Nutrient overload induces constitutive S6K1 (S6 kinase 1) activation, which leads to insulin resistance by suppressing insulin-induced class I PI3K (phosphoinositide 3-kinase) signalling [Um, Frigerio, Watanabe, Picard, Joaquin, Sticker, Fumagalli, Allegrini, Kozma, Auwerx and Thomas (2004) Nature 431, 200-205]. This finding gave rise to the question of the mechanism by which nutrients, such as AAs (amino acids), enter the mTOR (mammalian target of rapamycin)/S6K1 signalling pathway. Counter to the prevailing view, our recent studies have shown that the AA input into the mTOR/S6K1 signalling pathway is not mediated by the tumour suppressor TSC1 (tuberous sclerosis complex 1)/TSC2 or its target, the proto-oncogene Rheb (Ras homologue enriched in brain). Instead, we found that the AA input was mediated by class 3 PI3K, or hVps34 (human vacuolar protein sorting 34). In brief, ectopic expression of hVps34 drives S6K1 activation, but only in the presence of AAs, and this effect is blocked by small interfering RNAs directed against hVps34. Moreover, stimulation of cells with AAs increases hVps34 activity, as indicated by the production of PI3P (phosphatidylinositol 3-phosphate). PI3P mediates the recruitment of proteins containing FYVE (Fab1p, YOTB, Vac1p and EEA1) or PX (Phox homology) domains to endosomal membranes, with PI3P-rich micro-domains acting as signalling platforms. Additional evidence indicating hVps34 as the mediator of AA input to S6K1 came from experiments in which S6K1 activation was attenuated by ectopic expression of a cDNA containing two FYVE domains, which bind to PI3P, preventing binding of proteins containing either FYVE or PX domains [Nobukuni, Joaquin, Roccio, Dann, Kim, Gulati, Byfield, Backer, Natt, Bos, Zwartkruis and Thomas (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 14238-14243].

The mammalian Target of Rapamycin (mTOR) protein is a serine-threonine kinase that regulates cell-cycle progression and growth by sensing changes in energy status. We demonstrated that mTOR signaling plays a role in the brain mechanisms that respond to nutrient availability, regulating energy balance. In the rat, mTOR signaling is controlled by energy status in specific regions of the hypothalamus and colocalizes with neuropeptide Y and proopiomelanocortin neurons in the arcuate nucleus. Central administration of leucine increases hypothalamic mTOR signaling and decreases food intake and body weight. The hormone leptin increases hypothalamic mTOR activity, and the inhibition of mTOR signaling blunts leptin's anorectic effect. Thus, mTOR is a cellular fuel sensor whose hypothalamic activity is directly tied to the regulation of energy intake.

During the evolution of metazoans and the rise of systemic hormonal regulation, the insulin-controlled class 1 phosphatidylinositol 3OH-kinase (PI3K) pathway was merged with the primordial amino acid-driven mammalian target of rapamycin (mTOR) pathway to control the growth and development of the organism. Insulin regulates mTOR function through a recently described canonical signaling pathway, which is initiated by the activation of class 1 PI3K. However, how the amino acid input is integrated with that of the insulin signaling pathway is unclear. Here we used a number of molecular, biochemical, and pharmacological approaches to address this issue. Unexpectedly, we found that a major pathway by which amino acids control mTOR signaling is distinct from that of insulin and that, instead of signaling through components of the insulin/class 1 PI3K pathway, amino acids mediate mTOR activation by signaling through class 3 PI3K, hVps34.

Addition of fresh medium to stationary cells of Arabidopsis suspension culture induces increased phosphorylation of the S6 ribosomal protein and activation of its cognate kinase, AtS6k. Analysis of the activation response revealed that medium constituents required for S6 kinase activation were the phytohormones 1-naphthylacetic acid (auxin) and kinetin. Pretreatment of cells with anti-auxin or PI3-kinase drugs inhibited this response. Consistent with these findings, LY294002, a PI3-kinase inhibitor, efficiently suppressed phytohormone-induced S6 phosphorylation and translational up-regulation of ribosomal protein S6 and S18A mRNAs without affecting global translation. These data indicate that (1) activation of AtS6k is regulated by phytohormones, at least in part, via a lipid kinase-dependent pathway, that (2) the translational regulation of ribosomal proteins appears to be conserved throughout the plant and animal kingdom, and that (3) these events are hallmarks of a growth-related signal transduction pathway novel in plants.

Earlier studies have shown that mTOR plays a key role in ribosome biogenesis. In bacteria, amino acids and ATP levels independently control ribosome biogenesis. Here, we describe recent findings demonstrating that homeostatic levels of amino acids, most notably branched-chain amino acids, and ATP, independently regulate the activity of mTOR. Unlike the effects of amino acids, the effects of ATP appear to be direct. Based on these findings we propose a model by which tumor cells existing in the anaerobic environment may have an advantage in growth by exploiting the rapid, although less efficient, production of ATP to drive growth via the mTOR signaling pathway.

Ribosomal S6 kinases (S6Ks) have been depicted as critical effectors downstream of growth factor pathways, which play an important role in the regulation of protein synthesis by phosphorylating the ribosomal protein, S6. The goal of this study was to determine whether S6Ks regulate heart size, are critical for the induction of cardiac hypertrophy in response to a pathological or physiological stimulus, and whether S6Ks are critical downstream effectors of the insulin-like growth factor 1 (IGF1)-phosphoinositide 3-kinase (PI3K) pathway. For this purpose, we generated and characterized cardiac-specific S6K1 and S6K2 transgenic mice and subjected S6K1(-/-), S6K2(-/-), and S6K1(-/-) S6K2(-/-) mice to a pathological stress (aortic banding) or a physiological stress (exercise training). To determine the genetic relationship between S6Ks and the IGF1-PI3K pathway, S6K transgenic and knockout mice were crossed with cardiac-specific transgenic mice overexpressing the IGF1 receptor (IGF1R) or PI3K mutants. Here we show that overexpression of S6K1 induced a modest degree of hypertrophy, whereas overexpression of S6K2 resulted in no obvious cardiac phenotype. Unexpectedly, deletion of S6K1 and S6K2 had no impact on the development of pathological, physiological, or IGF1R-PI3K-induced cardiac hypertrophy. These studies suggest that S6Ks alone are not essential for the development of cardiac hypertrophy.

The mitogen-stimulated protein kinase p70 ribosomal protein S6 kinase 1 (S6K1) is a key enzyme in the regulation of cell growth and proliferation. Activation of S6K1 requires a complex, ordered series of conformational changes and phosphorylation reactions. While the role of sequential, multi-site phosphorylation has been extensively detailed, characterization of the priming step required to initiate this cascade has remained elusive. In the present study we show for the first time that this priming process is dependent on calcium. Calcium-dependent regulation of S6K1 did not specifically target Thr-229 and Thr-389, the key regulatory phosphorylation sites; rather, calcium chelation resulted in a global inhibition of S6K1 phosphorylation. Mutation of individual phosphorylation sites in the auto-inhibitory and hydrophobic domains to acidic residues (to mimic phosphorylation) yields a kinase that remains sensitive to calcium chelation, while the combined mutations alleviate the requirement for calcium. Furthermore, deletion of the C-terminal residues (398-502) also renders the kinase insensitive to calcium. We hypothesize that the initial calcium-dependent process is required to release an inhibitory interaction between the C- and N-termini of S6K1, thus allowing phosphorylation of these key domains. The requirement for this priming step can only be overcome by mutations mimicking the phosphorylation of both the auto-inhibitory and hydrophobic domains. We further propose that the priming event involves formation of a calcium-dependent protein complex that releases the interaction between the N- and C-termini. S6K1 is then accessible for activation by the kinases that target the known regulatory phosphorylation sites. Consistent with this hypothesis, serum stimulation of S6K1 activity is associated with its incorporation into a calcium-dependent high-molecular-mass complex.

Insulin inhibits the expression of the hepatic insulin-like growth factor-binding protein-1 (IGFBP-1) and glucose-6-phosphatase (G6Pase) genes. The signaling pathway that mediates these events requires the activation of phosphatidylinositol 3-kinase, whereas transfection studies have suggested an involvement of Akt (protein kinase B) and FKHR, a transcription factor regulated by Akt. We now demonstrate that insulin repression of endogenous IGFBP-1 gene transcription was blocked by rapamycin or by amino acid starvation. Rapamycin inhibited the mammalian target of rapamycin (mTOR) and the subsequent activation of p70/p85 S6 protein kinase-1 (S6K1) by insulin, whereas amino acid depletion prevented insulin induction of these signaling molecules. Importantly, we demonstrate that insulin regulation of the thymine-rich insulin response element of the IGFBP-1 promoter was also inhibited by rapamycin. However, sustained activation of S6K1 did not repress this promoter. In addition, rapamycin did not affect insulin regulation of G6Pase expression or Akt activation. We propose that these observations indicate that an mTOR-dependent, but S6K-independent mechanism regulates the suppression of IGFBP-1 (but not G6Pase) gene expression by insulin. Therefore, although the insulin-responsive sequence of the G6Pase gene promoter is related to that of the IGFBP-1 promoter, the signaling pathways that mediate suppression of these genes are distinct.

A critical step in S6 kinase 1 (S6K1) activation is Thr(229) phosphorylation in the activation loop by the phosphoinositide-dependent protein kinase (PDK1). Thr(229) phosphorylation requires prior phosphorylation of the Ser/Thr-Pro sites in the autoinhibitory domain and Thr(389) in the linker domain, consistent with PDK1 more effectively catalyzing Thr(229) phosphorylation in a variant harboring acidic residues in these positions (S6K1-E389D(3)E). S6K1-E389D(3)E has high basal activity and exhibits partial resistance to rapamycin and wortmannin, and its activity can be further augmented by mitogens, effects presumably mediated by Thr(229) phosphorylation. However, PDK1-induced Thr(229) phosphorylation is reported to be constitutive rather than phosphatidylinositide 3,4,5-trisphosphate-dependent, suggesting that S6K1-E389D(3)E activity is mediated through a distinct site. Here we use phosphospecific antibodies to show that Thr(229) is fully phosphorylated in S6K1-E389D(3)E in the absence of mitogens and that regulation of S6K1-E389D(3)E activity by mitogens, rapamycin, or wortmannin parallels Ser(371) phosphorylation. Consistent with this observation, a dominant interfering allele of the mammalian target of rapamycin, mTOR, inhibits mitogen-induced Ser(371) phosphorylation and activation of S6K1-E389D(3)E, whereas wild type mTOR stimulates both responses. Moreover, in vitro mTOR directly phosphorylates Ser(371), and this event modulates Thr(389) phosphorylation by mTOR, compatible with earlier in vivo findings.

The genes that encode the proteins composing the tuberous sclerosis complex (TSC) are tumor suppressors. Experiments in the model organism Drosophila melanogaster have provided insight into the identity of these genes and their functions in regulating cell size and proliferation. Montagne et al . describe the various genetic interactions that show TSC to be a regulator of the insulin signaling pathway and a regulator of progression through the cell cycle, which explains its effects on cell size and tissue and tumor growth.

The bacterial macrolide rapamycin is an efficacious anticancer agent against solid tumors. In a hypoxic environment, the increase in mass of solid tumors is dependent on the recruitment of mitogens and nutrients. When nutrient concentrations change, particularly those of essential amino acids, the mammalian Target of Rapamycin (mTOR) functions in regulatory pathways that control ribosome biogenesis and cell growth. In bacteria, ribosome biogenesis is independently regulated by amino acids and adenosine triphosphate (ATP). Here we demonstrate that the mTOR pathway is influenced by the intracellular concentration of ATP, independent of the abundance of amino acids, and that mTOR itself is an ATP sensor.

Meeting Report1 July 2000free access The ins and outs of protein phosphorylation Workshop report: Control of signaling by protein phosphorylation George Thomas George Thomas Friedrich Miescher Institute, Maulbeerstrasse 66, 4058 Basel, Switzerland Search for more papers by this author Flora de Pablo Flora de Pablo Centro de Investigaciones Biológicas (CSIC), Velázquez 144, 28006 Madrid, Spain Search for more papers by this author Joseph Schlessinger Joseph Schlessinger Department of Pharmacology and Skirball Institute, New York University Medical School, New York, NY, 10016 USA Search for more papers by this author Jorge Moscat Corresponding Author Jorge Moscat Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain Search for more papers by this author George Thomas George Thomas Friedrich Miescher Institute, Maulbeerstrasse 66, 4058 Basel, Switzerland Search for more papers by this author Flora de Pablo Flora de Pablo Centro de Investigaciones Biológicas (CSIC), Velázquez 144, 28006 Madrid, Spain Search for more papers by this author Joseph Schlessinger Joseph Schlessinger Department of Pharmacology and Skirball Institute, New York University Medical School, New York, NY, 10016 USA Search for more papers by this author Jorge Moscat Corresponding Author Jorge Moscat Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain Search for more papers by this author Author Information George Thomas1, Flora de Pablo2, Joseph Schlessinger3 and Jorge Moscat 4 1Friedrich Miescher Institute, Maulbeerstrasse 66, 4058 Basel, Switzerland 2Centro de Investigaciones Biológicas (CSIC), Velázquez 144, 28006 Madrid, Spain 3Department of Pharmacology and Skirball Institute, New York University Medical School, New York, NY, 10016 USA 4Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain *Corresponding author. Tel: +34 91 397 8039; Fax: +34 62 969 0055; E-mail: [email protected] EMBO Reports (2000)1:11-15https://doi.org/10.1093/embo-reports/kvd014 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The Centre for International Meetings on Biology was established in 1992 as a component of a private foundation: Fundación Juan March. Every year, it organizes a range of workshops and scientific activities covering different aspects of modern biology. At these meetings, scientists actively involved in the different fields present their latest results in a meeting format with about 22 invited speakers and a relatively small number of participants. This promotes discussion and a rich exchange of results and ideas. Introduction Dissecting the interrelationships among components of the molecular signaling pathways involved in the control of key physiological processes such as cell growth, differentiation, survival and development is a timely issue in biology. The activation of these pathways is generally triggered at the plasma membrane by specific agonists acting through their cognate receptors. The activated receptors in turn initiate signaling cascades that ultimately control the activities of specific intracellular effectors. Much of the regulation of these pathways is achieved by manipulating the phosphorylation states of various components of these cascades. The kinases and phosphatases that mediate this control were the topic of a recent Juan March Workshop held in Madrid (March 13–15, 2000). It became clear during this meeting that the vast amounts of data generated from cell culture experiments must be validated in more complex in vivo systems to determine the contribution of each signaling pathway to a given function. Mitogenic and survival signaling Many of the talks at this meeting revolved around the roles of the phosphoinositide-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways that participate downstream of receptor tyrosine kinases in regulating cell growth, proliferation and survival. An illustration of how these pathways are thought to branch from the receptor kinase, and the output of each branch is shown in Figure 1. Figure 1.Pathways controlling cell cycle progression and survival. Two signaling cascades emanate from the IRS molecules. One may signal survival, cell growth, and cell cycle progression through the PI3K branch and another, which constitutes a cooperative or overlapping pathway, operates through the MAPK cascade. The former can be down-regulated by the lipid phosphatase PTEN and acts independently of the MAPK pathway. Its effects on S6K1 regulate the translational machinery, influencing cell growth and cell division. The MAPK pathway may use different CREB kinases in a context-specific manner. Download figure Download PowerPoint The role of the PI3K pathway in mitogenic signaling was discussed by several speakers. T. Hunter (La Jolla, CA) showed that mice harboring a c-Kit receptor point mutation that prevents docking and activation of PI3K, but does not affect signaling mediated by other effectors, had normal hematopoiesis and pigmentation, although these processes are aberrant in c-Kit-deficient mice. Instead, loss of PI3K activation alone resulted in defects in spermatogonial stem cell survival. These results indicate that the PI3K-mediated component of c-Kit signaling is essential for effecting only a subset of c-Kit-mediated responses. L. Cantley (Boston, MA) discussed the role of PI3K in insulin signaling based on experiments in which the mouse p85α subunit of PI3K was deleted. Mice deficient for this protein mostly died at birth, but outbreeding the few surviving mice resulted in 20–30% viability. Such mice were hypoglycemic and had low levels of circulating insulin, but were hypersensitive for glucose uptake and Glut4 translocation in peripheral tissues. Cantley suggested that this hypersensitivity could result from breaking a negative feedback loop that might normally desensitize insulin receptor substrates (IRSs), the link between the insulin receptor and PI3K. Further downstream along the PI3K pathway lie phosphoinositide-dependent protein kinase (PDK)-1, protein kinase B (PKB, also known as Akt) and p70 ribosomal S6 kinase (S6K)-1. Several talks focused on the relationships among these molecules. G. Thomas (Basel, Switzerland) presented data from a genetic screen in Drosophila, which his group is using to search for effectors of S6K1. Results from their studies are consistent with the idea that S6K1 acts independently of PKB, with the two proteins representing a bifurcation of the PI3K pathway downstream of PDK1 (Figure 1). PDK1 has been proposed to be a key regulator of ribosomal subunit kinase (RSK) proteins as well as of S6K1 and PKB. The report by P. Cohen (Dundee, UK) that mouse ES cells homozygous mutant for PDK1 are viable and have normal growth rates was therefore unexpected, especially in light of the fact that PKB, S6K1 and all RSK family proteins fail to become activated in these cells. These cells showed robust CREB phosphorylation in the absence of RSK (shown to be the CREB kinase in other systems) activity, whereas ES cells lacking mitogen- and stress-activated protein kinase (MSK)-1 failed to phosphorylate CREB, even in the presence of active RSK. It therefore appears that in this system MSK1, rather than RSK, is the CREB kinase. Whether or not these results simply reflect cell type specificity awaits further study. Consistent with prevailing knowledge of the PI3K pathway, MAPK signaling was unaffected in the PDK1-deficient ES cells. In contrast to the data presented above, evidence presented by E. Hafen (Zürich, Switzerland) implied cross-talk between the MAPK and PI3K pathways. A modifier screen in Drosophila identified a line overexpressing PDK1 as a suppressor of phenotypes generated by mutations that upregulate the MAPK pathway. Hafen went on to report that flies deficient for dPDK1 are smaller in size, as is the case for the IRS loss-of-function phenotype previously described by Hafen's group. Hence, PDK1 seems to have some role in growth regulation. The presentation of M. Cobb (Dallas, TX) focused on the MAPK branch of the pathway. She reported on the effects of constitutively active mutants of one MAPK, the extracellular signal-regulated kinase (ERK)-2, in pancreatic β cell signaling. Activated ERK2 induces insulin transcription, whereas dominant-negative variants of ERK2 or specific inhibitors of the MAPK signaling pathway block this response. Using these tools, Cobb's group identified Beta2/NeuroD1, a transcription factor known to bind to the insulin promoter, as a direct ERK2 target involved in glucose-induced insulin transcription. E. Van Obberghen (Nice, France) reported that the activated insulin receptor recruits the protein signal transducer and activator of transcription (Stat)-5b, in addition to its other targets. This recruitment leads to activation and nuclear translocation of Stat5b, and induction of a number of genes containing Stat5b recognition motifs. In parallel, Stat5b activates suppressor of cytokine signaling (SOCS)-3, which binds to the same site on the insulin receptor, potentially providing an elegant feedback mechanism for regulating insulin signaling. F. de Pablo (Madrid, Spain) reported that regulated expression of proinsulin mRNA occurs prior to the expression of insulin-like growth factor (IGF)-1 during chick embryo neurulation and retinal neurogenesis. She went on to explain that insulin/IGF-1 hybrid receptors bind proinsulin and insulin with high affinity during early neural development, and treatment with antisense oligonucleotides or antibodies against proinsulin/insulin or the insulin receptor, but not against IGF-1, results in increased apoptosis in the locations in the neuroretina or the neurulating embryo where it occurs naturally. Together, these findings demonstrate that insulin, most likely in the form of proinsulin, signals prior to IGF-1, in survival pathways used during development. C. Marshall (London, UK) discussed the connection between cell cycle progression and early mitogenic signaling. He reported that the lack of the cell cycle regulator pRb105 significantly reduces the requirement for ERK during S-phase entry, suggesting a role for this MAPK in the activation of cyclin D-dependent kinases (cdks) that phosphorylate and inactivate pRb105. Furthermore, blocking the ERK pathway inhibits the activation of cdk4–cyclinD1 complexes, but not of cdk2–cyclinE complexes. The cell cycle was also covered by M. Barbacid (Madrid, Spain), who reported that mice deficient for cdk4 develop insulin-dependent diabetes. In contrast, those harboring activated alleles overproduce insulin. However, these defects are probably not directly related to insulin signaling, but rather result from proliferation defects during pancreatic development. J. Downward (London, UK) focused on the role of the MAPK pathway in cell survival. He showed that apoptosis induced by either transforming growth factor (TGF)-β or tumor necrosis factor (TNF)-α can be blocked by c-Raf. The precise mechanism whereby c-Raf is able to block apoptosis induced by such different stimuli remains unclear, but it must affect a common step in the two pathways. These studies further suggest that ERK, which is downstream of c-Raf, is an anti-apoptotic kinase, as has long been proposed by others. The above constitutes a large body of complex data. However, an important generalization emerges from it. In different cell systems, mitogenic signaling through a single receptor type can be mediated by different pathways acting in concert. The variations between systems may reflect context specificity, or possibly feedback loops. Studies combining vertebrate animal models such as knock-out mice with genetically simpler systems like Drosophila and Caenorhabditis elegans, as well as experiments in cell lines, will provide a better understanding of the real contribution of each pathway. Cytokine signal transduction Various talks centered on paradigms for cytokine signaling, with many focusing on the regulation of NF-κB activation. Figure 2 illustrates how a number of signaling components and adapters are thought to regulate the phosphorylation and degradation of IκB to release NF-κB from its sequestered, cytosolic state, ultimately allowing for its nuclear translocation. The kinase that phosphorylates IκB (IKK) consists of two catalytic subunits (IKKα and IKKβ) and an essential adapter protein (IKKγ or Nemo). Figure 2.Different signaling adapters involved in IKK and NF-κB activation. The signaling pathways that activate NF-κB in response to IL-1 or TNF-α involve functionally similar proteins. The TNFR1 recruits TRADD and TRAF2, along with RIP, which interacts with the inhibitory protein RIP3 and the adapter p62. The activation of IL-1 leads to the recruitment of MyD88 and IRAK, which together with TRAF6 are essential components in the activation of the IKK signaling complex. The IKK signalsome can be also activated by PMA, most probably through a classical PKC isoform such as αPKC, and by dsRNA through PKR. Download figure Download PowerPoint M. Karin (La Jolla, CA) showed data that suggest that IKKα is not essential for IKK activity. IKK activity is intact in mice deficient for IKKα, whereas the activation of both IKK and NF-κB is abolished by the deletion of IKKβ. Indeed, the phenotype of IKKβ-deficient mice resembles that of mice deficient for RelA, a critical component of the NF-κB complex. Karin went on to show that the double-stranded RNA activated kinase, PKR, is able to activate IKK in vitro and in vivo even when devoid of enzymatic activity. This raises the possibility that phosphorylation of IKK may not be necessary for its activation. J. Moscat (Madrid, Spain) reported that the overexpression of ζ-protein kinase C (ζPKC) is sufficient to activate IKKβ. However, in contrast to activation by PKR, activation by ζPKC is phosphorylation dependent, as a ζPKC kinase-inactive dominant interfering mutant severely impairs IKKβ activation. Moscat further showed that the ζPKC-interacting protein, p62, is required for the activation of NF-κB by TNF-α and interleukin (IL)-1. The activation of IKK by TNF-α was inhibited in p62-deficient cells, whereas ERK activation by epidermal growth factor (EGF) was unaffected, demonstrating that p62 activation has a restricted target range and may be specific to the NF-κB pathway. Interestingly, p62 binds to both receptor interacting protein (RIP) and tumor necrosis factor receptor-associated factor (TRAF)-6, two important intermediaries of TNF and IL-1 signaling, respectively. These interactions allow the two pathways to feed into NF-κB signaling. V. Dixit (San Francisco, CA) discussed another level of control that is mediated by RIP3, a RIP homolog which is a potent inhibitor of NF-κB activation. RIP3 interacts with the intermediary domain of RIP, and might thereby displace an effector of the NF-κB pathway, perhaps p62. The fact that PKR does not require its enzymatic activity to activate IKK and that it can dimerize in response to dsRNA, suggests that oligomerization of the different adapters may be sufficient to trigger activation of the IKK complex. Indeed, RIP, TRAF6, p62 and the atypical PKCs all have the capacity to dimerize and to form large oligomeric complexes. The IL-1 receptor-associated kinase (IRAK) and RIP are also examples of kinases that activate NF-κB independently of their enzymatic activity. The theme of cytokine signaling was continued by J. Ihle (Memphis, TN), who discussed erythropoietin (Epo) signaling through the Janus kinase (JAK)–Stat5 pathway. While JAK2-deficient mice are embryonic lethal and exhibit failure of fetal liver erythropoiesis, Stat5a/b-deficient mice were found to have normal erythropoiesis in spite of exhibiting other defects. Potential roles of the proto-oncogene myc and the anti-apoptotic factor Bcl-Xl as downstream targets of JAK2 were explored. The findings that Epo fails to induce Bcl-Xl in JAK2-deficient cells, and that ectopic expression of Bcl2 in JAK2-deficient cells blocks apoptosis and rescues fetal liver erythropoiesis in JAK2-deficient mice, suggest that JAK2 normally induces Bcl-Xl in a Stat5-independent manner. The presentation by Ihle is a nice example of how the combination of different in vivo animal models gives the most definitive characterization of the relative roles of different signaling components in a given process. Regarding the NF-κB pathway, it is clear that different signaling proteins must be recruited to the TNF receptor complex to activate the IKK conglomerate. A key issue now is the identification of the proximal cause of IKK activation. Whether this relies on kinase activation, on the multimerization of adapter proteins, or on a combination of both events awaits further studies in which various knock-out mice are crossbred, and endogenous signaling complexes analyzed in cytokine-activated cells derived from these animals. Development and differentiation The theme of developmental biology was the central issue in the following presentations. The first, by M. Greenberg (Cambridge, MA), focused on a primary cerebellar granule cell culture system used to analyze the role of ephrin B receptors in the formation of excitatory synapses. Oligomerized, soluble ephrin (Eph) B1 stimulated Eph B2 receptor clustering, followed by NMDA receptor-1 clustering. This stimulatory effect depends on the extracellular domain of the Eph B2 receptor rather than on kinase activity, since a kinase-dead receptor or even a construct lacking the cytoplasmic domain still induced clustering. However, receptor kinase activity was not entirely dispensable for the function of this molecule, as it was still found to be essential for promoting synapse formation. This example illustrates the importance of understanding the systems that are used to tease apart complex signaling pathways well enough to design the appropriate assays for testing a given hypothesis. Another talk that highlighted the central importance of phosphorylation to signaling processes involved in developmental regulation was presented by E. Wieschaus (Princeton, NJ). Wieschaus discussed a genetic approach for the identification of zygotic genes required for the mid-blastula transition during Drosophila embryogenesis. The main characteristic of this process is the ordered cellularization of a multinuclear syncytium. A systematic screening of embryos deficient for chromosomal segments identified a gene encoding a protein kinase whose expression must be downregulated for the mid-blastula transition to occur. Wieschaus also discussed work on the Armadillo (Arm)/β-catenin pathway. In Drosophila, Arm functions both as a component of the adhesive junctions and as a transcription factor. Its levels are upregulated by Wnt signaling and its vertebrate homologs are downregulated by the adenomatous polyposis coli protein (APC). The nuclear accumulation of Arm plays an important role in mediating Wnt signaling. Again employing a genetic approach, Wieschaus has identified two Drosophila homologs of APC that affect the nuclear localization of Arm. Eric Wieschaus Novel structure–function relationships At the molecular level, kinases and phosphatases share many common attributes. However, they also have distinct properties that explain their selective modes of action. S. Harrison (Cambridge, MA) explored this issue by comparing the X-ray structure of the Ser/Thr kinase p21-activated kinase (Pak)-1 with that of Src. In both proteins, the inactive state is characterized by regions of the polypeptide chain outside of the kinase domain stabilizing a conformation of the small lobe of the kinase that disrupts the catalytic site. In Src, an SH2 domain binds to a tyrosine residue in the C-terminal tail of the protein, and an SH3 domain binds to a proline-containing linker region connecting the SH2 domain to the protein tyrosine kinase core. In Pak1, the kinase domain is maintained in an inactive state by dimerization of CRIB domains. This CRIB domain is regulated by Pak1 activators and, consistently, its structure is similar to that of the corresponding region in WASP, which leads to regulation by its binding partners. J. Dixon (Ann Arbor, MI) turned the discussion to phosphatases with a talk overviewing the recent structural analysis of the tumor suppressor protein PTEN. This protein functions as a phosphatase for 3-phosphorylated phosphoinositides such as PtdIns(3,4,5)P3 (PIP3). Variation in the levels of PTEN expression and changes in its phosphatase activity regulate PKB function and ultimately control cell survival. Dixon described the crystal structure of PTEN, comparing its active site to those of protein tyrosine phosphatases and dual-specificity phosphatases. He explained that the active site of PTEN is modified to accommodate the lipid moiety of PIP3. Structures like that presented here provide a general model for understanding the specificities of phosphatases towards their cellular substrates. J. Schlessinger (New York, NY) focused on critical events upstream of kinases and phosphatases that set the stage for their activation. In one example, he described the crystal structure of fibroblast growth factor (FGF)-2 in complex with the extracellular ligand binding domain of FGF receptor (FGFR)-1. This structure shows that FGF has primary and secondary binding sites for FGFR, and that receptor dimerization is stabilized by both receptor–receptor interactions and the binding of heparin sulfate proteoglycans to a positively charged canyon of exposed basic residues. This structural information may provide a framework for understanding the deleterious effects of FGFR mutations that lead to numerous forms of human skeletal disorders. Conclusion In conclusion, this meeting addressed the most recent evidence regarding the roles of kinases and phosphatases, and the mechanisms whereby they function during cell signaling. It is becoming increasingly apparent that much redundancy and cross-talk exists between separate pathways. Also, there is some evidence that enzymatic activity may not always be necessary for a particular kinase protein to carry out all of its functions. The use of genetic models, including mutant and transgenic mice and flies, is helping to determine which of the known components in the different signaling cascades are essential for a given function, and is proving useful for finding novel, and often unanticipated, roles for known signaling molecules. Biography George Thomas, Flora de Pablo, Joseph Schlessinger and Jorge Moscat References Datta S.R., Brunet A. and Greenberg M.E. (1999) Cellular survival: a play in three Akts. Genes Dev., 13, 2905–2927.CrossrefCASPubMedWeb of Science®Google Scholar Edgar B.A. (1999) From small flies come big discoveries about size control. Nature Cell Biol., 1, E191–E193.CrossrefCASPubMedWeb of Science®Google Scholar Hunter T. (2000) Signaling-2000 and beyond. Cell, 100, 113–127.CrossrefCASPubMedWeb of Science®Google Scholar Rothwarf D.M. and Karin M. (1999) The NF-κB activation pathway: a paradigm in information transfer from membrane to nucleus. Science's STKE, 26 October 1999 (http://www.stke.org).Google Scholar Previous ArticleNext Article Volume 1Issue 11 July 2000In this issue FiguresReferencesRelatedDetailsLoading ...

Because ribosome biogenesis plays an essential role in cell proliferation, control mechanisms may have evolved to recognize Lesions in this critical anabolic process. To test this possibility, we conditionally deleted the gene encoding 40S ribosomal protein S6 in the Liver of adult mice. Unexpectedly, Livers from fasted animals deficient in S6 grew in response to nutrients even though biogenesis of 40S ribosomes was abolished. However, Liver cells failed to proliferate or induce cyclin E expression after partial hepatectomy, despite formation of active cyclin D-CDK4 complexes. These results imply that abrogation of 40S ribosome biogenesis may induce a checkpoint control that prevents cell cycle progression.

The developmental process of an organism can be considered as two intricate programs, one of which controls the correct patterning of the various organs and the other which regulates the growth of the constitutive tissues. The latter integrates cell growth and proliferation so that mature differentiated cells have an appropriate size. Cell growth depends on the activation of protein synthesis and has been shown to be dominant over cell proliferation, as a cell needs to reach a certain size before it divides. During late G1, cells become committed to complete the cell cycle. In yeast, this restriction point is called START [1]. The number of ribosomes act as a potential sensor for triggering START, as yeast studies have shown that the level of ribosomes is critical for the translation of the START-specific cyclin Cln3 [2]. In mammalian cells, the activation of the 40S ribosomal protein S6 kinase, S6K, has been demonstrated to mediate the translational upregulation of components of the protein synthetic apparatus (See Jaschke and Thomas, this volume), placing the activation of the S6K as a key component in the regulation of cell growth. To analyze the role of the S6K in the whole organism, two similar approaches have provided valuable insights. Firstly, the deletion of the murine S6K1 gene emphasized S6K1's role in growth control, however the discovery of a second compensatory homologous gene product, S6K2, meant that, as yet, the phenotype of a complete murine S6K loss-of-function phenotype has not been determined. Secondly, cloning of the homologous Drosophila S6K gene, has allowed the identification of mutants. These mutants are delayed in development and their body size is significantly reduced due to a reduction in cell size and not in cell number. These results indicate that S6K integrates the control of cell growth with cell size.

In recent times, interest in how signal transduction pathways, which control cell growth, integrate, with those, that control cell cycle progression has intensified. Upregulation of the activity of the translational machinery is a necessary step in both cell growth and cell proliferation (see Montagne et al., this volume). The 40S ribosomal protein S6 kinase (S6K), through phosphorylation of its substrate, has been implicated in the regulation of the translation of 5'TOP mRNAs, a family of transcripts encoding mainly for components of the protein synthetic apparatus. Inhibition of mitogen-induced S6K1 activation in vivo with either neutralising antibodies or by treatment with the immunosuppressant rapamycin inhibits G1 progression [1,2]. Cloning of S6K from mice revealed two isoforms. A shorter, cytoplasmic form (p70(S6K)) and a larger nuclear form (p85(S6K)) [3]. Both isoforms are generated from alternative translation start sites from the same transcript and appear to be largely co-ordinately regulated. Homozygous disruption of the S6K 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 S6K deficient mice proceeds normally in response to mitogen stimulation. Furthermore serum induced S6 phosphorylation and the translational upregulation of 5'TOP mRNAs, was as sensitive to the inhibitory effects of the immunosuppressant rapamycin in mouse embryo fibroblasts derived from S6K deficient mice as from wild-type mice. The observation that deletion of the S6K gene in mice did not impair either S6 phosphorylation or 5'TOP mRNA translation, led to the identification of a second S6 kinase gene which is highly homologous to the first gene [4-6]. For simplicity p70(S6K)/p85(S6K) and the new S6 kinase gene have been termed S6K1 and S6K2, respectively. Both isoforms appear to be ubiquitously expressed and in S6K1 deficient mice the S6K2 gene is upregulated in all tissues examined, especially in thymus, a main target of rapamycin action. This lecture will mainly focus on the activation mechanism of S6K1, its role in translation and the pathways regulating its activity.

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. In this review we set out to discuss the present state of knowledge regarding upstream signaling components which have been implicated in the control of S6K1 activation and the role of the kinase in controlling cell growth through regulating ribosome biogenesis at the translational level.

Cell proliferation requires cell growth; that is. cells only divide after they reach a critical size. However, the mechanisms by which cells grow and maintain their appropriate size have remained elusive. Drosophila deficient in the S6 kinase gene (dS6K) exhibited an extreme delay in development and a severe reduction in body size. These flies had smaller cells rather than fewer cells. The effect was cell-autonomous, displayed throughout Larval development, and distinct from that of ribosomal protein mutants (Minutes). Thus, the dS6K gene product regulates cell size in a cell-autonomous manner without impinging on cell number.

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. Our 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 could 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 p70(s6k) 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 p70(s6k) 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 p70(s6k) that regulates a crucial G1 checkpoint in T lymphocytes.

Recent studies have shown that the p70(s6k)/p85(s6k) 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. Here we demonstrate that homozygous disruption of the p70(s6k)/p85(s6k) gene does not affect viability or fertility of mice, but that it has a significant effect on animal growth, especially during embryogenesis. Surprisingly, S6 phosphorylation in liver or in fibroblasts from p70(s6k)/p85(s6k)-deficient mice proceeds normally in response to mitogen stimulation. Furthermore, serum-induced S6 phosphorylation and translational up-regulation of 5'TOP mRNAs were equally sensitive to the inhibitory effects of rapamycin in mouse embryo fibroblasts derived from p70(s6k)/p85(s6k)-deficient and wild-type mice. A search of public databases identified a novel p70(s6k)/p85(s6k) homolog which 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 p70(s6k)/p85(s6k)-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 p70(s6k)/p85(s6k) function, underscores the importance of S6K function in cell growth.

Here we have employed p70(s6k) truncation and point mutants to elucidate the role played by the carboxyl-terminal autoinhibitory domain <(S/T)under bar>P phosphorylation sites in kinase activation. Earlier studies showed that truncation of the p70(s6k) amino terminus severely impaired kinase activation but that this effect was reversed by deleting the carboxyl terminus, which in parallel led to deregulation of Thr(229) phosphorylation in the activation loop (Dennis, P. B., Pullen, N., Kozma, S. C., and Thomas, G. (1996) Mol. Cell. Biol. 16, 6242-6251). In this study, substitution of acidic residues for the four autoinhibitory domain <(S/T)under bar>P sites mimics the carboxyl-terminal deletion largely by rescuing kinase activation caused by the amino-terminal truncation. However, these mutations do not deregulate Thr(229) phosphorylation, suggesting the involvement of another regulatory element in the intact kinase. This element appears to be Thr(389) phosphorylation, because substitution of an acidic residue at this position in the p70(s6k) variant containing the <(S/T)under bar>P mutations leads to a large increase in basal Thr(229) phosphorylation and kinase activity. In contrast, an alanine substitution at Thr(389) blocks both responses. Consistent with these data, we, show that a mutant harboring the acidic <(S/T)under bar>P and Thr(389) substitutions is an excellent in vitro substrate for the newly identified Thr(229) kinase, phosphoinositide-dependent kinase-1 (Pullen, N., Dennis, P. B., Andjelkovic, M., Dufner, A., Kozma, S., Hemmings, B. A., and Thomas, G. (1998) Science 279, 707-710), whereas phosphoinositide-dependent kinase-l poorly utilizes the two p70(s6k) variants that have only one set of mutations. These findings indicate that phosphorylation of the <(S/T)under bar>P sites, in cooperation with Thr(389) phosphorylation, controls Thr(229) phosphorylation through an intrasteric mechanism.