RPS6-p70-protein kinase


Functional characterization of Drosophila Sk6

To determine whether the Drosophila S6k cDNA encodes a protein product equivalent to the endogenous protein, Drosophila Schneider line 2 (S2) cells were transiently transfected with a Drosophila S6k construct containing an myc epitope-tag at its N terminus. Following transfection, protein products were visualized by Western blot analysis, employing the monoclonal antibody 9E10. A protein band with Mr 70 kDa was specifically detected in extracts from transiently transfected cells, but was absent in extracts from control cells. To determine whether an equivalent protein could be detected in extracts of S2 cells, a rabbit polyclonal antibody (D20) was generated against a peptide representing amino acids 1-19 of the Drosophila S6k N terminus. This antibody specifically recognizes an endogenous protein of Mr 70 kDa in S2 cells, which comigrates with the kinase produced ectopically from transient transfection of the cDNA in S2 cells that is detected with either the D20 antiserum or the 9E10 antibody. Longer exposures of the film do not reveal additional bands that might be suggestive of an isoform equivalent to the mammalian p85 s6k. Preincubation of the D20 antiserum with the antigenic peptide prevents binding to Drosophila S6k. Thus, the Drosophila S6k cDNA encodes a protein that is antigenically equivalent to a Drosophila protein that migrates at a similar Mr (Stewart, 1996).

Activation of the rat p70 s6k is associated with phosphorylation of 10 residues, 5 of which are conserved in Drosophila S6k. By mutational analysis, 3 of the 10 sites, T229,S371, and T389, have been argued to be critical for p70 s6k activation. These sites are conserved in Drosophila S6k as T238, S380, and T398. In addition, the principal target of rapamycin-induced p70 s6k dephosphorylation and inactivation is T389, with T229 acting as a secondary target. However, for rapamycin to exert this inhibitory response on mammalian p70 s6k, the macrolide requires the acidic residues at the N terminus. This may be a feature also conserved in Drosophila S6k. To examine whether S2 cells contain S6 kinase activity and, if so, whether this activity is sensitive to rapamycin, mammalian 40S ribosomes were incubated with extracts from S2 cells treated with cycloheximide, an agent known to activate mammalian p70 s6k, in the absence or presence of the macrolide. Mammalian 40S ribosomes were employed in these assays because they were found to be as good a substrate as their Drosophila counterparts. Cycloheximide treatment of S2 cells increases S6 kinase activity 2.2-fold over basal levels and rapamycin pre-treatment prevents this increase, reducing activity below basal levels. However, a significant amount of rapamycin insensitive kinase activity toward S6 is still apparent (Stewart, 1996).

To distinguish between these rapamycin sensitive and insensitive S6 kinase activities and to determine whether either represents Drosophila S6k, extracts from cycloheximide-stimulated S2 cells, pretreated with or without rapamycin, were fractionated by Mono Q chromatography. The fractions eluted from the Mono Q column were monitored by A280 and subjected to an in vitro S6 kinase assay. Two peaks of S6 kinase activity emerged from the column. The first peak containing less S6 kinase activity eluted at 0.13 M NaCl, and was rapamycin insensitive. The second peak of activity eluted at 0.29 M NaCl, contained approximately 15-fold more S6 kinase activity, and was completely abolished by rapamycin pretreatment. To assess whether the S6 kinase activity in this peak is attributable to Drosophila S6k, Fast Flow Q Sepharose was used to concentrate the fractions 16-18 containing the rapamycin sensitive S6 kinase activity. The concentrated samples from cells treated with cycloheximide either in the absence or presence of rapamycin pretreatment, were either immunoprecipitated and assayed for S6 kinase activity or analyzed on Western blots employing the D20 antibody. The D20 antibody specifically immunoprecipitates native Drosophila S6k kinase activity from the sample stimulated with cycloheximide, but no activity could be detected from the sample pretreated with rapamycin prior to the addition of cycloheximide. Western blot analysis of these fractions reveals a slower electrophoretic mobility on SDSy PAGE for the active Drosophila S6k as compared with the inactive kinase. This altered electrophoretic mobility is similar to the mobility shift induced by phosphorylation of the mammalian p70 s6k, an effect that is ablated by rapamycin treatment. Thus, cycloheximide treatment induces the activation of Drosophila S6k, which is apparently regulated by phosphorylation and is rapamycin sensitive (Stewart, 1996).

To determine whether Drosophila S6k is regulated in a manner similar to the mammalian enzyme, HA-epitope-tagged Drosophila S6k was transiently expressed in NIH 3T3 cells. The addition of PDGF led to the decreased electrophoretic mobility of HA-S6k as determined by immunoblot analysis. The kinetics of the mobility shift are similar to that of mammalian p70S6k, which exhibits multiple size forms in response to PDGF that are attributable to phosphorylation. This mobility shift coincides with the activation of HA-S6k, as measured by S6k's ability to phosphorylate RPS6. Futhermore, pretreatment with wortmannin or RAP potently inhibits PDGF-dependent phosphorylation and activation of HA-S6K. Thus these results indicate that the upstream regulatory elements present in mammalian cells are capable of activating Drosophila S6K and that activated S6K can use mammalian RPS6 as a substrate. Expression of another epitope-tagged S6k (myc-S6k) in COS cells confirms that the components required for the regulation of Drosophila S6K are present in other mammalian cell types (Watson, 1996).

Scylla decreases S6K but not PKB activity

Diverse extrinsic and intrinsic cues must be integrated within a developing organism to ensure appropriate growth at the cellular and organismal level. In Drosopohila, the insulin receptor/TOR/S6K signaling network plays a fundamental role in the control of metabolism and cell growth. scylla and charybdis (a. k. a. charybde), two homologous genes identified as growth suppressors in an EP (enhancer/promoter) overexpression screen, act as negative regulators of growth. The genes are named after mythological monsters that lived in the Strait of Messina between Sicily and Italy, posing a threat to the passage of ships. The simultaneous loss of both genes generates flies that are more susceptible to reduced oxygen concentrations (hypoxia) and that show mild overgrowth phenotypes. Conversely, either scylla or charybdis overactivation reduces growth. Growth inhibition is associated with a reduction in S6K but not PKB/Akt activity. Together, genetic and biochemical analysis places Scylla/Charybdis downstream of PKB and upstream of TSC1. Furthermore, scylla and charybdis are induced under hypoxic conditions and scylla is a target of Drosopohila HIF-1 (hypoxia-inducible factor-1: Similar) like its mammalian counterpart RTP801/REDD1, thus establishing a potential cross-talk between growth and oxygen sensing (Reiling, 2004).

To test whether the placement of Scylla between PKB and TSC can be corroborated biochemically, the effect of scylla overexpression on PKB and S6K activity was examined. PKB activity of adult female heads overexpressing scylla or charybdis was tested in conjunction with PKB and PDK1 under control of the GMR-Gal4 enhancer. The same experimental setup has previously been used to demonstrate that PDK1 increases PKB activity. Total fly head protein was extracted and PKB activity was assayed by incorporation of 32P-labeled phosphate into a synthetic PKB substrate (Crosstide, CT). Although scylla/charybdis overexpression substantially suppresses the PKB/PDK1-induced bulging eye phenotype, PKB activity is not reduced in these eyes. Moreover, PKB activity is also unaffected in a scylla-/- background (Reiling, 2004).

These results are consistent with the placement of Scylla downstream of PKB. To test the effect of Scylla on S6K activity, second instar larvae expressing scylla under the control of Act5C-Gal4 were collected, and larval extracts were assayed for S6K activity. On average, S6K activity was down-regulated by >50%. Although there may also be a slight reduction in total S6K protein levels, this effect cannot account for the much stronger reduction in S6K activity. Taken together with the genetic evidence, these results strongly support the argument that Scylla acts between PKB and TSC to regulate S6K activity. Furthermore, Brugarolas (2004) provide direct biochemical evidence that a functional TSC complex is required for RTP801/REDD1 to affect S6 phosphorylation. Altogether, these data indicate that Scylla functions upstream of TSC (Reiling, 2004).

dTOR and PDK1 and their interaction with Sk6

Genetic studies in Drosophila underscore the importance of the insulin-signaling pathway in controlling cell, organ and animal size. Effectors of this pathway include Chico (the insulin receptor substrate homolog), PI(3)K, PKB, PTEN, and S6k. Mutations in any of these components have a striking effect on cell size and number, with the exception of S6k. Mutants in S6k affect cell size but not cell number, seemingly consistent with arguments that S6k is a distal effector in the signaling pathway, directly controlled by Target of rapamycin (Tor), a downstream effector of PI(3)K and PKB. Unexpectedly, recent studies showed that S6k activity is unimpaired in chico-deficient larvae, suggesting that S6k activation may be mediated through the PI(3)K docking sites of the Drosophila insulin receptor. It has been shown genetically, pharmacologically and biochemically that S6k resides on an insulin signaling pathway distinct from that of PKB, and surprisingly also from that of PI(3)K. More striking, despite PKB-PI(3)K-independence, S6k activity is dependent on the Drosophila homolog of the phosphoinositide-dependent protein kinase 1, PDK1, demonstrating that both PDK1 (as well as Tor) mediated S6K activation is phosphatidylinositide-3,4,5-trisphosphate (PIP3)-independent (Radimerski, 2002a).

Regulation of cellular growth by the drosophila target of rapamycin dTOR

The TOR protein kinases (TOR1 and TOR2 in yeast; mTOR/FRAP/RAFT1 in mammals) promote cellular proliferation in response to nutrients and growth factors, but their role in development is poorly understood. The Drosophila TOR homolog dTOR is required cell autonomously for normal growth and proliferation during larval development, and for increases in cellular growth caused by activation of the phosphoinositide 3-kinase (PI3K) signaling pathway. As in mammalian cells, the kinase activity of dTOR is required for growth factor-dependent phosphorylation of S6 kinase in vitro, and overexpression of S6k in vivo can rescue dTOR mutant animals to viability. Loss of dTOR also results in cellular phenotypes characteristic of amino acid deprivation, including reduced nucleolar size, lipid vesicle aggregation in the larval fat body, and a cell type-specific pattern of cell cycle arrest that can be bypassed by overexpression of the S-phase regulator cyclin E. These results suggest that dTOR regulates growth during animal development by coupling growth factor signaling to nutrient availability (Zhang, 2000).

The Drosophila genome encodes four members of the PIK-related family: mei41, which encodes an ATR/ATM homolog required for cell cycle arrest in response to DNA damage (Hari, 1995); CG6535, a second ATM-related gene of unknown function; CG4549, whose closest relative encodes the nonsense-mediated mRNA decay protein SMG-1 in C. elegans, and the dTOR. A fifth member of this family, the DNA-dependent protein kinase, is not found in Drosophila or C. elegans (Zhang, 2000).

Using a combination of cDNA library screening and RACE (5' rapid amplification of cDNA ends), overlapping cDNAs have been isolated that together contain a large ORF of 2471 amino acids with strong similarity to mammalian mTOR and to TOR1 and TOR2 from budding yeast. Subsequently, the identical ORF was identified by computational analysis of the annotated Drosophila genome (CG5092). Sequence comparisons reveal that dTOR is 56% and 38% identical to human mTOR and yeast TOR2, respectively, with the highest levels of identity in the carboxy-terminal region containing the putative kinase and rapamycin/FKBP12-binding domains (73% identity with mTOR over the carboxy-terminal 675 amino acids). Additional structural motifs were also found to be well-conserved, including a series of HEAT repeats in the amino-terminal half of the protein, a domain shown to bind the peripheral membrane protein gephyrin, and a short sequence at the extreme carboxyl terminus of essential but unknown function that is highly conserved amongst PIK-related family members. Interestingly, sites of autophosphorylation and phosphorylation by Akt/PKB are not conserved in dTOR (Zhang, 2000).

To begin a mutational analysis of dTOR, the Berkeley Drosophila Genome Project P-element database was searched and two independent homozygous lethal lines bearing P insertions in the dTOR gene were identified (designated here as dTORP1 and dTORP2). Mobilization of these elements restores viability to each chromosome, indicating that the insertions are responsible for the associated lethality. Comparison of the insertion sites of dTORP1 and dTORP2 with the dTOR transcription unit reveal that the P-elements are inserted at 24 and 74 bases downstream of the dTOR transcription start site, respectively, and would likely interfere with normal dTOR expression (Zhang, 2000).

To generate additional dTOR alleles, a series of deletions spanning the dTOR gene was generated by imprecise mobilization of the P-elements. One such mutant, designated dTORDeltaP, was selected for further analysis. Sequence analysis reveals that the dTORDeltaP deletion originates at the dTORP2 insertion site and extends 3514 bp downstream, removing the dTOR translation start site and amino-terminal 902 codons, and thus likely represents a null allele of dTOR. This was confirmed by the absence of detectable dTOR protein in immunoblots of dTORDeltaP larval extracts. A 9.4-kb genomic rescue construct encompassing the dTOR gene and no other predicted transcription units restores full viability and fertility to dTORDeltaP homozygotes (Zhang, 2000).

dTORDeltaP homozygotes hatch at normal rates, but grow more slowly than normal and eventually arrest during larval development, reaching only 24% the mass of wild-type controls. Larvae homozygous for dTORP1 or dTORP2 alleles display a less severe phenotype, eventually growing to approximately 40% and 79% the mass of wild type, respectively, indicating that these alleles likely retain partial dTOR function. In each case, the mutants remain viable and active during an extended larval period of ~30 d, and eventually die without pupating. Larvae heterozygous for dTOR grow at a rate indistinguishable from wild-type controls under normal culture conditions, but are hypersensitive to low concentrations of rapamycin. Thus, dTOR encodes a rapamycin-sensitive protein required for normal growth during larval development (Zhang, 2000).

Overall growth of an organism is generally accompanied by increases in cell number (proliferation), cell size (hypertrophy), or both. To determine how mutations in dTOR inhibit growth, these parameters were examined in a number of tissues. The effect of dTOR on cell size was analyzed in marked clones of dTORDeltaP homozygous cells, which were generated by FLP/FRT-mediated mitotic recombination in dTORDeltaP heterozygous animals. Examination of adult cuticular structures reveal that dTOR homozygous mutant cells are markedly reduced in size. For example, bristles of the wing margin that lack dTOR were both thinner and shorter than adjacent wild-type cell. Area measurements of mutant clones in the wing epithelium show that dTORDeltaP mutant cells are approximately half (56%) the size of controls. Similar effects have been observed in the eye, abdomen, and notum (Zhang, 2000).

To determine whether loss of dTOR affects the size of actively proliferating cells, dTOR mutant clones were examined in the developing imaginal discs, epithelial primordia that proliferate mitotically to give rise to adult structures. Imaginal wing discs containing GFP-marked clones of dTORDeltaP homozygous cells were dissociated into single cells, which were then analyzed by flow cytometry. The mean forward light scatter value (a measure of cell size) of dTOR mutant cells is decreased by 30% compared to wild-type control cells from the same discs. This decrease in cell size is observed in all phases of the cell cycle. Thus, loss of dTOR causes a cell autonomous reduction in the size of both proliferating and postmitotic cells (Zhang, 2000).

Fluorescence-activated cell sorter (FACS) analysis has also revealed that the cell cycle phasing of dTOR cells differs significantly from that of controls, with relatively more cells in G1, and fewer in S and G2 phases. This is consistent with the ability of rapamycin to induce G1 arrest in yeast and in mammalian cell culture. To measure proliferation rates of dTOR mutant cells, the number of cells in dTORDeltaP clones were compared with that of their wild-type sister clones (twin spots). Clones of dTORDeltaP mutant cells are similar in size to their twin spots at 48 h after induction, but by 72-96 h they contain significantly fewer cells, indicating that loss of dTOR leads to a reduced rate of cell proliferation. In addition, lone twin spots lacking a corresponding mutant sister clone have occasionally been observed at 96 h after induction, indicating that at some frequency dTORDeltaP homozygous cells are eliminated from the disc epithelium. Because dTORDeltaP cells remain viable for weeks in the context of a homozygous mutant animal, the loss of dTORDeltaP mutant clones is likely the result of cell competition with adjacent wild-type cells, which is known to occur for cells with a growth disadvantage (Zhang, 2000).

Growth properties of cells in the salivary glands of homozygous dTORDeltaP larvae were also examined. The salivary gland is comprised of two cell types: polytene gland cells that undergo multiple rounds of endoreduplication to generate giant nuclei with a ploidy of up to 2048 C, and imaginal ring cells that remain diploid and cycle mitotically. Loss of dTOR affects both cell types. The endoreplicative cells in dTORDeltaP salivary glands undergo only four to five rounds of replication before entering quiescence, reaching a ploidy of 16-32C and a size ~10% that of wild type. The imaginal rings in dTORDeltaP larvae contain approximately fivefold fewer cells than wild type. Together, these results indicate that dTOR is required to promote cell cycle progression in both mitotic and endoreplicative cells, and acts primarily at the G1/S transition (Zhang, 2000).

The cell autonomous reduction in the size of dTOR mutant cells is reminiscent of mutations in components of the PI3K/S6K signaling pathway. Mutations in Pten, the fly homolog of the PTEN tumor suppressor, cause activation of this pathway, leading to increased cell growth. To determine whether dTOR is required for PI3K-dependent signaling, the growth properties of cells lacking both Pten and dTOR were examined. Clonal loss of Pten causes enlargement of imaginal and adult cells, and increases the percentage of cells in the S and G2 phases of the cell cycle. In contrast, cells carrying null alleles of both Pten and dTOR are indistinguishable from cells lacking dTOR alone, with a similar reduction in cell size and accumulation in G1. Loss of dTOR also prevents the increased proliferation caused by mutations in Pten. Cells mutant for weaker alleles of Pten and dTOR (MGH1 and P2, respectively) are intermediate in size, indicating that dTORP2 cells retain partial signaling function. It is concluded that dTOR is epistatic to Pten, and therefore, that dTOR functions at a step downstream of or in parallel to PI3K signaling (Zhang, 2000).

In further tests of dTOR's role in PI3K/S6k signaling, it was found that rapamycin inhibits the serum-dependent phosphorylation of Drosophila p70S6k (S6k) expressed in S2 cells. Dephosphorylation of S6k by rapamycin is prevented by cotransfection of a dTOR point mutant containing a Ser1956 to Thr substitution (dTORRR, which confers rapamycin resistance to mammalian and yeast TOR proteins. Expression of dTORRR carrying an additional point mutation in a residue crucial for kinase activity (dTORRRKD fails to protect S6k from rapamycin-induced dephosphorylation, indicating that the kinase function of dTOR is required to maintain S6k phosphorylation (Zhang, 2000).

To determine whether these biochemical interactions between dTOR and S6k are relevant to their functions in vivo, tests were performed for genetic interactions between them. Remarkably, constitutive overexpression of Drosophila S6k or human p70S6K1 is able to rescue dTORP2/P2 and dTORP1/P2 flies to viability. The greatest degree of rescue is provided by a mutant version of p70S6K1, in which four mitogen-induced phosphorylation sites are mutated to aspartate and glutamate residues (mutant D4). Expression of this construct allowed 74% of expected dTORP1/P2 progeny to survive to adulthood, whereas no dTORP1/P2 animals survived in the absence of S6k overexpression. dTOR flies rescued by S6k overexpression are slightly smaller than wild-type controls, but are fertile and develop at a similar rate as wild type. Although overexpression of S6k does not rescue dTORDeltaP null mutants to adulthood, it does enable them to progress to the pupal stage. Overexpression of S6k in wild-type larvae also confers significant resistance to rapamycin. Again, constitutively active p70S6K1 provides the greatest degree of rapamycin resistance. Together, these results indicate that a major function of dTOR is to maintain levels of active S6k sufficient for normal growth (Zhang, 2000).

Like dTOR mutants, wild-type larvae deprived of amino acids enter an extended larval period with little or no growth. Amino acid deprivation also causes a series of distinctive cellular phenotypes including a reduction in nucleolar area, changes in morphology of the larval fat body, and a cell type-specific cell cycle arrest. Since TOR proteins have been proposed to be regulated in response to amino acid levels, it was of interest to examine whether loss of dTOR mimics these cellular effects (Zhang, 2000).

The nucleolus is the major cellular site of ribosomal assembly, and its size has been shown to correlate with protein synthetic capacity and proliferation rate. To measure nucleolar size in wild-type and dTOR mutant cells, wing imaginal discs containing clones of dTORDeltaP homozygous cells were labeled with an antibody against the nucleolar protein fibrillarin, and examined by confocal sectioning. The nucleolar area in clones of dTOR mutant cells in the wing imaginal disc is approximately half that of surrounding wild-type cells, consistent with a role for dTOR in ribosome biogenesis (Zhang, 2000).

During metamorphosis or starvation, stores of protein, lipid, and glycogen are mobilized from adipose cells of the larval fat body and are used by other tissues as an energy source in place of dietary nutrients. These metabolic effects are visible as changes in appearance of fat body cells. The major visible change in fat body cells in larvae deprived of amino acids is an aggregation of lipid vesicles, and this effect is indistinguishable from that caused by loss of dTOR (Zhang, 2000).

Within 48-72 h of amino acid withdrawal, endoreplicative cells become quiescent, whereas mitotic neuroblasts of the central nervous system continue to cycle for at least 8 d in the absence of amino acids. This pattern of cell cycle responses is distinct from that caused by complete inhibition of protein synthesis, which causes all larval cells to arrest DNA synthesis. To determine whether loss of dTOR causes a cell cycle response similar to that elicited by starvation, cell cycle behavior of these cell types was examined in dTORDeltaP larvae at multiple stages of development. At 3-4 d after egg deposition (AED), both endoreplicative and mitotic tissues were found to cycle normally in dTORDeltaP homozygotes, as measured by incorporation of the nucleotide analog BrdU. In contrast, by 5-6 d AED all endoreplicative tissues including the gut, fat body, and salivary glands fail to incorporate BrdU, whereas neuroblasts continued to cycle. A similar pattern is observed at 10 d AED. Presumably the appearance of this cell cycle arrest at 4-5 d AED results from the perdurance of maternal stores of dTOR mRNA or protein until this time. The cell cycle arrest of dTOR endoreplicative cells can be bypassed by overexpression of the G1/S regulators cyclin E or dE2F/dDP, as has been demonstrated previously for endoreplicative cells arrested by amino acid withdrawal. Thus, amino acid insufficency and loss of dTOR each cause similar growth arrests, changes in cell morphology, and cell type-specific patterns of G1 arrest (Zhang, 2000).

In budding yeast, TOR proteins govern S-phase entry in response to nutrient levels by regulating translation of the G1/S regulator Cln3 (Barbet, 1996 and Polymenis, 1997). Drosophila cyclin E has been proposed to play a role analogous to Cln3, and its abundance increases in response to growth stimuli such as overexpression of activated Ras. Because cyclin E overexpression is able to bypass the cell cycle arrest in dTOR mutants, whether loss of dTOR affects cyclin E expression was examined. Immunoblot analysis of whole larval extracts has revealed that the level of cyclin E protein is reduced ~30-fold in dTORDeltaP mutants, as compared to wild-type larvae of similar stage (Zhang, 2000).

Clonal induction of cells lacking cyclin E in the wing imaginal disc results in a G1 arrest within one to two cell divisions. In contrast, cells mutant for dTOR continue to cycle slowly for several days, and give rise to clones containing multiple cells, indicating that dTOR mutant cells retain at least partial cyclin E activity. Accordingly, cyclin E protein is reduced but not eliminated in dTOR mutant clones in the wing disc. Although 72-h dTORDeltaP clones containing little or no detectable cyclin E immunoreactivity are often observed, many dTOR clones were found containing cells with apparently normal cyclin E levels. It is concluded that the observed reduction in cyclin E protein levels in dTORDeltaP larval extracts is due largely to reduced cyclin E levels in endoreplicating cells, which comprise the majority of larval mass, resulting in a G1 arrest in this cell type. In contrast, dTOR may be required in imaginal cells to maintain normal rates of cyclin E accumulation, rather than absolute levels, consistent with the reduced rate of cell division and extended G1 phase observed in dTOR mutant clones (Zhang, 2000).

dTOR mutants differ from the known PI3K pathway mutants in several important respects. (1) The growth defects caused by loss of dTOR are more severe than those arising from mutations in components of the PI3K signaling pathway. (2) Null mutations in the PI3K subunits Dp110 or p60 allow growth to the third instar larval stage, and chico (IRS-1) and S6k mutants survive to adulthood. At least in the case of chico and S6k, many cells are able to cycle normally throughout development. In contrast, animals lacking dTOR reach only the size of second instar larvae, at which point they undergo cell cycle arrest. (3) Whereas overexpression of Dp110, Akt, or S6k leads to increased growth rate and cell size, dTOR overexpression inhibits growth and reduces cell size. Although variations in genetic background may partially account for some of these phenotypic differences, these results are inconsistent with dTOR acting as an integral component of a linear PI3K/Akt/S6k pathway, and instead argue that dTOR may converge upon this pathway in response to a distinct set of cues (Zhang, 2000 and references therein).

In yeast, TOR1 and TOR2 regulate cell growth directly in response to levels of nutrients such as amino acids, rather than in response to intercellular signals. Similarly, human mTOR may also be regulated by nutrient levels. These considerations prompted a comparison of the phenotypes of dTOR mutant animals with the physiological changes caused by nutrient deprivation. By the three criteria examined -- nucleolar size, fat body vesicle formation, and endoreplicative cell cycle arrest -- loss of dTOR precisely mirrors the effects of starvation. An efficient explanation of these results is that dTOR is required for normal responses to changes in nutrient levels. This would be consistent with a model in which full activation of growth targets such as S6k requires two distinct inputs: growth factor-mediated intercellular signals through PI3K, and nutrient-sensing signals through TOR. In this view, TOR proteins may act as part of a checkpoint to attenuate growth factor signaling when local conditions are unfavorable for cell growth (Zhang, 2000).

How TOR proteins might be regulated by nutrient levels is unclear. In the case of amino acids, recent evidence suggests that the primary signal may be uncharged tRNA, which increases in abundance when amino acid levels are low. Interestingly, other members of the PIK-related kinase family such as ATM and DNA-PK also act as checkpoint proteins that are regulated by specific nucleic acid structures. In addition, a recently described member of this family, SMG-1, is involved in the degradation of aberrant mRNAs containing inappropriate nonsense codons. Thus, regulation by nucleic acid may be a common feature of this kinase family (Zhang, 2000 and references therein).

Activation of p70S6K is a common response to virtually all mitogenic stimuli. Phosphorylation of the target of p70S6K, ribosomal protein S6, leads to the selective increase in translation of a subset of transcripts that contain an oligopyrimidine tract at their 5' termini (5' TOP). This class of messages encodes ribosomal proteins and translation elongation factors, and thus p70S6K activation leads to increased ribosomal biogenesis. The demonstration in this study that overexpression of S6k can rescue dTOR mutants to viability indicates that S6k is a critical effector of dTOR, and that one essential function of dTOR is to regulate the activity of S6k (Zhang, 2000).

Despite the central role of S6k in mediating the functions of dTOR, several lines of evidence indicate that dTOR has additional S6k-independent roles. (1) dTOR mutant phenotypes are more severe than those of S6k; lack of dTOR results in growth arrest at the second instar larval stage, whereas S6k mutant animals survive to adulthood, albeit with a delayed development and decreased body size. (2) Although S6k overexpression suppresses the lethality of dTOR mutants, this is only the case for hypomorphic dTOR allelic combinations; dTOR null animals expressing S6k advance only to the pupal stage. (3) dTOR flies rescued by S6k do not grow to the full size of wild-type controls. Thus, some dTOR functions are not fully rescued by S6k. As noted above, in mammalian cells mTOR also stimulates translation through phosphorylation and inactivation of 4E-BP1, an inhibitory binding factor of the translation initiation factor eIF4E. Drosophila eIF4E mutants display a severe growth arrest phenotype, and thus aspects of dTOR function that are not rescued by S6k may reflect diminished eIF4E activity. However, neither overexpression of eIF4E nor mutations in 4E-BP1 detectably alleviate dTOR mutant phenotypes (Zhang, 2000).

In addition to effects on translation, studies in budding yeast have found that inactivation of TOR modulates the level of a number of growth-related or nutrient-regulated transcripts, including those encoding ribosomal proteins (Barbet, 1996; Zaragoza, 1998; Beck, 1999; Cardenas, 1999; Hardwick, 1999; Powers, 1999). A transcriptional role for TOR in higher eukaryotes has been reported as well. Moreover, recent studies have found that mTOR can interact with a number of additional signaling factors including STAT3, protein kinase C, c-Abl, and 14-3-3 (Parekh, 1999; Kumar, 2000a and b; Mori, 2000; Yokogami, 2000). Rapamycin has also recently been shown to disrupt microtubule assembly and function in yeast, independent of its effects on translation (Choi, 2000). Thus, the inability of S6k overexpression to fully supplant dTOR may be due to a requirement for dTOR in multiple cellular functions (Zhang, 2000).

The reduced growth of dTOR mutants reflects reductions in both cell size and cell number. Because direct inhibition of proliferation increases rather than decreases cell size, it is proposed that the primary function of dTOR is to promote cell growth, and that the decreased proliferation of dTOR mutant cells is a secondary effect in response to their reduced rate of growth. The accumulation of dTOR mutant cells in the G1 phase of the cell cycle suggests that the G1/S transition is particularly sensitive to growth rate. This is consistent with previous observations that stimulation of cell growth by overexpression of dMyc, PI3K, or activated Ras promotes progression through the G1/S but not G2/M transition (Zhang, 2000 and references therein).

A factor likely to be involved in coupling growth and division rates is the G1/S regulator cyclin E. Cyclin E is rate limiting for G1/S progression in imaginal discs, and its levels increase by a post-transcriptional mechanism in response to stimulation of cell growth. The demonstration that dTOR is required for normal accumulation of cyclin E suggests that translational control is likely to play an important role in cyclin E regulation. In budding yeast, translation of the G1 cyclin Cln3 is regulated by a leaky scanning mechanism involving a small upstream ORF (Polymenis, 1997). In this regard, it is interesting to note that the 5'-untranslated region of the Drosophila cyclin E message contains multiple upstream ORFs. Thus, the mechanisms connecting G1/S progression to cell growth may be conserved between yeast and multicellular organisms (Zhang, 2000).

Gigas negatively regulates S6k

Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in one of two tumor suppressor genes, TSC1 and TSC2. Absence of Drosophila Tsc1 and/or Tsc2 (Gigas) leads to constitutive S6k activation and inhibition of PKB, the latter effect being relieved by loss of S6K. In contrast, the Pten tumor suppressor, a negative effector of PI3K, has little effect on S6k, but negatively regulates PKB (Akt1). More importantly, reducing S6k signaling rescues early larval lethality associated with loss of Tsc1/2 function, arguing that the S6k pathway is a promising target for the treatment of TSC (Radimerski, 2002b).

To determine whether loss of Tsc1/2 or Pten directly affected S6k activity, each was depleted in Drosophila Kc167 cells by dsRNAi. Quantitative Real Time PCR showed that such treatment strongly reduced levels of both transcripts. Compared with control cells, depletion of Tsc1 increases S6k activity and T398 phosphorylation, consistent with the reduced electrophoretic mobility of S6k. These results are in agreement with recent findings in TSC1 null mammalian cells (Kwiatkowski, 2002). Insulin treatment of either control cells or Tsc1-depleted cells did not significantly increase these responses beyond that of Tsc1 depletion alone, indicating that loss of Tsc function leads to full S6k activation. RAD001, a rapamycin derivative, blocks S6k activity in both control and Tsc1-depleted cells treated with insulin. However, it was consistently noted that the RAD001 block of insulin-induced S6k activation is not as strong in Tsc1-depleted cells, suggesting that not all the effects of Tsc on S6k are dependent on Tor, the Drosophila target of rapamycin. Similar results to those described here were obtained by Tsc2 depletion. In addition, the effects appear specific, since Tsc1 depletion has no effect on the basal activity of other AGC-kinase family members, such as PKB or Drosophila atypical PKC. However, insulin-induced PKB activation and S505 phosphorylation are repressed in Tsc1-depleted cells as compared with control cells, consistent with S6k acting in a negative feedback loop to dampen PKB signaling. In contrast to loss of Tsc1, depletion of Pten has little effect on S6k activity and T398 phosphorylation, whereas it leads to elevated levels of both basal and insulin-stimulated PKB activity and S505 phosphorylation. Thus, loss of Tsc1/2, but not Pten, leads to constitutive S6k activation (Radimerski, 2002b).

To determine whether the findings above could be corroborated in the animal, S6k activity was measured in extracts of Tsc1, Pten, and S6k null larvae. The results show that S6k activity in extracts derived from Tsc1 null larvae is strongly increased over that of wild-type larvae, whereas it is slightly increased in larvae lacking Pten. The opposite was found for PKB activity, which is strongly repressed in Tsc1 null larvae, and up-regulated in Pten-deficient larvae. Hence, it cannot be excluded that reduced PKB activity contributes to larval lethality of Tsc mutants. Given that loss of Tsc function leads to increased S6k activity, it was reasoned that ectopic expression of Tsc1/2, but not Pten, would inhibit S6k activity. To test this hypothesis, both tumor suppressors were expressed ubiquitously in larvae using the GAL4/UAS system, such that the GAL4 promoter chosen in each case led to developmental arrest at late larval second instar. Extracts from larvae overexpressing Tsc1/2 display strongly reduce S6k activity, whereas those from Pten overexpressing larvae have normal levels of S6k activity. In contrast, PKB activity is strongly suppressed in Pten overexpressing larvae and little affected in extracts from larvae overexpressing Tsc1/2. These data corroborate previous findings that S6k and PKB act in parallel signal transduction pathways (Radimerski, 2002a), and provide compelling evidence that they are negatively controlled by distinct tumor suppressor genes (Radimerski, 2002b).

Despite the fact that S6k and PKB act in parallel signaling pathways, loss of Tsc1/2 function leads to inhibition of PKB activity, suggesting cross-talk between the two pathways. Compatible with such a model, recent studies have shown that rapamycin treatment of adipocytes inhibits a negative feedback loop, which normally functions to dampen insulin-induced PKB activation. Since RAD001 inhibits S6k activity (Radimerski, 2002a) and increases PKB activity (Radimerski, 2002a), it raised the possibility that the effects of Tsc mutants on PKB are mediated through S6k. Consistent with this hypothesis, inhibition of PKB activity due to loss of Tsc function was relieved in the absence of S6k. Similar results were obtained by using dsRNAi in cell culture. Thus, the suppression of PKB by loss of Tsc function requires S6k (Radimerski, 2002b).

To genetically test the specificity of Tsc1/2 and Pten tumor suppressor function, either Tsc1 or Pten were removed in cells giving rise to the adult eye structure, by inducing mitotic recombination with the FLP/FRT system under the control of the eyeless promoter. In a wild-type genetic background, loss of either Tsc1 or Pten within the developing eye causes strong overgrowth of the head. Eye overgrowth by removal of Tsc1 is strongly suppressed in a genetic background null for S6k, as is ommatidia size, in agreement with a previous report analyzing double mutant clones of Tsc2 and S6k in the eye (Potter, 2001). In contrast, removal of Pten in the eyes of S6k null flies still induces overgrowth of clones with enlarged ommatidia. These findings are supported by results showing that eye overgrowth by removal of Tsc1 is still observed in clones devoid of PKB function (Potter, 2001) and overgrowth by removal of Pten is suppressed in a viable PKB mutant genetic background (Stocker, 2002). Thus, Tsc1/2 appears to be specific for the S6k-signaling pathway, whereas Pten antagonizes PI3K signaling to counteract PKB activation by decreasing PIP3 levels (Radimerski, 2002b).

Since Tsc1/2 loss-of-function overgrowth in clones is suppressed by removing S6k, it was reasoned that reducing increased S6k activity in Tsc1 loss-of-function larvae might rescue second larval instar lethality. Consistent with this, feeding Tsc1 null larvae low doses of RAD001, which induces a developmental delay of 3 d in wild-type larvae, allowed them to reach late wandering third larval instar. The Tsc1 null larvae died shortly after pupation, presumably because wandering third instar larvae stop feeding and thus failed to receive the drug during pupal stages. To circumvent the problem of feeding, attempts were made to reduce S6k signaling by reducing the dosage of the gene. Compared with wild-type pupae, S6k null larvae are significantly reduced in size, and lack of one allele of Tsc1 in this background has no significant effect on the S6k null phenotype. Strikingly, the second instar lethality caused by lack of both Tsc1 alleles is rescued to early pupal stages in the S6k null background; however, these larvae are still small and severely delayed in development. In contrast, larvae in which one allele of S6k has been removed in a Tsc1 null background develop to early pupal stages with little developmental delay, although they are now significantly larger than wild type. On the basis of these latter findings, it was reasoned that further reduction of S6k signaling, but not its abolishment, may allow Tsc1 null animals to develop beyond early pupal stages. To test this in the Tsc1 null background, either one allele of Tor bearing a mutation in the kinase domain was used alone or in combination with one null allele of S6k. Tsc1 null larvae bearing one kinase mutant Tor allele survived with higher frequency to pupae than animals with one null allele of S6k, with a few emerging as adults. However, genetically lowering S6k signaling further by combining the Tor and S6k loss-of function alleles, results in more than 60% of animals surviving to the adult stage. The rescued females and males were slightly larger than wild-type flies, with overall patterning appearing normal. Furthermore, the rescued females were semifertile when crossed to wild-type males, whereas the rescued males were fully fertile when crossed to wild-type females. Similarly, animals lacking Tsc2 function were rescued to viability by the same genetic approach applied above. Importantly, flies lacking one S6k allele and bearing one kinase mutant Tor allele display no obvious mutant phenotype. Therefore, lowering but not abolishing S6k signaling is sufficient to allow development of Drosophila lacking Tsc function (Radimerski, 2002b).

Taken together, these results demonstrate that the tumor suppressor Tsc1/2 is a critical component in controlling S6k activation. Interestingly, this effect may be Tor independent, as insulin-induced S6k activation is more elevated in Tsc1/2-depleted cells pretreated with RAD001 than in control cells, and in preliminary studies, clonal overgrowth in the eye induced by loss of Tsc1 is not suppressed in a semiviable, heterorallelic Tor mutant background. Overexpression of Tsc1/2 selectively suppresses the S6k-signaling pathway, whereas Pten operates on the dPI3K-signaling pathway. Double mutations for Pten and Tsc1 are additive for clonal overgrowth, compatible with S6k and PKB independently mediating growth. Nevertheless, inhibition of PKB by loss of Tsc function shows that there is negative cross-talk between the two signaling pathways. Given this negative cross-talk, the observation that in double mutant clones growth is additive, suggests that in the absence of Pten, inhibition of PKB by loss of Tsc is circumvented. However, despite the observation that double mutations for Pten and Tsc1 are additive for clonal overgrowth, overgrowth induced by absence of Pten is suppressed in clones mutant for Tor. Since S6k does not prevent such overgrowth, it is possible that this suppression actually represents an intermediate phenotype, or that Pten negatively acts on a Tor target distinct from S6k. At this point, it is important to gain a deeper knowledge of the molecular mechanisms by which Tsc1/2 acts to suppress S6k function and how the signaling components of these two pathways cross-talk with one another (Radimerski, 2002b).

Recently, a successful Phase I clinical trial was completed for a rapamycin analog in the treatment of solid tumors. The results of the trial demonstrated that the drug was efficacious at subtoxic doses, and suggested that specific tumor types may be more sensitive to inhibition by rapamycin than others. The question that arose from the trial is, which tumors would be susceptible to rapamycin treatment? Here, it has been demonstrated for the first time in vivo that a mild reduction in S6k signaling, which alone has no blatant phenotype, is sufficient to restore viability of flies devoid of Tsc function. Thus, these findings imply that rapamycin or its derivatives might be very promising pharmaceutical agents in the treatment of tumors arising from TSC (Radimerski, 2002b).

Rheb, a target of Gigas, functions upstream of S6K

Insulin signalling is a potent inhibitor of cell growth and has been proposed to function, at least in part, through the conserved protein kinase TOR (target of rapamycin). Recent studies suggest that the tuberous sclerosis complex Tsc1-Tsc2 may couple insulin signalling to Tor activity. However, the regulatory mechanism involved remains unclear, and additional components are most probably involved. In a screen for novel regulators of growth, Rheb (Ras homolog enriched in brain), a member of the Ras superfamily of GTP-binding proteins, was identified. Increased levels of Rheb in Drosophila promote cell growth and alter cell cycle kinetics in multiple tissues. In mitotic tissues, overexpression of Rheb accelerates passage through G1-S phase without affecting rates of cell division, whereas in endoreplicating tissues, Rheb increases DNA ploidy. Mutation of Rheb suspends larval growth and prevents progression from first to second instar. Genetic and biochemical tests indicate that Rheb functions in the insulin signalling pathway downstream of Tsc1-Tsc2 and upstream of TOR. Levels of rheb mRNA are rapidly induced in response to protein starvation, and overexpressed Rheb can drive cell growth in starved animals, suggesting a role for Rheb in the nutritional control of cell growth (Saucedo, 2003).

To examine how Rheb interfaces with TOR, advantage was taken of a biochemical readout of TOR function: S6K phosphorylation. Tagged S6K and Rheb were transfected into Drosophila S2 cells and activation of S6K was measured using a phospho-specific antibody (Thr 398) that correlates with kinase activity. Overexpression of Rheb increases S6K phosphorylation. Although S6K is normally inactivated in response to amino-acid starvation, Rheb-mediated phosphorylation of S6K persists in the absence of amino acids. Loss of Tsc1 or Tsc2 has been shown to cause a similar, nutrient-insensitive increase in S6K activity. RNA interference (RNAi) was used to examine the relationship between Tsc2 and Rheb in modulating S6K function. Whereas loss of Tsc2 results in persistent phosphorylation of S6K in media free of amino acids, loss of Rheb abolishes S6K phosphorylation regardless of the presence of amino acids. In the absence of both Tsc2 and Rheb, S6K remains dephosphorylated. These experiments indicate that Rheb is epistatic to Tsc2, required for S6K phosphorylation, and, presumably, S6K activity (Saucedo, 2003).

Signaling from Akt to FRAP/TOR targets both 4E-BP and S6K in Drosophila melanogaster

The eIF4E-binding proteins (4E-BPs) interact with translation initiation factor 4E to inhibit translation. Their binding to eIF4E is reversed by phosphorylation of several key Ser/Thr residues. In Drosophila, S6 kinase (dS6K) and a single 4E-BP (d4E-BP) are phosphorylated via the insulin and target of rapamycin (TOR) signaling pathways. Although S6K phosphorylation is independent of phosphoinositide 3-OH kinase (PI3K) and serine/threonine protein kinase Akt, that of 4E-BP is dependent on PI3K and Akt. This difference prompted an examination of the regulation of d4E-BP/Thor in greater detail. Analysis of d4E-BP phosphorylation using site-directed mutagenesis and isoelectric focusing-sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the regulatory interplay between Thr37 and Thr46 of d4E-BP is conserved in flies and that phosphorylation of Thr46 is the major phosphorylation event that regulates d4E-BP activity. RNA interference (RNAi) was used to target components of the PI3K, Akt, and TOR pathways. RNAi experiments directed at components of the insulin and TOR signaling cascades show that d4E-BP is phosphorylated in a PI3K- and Akt-dependent manner. Surprisingly, RNAi of dAkt also affects insulin-stimulated phosphorylation of dS6K, indicating that dAkt may also play a role in dS6K phosphorylation (Miron, 2003).

Is d4E-BP regulated by a PI3K/Akt-independent pathway similar to that described for dS6K? Analysis of signaling to d4E-BP using RNAi indicates that it is not. It is more likely that d4E-BP is a direct downstream target of the dInR-dPI3K-dPTEN-dAkt-dTSC-dTOR signaling cascade. Thus, a linear pathway from InR to Akt that is important for 4E-BP regulation is conserved between Drosophila and mammals (Miron, 2003)

dPDK1 is critical for regulating growth by phosphorylating dAkt and dS6K. RNAi of dPDK1 does not significantly affect insulin-induced phosphorylation of d4E-BP. However, consistent with the direct phosphorylation of dS6K by dPDK1, the phosphorylation of dS6K at Thr398 is completely blocked by RNAi of PDK1. Thus, the results favor a model in which d4E-BP regulation is effected through dAkt, even when dPDK1 levels are dramatically reduced, whereas dS6K requires both dAkt and dPDK1. The differential effects of dPDK1 RNAi on d4E-BP and dS6K phosphorylation can be explained as follows: dPDK1 levels may be reduced below a threshold that is required to phosphorylate dS6K but is still adequate to activate dAkt, allowing d4E-BP phosphorylation. Since dS6K requires direct phosphorylation by dPDK1, it may be more susceptible to variations in its levels. In contrast, d4E-BP, which relies on a signal relayed by dAkt, may be less affected by variations in dPDK1. In mammalian PDK1-hypomorphic mutants, a kinase activity that is 10-fold lower than normal still results in normal Akt and S6K1 activation, yet these animals are greatly reduced in size. This observation supports the notion that reduced PDK1 activity may differentially activate downstream targets (Miron, 2003).

In Drosophila, coexpression of dS6K with dPI3K does not cause additive cellular overgrowth, unlike coexpression of dAkt and dPI3K. RNAi of dPTEN in Kc 167 cells and overexpression of dPTEN in Drosophila larvae had little effect on dS6K activity. Moreover, removal of both dS6K and dPTEN in cell clones does not prevent the dPTEN-dependent overgrowth phenotype. Together, these results and the results of dPI3K and dPTEN RNAi experiments would seemingly support the notion that dS6K-dependent cell growth is not influenced by dPI3K and dPTEN. However, a different effect of dPTEN RNAi on dS6K has been reported in another study: increase in dS6K phosphorylation following RNAi of dPTEN. Consistent with this observation RNAi directed against dPI3K and dPTEN has been shown to modulate dS6K phosphorylation. A reasonable explanation for these discrepancies is that the knockdown of dPI3K and dPTEN achieved in the current experiments was not sufficient to completely deplete these proteins and affect dS6K phosphorylation (Miron, 2003 and references therein).

The role of dAkt in regulating dS6K is subject to debate. In Drosophila, Akt plays a predominant role in mediating the effects of increased PIP3 levels, and all Akt-mediated growth signals are thought to be transduced via Tsc1/2. Tsc2 is directly phosphorylated by Akt, implying that S6K is downstream of Akt in the PI3K signaling pathway. The observation that RNAi of dAkt reduces dS6K phosphorylation at Thr398 supports a direct link among dAkt, dTSC, and dS6K but contradicts the finding that TSC modulates dS6K activity in a dAkt-independent manner. Recent data also support the conclusion of a link between dAkt and dS6K. Clones of cells doubly mutant for dPTEN and dTsc1 display an additive overgrowth phenotype, suggesting that the tumor suppressors act on two independent pathways, from dPTEN to dAkt and from dTSC to dS6K. The findings demonstrate clear effects of dPTEN, dAkt, and dTSC on d4E-BP, which does not preclude the possibility that two pathways regulate d4E-BP; however, a simpler interpretation is that a single pathway is important for its regulation. A possibility is that d4E-BP requires higher dAkt activity than dS6K in order to be phosphorylated. In circumstances of low PI3K activation, low levels of PIP3 are produced, resulting in weaker dAkt activity that is sufficient for dS6K activation but not for d4E-BP phosphorylation. A differential threshold of activation could be the source of the discrepancies between the current results and those of others. This model is strongly supported by recent data showing that in cells lacking both Akt1 and Akt2 isoforms, the low level of Akt activity remaining is sufficient for robust S6K1 phosphorylation, but phosphorylation of 4E-BP1 is dramatically reduced (Miron, 2003 and references therein).

Alternatively, the results could also be explained by the existence of a negative feedback loop between dPI3K and dS6K that dampens insulin signaling by suppressing dAkt activity. This negative feedback loop has been described. Similar observations were made in mammals; insulin-induced activation of Akt is inhibited in Tsc2-deficient mouse embryonic fibroblasts. Thus, depletion of dAkt may trigger this negative feedback loop, which diminishes dS6K phosphorylation and activation. Interestingly, engagement of this feedback mechanism can also provide an explanation for the reduction in total d4E-BP levels observed in dPDK1 RNAi-treated cells. Under these conditions, the reduction of dS6K signaling is accompanied by a concomitant reduction in growth signaling on the dPI3K-dAkt branch of the pathway. Thus, a reduced level of d4E-BP is required to accommodate the reduced need for deIF4E inhibition (Miron, 2003).

MAP4K3 regulates body size and metabolism in Drosophila

The TOR pathway mediates nutrient-responsive regulation of cell growth and metabolism in animals. TOR Complex 1 activity depends, among other things, on amino acid availability. MAP4K3 was recently implicated in amino-acid signaling in cell culture. This study reports the physiological characterization of MAP4K3 mutant flies. Flies lacking MAP4K3 have reduced TORC1 activity detected by phosphorylation of S6K and 4EBP. Furthermore MAP4K3 mutants display phenotypes characteristic of low TORC1 activity and low nutrient availability, such as reduced growth rate, small body size, and low lipid reserves. The differences between control and MAP4K3 mutant animals diminish when animals are reared in low-nutrient conditions, suggesting that the ability of TOR to sense amino acids is most important when nutrients are abundant. Lastly, physical interaction is shown between MAP4K3 and the Rag GTPases raising the possibility they might be acting in one signaling pathway (Bryk, 2010).

The multiprotein complex TORC1, containing TOR kinase, is a central regulator of cellular growth and metabolism in animals. It is activated by a number of inputs relating to cellular energy and nutrient status. These include insulin, glucose, cellular energy levels and amino acid availability. In response, TORC1 activates protein synthesis via a number of mechanisms including activation of the ribosomal S6 kinase (S6K), repression of the translational inhibitor 4E-BP, and promotion of ribosome biogenesis via myc. In particular since TORC1 is a master regulator of protein biosynthesis, its regulation by amino acids, the building blocks of proteins, likely constitutes an important regulatory feedback mechanism. Furthermore, the importance of amino acid signaling to TOR is highlighted by the observation that circulating amino acids are elevated in humans with obesity, where they have been shown to activate TORC1 activity and modulate glucose metabolism. Despite this, understanding of the molecular mechanism by which amino acids regulate TOR remains fragmentary (Bryk, 2010).

Three protein complexes have recently been implicated in the activation of TORC1 in response to amino acids. The human class III PI3K (phosphoinositide 3-kinase) hVps34 is activated by amino acids via a calcium dependent mechanism (Gulati, 2008). This leads to accumulation of phosphatidylinositol 3-phosphate (PI(3)P) in cells, which is thought to cause the recruitment of proteins recognizing PI(3)P to early endosomes, forming an intracellular signaling platform that leads to TORC1 activation. This feature of the pathway may be specific for vertebrates, as flies mutant for Vps34 have been reported to not have TORC1 signaling defects. Recently, two groups discovered that Rag GTPases mediate amino acid signaling to TORC1. The emerging picture is that amino acids change the GDP/GTP loading of the Rag GTPases, thereby stimulating the binding of Rag heterodimeric complexes to TORC1. This in turn causes TORC1 to change its intracellular localization, perhaps relocalizing it to vesicles containing the activator Rheb. This mechanism appears to be evolutionarily conserved from flies to humans (Bryk, 2010).

The third protein recently identified as a mediator of amino acid signaling to TOR is MAP4K3 (Findlay, 2007); in HeLa cells the kinase activity of MAP4K3 is activated in the presence of amino acids. In turn, MAP4K3 is required for TOR to phosphorylate its targets S6K and 4E-BP1 in response to amino acid sufficiency. The cell-culture data also suggest this mechanism is conserved from flies to humans as knockdown of Drosophila MAP4K3 causes a reduction in TOR activity (Bryk, 2010).

Although considerable progress has been made, important questions remain unanswered. The role of TORC1 activity in vivo has been well studied in flies and mice, but fundamental issues regarding the regulation of TOR by amino acids have thus far only been explored in vitro in cell culture. To assess the functional significance of the ability of TORC1 to sense amino acids in the organismal context, use has been made of a Drosophila mutant for MAP4K3 (CG7097). dMAP4K3 mutant flies have reduced TOR activity, detected by phosphorylation of TOR targets. dMAP4K3 mutants are viable but display physiological aberrations emblematic of animals starved of nutrients: MAP4K3 mutant flies have retarded growth, reduced size, and low lipid reserves. Both the biochemical results and the phenotypes indicate that MAP4K3 modulates, but is not absolutely required, for TOR activity in vivo. This is similar to what is observed with other modulators of the pathway, such as Melted. Unexpectedly, the function of MAP4K3 is most required when nutrient conditions are rich (Bryk, 2010).

Recent reports have shown that not all components identified in cell culture as regulators of TORC1 activity also affect TORC1 in vivo in an animal model. The purpose of this study was two-fold: (1) to analyze whether MAP4K3 regulates TORC1 activity in vivo in the fly, and (2) to study the physiological consequences for the organism when the ability of TORC1 to sense amino acids is impaired (Bryk, 2010).

Biochemical evidence is presented that TORC1 activity is reduced in MAP4K3 mutant animals, consistent with published cell-culture data showing that MAP4K3 is required for full TORC1 activation (Findlay, 2007). Furthermore, MAP4K3 mutants have defects typical of reduced TORC1 activity. They are delayed in their development due to a reduced rate of growth. They eventually pupate leading to adults of reduced size and their tissues are comprised of cells that are smaller than normal. Furthermore, MAP4K3 mutants have significantly reduced triglyceride stores compared to controls. These physiological effects are similar to the phenotypes observed with mutants for other regulators of TOR, such as Melted. Melted mutant flies are also 10% smaller than controls and are significantly leaner (Bryk, 2010).

As a whole, the MAP4K3- mutant phenotypes emulate the physiological effects observed when flies are grown on conditions of limiting food. When wildtype larvae are put on a low-nutrient diet, they are delayed in pupation and yield animals of small size that are lean. Thus loss of MAP4K3 activity phenocopies a reduced nutrient environment, consistent with MAP4K3 playing a role in the ability of animals to sense their nutrient conditions. This suggests the ability of TORC1 to sense amino acids is most important when nutrient conditions are rich, allowing animals to accelerate their growth accordingly. In contrast, on a low-nutrient diet, control and MAP4K3 mutant flies grow equally slowly consistent with TOR activity being low in both groups. This parallels nicely the results reported in cell culture by (Findlay, 2007): In the absence of amino acids, both control and MAP4K3 knockdown cells have low TOR activity whereas in the presence of amino acids, TOR is activated strongly in control cells but only weakly in MAP4K3 knockdown cells (Bryk, 2010).

Unexpectedly, it was found that MAP4K3 mutant animals are viable, although they have an elevated mortality rate compared to controls. This suggests that the amino acid sensing pathway might only modulate TORC1 activity. If TORC1 activity were completely blunted in MAP4K3 mutants, the animals would be dead, as is the case for TOR or Rheb mutants. Consistent with this, residual TORC1 activity was observed in MAP4K3 mutants, as detected by phosphorylation of the TORC1 targets S6K and 4EBP. This also parallels results from cell culture. The results presented in Findlay (2007) are obtained with cells starved of serum and consequently of insulin signaling. In the presence of insulin signaling, which resembles the physiological situation more closely, MAP4K3 mutant cells still retain residual TOR activity, similar to what was observed in vivo in this study. Consistent with these findings, it was observed that the Rheb expression is able to drive tissue growth also in the absence of MAP4K3 (Bryk, 2010).

Both MAP4K3 and the Rag GTPases have recently been shown to be required for amino acids to stimulate TORC1 activity. While studying dMAP4K3, it was noticed that dMAP4K3 binds physically to the Rag GTPases, suggesting they might act together as components of a single signaling pathway. This interaction is likely specific for several reasons: (1) no binding of MAP4K3 to another GTPase, Rheb, could be detected, (2) binding of MAP4K3 to the RagA/C complex was significantly stronger than binding of an unrelated HA-tagged protein, HA-medea (3) MAP4K3 bound FLAG-RagC significantly stronger than FLAG-RagA showing that MAP4K3 distinguishes between two Rag proteins and (4) binding of MAP4K3 to RagC depended on its GDP/GTP state (Bryk, 2010).

Further studies will be required to test whether this interaction is important for TORC1 to sense amino acids. The data suggest that MAP4K3 might be functioning upstream of the Rag GTPases, and not downstream since activated RagA does not require MAP4K3 to promote tissue growth in vivo. This raises the possibility that the Rag GTPases may be substrates for MAP4K3 phosphorylation. Indeed, RagC is phosphorylated in vivo in Kc167 cells on Ser388. If MAP4K3 were to phosphorylate RagC, this would provide a mechanism for regulation of the Rag GTPases, which to date is mysterious. Work in the near future should shed further light on this issue (Bryk, 2010).

In summary, this study has characterized the physiological function of MAP4K3 in Drosophila, and shown that it modulates TORC1 activity, tissue growth and lipid metabolism in the animal. Physical interaction data hints at a possible link between MAP4K3 and the Rag GTPases. The organismal function of amino acid sensing by TORC1 is mainly required to spur growth when nutrient conditions are rich (Bryk, 2010).

ATG1, an autophagy regulator, inhibits cell growth by negatively regulating S6 kinase

It has been proposed that cell growth and autophagy are coordinated in response to cellular nutrient status, but the relationship between them is not fully understood. This study characterized the fly mutants of Autophagy-specific gene 1 (ATG1), an autophagy-regulating kinase, and it was found that ATG1 is a negative regulator of the target of rapamycin (TOR)/S6 kinase (S6K) pathway. The Drosophila studies have shown that ATG1 inhibits TOR/S6K-dependent cell growth and development by interfering with S6K activation. Consistently, overexpression of ATG1 in mammalian cells also markedly inhibits S6K in a kinase activity-dependent manner, and short interfering RNA-mediated knockdown of ATG1 induces ectopic activation of S6K and S6 phosphorylation. Moreover, ATG1 specifically inhibits S6K activity by blocking phosphorylation of S6K at Thr 389. Taken together, these genetic and biochemical results strongly indicate crosstalk between autophagy and cell growth regulation (Lee, 2007).

Previous studies have demonstrated that homozygous EP3348 flies, in which a single P element is inserted into the 5' untranslated region of DmATG1, show larval/pupal lethality. However, after the genomic background of the EP3348 line was cleared by four backcrosses with w1118 flies, about 30% of homozygous mutants were found to develop to adults. To confirm that the P-element insertion in EP3348 hampers transcription of DmATG1 (Scott, 2004), quantitative real-time reverse transcriptase-PCR (qRT-PCR), was performed. This showed highly reduced DmATG1 expression in the mutant. Therefore, this cleared EP3348 allele was named DmATG11.

The mitotic phenotypes in the larval brain and imaginal discs of DmATG1 mutants, DmATG11 and DmATG1Δ3d (a null allele of DmATG1 were examined. However, no notable mitotic defects could be found in the mutants compared with the wild-type control (w1118. Consistently, DmATG1 mutants had no gross chromosomal abnormalities in the neuroblast cells of third instar larval brains (Lee, 2007).

Next, defects in autophagy in DmATG11 flies were examined using toluidine blue-azure II staining and transmission electron microscopy (TEM) analyses. As expected, DmATG11 flies showed marked defects in the induction of autophagy under conditions of starvation. This is highly consistent with the previous study using DmATG1Δ3d (Scott, 2004). These results strongly supported that DmATG11 is a new hypomorphic mutant allele of DmATG1 (Lee, 2007).

To understand further the in vivo roles of ATG1 in Drosophila, double-mutant lines were generated for dTORP1 (a loss-of-function mutant allele of dTOR and DmATG1 mutants (DmATG11, DmATG1Δ3d and EP3348). Homozygous dTORP1 mutants showed growth arrest in the second/early third instar larval stage and markedly delayed growth. Surprisingly, homozygous dTORP1 larvae with a heterozygous genetic background of DmATG11 or DmATG1Δ3d not only grew faster than homozygous dTORP1, but also extended their developmental stage to the mid-late third instar larval stage. Furthermore, the double-homozygous mutants between dTORP1 and various DmATG1 mutants survived up to the mid-late third instar larval stage, which is inconsistent with the previous results (Scott, 2004; Lee, 2007).

In addition, it was found that another phenotype of dTORP1 mutants, lipid vesicle aggregation in the fat body, was also suppressed by a reduction of the gene dosage of DmATG1. These results implicated that ATG1 negatively mediates the developmental and physiological roles of TOR in Drosophila (Lee, 2007).

Since dTOR regulates cell growth in a cell-autonomous manner, whether DmATG1 is also involved in this role of dTOR was examined. The cell and nuclear sizes of the salivary gland cells were markedly reduced in dTORP1 larvae. Intriguingly, the heterozygous genetic background of DmATG11 or DmATG1Δ3d partly rescued the reduced cell and nuclear size phenotype of dTORP1, strongly implicating that DmATG1 mediates the crucial function of dTOR in cell growth regulation. This is further supported by recent results, that DmATG1-null cells have a relative growth advantage over wild-type cells when treated with rapamycin, a specific inhibitor of dTOR (Lee, 2007).

To examine the possibility that the suppression of dTORP1 phenotypes by DmATG1 mutation resulted from altered auto-phagic activities, the genetic interactions were investigated between dTOR and other autophagy-related genes, such as ATG6 (Beclin1) and UVRAG (UV radiation resistance associated gene), which are known to form a complex regulating autophagosome formation (Liang, 2006). As a result, ATG6 and UVRAG mutations did not suppress the developmental delay and cell growth defects of dTOR mutants, showing that the interaction between dTOR and DmATG1 is not caused indirectly by uncontrolled regulation of autophagy (Lee, 2007).

To determine the functional interaction between ATG1 and S6K, a downstream effector of TOR, double-mutant lines were generated between DmATG11 and dS6K mutants -- a hypomorphic allele, dS6K07084, and a null allele, dS6Kl−1. Using TEM analyses, it was observed that a reduction of dS6K gene dosage did not rescue the defects in autophagosome formation in starved DmATG11 homozygous larvae. However, surprisingly, the reduced gene dosage of dS6K increased the eclosion rate of homozygous DmATG11 in a dS6K gene dosage-dependent manner. Although the possibility that S6K promotes autophagy as reported previously (Scott, 2004) cannot be excluded, these data indicate that dS6K has an important role in DmATG1-dependent developmental processes (Lee, 2007).

Next, a biochemical analysis was conducted to examine the effect of loss of DmATG1 on dS6K activation. As a result, it was found that dS6K was markedly activated (~threefold increase) in homozygous DmATG11 larvae and pupae, measured by the phosphorylation of dS6K at the Thr 398 site, compared with the wild-type controls. Notably, the increased level of dS6K phosphorylation in DmATG11 mutants was about one-third of that in flies overexpressing Rheb. Consistent with this, DmATG1 overexpression almost completely inhibited dS6K Thr 398 phosphorylation in Drosophila (Scott, 2007). These results strongly suggest an important role of ATG1 in the regulation of S6K (Lee, 2007).

To extend these findings to the mammalian system and also to investigate further the molecular mechanism of the interaction between ATG1 and TOR/S6K, the effect of ATG1 on the activity of S6K was examined in mammalian cells. There are two isoforms of ATG1 in mammals, which are named UNC-51-like kinase (ULK) 1 and 2. However, according to the agreement on gene nomenclature made by researchers in the field of autophagy, they were renamed ATG1α and ATG1β, respectively. Nutrient deprivation of HEK293T cells abolished the phosphorylation of S6K at both Thr 229 and Thr 389 sites, which represents the activation status of S6K . When nutrients including amino acids and glucose (DMEM) were added back to the cells, the phosphorylation of both sites in S6K was strongly induced. However, co-expression of wild-type mouse ATG1α (ATG1α WT) strongly inhibited S6K activity induced by DMEM. On the contrary, a kinase-dead form of ATG1α (ATG1α KI) was not able to block the nutrient-induced activation of S6K, showing that ATG1α inhibits S6K in a kinase activity-dependent manner. Consistently, epidermal growth factor (EGF)-stimulated S6K activation was also inhibited by ATG1α. Furthermore, ATG1β, another isoform of ATG1, has the same inhibitory effect on S6K phosphorylation as ATG1α. These data strongly suggest that ATG1 regulates the activities of upstream kinases or phosphatases of S6K, which affect both Thr 229 and Thr 389 phosphorylation (Lee, 2007).

As ATG1 is a crucial regulator of autophagy in yeast and Drosophila, whether overexpression of ATG1 can induce autophagy in mammalian cells was tested. Nutrient deprivation was able to induce autophagy in MCF-7 cells, whereas overexpression of ATG1 did not induce autophagy in MCF-7 and HEK 293T cells, indicating that the inhibition of TOR/S6K by ATG1 is not an indirect consequence of an ectopic induction of autophagy. This was further supported by the observation that overexpression of ATG6 and UVRAG did not inhibit the phosphorylation of S6K , which is also highly consistent with the above Drosophila data (Lee, 2007).

Then, short interfering RNA (siRNA) targeting was used for ATG1α and ATG1β messenger RNA to confirm that ATG1 inhibits S6K activity. The efficacy of siRNA was verified by qRT–PCR using ATG1-specific primers. Transfection of ATG1α and ATG1β siRNA to HEK 293T cells led to increased phosphorylation of S6K Thr 389 and S6 (the only proven in vivo substrate of S6K) Thr 235/236. This result was further supported by immunocytochemistry by using phosphospecific S6 antibody; ATG1 siRNA transfection alone induced phosphospecific immunostaining of S6 in starved cells. Taken together, these results clearly demonstrate that ATG1 inhibits S6K and S6 in vivo (Lee, 2007).

Notably, the level of S6K activation by ATG1 siRNA was about 5% of that by nutritional stimulation. This weak activation of S6K resulted from partial gene knockdown by RNAi. Consistent with this conclusion, more pronounced activation of dS6K was observed in DmATG1 mutants in Drosophila, which contains only a single orthologue of ATG1 (Lee, 2007).

S6K is in the AGC kinase family, which also includes Akt and p90 ribosomal S6 kinase (RSK). These kinases are regulated by a similar mechanism in which both phosphorylation at their activation loop and a hydrophobic motif next to the kinase domain are required for full activation. 3-Phosphoinositide-dependent kinase 1 (PDK1) is a kinase responsible for phosphorylation at the activation loop of AGC kinases. In the case of S6K, PDK1 directly phosphorylates the Thr 229 residue at the activation loop of S6K, which is strictly dependent on the previous phosphorylation of Thr 389 at the hydrophobic motif. These motifs are well conserved among the family members in different species. Therefore, whether ATG1α also affects the phosphorylation of Akt and RSK was examined. Interestingly, the phosphorylation of Akt and RSK was not affected by ATG1α, with or without stimulation by insulin and EGF. These data indicate that ATG1α specifically modulates S6K activity (Lee, 2007).

Next, to understand the molecular mechanism of the specific regulation of S6K by ATG1, whether ATG1 affects the phosphorylation of Thr 229 in S6K was investigated by using an S6K mutant that specifically mimics the phosphorylated form of S6K, Thr 389 Glu. As a result, Thr 229 phosphorylation of the S6K Thr 389 Glu mutant was not affected by wild-type ATG1. Because Akt was not inhibited by ATG1, it is unlikely that ATG1 regulates Thr 389 phosphorylation of S6K by inhibiting the PDK1/Akt signalling module. Therefore, it is believed that ATG1 modulates S6K activity by affecting S6K Thr 389-specific kinases or phosphatases (Lee, 2007).

In summary, under nutrient-rich conditions, activation of TOR leads to inhibition of ATG1, which facilitates S6K Thr 389 phosphorylation and the subsequent phosphorylation of Thr 229 by PDK1 to activate S6K fully. Consequently, activated S6K promotes cell growth. However, under conditions of starvation, TOR becomes quiescent and ATG1 can inhibit S6K by blocking Thr 389 phosphorylation. This nutrient-dependent signalling switch operated by TOR and ATG1 is highly consistent with that in yeast. The observations described in this study clearly show the presence of crosstalk between ATG1 and S6K signalling, in which ATG1 specifically inhibits S6K. This study also showed that this is evolutionarily conserved between Drosophila and mammals. It is believed that these biochemical data and the fly system will be useful in future studies that address the detailed molecular mechanism of crosstalk between the two nutrition-dependent physiological processes -- autophagy and cell growth (Lee, 2007).

AKT and TOR signaling set the pace of the circadian pacemaker

The circadian clock coordinates cellular and organismal energy metabolism. The importance of this circadian timing system is underscored by findings that defects in the clock cause deregulation of metabolic physiology and result in metabolic disorders. On the other hand, metabolism also influences the circadian clock, such that circadian gene expression in peripheral tissues is affected in mammalian models of obesity and diabetes. However, to date there is little to no information on the effect of metabolic genes on the central brain pacemaker which drives behavioral rhythms. This study found that the AKT and TOR-S6K pathways, which are major regulators of nutrient metabolism, cell growth, and senescence, impact the brain circadian clock that drives behavioral rhythms in Drosophila. Elevated AKT or TOR activity lengthens circadian period, whereas reduced AKT signaling shortens it. Effects of TOR-S6K appear to be mediated by SGG/GSK3beta, a known kinase involved in clock regulation. Like SGG, TOR signaling affects the timing of nuclear accumulation of the circadian clock protein Timeless. Given that activities of AKT and TOR pathways are affected by nutrient/energy levels and endocrine signaling, these data suggest that metabolic disorders caused by nutrient and energy imbalance are associated with altered rest:activity behavior (Zheng, 2010).

There are several possible mechanisms by which nutrient and energy metabolism could affect peripheral clocks. Local physiological factors dependent on metabolic activity could influence the expression of core clock components and of nuclear receptors that regulate clock gene expression. Indeed, cellular redox state, AMPK activity, NAD+ levels, and SIRT1 activities appear to feed into the circadian clock in peripheral tissues such as the liver. AMPK, which acts upstream of TSC in mammals, directly phosphorylates Cryptochrome in peripheral tissues. However, prior to this work, there was no known mechanism for the modulation of the central pacemaker by nutrient-sensing pathways. This study identifies such a mechanism by demonstrating that metabolic genes such as AKT and TOR-S6K act in the central pacemaker cells in the brain. The lengthened circadian period caused by high-fat diet in mammals is likely mediated by these molecules. This conclusion is further supported by a recent cell-culture-based genome-wide RNAi study that implicated the PI3K-TOR pathway in the regulation of circadian period. In addition, another ribosomal S6 kinase (S6KII) was found to influence the circadian clock through its interaction with casein kinase 2β. Importantly, daily fasting:feeding cycles driven by the central clock regulate circadian gene transcription in the liver, whereas clock function in the liver contributes to energy homeostasis. It is speculated that metabolic stress or energy imbalance affects AKT and TOR-S6K signaling, resulting in general circadian disruption, which in turn exacerbates metabolic deregulation and, consequently, facilitates the development of metabolic syndromes prevalent in modern society (Zheng, 2010).

S6 kinase localizes to the presynaptic active zone and functions with PDK1 to control synapse development

The dimensions of neuronal dendrites, axons, and synaptic terminals are reproducibly specified for each neuron type, yet it remains unknown how these structures acquire their precise dimensions of length and diameter. Similarly, it remains unknown how active zone number and synaptic strength are specified relative the precise dimensions of presynaptic boutons. This paper demonstrates that S6 kinase (S6K) localizes to the presynaptic active zone. Specifically, S6K colocalizes with the presynaptic protein Bruchpilot (Brp) and requires Brp for active zone localization. Evidence is provided that S6K functions downstream of presynaptic PDK1 to control synaptic bouton size, active zone number, and synaptic function without influencing presynaptic bouton number. It was further demonstrated that PDK1 is also a presynaptic protein, though it is distributed more broadly. A model is presented in which synaptic S6K responds to local extracellular nutrient and growth factor signaling at the synapse to modulate developmental size specification, including cell size, bouton size, active zone number, and neurotransmitter release (Cheng, 2011).

Dendrite diameter, the size of a dendritic field, and the area of the presynaptic nerve terminal are all reproducibly specified for a given cell type. The size and complexity of a neuron far exceeds that of any other cell type, suggesting that there may be unique solutions to the challenge of controlling and coordinating the growth of the many different and distinct features of neuronal architecture. Specification of neuronal dimension is further complicated because it is intimately associated with the electrochemical function of the neuron. Indeed, a specific form of neuronal growth, isoelectronic growth, has been observed in both invertebrates and vertebrates in which dendrite diameter grows precisely as the square of dendrite length, thereby maintaining the cable properties of individual dendrites. This type of growth is fundamentally different from most types of neuronal growth that are characterized experimentally, including axon extension, dendrite branching, spine formation, and synapse expansion. It remains unknown how the dimensions of individual neuronal compartments can be precisely specified and how these growth-related parameters are coordinated with neuronal function. Mechanistically, very few studies report the identification of genes that specifically control neuronal dimensions without otherwise perturbing the ability of these structures to form properly (Cheng, 2011).

Candidates for the control of neuronal dimension are genes associated with the regulation of cell size, including mammalian target of rapamycin, PDK1, and S6 kinase (S6K). S6K is necessary for long-term facilitation, the early phase of long-term potentiation, learning, and activity-dependent neuronal sprouting during epilepsy. S6K also influences the growth of dendritic arbors in cultured hippocampal neurons. However, there are conflicting studies regarding whether S6K influences bouton number at the Drosophila melanogaster neuromuscular junction (Cheng, 2011).

The formation of complex neural circuitry is largely determined by the sequential processes of axon guidance, target recognition, and the activity-dependent refinement of synaptic connectivity. However, the dimensions of individual neuronal compartments, including the length and diameter of dendrites, axons, and presynaptic boutons are also reproducibly specified for each cell type, and these parameters strongly influence neuronal function. It was previously established that PDK1 and S6K control cell size, but it has remained unclear how these potent signaling molecules influence synaptic growth. This study provides evidence that signaling through PDK1 and S6K is specifically required to control synaptic bouton size without strongly influencing the proliferation of bouton number or the length of the nerve terminal. Many genes have been identified that perturb synapse morphology at the Drosophila NMJ when mutated. In most cases, however, the gross morphology of the synapse is perturbed, indicating that the cellular mechanics of nerve terminal extension are altered. In contrast, signaling via PDK1 and S6K primarily control the dimensions of individual synaptic boutons without otherwise altering the appearance of the presynaptic terminal (Cheng, 2011).

How are changes in compartment dimensions (cell diameter, axon diameter, and bouton diameter) mechanistically executed downstream of PDK1 and S6K? It is particularly interesting that both PDK1 and S6K are distributed throughout the cell, being present in axons, presynaptic terminals, and in the case of S6K, at the active zone. This distribution suggests that PDK1 and S6K could exert local effects that contribute to the specification of cell shape. Indeed, there is a correlation between impaired S6K localization at the active zone in the brp mutant and decreased bouton size as well as decreased axon size. Future experiments will be necessary to determine whether the changes in compartment size are a direct consequence of S6K mislocalization or whether size is influenced by altered synaptic transmission in the brp mutant. It is interesting to speculate about the cell biological processes that might function downstream of S6K to influence cellular dimensions. A previous study has provided evidence that the submembranous skeleton composed of spectrin and ankyrin provides structural integrity and modulates the shape of axons and presynaptic terminals, in part through organization of the underlying microtubule cytoskeleton (Pielage, 2008). However, a connection between PDK1, S6K, and the submembranous spectrin/ankyrin skeleton remains to be identified. In other systems, S6K has been shown to interact with spinophillin/neurabin, which is an F-actin- and protein phosphatase 1-binding protein linked to the control of dendritic spine size in vertebrates and active zone integrity in Caenorhabditis elegans. A link between S6K and neurabin/spinophillin could provide a mechanism to link local nutrient detection to the modulation of the neuronal cytoskeleton and cell shape (Cheng, 2011).

It remains unknown how active zone number is specified for a given cell type. The neuromuscular synapse, the Calyx of Held, and other large, powerful synapses harbor a robust and reproducibly large number of active zones. In contrast, other neuronal cell types make synaptic connections composed of fewer active zones, many forming a single release site within a synaptic bouton. Genes have been identified that negatively regulate active zone assembly. Other genes have been identified that are necessary for correct placement of active zones, active zone assembly, and active zone dimension. In contrast, the specification of total active zone number has remained less well defined. Target-derived growth factors clearly influence the growth and development of the presynaptic nerve terminal and have been shown to influence total active zone number. These signaling systems will activate downstream intracellular signaling cascades, such as MAPK signaling, recently implicated in the specification of active zone number at the Drosophila NMJ (Wairkar, 2009). However, it also seems likely that the control of active zone number during development will be more complex than simple specification based upon the quantity of a target-derived trophic signal. It is speculated that there will exist cell type-specific programs that interface with growth factor signaling to determine characteristic active zone densities. Cellular metabolic signaling might be one such cell type-specific parameter, including the actions of S6K and PDK1 (Cheng, 2011).

It is particularly remarkable that presynaptic overexpression of S6K and PDK1 are sufficient to increase active zone number at the NMJ. These data indicate that synapse assembly during neuromuscular development is a process that can be driven by signaling that originates within the motoneuron. It is speculated that the localization of S6K at the active zone may be critical in this regard. In some respects, it seems counterintuitive that a signaling system coupled to the metabolism of the motoneuron (PDK1-S6K) would be able to determine active zone number and, therefore, the level of postsynaptic excitation. Therefore, in keeping with well-established trophic mechanisms, it is predicted that S6K and PDK1 normally function downstream of muscle-derived factors, including nutrients and growth factors, that couple the needs of the muscle to the insertion of active zones by the motoneuron (Cheng, 2011).

There is increasing evidence that local protein synthesis plays a prominent role within postsynaptic dendrites. In contrast, the evidence for presynaptic protein translation is less abundant, including an apparent absence of polyribosomes within axons and nerve terminals. The most convincing evidence for local presynaptic protein translation is observed in Aplysia, in which serotonin-dependent long-term facilitation can be induced in synaptic compartments separated from the soma. Interestingly, two recent studies implicate S6K in presynaptic, translation-dependent long-term facilitation in Aplysia. The localization of S6K to the presynaptic active zone, where additional RNA-interacting signaling molecules have recently been identified, is intriguing. In Drosophila, the translational repressors nanos and pumillio have been implicated in the regulation of neuromuscular growth, membrane excitability, and postsynaptic glutamate receptor abundance. Nanos is present presynaptically, and recent data demonstrate that loss of nanos leads to an increase in total bouton number and an increase in the total active zone number. In a separate study, the IGF-II RNA-binding protein (Imp-GFP) was observed to traffic to the presynaptic terminal, and loss of Imp caused a decrease in bouton number. Although none of these data provide direct evidence for local presynaptic protein translation being important for presynaptic development or function, there is an accumulation of data suggesting that this may be a realistic possibility. Perhaps local protein translation, downstream of nutrient and growth factor signaling, could help independently shape the dimensions of each neuronal compartment (soma, axon, dendrite, and nerve terminal) and, thereby, fine tune the input-output properties of neurons during development. In this regard, it is particularly interesting that the synaptic localization of S6K by Brp seems to be important for size regulation at the synapse and perhaps throughout the cell (Cheng, 2011).

A mitochondrial ATP synthase subunit interacts with TOR signaling to modulate protein homeostasis and lifespan in Drosophila

Diet composition is a critical determinant of lifespan, and nutrient imbalance is detrimental to health. However, how nutrients interact with genetic factors to modulate lifespan remains elusive. This study investigated how diet composition influences mitochondrial ATP synthase subunit d (ATPsyn-d) in modulating lifespan in Drosophila. ATPsyn-d knockdown extends lifespan in females fed low carbohydrate-to-protein (C:P) diets but not the high C:P ratio diet. This extension is associated with increased resistance to oxidative stress; transcriptional changes in metabolism, proteostasis, and immune genes; reduced protein damage and aggregation, and reduced phosphorylation of S6K and ERK in TOR and mitogen-activated protein kinase (MAPK) signaling, respectively. ATPsyn-d knockdown did not extend lifespan in females with reduced TOR signaling induced genetically by Tsc2 overexpression or pharmacologically by rapamycin. These data reveal a link among diet, mitochondria, and MAPK and TOR signaling in aging and stresses the importance of considering genetic background and diet composition in implementing interventions for promoting healthy aging (Sun, 2014).

Dietary nutrients are among the most critical environmental factors that modulate healthspan and lifespan. Nutrient imbalance is a major risk factor to human health and common among old people. Dietary restriction (DR), by reducing the amount of all or specific nutrients, is a potent nongenetic intervention that promotes longevity in many species. In general, protein restriction is more effective in influencing lifespan than sugar or calorie restriction in Drosophila. However, increasing evidence indicates that the composition of dietary nutrients, such as carbohydrate-to-protein (C:P) ratio, is more critical than individual nutrients in affecting health and lifespan. Optimal lifespan peaks at the C:P ratio 16:1 in Drosophila and 9:1 in Mexican fruit fly. A recent study in mice shows that lifespan is primarily regulated by the C:P ratio in the diet and tends to be longer with higher C:P ratios. Diet composition is also critical for DR to promote longevity in nonhuman primate rhesus monkeys. Two major nutrient-sensing pathways are known to modulate lifespan. One is target-of-rapamycin (TOR) signaling that mostly senses cellular amino acid content and the other is insulin/insulin-like signaling that primarily responds to circulating glucose and energy levels. Excessive carbohydrate and protein intake both contribute to development of insulin resistance and diabetes in animal models and humans. Dietary macronutrients, such as sugar, protein, and fat, may interact with each other to influence nutrient-sensing pathways and consequently health outcome. It is thus critical to take into account diet composition in elucidating molecular mechanisms of aging and in developing effective interventions for promoting healthy aging (Sun, 2014).

Aging is associated with transcriptional and translational changes in many genes and proteins. Some age-related changes are evolutionarily conserved, and many function in nutrient metabolism, such as mitochondrial electron transfer chain (ETC) genes, many of which are downregulated with age in worms, flies, rodents, and humans. Knocking down ETC genes affects lifespan in yeast, worms, and flies. Mitochondrial genes also play a key role in numerous age-related diseases, such as Parkinson's and Alzheimer's disease. However, how mitochondrial genes interact with nutrients to modulate lifespan and health-span remains incompletely elucidated. Understanding gene-environment interactions will be a key to tackle aging and age-related diseases (Sun, 2014).

ATP synthase subunit d (ATPsyn-d) is a component of ATP synthase, ETC complex V, and is known to modulate lifespan in C. elegans. How ATPsyn-d modulates lifespan and whether it functions in modulating lifespan in other species remain to be determined (Sun, 2014).

Given the importance of nutrients as environmental factors in modulating lifespan, this study has investigated whether and how ATPsyn-d interacts with dietary macronutrients to modulate lifespan in Drosophila. ATPsyn-d was found to interact with dietary macronutrients to influence accumulation of oxidative damage and protein aggregates; resistance to oxidative stress; and expression of numerous genes involved in metabolism, proteolysis, and innate immune response and more importantly to modulate lifespan. Moreover, ATPsyn-d affects mitogen-activated protein (MAP) kinase (MAPK) signaling and genetically interacts with TOR signaling to influence lifespan of flies in a diet-composition-dependent manner. This study reveals the critical interaction between mitochondrial genes and nutritional factors and the underlying mechanisms involving TOR signaling in modulating lifespan (Sun, 2014).

Considering the essential role of mitochondrial function in metabolism and aging, this study investigated how diet composition influences the function of ATPsynd, a component of mitochondrial ATP synthase, in aging and the underlying mechanisms. ATPsyn-d knockdown extends lifespan in Drosophila under low sugar-high protein diets, but not under a high sugar-low protein diet. Lifespan extension induced by ATPsyn-d knockdown is associated with increased resistance to oxidative stress and improved protein homeostasis. Furthermore, evidence is provided suggesting ATPsyn-d modulates lifespan through genetically interacting with TOR signaling. Knocking down of atp-5, the worm ATPsynd, extends lifespan in C. elegans, along with the current data suggesting a conserved role of ATPsyn-d in modulating lifespan. Altogether, these findings reveal a connection among diet, mitochondrial ATP synthase, and MAPK and TOR signaling in modulating lifespan and shed light on the molecular mechanisms underlying the impact of diet composition on lifespan (Sun, 2014).

The following model is proposed to explain how ATPsyn-d interacts with dietary macronutrients to modulate lifespan, considering the genetic interaction between ATPsyn-d and TOR signaling and the fact that suppression of TOR signaling by altering expression of its components, such as Tsc1/Tsc2, S6K, and 4E-BP, activates autophagy, improves proteostasis, and promotes longevity in high-protein diets, but not necessarily low-protein diets. It is postulated that TOR signaling is regulated by ATPsyn-d and perhaps other mitochondrial proteins. ATPsyn-d knockdown reduces MAPK signaling and probably affects other signaling pathways, which may consequently decrease TOR signaling to extend lifespan in Drosophila fed high-protein diets, such as SY1:9 and SY1:1, but not low protein diets. It is possible that diet-dependent response is due to knockdown of ATPsyn-d protein to different extent by RNAi under different dietary conditions. This is not likely the case. The amount of ATPsyn-d knockdown is not much different between flies on sugar (S) and yeast (Y) SY1:9 and SY9:1, although lifespan is not increased by ATPsyn-d knockdown for flies under SY9:1. Therefore, variations in ATPsyn-d knockdown under current experimental conditions unlikely contribute significantly to diet-dependent lifespan extension. Consistent with this model, ATPsyn-d knockdown increases resistance to acute oxidative stress, reduces cellular oxidative damage, and improves proteostasis in Drosophila. Reduced oxidative damage by ATPsynd knockdown may lead to decreased MAPK signaling, which in turn modulates TOR signaling and proteostasis (Sun, 2014).

Another likely scenario would be that ATPsyn-d and TOR signaling form a positive but vicious feedback loop through MAPK signaling to induce molecular, metabolic, and physiological changes detrimental to lifespan. This vicious cycle can be disrupted by high-C:P diet, knockdown of mitochondrial genes, or suppression of TOR signaling pharmacologically by rapamycin or genetically by Tsc2 overexpression. Consistent with this possibility is that ATPsyn-d knockdown reduces phosphorylation of S6K, a component of TOR signaling, and increases expression of genes involved in maintaining proteostasis and possibly autophagy, which are regulated by TOR signaling. The level of pS6K reflects the strength of TOR signaling, and reduction- of-function mutants of S6K are known to extend lifespan in several species. ATPsyn-d may genetically interact with TOR signaling to modulate lifespan by influencing protein levels of both S6K and pS6K, although it does not necessarily affect the pS6K/S6K ratio, which may not be a reliable indicator for the strength of TOR signaling under the three SY diets due to the change of S6K level. Furthermore, ATPsyn-d knockdown reduces oxidative damage and polyubiquitinated protein aggregates, which are biomarkers of aging. Rapamycin reduces lifespan extension induced by ATPsyn-d knockdown, which may be due to exacerbation of some detrimental effects of reduced TOR signaling. However, this observation further supports the connection between ATPsyn-d and TOR signaling. Although both rapamycin and ATPsyn-d knockdown reduce pS6K level, ATPsyn-d knockdown, but not rapamycin, decreases S6K level, suggesting ATPsyn-d knockdown and rapamycin affect TOR signaling in different manners. Further studies are warranted to clarify the epistatic relationship between ATPsyn-d and TOR signaling (Sun, 2014).

Increasing evidence has demonstrated the importance of diet composition or carbohydrate to protein ratio in modulating lifespan and health. Nutrient geometry studies conducted in Drosophila have shown that C:P ratio in the diet is far more important in determining lifespan than calorie content or single macronutrient. A recent tour de force nutrient geometry study in mice has confirmed and expanded the view on the critical role of C:P ratio in regulating lifespan and cardiometabolic health to mammals. An important implication from nutrient geometric studies is that diet composition would have a significant impact on the effectiveness of inventions for promoting healthy aging by genetic, pharmaceutical, or nutraceutical approaches (Sun, 2014).

This indeed is the case, although evidence comes from only a handful of studies. Rapamycin feeding extends lifespan in yeast, worms, flies, and mice. Although rapamycin feeding has been shown to extend lifespan of flies under a broad range of diets, some studies have shown that rapamycin feeding does not extend lifespan in flies under high carbohydrate-low protein diets. Supplementation of a nutraceutical derived from cranberry extends lifespan in female flies under a high-C:P-ratio diet, but not a low-C:P-ratio diet. Suppression of TOR signaling by overexpression of Tsc1/Tsc2 extends lifespan in flies under relatively higher-protein diets, but not under low-protein diets, although those studies focused on the variation of protein concentration instead of C:P ratio. Consistent with the link between ATPsyn-d and TOR signaling, ATPsyn-d knockdown extends lifespan in female flies under low sugar-high protein diets, but not high sugar-low protein diet, likely due to the fact that TOR signaling is already low under the high sugar-low protein diet. It was further shown that rapamycin feeding extends lifespan in wild-type female flies, but not in ATPsyn-d knockdown flies (Sun, 2014).

Aging is associated with profound decline in protein homeostasis, and many longevity-related pathways, such as TOR and insulin-like signaling, modulate lifespan through improving proteostasis. Suppression of TOR signaling extends lifespan through decreasing protein translation and increasing autophagy, key processes for maintaining proteostasis. This study found that ATPsyn-d knockdown reduces the level of 4-HNE (an α, β-unsaturated hydroxyalkenal that is produced by lipid peroxidation) protein adducts; a biomarker for lipid protein oxidation; and the level and aggregation of polyubiquitinated protein, a biomarker for proteostasis and aging. ATPsyn-d is a key component of mitochondrial ATP synthase complex. Along with the link between ATPsyn-d and TOR signaling, these data suggest that mitochondrial ATP synthase is critical for maintaining proteostasis and modulating lifespan. This notion is further supported by a recent study showing that α-ketoglutarate, an intermediate in the TCA cycle, suppresses mitochondrial ATP synthase probably by binding to ATP synthase subunit b (ATPsyn-b) and also inhibits TOR signaling to extend lifespan in C. elegans. However, it remains to be determined whether suppression of ATP synthase by α-ketoglutarate results in inhibition of TOR signaling in C. elegans or any other species. It is also likely that ATPsyn-d and ATPsyn-b influences ATP synthase and TOR signaling through different mechanisms, because α-ketoglutarate reduces cellular ATP level in C. elegans, whereas ATPsyn-d knockdown does not significantly change or even increase ATP level in Drosophila. This also suggests that lifespan extension is not necessarily associated with decreased ATP level, which is supported by a study in Drosophila showing that any change of ATP level is not correlated with any change of lifespan induced by knockdown of a number of mitochondrial genes. Nevertheless, these studies suggest that ATP synthase is a key and conserved player linking dietary nutrients from TOR signaling to proteostasis and lifespan (Sun, 2014).

Similar to many longevity-related mutants, lifespan extension induced by ATPsyn-d knockdown is associated with reduced oxidative damage and increased resistance to oxidative stress. ATPsyn-d knockdown increases lifespan and resistance to paraquat, an acute oxidative stress response, under SY1:9 or SY1:1. However, ATPsyn-d knockdown increases resistance to paraquat but does not extend lifespan in female flies under SY9:1. In addition, ATPsyn-d knockdown decreases 4-HNE level, an indicator of accumulated oxidative damage, under SY1:9, but not SY1:1. These indicate that the effect of ATPsyn-d knockdown on oxidative damage and lifespan depends on diet composition, suggesting that oxidative stress resistance is at most partially responsible for lifespan extension. This should not be surprising because it is consistent with numerous studies in the literature showing that stress resistance does not always result in lifespan extension despite the strong link between oxidative stress and aging (Sun, 2014).

The role of mitochondrial genes in modulating lifespan is complex. Knockdown of some electron transfer chain (ETC) genes increases lifespan whereas knockdown of others decreases or does not alter lifespan in C. elegans and Drosophila. This study reveals another layer of complexity regarding the role of ETC genes in lifespan modulation, namely the impact of diet composition. These findings indicate that ATPsyn-d knockdown promotes longevity at least partially through TOR signaling. TOR signaling senses cellular amino acid content and regulates numerous biological processes, including translation, autophagy, and lifespan. 4E-BP, a translational repressor in TOR signaling, mediates lifespan extension induced by dietary restriction (Sun, 2014).

Activated 4E-BP suppresses general translation but selectively increases translation of some mitochondrial ETC genes, the latter of which results in increased mitochondrial biogenesis and potentially lifespan. Lifespan extension induced by dietary restriction is suppressed by knocking down ETC genes regulated by 4E-BP. The findings by Zid suggest that increased protein expression of some ETC genes is associated with lifespan extension induced by dietary restriction. However, unlike those ETC genes, ATPsyn-d knockdown extends instead of decreases lifespan under high-protein diets. Therefore, it is likely that ETC genes can be categorized into two groups: one selectively upregulated by activated 4E-BP and the other insensitive to activated 4E-BP, the latter of which may include ATPsyn-d. The two groups of ETC genes may interact with dietary macronutrients to modulate lifespan perhaps through different modes of action. Future studies are warranted to investigate the dichotomous role of translation of ETC genes in modulating lifespan (Sun, 2014)

Ribosomal protein S6 kinase: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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