective inhibition of p70 and eIF-4E BP1 phosphorylation by amino acid withdrawal resembles the response to rapamycin, which prevents p70 reactivation by amino acids, indicating that mTOR is required for the response to amino acids. A p70 deletion mutant, p702-46/CT104, that is resistant to inhibition by rapamycin (but sensitive to wortmannin) is also resistant to inhibition by amino acid withdrawal, indicating that amino acid sufficiency and mTOR signal to p70 through a common effector, which could be mTOR itself, or an mTOR-controlled downstream element, such as a protein phosphatase (Hara, 1998).
Tuberous sclerosis (TSC) is a autosomal dominant genetic disorder caused by mutations in either TSC1 or TSC2, and characterized by benign hamartoma growth. A murine model of Tsc1 disease was developed by gene targeting. Tsc1 null embryos die at mid-gestation from a failure of liver development. Tsc1 heterozygotes develop kidney cystadenomas and liver hemangiomas at high frequency, but the incidence of kidney tumors is somewhat lower than in Tsc2 heterozygote mice. Liver hemangiomas are more common, more severe and cause higher mortality in female than in male Tsc1 heterozygotes. Tsc1 null embryo fibroblast lines have persistent phosphorylation of the p70S6K (S6K) and its substrate S6; this phosphorylation is sensitive to treatment with rapamycin, indicating constitutive activation of the mTOR-S6K pathway due to loss of the Tsc1 protein, hamartin. Hyperphosphorylation of S6 is also seen in kidney tumors in the heterozygote mice, suggesting that inhibition of this pathway may have benefit in the control of TSC hamartomas (Kwaitkowski, 2002).
The S/T-protein kinases activated by phosphoinositide 3-kinase (PI3K) regulate a myriad of cellular processes. An approach using a combination of biochemistry and bioinformatics can identify substrates of these kinases. This approach identifies the tuberous sclerosis complex-2 gene product, tuberin, as a potential target of Akt/PKB (see Drosophila Akt1). Upon activation of PI3K, tuberin is phosphorylated on consensus recognition sites for PI3K-dependent S/T kinases. Moreover, Akt/PKB can phosphorylate tuberin in vitro and in vivo. S939 and T1462 of tuberin are PI3K-regulated phosphorylation sites and T1462 is constitutively phosphorylated in PTEN-/- tumor-derived cell lines. Finally, a tuberin mutant lacking the major PI3K-dependent phosphorylation sites can block the activation of S6K1, suggesting a means by which the PI3K-Akt pathway regulates S6K1 activity (Manning, 2002).
Serine/threonine (S/T) protein kinases can account for much of the functional diversity of PI3K signaling. Akt/protein kinase B and the 70 kDa-S6 kinase 1 (S6K1) are the best characterized of the PI3K-regulated S/T kinases. The mitogen-stimulated activation of both of these kinases is blocked by PI3K-specific inhibitors. Akt contains a PH domain that is specific to PtdIns-3,4P2 and PtdIns-3,4,5P3. Akt is thereby recruited to these PI3K-generated second messengers and to the PDK1 protein kinase, which also specifically binds to these lipids. PDK1 then phosphorylates and activates Akt (Manning, 2002).
The regulation of S6K1 is much more complex, with both PI3K-dependent and -independent signaling pathways involved in its activation. Several PI3K-regulated effectors are known to participate in the activation of S6K1, including PDK1, PKCzeta/lambda, Cdc42, Rac1, and Akt. However, the molecular mechanism of how these contribute to S6K1 activation remains unclear. In addition to mitogen-regulated signaling to S6K1, the metabolic state of the cell and the availability of nutrients control S6K1 activation through the mammalian target of rapamycin (mTOR, also known as FRAP, RAFT, and RAPT). Recent studies suggest that mTOR is also regulated by mitogenic signals. Interestingly, it has been suggested that the point of convergence of the mitogenic and nutrient-sensing signals in the regulation of S6K1 may be at the level of Akt directly phosphorylating mTOR. However, this phosphorylation does not appear to affect mTOR activity or S6K1 activation. Thus, of the PI3K-regulated effectors thought to participate in S6K1 activation, the molecular basis of how Akt regulates S6K1 remains the least well understood (Manning, 2002).
Akt itself has been implicated in many of the PI3K-regulated cellular events, and several substrates have been shown to be phosphorylated in vitro and/or in vivo by Akt. Therefore, the total cellular effect of PI3K activation and subsequent activation of Akt is mediated through a variety of different targets. However, it seems unlikely that the large array of processes controlled by the PI3K-Akt pathway can be accounted for by current knowledge of downstream targets (Manning, 2002).
An approach has been developed to screen for substrates of PI3K-dependent S/T kinases, such as Akt. This approach uses phospho-specific antibodies generated against a phosphorylated protein kinase consensus recognition motif in combination with a protein database motif scanning program called Scansite (http://scansite.mit.edu). Scansite is a web-based program that searches protein databases for optimal substrates of specific protein kinases and for optimal binding motifs for specific protein domains with data generated by peptide library screens. The phospho-motif antibody is used to recognize proteins phosphorylated specifically under conditions in which the kinase of interest is active. Scansite is then used to identify candidate substrates of this protein kinase that have the predicted molecular mass of the proteins recognized by the phospho-motif antibody. This approach successfully identifies known substrates of Akt. The tuberous sclerosis complex-2 (TSC2) tumor suppressor gene product, tuberin, is also identified and characterized as an Akt substrate. Furthermore, it is found that overexpression of a tuberin mutant lacking the major Akt phosphorylation sites can inhibit growth factor-induced activation of S6K1. These results provide a biochemical link between the PI3K-Akt pathway and regulation of S6K1 and also indicate a biochemical basis for the disease tuberous sclerosis complex (TSC) (Manning, 2002).
Expression in human cells of the tuberinS939A/T1462A mutant, which lacks the major PI3K-dependent phosphorylation sites, at levels comparable to endogenous tuberin leads to a decrease in growth factor-induced S6K1 phosphorylation and activity. This phosphorylation and subsequent activation of S6K1 has been previously demonstrated to be dependent on PI3K. These results, along with those from genetic studies in other systems, are consistent with a model in which growth factors activate PI3K leading to the phosphorylation of tuberin by Akt. This phosphorylation inhibits the tuberin-hamartin complex, thereby relieving its inhibition of S6K1. In this model, expression of the tuberinS939A/T1462A mutant, which would not be phosphorylated and inhibited, would have a dominant effect over endogenous tuberin and block growth factor-induced S6K1 activation. It will be of great interest to determine the molecular nature of S6K1 inhibition by the tuberin-hamartin complex in the absence of mitogenic stimuli. It is possible that the complex does so upstream of mTOR, because the constitutive activation of S6K1 in TSC1-/- MEFs is sensitive to rapamycin. Alternatively, mTOR might regulate S6K1 in a nutrient-sensitive pathway parallel to the mitogen-sensitive PI3K-Akt-tuberin pathway (Manning, 2002).
Recent studies have suggested that S6K1 activation can occur independent of PI3K and Akt. These studies demonstrate the existence of multiple pathways regulating S6K1 and that the tuberin-hamartin complex might integrate signals from many different inputs. The identification of tuberin as a direct downstream target of the PI3K-Akt pathway provides the missing link between this signaling cascade and control of S6K1 activity (Manning, 2002).
The identification of this biochemical relationship between the mammalian TSC tumor suppressor gene products and the oncogenic PI3K-Akt pathway could have important implications in human diseases. For instance, in approximately 10%-15% of patients diagnosed with TSC, mutations in TSC1 or TSC2 have not been detected. Based on the findings of this study, it is possible that mutations leading to aberrant activation of the PI3K-Akt pathway, such as PTEN mutations, could inhibit the function of the tuberin-hamartin complex by causing constitutive phosphorylation of tuberin. It will be interesting to examine the phosphorylation state of tuberin within hamartomas from such TSC patients. Indeed, in PTEN-/- cell lines derived from both glioblastoma and prostate tumors, growth factor-independent phosphorylation of tuberin on the PI3K-dependent T1462 site is detected (Manning, 2002).
Germline mutations in either PTEN or the TSC genes cause autosomal dominant diseases that are characterized by the occurrence of widespread hamartomas due to loss of heterozygosity at these loci. However, the tissue distribution of these benign tumors varies between patients with loss of PTEN and those with TSC. These differences might be explained by a model in which the tuberin-hamartin complex is the primary growth-inhibiting target of the PI3K-Akt pathway in tissues affected in TSC patients. In other tissues, such as those affected in patients with PTEN mutations, this complex might be one of many targets of the PI3K-Akt pathway. Interestingly, though, recent studies have suggested that mTOR activity is essential for oncogenic transformation of cells by activated PI3K or Akt and for growth of PTEN-/- tumors. Therefore, aberrant phosphorylation and inhibition of the tuberin-hamartin complex, and subsequent increased activity of mTOR and/or S6K1, would likely contribute to tumorigenesis caused by mutations that activate the PI3K-Akt pathway. Future studies using crosses between PTEN and TSC knockout mice should help determine the contribution of the tuberin-hamartin complex in prevention of the variety of tumors caused by uncontrolled signaling through the PI3K-Akt pathway. Finally, the elucidation of a PI3K-Akt-tuberin pathway controlling S6K1 activity will have important implications in the understanding and treatment of the prevalent TSC disease (Manning, 2002).
The mammalian target of rapamycin (mTOR) regulates cell growth and proliferation via the downstream targets ribosomal S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1). Phosphatidic acid (PA) has been identified as a mediator of mitogenic activation of mTOR signaling. This study tests the hypotheses that phospholipase D 1 (PLD1) is an upstream regulator of mTOR and that the previously reported S6K1 activation by Cdc42 is mediated by PLD1. Overexpression of wild-type PLD1 is found to increase S6K1 activity in serum-stimulated cells, whereas a catalytically inactive PLD1 exerts a dominant-negative effect on S6K1. More importantly, eliminating endogenous PLD1 by RNAi leads to drastic inhibition of serum-stimulated S6K1 activation and 4E-BP1 hyperphosphorylation in both HEK293 and COS-7 cells. Knockdown of PLD1 also results in reduced cell size, suggesting a critical role for PLD1 in cell growth control. Using a rapamycin-resistant S6K1 mutant, Cdc42's action has been demonstrated to be through the mTOR pathway. When Cdc42 is mutated in a region specifically required for PLD1 activation, its ability to activate S6K1 in the presence of serum is hindered. However, when exogenous PA is used as a stimulus, the PLD1-inactive Cdc42 mutant behaves similarly to the wild-type protein. These observations reveal the involvement of PLD1 in mTOR signaling and cell size control, and provide a molecular mechanism for Cdc42 activation of S6K1. A new cascade is proposed to connect mitogenic signals to mTOR through Cdc42, PLD1, and PA (Fang, 2003).
The tuberous sclerosis tumor suppressors TSC1 and TSC2 regulate the mTOR pathway to control translation and cell growth in response to nutrient and growth factor stimuli. The stress response REDD1 gene has been identified as a mediator of tuberous sclerosis complex (TSC)-dependent mTOR regulation by hypoxia. REDD1 (Drosophila homologs Scylla and Charybde) inhibits mTOR function to control cell growth in response to energy stress. Endogenous REDD1 is induced following energy stress, and REDD1-/- cells are highly defective in dephosphorylation of the key mTOR substrates S6K and 4E-BP1 following either ATP depletion or direct activation of the AMP-activated protein kinase (AMPK). REDD1 likely acts on the TSC1/2 complex, because regulation of mTOR substrate phosphorylation by REDD1 requires TSC2 and is blocked by overexpression of the TSC1/2 downstream target Rheb but is not blocked by inhibition of AMPK. Tetracycline-inducible expression of REDD1 triggers rapid dephosphorylation of S6K and 4E-BP1 and significantly decreases cellular size. Conversely, inhibition of endogenous REDD1 by short interfering RNA increases cell size in a rapamycin-sensitive manner, and REDD1-/- cells are defective in cell growth regulation following ATP depletion. These results define REDD1 as a critical transducer of the cellular response to energy depletion through the TSC-mTOR pathway (Sofer, 2005).
Tumor suppressor genes evolved as negative effectors of mitogen and nutrient signaling pathways, such that mutations in these genes can lead to pathological states of growth. Tuberous sclerosis (TSC) is a potentially devastating disease associated with mutations in two tumor suppressor genes, TSC1 and 2 (See Drosophila Gigas), that function as a complex to suppress signaling in the mTOR/S6K/4E-BP pathway. However, the inhibitory target of TSC1/2 and the mechanism by which it acts are unknown. Evidence is provided that TSC1/2 is a GAP for the small GTPase Rheb (see Drosophila Rheb) and that insulin-mediated Rheb activation is PI3K dependent. Moreover, Rheb overexpression induces S6K1 phosphorylation and inhibits PKB phosphorylation, as do loss-of-function mutations in TSC1/2, but contrary to earlier reports Rheb has no effect on MAPK phosphorylation. Finally, coexpression of a human TSC2 cDNA harboring a disease-associated point mutation in the GAP domain, failed to stimulate Rheb GTPase activity or block Rheb activation of S6K1 (Garami, 2003).
The tuberous sclerosis complex 2 (TSC2) tumor suppressor gene product is a negative regulator of protein synthesis upstream of the mTOR and ribosomal S6 kinases. Because of the homology of TSC2 with GTPase-activating proteins for Rap1, whether a Ras/Rap-related GTPase might be involved in this process was examined. TSC2 was found to bind to Rheb-GTP in vitro and to reduce Rheb GTP levels in vivo. Over-expression of Rheb but not Rap1 promotes the activation of S6 kinase in a rapamycin-dependent manner, suggesting that Rheb acts upstream of mTOR. The ability of Rheb to induce S6 phosphorylation is also inhibited by a farnesyl transferase inhibitor, suggesting that Rheb may be responsible for the Ras-independent anti-neoplastic properties of this drug (Castro, 2003).
Tuberous sclerosis complex is a genetic disease caused by mutation in either TSC1 or TSC2. The TSC1 and TSC2 gene products form a functional complex and inhibit phosphorylation of S6K and 4EBP1. These functions of TSC1/TSC2 are likely mediated by mTOR. TSC2 is a GTPase-activating protein (GAP) toward Rheb, a Ras family GTPase. Rheb stimulates phosphorylation of S6K and 4EBP1. This function of Rheb is blocked by rapamycin and dominant-negative mTOR. Rheb stimulates the phosphorylation of mTOR and plays an essential role in regulation of S6K and 4EBP1 in response to nutrients and cellular energy status. These data demonstrate that Rheb acts downstream of TSC1/TSC2 and upstream of mTOR to regulate cell growth (Inoki, 2003).
The small G protein Rheb (Ras homolog enriched in brain) is a molecular target of TSC1/TSC2 that regulates mTOR signaling. Overexpression of Rheb activates 40S ribosomal protein S6 kinase 1 (S6K1) but not p90 ribosomal S6 kinase 1 (RSK1) or Akt. Furthermore, Rheb induces phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and causes 4E-BP1 to dissociate from eIF4E. This dissociation is completely sensitive to rapamycin (an mTOR inhibitor) but not wortmannin (a phosphoinositide 3-kinase [PI3K] inhibitor). Rheb also activates S6K1 during amino acid insufficiency via a rapamycin-sensitive mechanism, suggesting that Rheb participates in nutrient signaling through mTOR. Moreover, Rheb does not activate a S6K1 mutant that is unresponsive to mTOR-mediated signals, confirming that Rheb functions upstream of mTOR. Overexpression of the Tuberin-Hamartin heterodimer inhibits Rheb-mediated S6K1 activation, suggesting that Tuberin functions as a Rheb GTPase activating protein (GAP). Supporting this notion, TSC patient-derived Tuberin GAP domain mutants were unable to inactivate Rheb in vivo. Moreover, in vitro studies reveal that Tuberin, when associated with Hamartin, acts as a Rheb GTPase-activating protein. Finally, it is shown that membrane localization of Rheb is important for its biological activity because a farnesylation-defective mutant of Rheb stimulated S6K1 activation less efficiently. It is concluded that Rheb acts as a novel mediator of the nutrient signaling input to mTOR and is the molecular target of TSC1 and TSC2 within mammalian cells (Tee, 2003).
To examine whether Rheb overexpression could modulate S6K1 activity, S6K1 was coexpressed with Rheb at two different expression levels in HEK293E cells and kinase activity was assayed by using GST-S6 as a substrate. Coexpression of Rheb significantly increased the basal and insulin-stimulated activity of S6K1. Higher levels of Rheb expression enhanced the basal and insulin-stimulated activity of S6K1 by 5.6- and 1.7-fold, respectively, and this activity level is more potent than the S6K1 activity observed in the presence of lower levels of Rheb protein. These results indicate that Rheb activates signaling cascades that result in S6K1 activation (Tee, 2003).
Given that the activity of S6K1 is enhanced upon cell signaling through mTOR, PI3K, and mitogen-activated protein kinase (MAPK) and protein kinase C (PKC)-mediated pathways, it was determined which signaling pathway was activated upon Rheb overexpression. To investigate PI3K-mediated signaling, Rheb, along with Akt, a downstream target of PI3K, was expressed within HEK293E cells and kinase activity was assayed. Whereas EGF stimulation led to a 4-fold increase in Akt activity, Rheb overexpression did not enhance basal or EGF-stimulated Akt activity. In contrast, Rheb potently activated S6K1 by 11-fold when assayed in parallel. Wortmannin was used as a control to show that PI3K-mediated activation of Akt upon stimulation with EGF was specifically measured. To examine whether Rheb enhanced MAPK-mediated signaling, Rheb was coexpressed with RSK1, a known downstream signaling component of MAPK, within HEK293E cells. Although EGF stimulation led to a 12-fold increase in RSK1 activity, Rheb did not augment the basal or EGF-induced activation of RSK1 assayed with GST-S6 as a substrate. In contrast, Rheb overexpression drastically increased S6K1 activity when assayed in parallel. To inhibit activation of ERK (extracellular signal-regulated kinase) through MEK (MAPK/ERK-kinase)-mediated signaling, cells were treated with the U0126 compound to specifically inhibit MEK. These findings suggest that Rheb does not function upstream of either PI3K/Akt or ERK/RSK1 signaling pathways (Tee, 2003).
The above data implies that Rheb may modulate mTOR signaling. Given that the loss of Rheb in yeast mimics nutrient starvation, Rheb overexpression may promote mTOR signaling through a nutrient-regulated signaling pathway. To address this possibility, whether Rheb promotes S6K1 activation was investigated in the absence of amino acids. During conditions of amino acid withdrawal, Rheb overexpression potently activated S6K1, which was completely blocked by rapamycin but only partially inhibited by wortmannin. Importantly, insulin stimulation of these amino acid-deprived cells potently activated Akt (as observed by Akt phosphorylation on Ser473) but only weakly activated S6K1. Therefore, unlike Rheb-mediated signaling, acute stimulation of PI3K and Akt is not sufficient to fully activate S6K1 during nutrient insufficiency. The modest insulin-induced activation of S6K1 during amino acid insufficiency was blocked by wortmannin, revealing that this activation is completely dependent on PI3K. Activation of S6K1 upon readdition of amino acids was enhanced when Rheb was overexpressed or when cells were stimulated with insulin). Interestingly, Rheb-induced S6K1 activation upon readdition of amino acids was completely inhibited by rapamycin but only partially inhibited by wortmannin. In contrast, insulin-induced S6K1 activity was markedly impaired by both rapamycin and wortmannin. These data convincingly reveal that Rheb potently activates S6K in the absence of nutrients through mTOR rather than PI3K-mediated signaling. Therefore, it is likely that Rheb enhances nutrient-mediated signaling through mTOR (Tee, 2003).
To decisively determine whether Rheb positively activates mTOR signaling, use was made of a rapamycin-resistant mutant of S6K1 (S6K1-F5A-DeltaCT). Unlike treatment with wortmannin, rapamycin treatment was unable to prevent insulin-induced activation of S6K1-F5A-DeltaCT, demonstrating that this mutant is responsive to PI3K signaling but not mTOR signaling. As a positive control, PDK1 and PKCζ, which are known to activate S6K1 through a PI3K-dependent input, were coexpressed. Overexpression of Rheb potently activates wild-type S6K1 basally (by 11-fold) and during insulin stimulation (by 17-fold) but does not enhance the activity of the S6K1-F5A-DeltaCT mutant. In contrast, increased PI3K-mediated signaling toward S6K1 by coexpression of PDK1 and PKCζ results in significantly enhanced activation of both wild-type S6K1 and S6K1-F5A-DeltaCT. These findings strongly suggest that Rheb induces S6K1 activation via a signaling input that is upstream of mTOR but not PI3K (Tee, 2003).
Overexpression of wild-type Rheb has been shown to lead to a significant increase in its activity and implies that the majority of the overexpressed Rheb must exist in the active GTP bound form. If this is true, then the RhebGAP activity must be a limiting factor. If Tuberin possesses RhebGAP activity, overexpression of Tuberin should switch Rheb from an active GTP bound state to an inactive GDP bound state. To indirectly measure Rheb activity, Rheb-induced S6K1 activation was analyzed within nutrient-deprived HEK293E cells. Coexpression of Hamartin and Tuberin completely block Rheb's ability to activate S6K1, implying that Tuberin may function as a RhebGAP (Tee, 2003).
If the GAP domain of Tuberin is essential for Rheb inactivation, then patient-derived TSC2 GAP domain point mutants should not block Rheb-induced S6K1 activation. To address this, three Tuberin mutants were generated that mimic patient-derived TSC2 mutations that occur within the GAP domain and these Tuberin mutants were coexpressed with Hamartin and S6K1. Under serum-starved conditions, Rheb potently activates S6K1, which was fully blocked by coexpression of wild-type Tuberin with Hamartin. In contrast, the three TSC2 GAP domain point mutants were unable to repress Rheb-induced S6K1 activation, revealing that the GAP domain of Tuberin is critical for Tuberin's ability to repress Rheb-mediated signaling (Tee, 2003).
Thus, Rheb functions upstream of mTOR within the nutrient signaling pathway. Rheb specifically activates mTOR-mediated signaling rather than cell signaling through MEK/ERK and PI3K, as shown by Rheb-mediated activation of S6K1 but not Akt or RSK1. Therefore, it is unlikely that Rheb activates PI3K and Raf, two downstream effectors of Ras. Rheb has previously been shown to interact with Raf in vitro, but the current data suggest that Raf is not an effector of Rheb in vivo. Additionally, Rheb overexpression does not increase the activity of the rapamycin-resistant S6K1 mutant that is unresponsive to mTOR signaling inputs but is activated in response to PI3K signaling. S6K1 activation is regulated by multiple signaling inputs, one of which is directed by PI3K. Therefore, these findings are important and confirm that Rheb overexpression specifically promotes mTOR rather than PI3K signaling. Furthermore, Rheb-induced 4E-BP1 phosphorylation is completely sensitive to rapamycin but not to wortmannin, which further strengthens the notion that Rheb acts upstream of mTOR rather than PI3K. 4E-BP1 dissociates from eIF4E upon Rheb overexpression, revealing that Rheb-mediated signaling through mTOR promotes cap-dependent translation (Tee, 2003).
Evidence has also been provided that Rheb functions within the nutrient signaling cascade upstream of mTOR, as shown by Rheb's ability to potently stimulate S6K1 activity during amino acid insufficiency. During amino acid withdrawal, acute insulin stimulation is still able to elicit high levels of Akt phosphorylation but poorly activates S6K1, showing that the nutrient-mediated mTOR signaling input is essential for optimal S6K1 activation. Therefore, Rheb overexpression supersedes the dependency of the nutrient input to mTOR, suggesting that Rheb is an activator of mTOR within the nutrient-signaling pathway. Interestingly, resupplying cells with amino acids further enhances the activity of S6K1 when Rheb is overexpressed, suggesting that amino acids may promote the activation of Rheb. This research has revealed that the Tuberin-Hamartin heterodimer functions as an inhibitor of nutrient signaling through mTOR. The Tuberin-Hamartin heterodimer inhibits Rheb-induced S6K1 activation during conditions of amino acid withdrawal. This work, therefore, extends these earlier studies, revealing that inhibition of Rheb is the mechanism by which the Tuberin-Hamartin heterodimer inhibits nutrient-mediated signaling. Importantly, the Rheb-inhibitory function of Tuberin-Hamartin heterodimers depends on an intact Tuberin GAP domain; patient-derived point mutations within the GAP domain of TSC2 prevented the Tuberin-Hamartin heterodimer from blocking Rheb-induced S6K1 activation. These data indicate that the GAP activity of Tuberin promotes inactivation of Rheb in vivo, presumably through increasing the intrinsic GTPase activity of Rheb. Confirming this hypothesis, in vitro Rheb GTPase activity assays have revealed that Tuberin enhances the intrinsic GTPase activity of Rheb. Interestingly, coexpression of Hamartin with Tuberin markedly enhances the GTPase activity of Rheb, implying that Hamartin promotes the GAP function of Tuberin toward Rheb. A model is proposed whereby Rheb promotes mTOR signaling when it is in an active GTP bound form, whereas the Tuberin-Hamartin heterodimer inhibits Rheb by converting it to an inactive GDP bound state. These findings reveal that the Tuberin-Hamartin heterodimer and Rheb respectively inhibit and activate the nutrient-signaling input to mTOR. Small G proteins are additionally regulated by guanine nucleotide exchange factors (GEFs). In this model it is proposed that a RhebGEF becomes activated during conditions of nutrient sufficiency, and its activation switches Rheb to an active GTP bound form. Therefore, identifying this Rheb-GEF will be of great importance and may provide new insights into how mTOR senses intracellular amino acids. However, at this point the possibility that Rheb- and nutrient-mediated signaling may function in parallel pathways upstream of mTOR cannot be ruled out. Further experiments will be carried out to investigate this possibility (Tee, 2003).
Dominant negative mutants of S. pombe Rheb (SpRheb) are reported. Screens of a randomly mutagenized SpRheb library yielded a mutant, SpRhebD60V, whose expression in S. pombe results in growth inhibition, G1 arrest, and induction of fnx1+, a gene whose expression is induced by the disruption of Rheb. Alteration of the Asp-60 residue to all possible amino acids by site-directed mutagenesis led to the identification of two particularly strong dominant negative mutants, D60I and D60K. Characterization of these dominant negative mutant proteins revealed that D60V and D60I exhibit preferential binding of GDP, while D60K lost the ability to bind both GTP and GDP. A possible use of the dominant negative mutants in the study of mammalian Rheb was explored by introducing dominant negative mutations into human Rheb. Transient expression of the wild type Rheb1 or Rheb2 causes activation of p70S6K, while expression of Rheb1D60K mutant results in inhibition of basal level activity of p70S6K. In addition, Rheb1D60K and Rheb1D60V mutants block nutrient- or serum-induced activation of p70S6K. This provides critical evidence that Rheb plays a role in the mTOR/S6K pathway in mammalian cells (Tabancay, 2003).
The eukaryotic translation initiation factor 4B (eIF4B) plays a critical role in recruiting the 40S ribosomal subunit to the mRNA. In response to insulin, eIF4B is phosphorylated on Ser422 by S6K in a rapamycin-sensitive manner. This study demonstrates that the p90 ribosomal protein S6 kinase (RSK; see Drosophila) phosphorylates eIF4B on the same residue. The relative contribution of the RSK and S6K modules to the phosphorylation of eIF4B is growth factor-dependent, and the two phosphorylation events exhibit very different kinetics. The S6K and RSK proteins are members of the AGC protein kinase family, and require PDK1 phosphorylation for activation. Consistent with this requirement, phosphorylation of eIF4B Ser422 is abrogated in PDK1 null embryonic stem cells. Phosphorylation of eIF4B on Ser422 by RSK and S6K is physiologically significant, as it increases the interaction of eIF4B with the eukaryotic translation initiation factor 3 (Shahbazian, 2006).
PGC-1α is a transcriptional coactivator that powerfully regulates many pathways linked to energy homeostasis. Specifically, PGC-1α controls mitochondrial biogenesis in most tissues but also initiates important tissue-specific functions, including fiber type switching in skeletal muscle and gluconeogenesis and fatty acid oxidation in the liver. S6 kinase, activated in the liver upon feeding, can phosphorylate PGC-1α directly on two sites within its arginine/serine-rich (RS) domain. This phosphorylation significantly attenuates the ability of PGC-1α to turn on genes of gluconeogenesis in cultured hepatocytes and in vivo, while leaving the functions of PGC-1α as an activator of mitochondrial and fatty acid oxidation genes completely intact. These phosphorylations interfere with the ability of PGC-1α to bind to HNF4α, a transcription factor required for gluconeogenesis, while leaving undisturbed the interactions of PGC-1α with ERRα and PPARα, factors important for mitochondrial biogenesis and fatty acid oxidation. These data illustrate that S6 kinase can modify PGC-1α and thus allow molecular dissection of its functions, providing metabolic flexibility needed for dietary adaptation (Lustig, 2011).
Growth-promoting signaling molecules, including the mammalian target of rapamycin complex 1 (mTORC1; see Drosophila Tor), drive the metabolic reprogramming of cancer cells required to support their biosynthetic needs for rapid growth and proliferation. Glutamine is catabolyzed to alpha-ketoglutarate (alphaKG), a tricarboxylic acid (TCA) cycle intermediate, through two deamination reactions, the first requiring glutaminase (GLS) to generate glutamate and the second occurring via glutamate dehydrogenase (GDH) or transaminases. Activation of the mTORC1 pathway has been shown previously to promote the anaplerotic entry of glutamine to the TCA cycle via GDH. Moreover, mTORC1 activation also stimulates the uptake of glutamine, but the mechanism is unknown. It is generally thought that rates of glutamine utilization are limited by mitochondrial uptake via GLS, suggesting that, in addition to GDH, mTORC1 could regulate GLS. This study demonstrates that mTORC1 positively regulates GLS and glutamine flux through this enzyme. mTORC1 controls GLS levels through the S6K1-dependent regulation of c-Myc (Myc; see Drosophila Myc). Molecularly, S6K1 enhances Myc translation efficiency by modulating the phosphorylation of eukaryotic initiation factor eIF4B (see Drosophila eIF4B), which is critical to unwind its structured 5' untranslated region (5'UTR). Finally, these data show that the pharmacological inhibition of GLS is a promising target in pancreatic cancers expressing low levels of PTEN (Csibi, 2014).
Phosphoinositide 3-kinase (PI3K) has been shown to regulate cell and organ size in Drosophila, but the role of PI3K in vertebrates in vivo is not well understood. To examine the role of PI3K in intact mammalian tissue, transgenic mice expressing constitutively active or dominant-negative mutants of PI3K in the heart have been created and characterized. Cardiac-specific expression of constitutively active PI3K results in mice with larger hearts, while dominant-negative PI3K results in mice with smaller hearts. The increase or decrease in heart size is associated with comparable increase or decrease in myocyte size. Cardiomyopathic changes, such as myocyte necrosis, apoptosis, interstitial fibrosis or contractile dysfunction, are not observed in either type of transgenic mouse. Thus, the PI3K pathway is necessary and sufficient to promote organ growth in mammals (Shioi, 2000).
What are the downstream targets of PI3K that are involved in the regulation of organ size? The available information does not provide a definitive answer to this question. However, Akt, a well characterized downstream target of PI3K, is likely to be one of the major mediators of this process. Akt is necessary and sufficient for phosphorylation and subsequent inactivation of 4E-BP1, a repressor of mRNA translation. Akt can also activate p70S6K in some contexts, although activation of p70S6K might not be solely dependent on Akt. p70S6K is upregulated in constitutively active PI3K hearts and downregulated in dominant negative PI3K hearts. The amount of phosphorylated S6 protein correlates with the activation of p70S6K. The inhibition of the p70S6K pathway by rapamycin at nanomolar concentrations selectively suppresses an increase in protein synthesis of cultured neonatal myocytes in response to growth factors. Interestingly, rapamycin does not inhibit other phenotypic changes associated with myocyte hypertrophy, such as re-activation of fetal genes and sarcomere organization. This raises the possibility that p70S6K may selectively regulate cell size via controlling the rate of protein synthesis. Gene disruption of p70S6K is known to result in smaller body size in mice. In Drosophila, deficiency of the S6K gene is associated with a reduction in body size associated with smaller cells (Shioi, 2000 and references therein).
The role of the PI3K-p70 S6 kinase (S6K) signaling cascade in the stimulation of endothelial cell proliferation has been examined. Inhibitors of the p42/p44 MAPK pathway (PD98059) and the PI3K-p70 S6K pathway (wortmannin, Ly294002, and rapamycin) all block thymidine incorporation stimulated by fetal calf serum in the resting mouse endothelial cell line 1G11. The action of rapamycin can be generalized, since it completely inhibits the mitogenic effect of fetal calf serum in primary endothelial cell cultures (human umbilical vein endothelial cells) and another established capillary endothelial cell line (LIBE cells). The inhibitory effect of rapamycin is only observed when the inhibitor is added at the early stages of G(0)-G(1) progression, suggesting an inhibitory action early in G(1). Rapamycin completely inhibits growth factor stimulation of protein synthesis, which perfectly correlates with the inhibition of cell proliferation. In accordance with its inhibitory action on protein synthesis, activation of cyclin D1 and p21 proteins by growth factors is also blocked by preincubation with rapamycin. Expression of a p70 S6K mutant partially resistant to rapamycin reverses the inhibitory effect of the drug on DNA synthesis, indicating that rapamycin action is via p70 S6K. Thus, in vascular endothelial cells, activation of protein synthesis via p70 S6K is an essential step for cell cycle progression in response to growth factors (Vinals, 1999).
In T lymphocytes, the hematopoietic cytokine interleukin-2 (IL-2) uses phosphatidylinositol 3-kinase (PI 3-kinase)-induced signaling pathways to regulate E2F transcriptional activity, a critical cell cycle checkpoint. PI 3-kinase also regulates the activity of p70(s6k), the 40S ribosomal protein S6 kinase, a response that is abrogated by the macrolide rapamycin. This immunosuppressive drug is known to prevent T-cell proliferation, but the precise point at which rapamycin regulates T-cell cycle progression has yet to be elucidated. Moreover, the effects of rapamycin on, and the role of p70(s6k) in, IL-2 and PI 3-kinase activation of E2Fs have not been characterized. The present results show that IL-2- and PI 3-kinase-induced pathways for the regulation of E2F transcriptional activity include both rapamycin-resistant and rapamycin-sensitive components. Expression of a rapamycin-resistant mutant of p70(s6k) in T cells can restore rapamycin-suppressed E2F responses. Thus, the rapamycin-controlled processes involved in E2F regulation appear to be mediated by p70(s6k). However, the rapamycin-resistant p70s6k could not rescue rapamycin inhibition of T-cell cycle entry, consistent with the involvement of additional, rapamycin-sensitive pathways in the control of T-cell cycle progression. The present results thus show that p70s6k is able to regulate E2F transcriptional activity and provide direct evidence for the first time for a link between IL-2 receptors, PI 3-kinase, and p70s6k that regulates a crucial G1 checkpoint in T lymphocytes (Brennan, 1999).
High-resistance exercise training results in an increase in muscle wet mass and protein content. To begin to address the acute changes following a single bout of high-resistance exercise, a new model has been developed. Training rats twice a week for 6 wk resulted in 13.9% and 14.4% hypertrophy in the extensor digitorum longus (EDL) and tibialis anterior (TA) muscles, respectively. Polysome profiles after high-resistance lengthening contractions suggest that the rate of initiation is increased. The activity of the 70-kDa S6 protein kinase (p70S6k), a regulator of translation initiation, is also increased following high-resistance lengthening contractions. Furthermore, the increase in p70S6k activity 6 h after exercise correlates with the percent change in muscle mass after 6 wk of training. The tight correlation between the activation of p70S6k and the long-term increase in muscle mass suggests that p70S6k phosphorylation may be a good marker for the phenotypic changes that characterize muscle hypertrophy and may play a role in load-induced skeletal muscle growth (Baar, 1999).
The mammalian target of rapamycin (mTOR) and phosphatidylinositol 3-kinase (PI3K) signaling pathways promote cell growth and cell cycle progression in response to nutritional, energy, and mitogenic cues. In mammalian cells, the ribosomal protein S6 kinases, S6K1 and S6K2, lie downstream of mTOR and PI3K, suggesting that translational control through the phosphorylation of S6 regulates cell growth. Interestingly, genetic experiments predict that a substrate that is specific to S6K1 but not S6K2 regulates cell growth. SKAR has been identified as a novel and specific binding partner and substrate of S6K1 but not S6K2. Serines 383 and 385 of human SKAR are insulin-stimulated and rapamycin-sensitive S6K1 phosphorylation sites. Quantitative mass spectrometry reveals that serine 383/385 phosphorylation is sensitive to RNA interference (RNAi)-mediated S6K1 reduction, but not S6K2 reduction. Furthermore, RNAi-mediated reduction of SKAR decreases cell size. SKAR is nuclear protein with homology to the Aly/REF family of RNA binding proteins, which has been proposed to couple transcription with pre-mRNA splicing and mRNA export. Database searches reveal SKAR homologs in mouse and rat with 91% amino acid identity and a potential homolog in Drosophila but not in lower eukaryotes. This study has identified a novel and specific target of S6K1, SKAR, which regulates cell growth. The homology of SKAR to the Aly/REF family links S6K1 with mRNA biogenesis in the control of cell growth (Richardson, 2004).
The regulated phosphorylation of ribosomal protein (rp) S6 has attracted much attention since its discovery in 1974, yet its physiological role has remained obscure. To directly address this issue, viable and fertile knock-in mice have been established, whose rpS6 contains alanine substitutions at all five phosphorylatable serine residues (rpS6P-/-). Contrary to the widely accepted model, this mutation does not affect the translational control of mRNAs characterized by an oligopyrimidine tract at their 5' terminus (TOP mRNAs) rpS6P-/- mouse embryo fibroblasts (MEFs) display an increased rate of protein synthesis and accelerated cell division, and they are significantly smaller than rpS6P+/+ MEFs. This small size reflects a growth defect, rather than a by-product of their faster cell division. Moreover, the size of rpS6P-/- MEFs, unlike wild-type MEFs, is not further decreased upon rapamycin treatment, implying that the rpS6 is a critical downstream effector of mTOR in regulation of cell size. The small cell phenotype is not confined to embryonal cells, since it also selectively characterizes pancreatic beta-cells in adult rpS6P-/- mice. These mice suffer from diminished levels of pancreatic insulin, hypoinsulinemia, and impaired glucose tolerance (Ruvinsky, 2005).
Enduring forms of synaptic plasticity and memory require new protein synthesis, but little is known about the underlying regulatory mechanisms. The role of MAPK signaling in these processes has been investigated. Conditional expression of a dominant-negative form of MEK1 in the postnatal murine forebrain inhibits ERK activation and causes selective deficits in hippocampal memory retention and the translation-dependent, transcription-independent phase of hippocampal L-LTP. In hippocampal neurons, ERK inhibition blocks neuronal activity-induced translation as well as phosphorylation of the translation factors eIF4E, 4EBP1, and ribosomal protein S6. Correspondingly, protein synthesis and translation factor phosphorylation induced in control hippocampal slices by L-LTP-generating tetanization are significantly reduced in mutant slices. Translation factor phosphorylation induced in the control hippocampus by memory formation is similarly diminished in the mutant hippocampus. These results suggest a crucial role for translational control by MAPK signaling in long-lasting forms of synaptic plasticity and memory (Kelleher, 2004).
Translation of eukaryotic mRNAs is primarily regulated at the level of initiation. Studies in mitotic cells have defined 5' cap recognition and ribosomal recruitment by translation initiation factors as key events in this multistep process. Cap recognition is accomplished by eukaryotic initiation factor 4E (eIF4E), and the eIF4E-associated factor eIF4G then recruits the 40S ribosomal subunit. Cap-dependent translation accounts for the synthesis of the vast majority of cellular proteins, since all mRNAs transcribed in the nucleus bear a 5' cap. The synthesis of the translation machinery itself is additionally regulated by an inhibitory cis-acting element, termed a 5' terminal oligopyrimidine tract (5' TOP), which occurs adjacent to the cap in mRNAs encoding ribosomal proteins and translation factors (Meyuhas, 2000).
Prior work in mitotic cells has identified the ERK-dependent kinase Mnk1 as the major eIF4E kinase, indicating a dominant role for ERK signaling in eIF4E phosphorylation. The complex pattern of inducible 4E-BP1 hyperphosphorylation appears to be mediated primarily by rapamycin-sensitive mTOR-dependent pathways, while some evidence has suggested ERK-dependent modulation of Ser65 phosphorylation. Studies on the mitogen-induced hyperphosphorylation of S6 have delineated a central role for mTOR-dependent activation of S6 kinase. The current findings demonstrate a major role for the ERK pathway in the neuronal activity-induced phosphorylation of S6, eIF4E, and 4E-BP1, with consistently greater effects on eIF4E relative to S6 across all levels of analysis. Interestingly, the phosphorylation of all three factors was also found to be highly sensitive to rapamycin, with the greatest effect on S6. These observations suggest that the ERK and mTOR pathways cooperate in the coordinate regulation of cap-dependent and 5' TOP-dependent translation. Hippocampal L-LTP and serotonin-induced LTF in Aplysia have been shown to be sensitive to rapamycin, implicating mTOR-dependent translation in these processes. Translational efficiency during the establishment of long-term synaptic plasticity and memory may therefore be determined through the functional interplay of ERK- and mTOR-dependent signaling mechanisms. General translational induction of a broad range of neuronal mRNAs by such activity-dependent mechanisms may provide the protein products required for the input-specific 'capture' of long-term synaptic plasticity by 'tagged' synapses (Meyuhas, 2000).
Genetic S6K1 (see Drosophila S6k) inactivation can induce apoptosis in PTEN-deficient cells (see Drosophila Pten). This study analyzed the therapeutic potential of S6K1 inhibitors in PTEN-deficient T cell leukemia and glioblastoma. Results revealed that the S6K1 inhibitor LY-2779964 was relatively ineffective as a single agent, while S6K1-targeting AD80 induced cytotoxicity selectively in PTEN-deficient cells. In vivo, AD80 rescued 50% of mice transplanted with PTEN-deficient leukemia cells. Cells surviving LY-2779964 treatment exhibited inhibitor-induced S6K1 phosphorylation due to increased mTOR-S6K1 (see Drosophila Tor) co-association, which primed the rapid recovery of S6K1 signaling. In contrast, AD80 avoided S6K1 phosphorylation and mTOR co-association, resulting in durable suppression of S6K1-induced signaling and protein synthesis. Kinome analysis revealed that AD80 coordinately inhibits S6K1 together with the TAM family tyrosine kinase AXL. TAM suppression by BMS-777607 or genetic knockdown potentiated cytotoxic responses to LY-2779964 in PTEN-deficient glioblastoma cells. These results reveal that combination targeting of S6K1 and TAMs is a potential strategy for treatment of PTEN-deficient malignancy (Liu, 2017).
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