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 p70^S6k, 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).

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 dTOR^P1 and dTOR^P2). 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 dTOR^P1 and dTOR^P2 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 dTOR^DeltaP, was selected for further analysis. Sequence analysis reveals that the dTOR^DeltaP deletion originates at the dTOR^P2 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 dTOR^DeltaP 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 dTOR^DeltaP homozygotes (Zhang, 2000).

dTOR^DeltaP 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 dTOR^P1 or dTOR^P2 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 dTOR^DeltaP homozygous cells, which were generated by FLP/FRT-mediated mitotic recombination in dTOR^DeltaP 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 dTOR^DeltaP 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 dTOR^DeltaP 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 dTOR^DeltaP clones were compared with that of their wild-type sister clones (twin spots). Clones of dTOR^DeltaP 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 dTOR^DeltaP homozygous cells are eliminated from the disc epithelium. Because dTOR^DeltaP cells remain viable for weeks in the context of a homozygous mutant animal, the loss of dTOR^DeltaP 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 dTOR^DeltaP 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 dTOR^DeltaP 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 dTOR^DeltaP 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 dTOR^P2 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 p70^S6k (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 (dTOR^RR, which confers rapamycin resistance to mammalian and yeast TOR proteins. Expression of dTOR^RR carrying an additional point mutation in a residue crucial for kinase activity (dTOR^RRKD 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 p70^S6K1 is able to rescue dTOR^P2/P2 and dTOR^P1/P2 flies to viability. The greatest degree of rescue is provided by a mutant version of p70^S6K1, in which four mitogen-induced phosphorylation sites are mutated to aspartate and glutamate residues (mutant D4). Expression of this construct allowed 74% of expected dTOR^P1/P2 progeny to survive to adulthood, whereas no dTOR^P1/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 dTOR^DeltaP 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 p70^S6K1 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 dTOR^DeltaP 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 dTOR^DeltaP 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 dTOR^DeltaP 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 dTOR^DeltaP 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 dTOR^DeltaP 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 dTOR^DeltaP 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 p70^S6K is a common response to virtually all mitogenic stimuli. Phosphorylation of the target of p70^S6K, 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 p70^S6K 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).

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