BIOLOGICAL OVERVIEW

The adaptation of growth in response to nutritional changes is essential for the proper development of all organisms. Described here is the identification of the Drosophila homolog of the target of rapamycin (TOR, often referred to as dTOR), a candidate effector for nutritional sensing. Genetic and biochemical analyses indicate that TOR impinges on the insulin signaling pathway by autonomously affecting growth through modulating the activity of Drosophila S6k. However, in contrast to other components in the insulin signaling pathway, partial loss of TOR function preferentially reduces growth of the endoreplicating tissues. TOR 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. The kinase activity of TOR is required for growth factor-dependent phosphorylation of S6 kinase. Loss of TOR 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 TOR regulates growth during animal development by coupling growth factor signaling to nutrient availability (Oldham, 2000b; Zhang, 2000).

During the development of unicellular and multicellular organisms, growth is dependent on the integration of diverse intracellular signals, which are triggered by patterning and/or environmental cues (Oldham, 2000a). The availability of nutrients strongly influences growth of single cells and multicellular organisms and in some cases specifies alternative developmental programs. For example, in yeast, nitrogen or carbon deprivation leads to withdrawal from vegetative growth, arrest in G₀, and up-regulation of autophagy. Likewise, in the nematode Caenorhabditis elegans, limiting nutrients or overcrowding elicits an alternative developmental program termed the dauer stage. In this stage, the animal remains sexually immature and stockpiles additional lipids for survival during unfavorable growth periods. In more complex organisms such as insects and mammals, an alternative developmental program is lacking. Instead, nutrient limitation is managed by delaying development and, in severe cases, reducing the final body size of the organism. In the case of Drosophila, when nutrients become limiting, available resources are mobilized toward maintaining the growth of the mitotic tissues, which during metamorphosis form smaller, yet still fertile, flies (Oldham, 2000b and references therein).

In metazoans, one component of the complex physiological response of energy homeostasis and growth control is the insulin and IGF signaling system. Insulin and IGF activate two main signaling pathways via the insulin receptor substrates (IRS1-4): the Ras/MAPK pathway, which is involved in proliferation, and the phosphatidyl-inositol 3-kinase (PI3K) signaling pathway, which is involved in cell growth, survival, and metabolic homeostasis. PI3K mediates its effects on downstream signaling components through the production of phosphatidylinositol 3,4,5-tris phosphate (PIP₃), which acts to recruit pleckstrin homology (PH) domain-containing proteins like protein kinase B (PKB). The actions of activated PI3K are antagonized by the 3'-phospho-inositol specific lipid phosphatase encoded by the tumor suppressor gene PTEN. An important protein implicated in the PI3K signaling pathway is the target of rapamycin (TOR). TOR has been reported to be regulated by PKB (Scott, 1998). Initially, TOR1 and TOR2 were identified in yeast as mutations that conferred resistance to the antiproliferative effects of rapamycin (Heitman, 1991). Rapamycin is an antibiotic that inhibits both yeast TOR and mammalian TOR (mTOR, also known as FRAP or RAFT) function by forming an inhibitory complex with the immunophilin, FK506 binding protein-12 (FKBP12), that binds to a region adjacent to the kinase domain termed the FKBP12-rapamycin binding domain (FRB; Brown, 1994; Sabatini, 1994; Thomas, 1997; Cutler, 1999). TOR is most related to the ATM/DNA-PK family of checkpoint protein kinases and is more distantly related to the PI3K family (Thomas, 1997; Cutler, 1999; Oldham, 2000b and references therein).

A key downstream target of mTOR function is protein synthesis. In part, mTOR positively mediates protein synthesis by modulating the activities of important translational components, including the translation initiation factor 4E binding proteins (4E-BP1-3) and the ribosomal protein S6 kinases (Chou, 1995; Lawrence, 1997; Dennis, 1999). Under conditions of reduced nutrients, such as amino acid limitation, mTOR negatively regulates protein synthesis and positively up-regulates autophagy (Dennis, 1999). Thus, mTOR may serve as a nutritional checkpoint for cell growth and ultimately, proliferation (Oldham, 2000b and references therein).

To initiate a genetic analysis of TOR in a multicellular organism, mutations were generated in Drosophila TOR. These mutants were used to study the role of TOR during development. The PI3K/Akt/p70S6K signaling module is conserved in Drosophila, where it acts to regulate cell, organ, and organismal growth (for review, see Coelho and Leevers 2000). Mutational inactivation of this pathway reduces cell size, hinders proliferation, and delays or arrests development, and its activation leads to autonomous increases in cell and organ size. Drosophila TOR mutant phenotypes are found to recapitulate aspects of both PI3K-dependent signaling and nutritional sensing, consistent with TOR acting at the junction of these pathways (Oldham, 2000b; Zhang, 2000).

A tissue-specific genetic screen has been carried out for recessive mutations affecting cell growth and proliferation in the Drosophila compound eye. In this screen, genetically mosaic flies are generated in which the eye and head capsule are homozygous for a randomly induced mutation, while the rest of the body and the germ line are heterozygous and, thus, phenotypically wild type. Remarkably, mosaic flies containing a mutation in a growth-promoting gene have eye and head structures that are strongly reduced in size relative to their wild-type sized heterozygous bodies and are termed pinhead flies. Two EMS-induced pinhead mutations (2L1 and 2L19) map to chromosomal position 34A, where the Drosophila homolog of TOR (dTOR) is located. These mutations fail to complement two lethal P-element insertions, EP(2)2353 and l(2)k17004, located 262 and 211 bp, respectively, upstream of the putative translation start site of dTOR. Sequence analysis of DNA from flies heterozygous for the EMS-induced dTOR mutations reveal two nucleotide substitutions. The lesion of dTOR^2L1 results in a change of a proline to a leucine at amino acid position 2303 (P2303L). The location of this mutation within a highly conserved region of the kinase domain implies that the kinase activity of dTOR is critical for its function, as has been shown for the yeast TORs. In contrast, the lesion in dTOR^2L19 is an arginine changed to a nonsense mutation at amino acid residue 248 (R248Stop), giving rise to a stop codon. This mutation would be predicted to result in a short, truncated protein and should thus be a complete loss-of-function mutation (Oldham, 2000b).

The phenotypes associated with the complete loss of dTOR function are remarkably similar to phenotypes associated with mutations in the Insulin-like receptor (Inr) pathway. (1) Strong dTOR mutants arrest development at a similar stage as do strong mutants in the Inr pathway or amino acid-starved larvae with little detectable imaginal tissue. (2) dTOR mutant clones have a significant proliferative disadvantage similar to Inr pathway mutant clones. Clones of dTOR null mutant cells, although severely affected, are not cell lethal. Similarly, in most mammalian cell types, rapamycin decreases but does not abolish cell growth, except for IL2-mediated T-cell proliferation. (3) The strict autonomous control of cell growth without disturbing the specification and differentiation is also seen with the dTOR mutants. Indeed, loss of dTOR function in clones of homozygous mutant cells in the adult eye show that only the mutant cells, as exemplified by the dark, circular rhabdomeres, are severely reduced in size. Analysis of imaginal wing disc cells at the end of the third larval instar by fluorescence-activated cell sorting (FACS), confirms that cells from the weak heteroallelic combination, dTOR^2L1/dTOR^l(2)k17004, are smaller than those of wild type. The effect on cell size is more pronounced in G₁ than G₂, consistent with mTOR function having a predominante role on cell growth during G₁ (Oldham, 2000b).

Despite this observation, there is no apparent difference between the distribution of dTOR mutant and wild-type cells within each phase of the cell cycle. Although the similarities of the loss-of-function mutant phenotypes of dTOR and other components of the Inr pathway are consistent with a model in which dTOR acts downstream of dPI3K, the analysis of partial loss-of-function dTOR mutants and the biochemical analysis of Drosophila S6K activity indicates a more complex relationship between dTOR and the Inr pathway (Oldham, 2000b).

Genetic interactions between dTOR mutants and other mutations in the insulin pathway were examined. Drosophila Pten encodes a negative effector of insulin signaling, and eyes and heads lacking Pten function are significantly larger than wild-type heads. Removal of dTOR function strongly reduces the size of Pten mutant heads, suggesting that dTOR is required for the increased growth generated by the loss of Pten function (Oldham, 2000b).

In vertebrates, S6K activity is blocked by rapamycin, an inhibitor of TOR. Therefore, Drosophila S6K activity was examined in immunoprecipitates of extracts from larvae mutant for dTOR, S6K, chico, and larvae treated with rapamycin or deprived of amino acids. A severe reduction in the phosphorylation of ribosomal protein S6 was observed in extracts from strong dTOR^2L1/dTOR^2L19 mutant larvae. This was not caused by a reduction in Drosophila S6k protein as shown by Western blotting of these extracts. In addition, the S6k protein is up-regulated in the dTOR mutant larvae and amino acid-starved larvae. In all cases, Western blot analysis has shown equivalent amounts of initiation factor 4E (eIF-4E). S6k activity is not detected in S6k^l-1 null mutants and is severely reduced when wild-type larvae are starved for amino acids or treated with rapamycin. Higher doses of rapamycin blocks development during early larval stages, leading to lethality. Analysis of the weak dTOR^2L1/dTOR^l(2)k17004 or dTOR^2L1/dTOR^EP(2)2353 heteroallelic combinations also reveal a reduction in S6k activity in the third larval instar and an up-regulation of the protein as compared with wild-type flies. The surprising fact that dTOR mutants and amino acid starvation result in an up-regulation of S6k levels suggests that dTOR and amino acids may negatively control the protein levels of S6k. Unexpectedly, S6k activity as well as protein levels are unaffected in chico mutants. It may be that Inr does not signal to S6k or that S6k resides on a parallel pathway that bifurcates upstream of Chico. In support of the latter possibility, Inr has been shown to genetically interact with PI3K independently of Chico, presumably through docking sites for the p60 adaptor of PI3K in the Inr C-terminal tail. This result suggests that there is a S6k independent pathway for growth control and that the reduced Inr-mediated PI3K signaling in a chico mutant is sufficient for S6k activation (Oldham, 2000b).

The biochemical differences between the ability of Chico and dTOR to activate S6k argue for a more complex relationship between the Inr pathway and dTOR. Given the low number of pharate adults, the weights of dTOR, S6k^l-1, and chico mutants were compared at an early pupal stage. The weight of the dTOR mutant pupae is more similar to S6k than to chico mutant pupae. Thus, in the absence of S6k function or the presence of reduced dTOR levels, cellular growth rates are diminished but larvae pupariate at a larger size as a result of a longer developmental delay. Importantly, S6k mutant flies have cells that are smaller but of the normal number. However, in chico mutants, pupariation is initiated at a much smaller size. The result is that chico mutants emerge after only a 2-d delay and are smaller than dTOR and S6k mutants because of fewer and smaller cells. Therefore, while insulin signaling controls cell size and cell number, S6k primarily controls cell size. It will be of interest to know whether dTOR is also limited to controlling only cell size (Oldham, 2000b).

Larvae are composed of mitotic cells, largely represented by the imaginal discs, and of endoreplicating tissues, which form larval structures like the gut, fat body, and salivary glands. An increase in DNA ploidy of larval cells is required for the ~200-fold increase in mass obtained by the larvae during the 5-d period between the completion of embryogenesis and the beginning of pupation. During starvation, larvae sacrifice their endoreplicating tissue to maintain the growth and proliferation of the mitotic cells that are required to form the reproductive adult. Furthermore, S6k activity is reduced in starved larvae and dTOR mutants. These observations prompted an analysis of the mitotic and endoreplicating tissues of dTOR, S6k, and chico mutant larvae just before pupariation. Strong dTOR and PI3K mutants, as well as amino acid-starved larvae, are incapable of growth and have barely detectable imaginal and endoreplicative tissues. Surprisingly, the wing discs of the weak dTOR heteroallelic combination are of approximately equivalent size to that of wild-type larvae, whereas those of S6k^l-1 mutants are reduced. However, the amount of endoreplicating tissue in the dTOR mutant as compared to wild-type larvae is severely decreased. This is clearly demonstrated by comparing the salivary glands of dTOR mutant and wild-type larvae. In contrast, the size of endoreplicating tissue and imaginal discs in S6k null mutants as well as chico null mutants is reduced to approximately the same extent. Staining of the salivary glands with DAPI and phalloidin reveals that the size of the nuclei and, thus, the degree of endoreplication is severely reduced in S6k, chico, and dTOR mutants. The difference in size between dTOR and S6k mutant salivary glands is largely caused by a very pronounced reduction in cytoplasmic volume in dTOR mutants. The nuclear to cytoplasmic ratio is higher in dTOR salivary glands than in y w, S6k, or chico mutant salivary glands. Thus, it appears that partial loss of dTOR function permits the growth of imaginal tissue to wild-type size, while endoreplicating tissue is disproportionally reduced, a phenotype distinct from S6k mutants. Consistent with this finding, the lethality of the different dTOR mutants could not be rescued by constitutive expression of a S6K1 variant, D₃E-E₃₈₉, which exhibits high basal activity in the absence of mitogens under the control of the alpha-tubulin promoter, which rescues all aspects of the S6k^l-1 null phenotype. Therefore, S6k-independent processes must contribute to the weak dTOR phenotype (Oldham, 2000b).

The effect of rapamycin and amino acids on translation in mammals is mediated through the S6Ks and the 4E-BPs. Unlike the other elements in the PI3K signaling pathway, absence of amino acids blocks both S6K activation and 4E-BP phosphorylation. Indeed, a mutant of S6K1, lacking a portion of both its amino and carboxyl termini, is resistant to rapamycin but still sensitive to the fungal metabolite wortmannin, an inhibitor of PI3K. This suggests that the PI3K-dependent signal to S6K activation does not involve TOR. This same mutant is also unaffected by amino acid withdrawal, consistent with the role of mTOR as an amino acid checkpoint in S6K activation. Although there is some controversy concerning the ability of mitogens to activate mTOR, the in vitro activity of mTOR from cultured cells toward either itself, S6K1, or 4E-BP1 is unaffected by mitogens. Thus, mTOR may act as a permissive signal that primes 4E-BP phosphorylation and S6K activation by the PI3K signaling pathway if amino acids, and possibly other nutrients, are at sufficient levels. Likewise, in Drosophila larvae, amino acids are necessary, but not sufficient, for imaginal disc and endoreplicating tissue proliferation, compatible with dTOR acting in a parallel pathway involved in amino acid sensing. The fact that chico mutant larvae have normal levels of S6k activity and that the dTOR larval phenotypes with respect to the imaginal discs and endoreplicating tissues are so distinct compared with other mutants in the Inr pathway, supports the possibility that dTOR is not responsive to insulin signaling (Oldham, 2000b and references therein).

It is well established in yeast that TOR is an important mediator of nutrient limitation, and it has been proposed that TOR acts as an amino acid effector to coordinate the response of yeast to different nutritional conditions (Barbet, 1996). Indeed, the similarities between dTOR mutant larvae and larvae deprived of amino acids are striking. Therefore, it is likely that dTOR also functions as an amino acid sensor in multicellular organisms. The fact that yeast and Arabidopsis do not have an insulin system suggests that TOR may be an ancestral and widespread nutritional sensor. To provide additional levels of control, it may have been integrated into the insulin system later to respond to different modes of nutrient deprivation with different developmental responses (Oldham, 2000b and references therein).

Insulin treatment of Drosophila Kc 167 cells induces the multiple phosphorylation of a Drosophila ribosomal protein, as judged by its decreased electrophoretic mobility on two-dimensional polyacrylamide gels. The extent to which insulin induces this response is potentiated by cycloheximide and blocked by pretreatment with rapamycin. Isolation and mass spectrometric analysis have revealed that the multiply phosphorylated protein is the larger of two Drosophila melanogaster orthologs of mammalian 40S ribosomal protein S6, termed here DS6A. Proteolytic cleavage of DS6A (derived from stimulated Kc 167 cells), with the endoproteinase Lys-C releases a number of peptides, one of which contains all the putative phosphorylation sites. Conversion of phosphoserines to dehydroalanines with Ba(OH)(2) shows that the sites of phosphorylation reside at the carboxy terminus of DS6A. The sites of phosphorylation have been identified by Edman degradation after conversion of the phosphoserine residues to S-ethylcysteine as Ser(233), Ser(235), Ser(239), Ser(242), and Ser(245). Phosphopeptide mapping of individual phosphoderivatives, isolated from two-dimensional polyacrylamide gels, indicate that DS6A phosphorylation, in analogy to mammalian S6 phosphorylation, appears to proceed in an ordered fashion (Oldham, 2000b and references therein).

GENE STRUCTURE

cDNA clone length - 7682 bp

Exons - 7

Bases in 3' UTR - 169

PROTEIN STRUCTURE

Amino Acids - 2470

Structural Domains

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). The dTOR genomic region spans ~10 kb and is composed of seven exons (7.4 kb cDNA) encoding a 2480-amino acid protein with a predicted M_w of 282 kD. The dTOR protein exhibits 35%, 38%, 35%, and 41% overall amino acid identity to yeast TOR1, TOR2, C. elegans TOR, and Arabidopsis thaliana TOR, respectively. The overall identity with mTOR is significantly higher (56%) and is especially conserved in the kinase and FRB domains (74% and 77%) (Oldham, 2000b).

date revised: 19 September 2003

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