chico

DEVELOPMENTAL BIOLOGY

Embryonic Development

Insulin/IGF signaling during development controls growth and size, possibly by coordinating the activities of the Ras and PI 3-kinase signaling pathways. In vertebrates, the IR and IGFR act through IRS1-IRS4 proteins, which are multifunctional adaptors that link insulin and IGF signaling to the Ras/MAPK and phosphoinositide 3'-kinase (PI 3-kinase) signaling pathways. The pleckstrin homology domain (PH) and phosphotyrosine binding domain (PTB) of the IRS proteins are believed to mediate binding to phosphoinositol phosphates and the juxtamembrane NPXY motif of IR/IGFR, respectively. Grb2 (Drosophila homolog Drk) is an adaptor protein containing SH2 and SH3 domains. It has been suggested that Grb2 may, via its binding to IRS, link insulin/IGF to the Ras/MAPK pathway and thereby control proliferation. The Drosophila homolog of the SH2 domain containing p85 PI 3-kinase adaptor subunit, p60, binds Chico/IRS and thereby recruits the p110 catalytic subunit of PI 3-kinase [which converts phosphoinositol(4,5)P₂ (PtdIns(4,5)P₂) into phosphoinositol(3,4,5)P₃ (PtdIns(3,4,5)P₃)] to the plasma membrane. The p110 PI 3-kinase belongs to the class I PI 3-kinases implicated in the metabolic effects of insulin. The classical effectors that mediate the biological outcomes of insulin and IGF downstream of IRS have been divided into two functional branches: the Ras/MAPK proliferation pathway, and the PI 3-kinase metabolic, growth and survival pathway (Oldham, 2002).

To analyze the role of the different domains of Chico/IRS under physiological conditions, a panel of effector site mutants was created in a genomic rescue construct for chico that disrupts the PH or the PTB domains or the putative binding sites of Drk/Grb2 and p60. The constructs include the cis-regulatory sequences that permit expression of chico in its normal spatial and temporal pattern. The wild-type chico construct fully restores the defects of chico homozygous null mutants. In this manner, the effector site mutants were assayed for the ability to rescue the three different phenotypes associated with complete loss of Chico function: body size reduction, female sterility and lipid alterations. The Drk/Grb2 consensus binding site mutant is able to fully rescue the reduced weight to the same extent as the wild-type rescue construct. Therefore, the presence of a functional Drk binding site in Chico and thus the link to the activation of the Ras/MAPK kinase pathway is not required for its wild-type function. In contrast, the PH and PTB domain mutants and the double p60 PI 3-kinase binding site mutant were unable to rescue the reduced body weight. The latter result is surprising because InR contains additional functional PI 3-kinase binding sites in its C-terminal tail, an extension shared only with the C. elegans InR homolog, Daf-2, and not the mammalian IR or IGFR. This suggests that the presence of additional p60 binding sites in the InR C-terminal tail is not sufficient in vivo to mediate wild-type levels of growth and proliferation in the absence of the Chico p60 PI 3-kinase binding sites and that the InR C-terminal tail may contribute only low levels of PI 3-kinase signaling. Although the PTB domain mutant fails to restore normal body weight, it rescues the female sterility associated with the loss of Chico function. With the exception of the full rescue of the lipid accumulation observed in Drk/Grb2 mutant, all the other effectors only partially restore the change in lipid accumulation (Oldham, 2002).

The inability of the p60 binding site mutant to rescue the size defect indicates that the Chico PI 3-kinase docking sites are necessary for InR/Chico (insulin/IGF) action in size control. However, the issue of whether recruitment of PI 3-kinase to Chico is sufficient to mediate the attainment of wild-type body size is unresolved. It has been reported that overexpression of PI 3-kinase and Akt in Drosophila is sufficient for increased growth but not proliferation. Loss of zygotic InR function results in embryonic lethality with some small arrested larvae, but loss of zygotic Chico function results in viable small flies. Two parsimonious hypotheses could explain this difference. (1) InR activates not only the PI 3-kinase pathway but also another, Chico-independent, signal transduction pathway, or (2) InR signals predominantly through PI 3-kinase, but loss of Chico does not block PI 3-kinase activation completely because of direct interaction of p60 with the InR C-terminal tail. This provides residual PI 3-kinase activation sufficient to rescue viability, but not wild-type size. If the latter hypothesis were true, then increasing PtdInsP₃ levels should be sufficient to rescue loss of InR function (Oldham, 2002).

Viable allelic combinations of insulin receptor pathway components result in at least three characteristic phenotypes: small body size, female sterility and increased lipid content. The results from the chico effector mutants permit the separation of the three different Chico phenotypes (Oldham, 2002).

(1) Size and fertility. Is there a causal link between the small body size and female sterility? The PTB domain mutant rescues the sterility, but not the size defect, thus separating the growth and the sterility phenotypes. It remains to be resolved whether different levels of PI 3-kinase activation are needed to restore growth and fertility or whether control of fertility involves at least in part the association of Chico with a different receptor which does not require the PTB domain. For the growth regulatory function of Chico, a functional PTB and PH domain are essential. This indicates that in vivo, in the absence of overexpression, these two domains serve non-redundant functions presumably in the localization to the membrane and binding to the insulin/IGF receptor (Oldham, 2002).

(2) Size and lipids. Does the small size cause the increased lipid levels? Chico mutant flies lacking functional p60/PI 3-kinase binding sites, PTB or PH domains are all small, yet the increase in lipid levels is less pronounced than in chico null mutant flies. Also, the Irs2-deleted mice and insulin pathway mutants in C. elegans are not small, yet display increased lipids. Therefore, there seems to be no direct correlation between developmental growth and energy stores in the adult (Oldham, 2002).

(3) Lipids and sterility. Like chico flies, mice mutant for IRS2 or lacking insulin receptor function in the brain (NIRKO) display increased lipids and are female sterile. Are the increased lipid levels a sign of metabolic dysfunction that leads to the female sterility? The chico PI 3-kinase, PTB and PH effector mutants have similar lipid increases, yet the PTB mutant is fertile while the PI 3-kinase mutant is not. Therefore, there appears to be no direct correlation between lipid accumulation and sterility in Drosophila (Oldham, 2002).

Collectively, these data firmly establish Drosophila as a valid model organism for the study of metabolic diseases like diabetes and obesity as well as for the study of growth disorders like cancer. Pten mutant flies are larger in size due to increased cell size and number, but have a corresponding decrease in energy stores, a situation completely opposite that of mutations in positive components of the insulin signaling pathway like InR, chico, PI 3-kinase, and dAkt. These large viable Pten mutants show that a reduction of PTEN function is sufficient for increased organism size. This fact suggests that the four-fold size difference between viable InR and Pten mutants can simply be controlled by the amount of PtdInsP₃ and this phenomenon may possibly be extended to vertebrate size regulation. Thus, in Drosophila, the InR/PI 3-kinase/PTEN pathway combines both metabolism and growth control into one pathway that later diverged into two separate, yet interacting systems in mammals (Oldham, 2002).

Effects of Mutation or Deletion

In a search for mutations causing a reduction in body size, a P element-induced mutation, fs(2)4¹, was identified that had been described previously as a female sterile mutation (Berg, 1991). Since homozygous fs(2)4¹ animals are severely reduced in body size, the mutation was renamed chico, which in Spanish means 'small boy'. Homozygosity for chico causes semilethality and an overall delay in development. Homozygous chico flies eclose 2-3 days after their heterozygous siblings. Under noncrowded culture conditions, homozygous chico mutant females can produce only a few viable progeny, all of which lack both maternal and zygotic chico function. To quantitate size differences in various mutants, the weight of individual flies was measured. Flies homozygous for the P element (chico¹) or the synthetic chico deletion (chico²) have a drastic weight reduction (by 65% in females and 55% in males) compared with wild-type control flies of the same age (wild type females average about 1.35 mg and wild type males are about 0.75 mg . Body size reduction is observed at all developmental stages but does not alter the overall proportions of the flies (Böhni, 1999).

The reduction in body and organ size in chico mutants could be due to a reduction in the number of cells and/or a reduction in the size of the individual cells. To distinguish between these alternatives, cell number and cell size were determined. In the wing, each epithelial cell secretes cuticle containing a single hair, so that counting the number of hairs and determining their density provides a direct measure of cell number and cell size in the wing. The 40% reduction in the size of chico mutant wings is caused by a reduction in both cell number and cell size. Reduction in cell number accounts for 68% of the total reduction in wing size. The remaining 32% of the reduction in wing size is due to a reduction in the average size of mutant cells. Similar results were obtained for the eye. In homozygous mutant chico flies, ommatidial number is reduced by approximately 40%: homozygous chico flies have only about 480 ommatidia per eye, whereas wild-type flies have approximately 780 ommatidia per eye. Therefore, loss of chico function reduces body size by means of reducing cell number and cell size (Böhni, 1999).

To test whether the reduction in the size of chico mutant cells is also observed during larval stages, third instar wing discs of larvae homozygous or heterozygous for chico were dissociated and the relative cell size of the two cell populations were determined by FACS analysis. A 10%-14% reduction in the mean of the forward scatter of homozygous chico cells compared with heterozygous cells indicates that the size of chico imaginal disc cells is also reduced (Böhni, 1999).

The effect of loss of chico function on the overall body and organ size could be due to a nonautonomous role of chico in humoral growth regulation or to an autonomous role in a tissue- and cell type-specific manner. To test the cell autonomy of the chico mutation, clones of genetically marked homozygous mutant chico cells were generated in a heterozygous background in the eye. In each ommatidium, the R1-R6 photoreceptor cells are arranged in an asymmetric trapezoid. The tall side of the trapezoid is formed by photoreceptors R1-R3, facing anteriorly. The centrally located R7 photoreceptor has a smaller rhabdomere than the six outer cells. Each of these morphological characteristics is retained in chico mutant ommatidia. Thus, loss of chico function does not impair the specification of cell fate. However, it is striking that the size of each mutant photoreceptor, and hence the entire ommatidial unit in the mutant clone, is reduced by more than 50%. On the periphery of the clones of homozygous mutant tissue, ommatidia consist of homozygous and heterozygous cells. The genotype of each photoreceptor can be assessed by the presence or absence of red pigment. Small homozygous mutant photoreceptor cells coexist with heterozygous cells in the same ommatidium. Remarkably, this does not significantly alter the shape of the ommatidia and the arrangement of the photoreceptors. Autonomy of cell size control is also observed in the wing. Therefore, final cell size is autonomously dependent on chico function in each individual cell (Böhni, 1999).

To test whether chico affects the size of organs and body parts autonomously, chico function was selectively removed in the eye imaginal disc using the ey-FLP technique. The eye imaginal disc gives rise to the compound eye and the head capsule but not to the proboscis. In embryos heterozygous for chico, mitotic recombination was selectively induced in the eye progenitor cells by using an FLP recombinase driven by the eyeless enhancer. Owing to the presence of a recessive mutation affecting cell survival on the chico⁺ chromosome, chico homozygous mutant cells have a proliferative advantage and contribute to the majority of cells in the eye and the head. Thus, flies have heads that are largely homozygous for chico, while the rest of the body is heterozygous. In such flies, the eyes and the head capsule are strongly reduced in size, while the proboscis and the rest of the body are of wild-type size. Thus, chico acts autonomously in the control of cell size and organ size (Böhni, 1999).

The reduction in cell number caused by the absence of chico function may be the result of a prolonged cell cycle time or of an increased rate of apoptosis during development. In order to analyze the behavior of chico mutant cells during development, genetically marked homozygous mutant cells were generated by mitotic recombination. This allowed a comparison of the behavior of homozygous mutant clones and their wild-type sister clones, called twin spots, generated during mitotic recombination. Three differences between mutant and wild-type twin clones are obvious: (1) chico mutant clones are rare; in approximately 90% of the clones, only the darkly pigmented wild-type twin spot can be detected. This is most likely due to the fact that small mutant clones encompass only a few ommatidia and escape detection. (2) When a nonpigmented mutant clone is detected, the clone is variable in size and often significantly smaller than the wild-type sister clone, and (3) there are regional differences in the frequency of mutant clones: clones are more frequently observed in the anterior half of the eye around the equator. The equator defines a line of dorsoventral mirror image symmetry in the orientation of the ommatidial units. It appears that mutant cells have a better chance to grow or survive in the center of the eye than on its periphery. The behavior of chico mutant clones is similar to that of Minute mutant clones and has been described as cell competition. It indicates that the development of chico mutant cells is selectively impaired compared with wild-type cells and that there are regional differences in the ability of mutant cells to grow or survive (Böhni, 1999).

By examining mutant clones in the eye and wing imaginal discs, a test was performed to see whether the reduced size of chico mutant clones observed in the adult is due to a growth disadvantage or to impaired cell survival during the final stages of differentiation As seen in the adult, the mutant clones are smaller than their twin spots and are variable in size. The clones often form a thin line. The fact that chico mutant clones in the third instar disc and in the adult eye exhibit a similar behavior argues against the possibility that homozygous mutant chico cells are eliminated during differentiation in the pupal stage (Böhni, 1999).

Apoptosis has been postulated to be a critical determinant of organ size through counterbalancing cell proliferation. In order to test whether programmed cell death contributes to size control by reducing cell number, discs containing either chico mutant clones or wild-type control clones were analyzed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL). No significant difference in occurrence of apoptotic cells are observed between wild-type and mutant clones. Since chico mutant clones are rather small, mutant clones were also induced in a Minute background. Even though such clones are greatly enlarged due to their growth advantage, they also do not reveal enhanced apoptosis when compared with wild-type control clones in a Minute background. No increase in morphological signs of programmed cell death, such as enlarged cells or cells with picnotic nuclei in chico mutant clones, are observed in the imaginal discs or in the adult eye. These results are also consistent with the FACS analysis of heterozygous and homozygous chico mutant wing disc cells. No significant difference in the apoptotic sub-G1 fraction of homozygous chico mutant cells is observed, as compared with heterozygous cells. Therefore, it is concluded that chico function is not necessary for cell survival but is required for cell growth and cell proliferation throughout development. Homozygous chico mutant cells have a selective growth disadvantage: they grow more slowly than wild-type cells, as indicated by their underrepresentation in discs and in the adult eye, and they cannot reach the normal size of wild-type cells. However, the cell cycle profiles of heterozygous and homozygous chico mutant wing disc cells are similar, suggesting that the increased cell cycle time of chico mutant cells is caused by proportional expansion of the G1, S, and G2 phase of the cell cycle (Böhni, 1999).

The human tumor suppressor gene PTEN gets its name from its biochemical function, its domain structure and its chromosomal location: PTEN stands for the combination of phosphatase and tensin homolog on chromosome 10. The lipid phosphatase function of PTEN places it in the middle of the insulin pathway, known to involve lipid signaling (Goberdhan, 1999; Huang, 1999). Drosophila Pten modulates cell size and consequently tissue mass by acting antagonistically to the lipid modifiying enzyme Phosphotidylinositol 3 kinase 92E, also known as Dp110, and its upstream activator Chico, an insulin receptor binding and signal transduction protein. All signals from the insulin receptor can be antagonized by Pten. In terms of its protein phosphatase function, mammalian PTEN targets focal adhesion kinase, a major effector of cytoskeletal function. Overexpression of wild-type mammalian PTEN and mutant PTEN that lacks lipid phosphatase activity can reduce levels of focal adhesion kinase (FAK: see Drosophila Focal adhesion kinase-like) phosphorylation and the formation of focal adhesions, thereby inhibiting cell migration and invasiveness (Goberdhan, 1999 and references therein).

Drosophila Pten was identified in two laboratories using two different approaches, one based on homology to mammalian PTEN (Huang, 1999) and the second based on intensive analysis of a chromosomal region containing several other genes coding for signaling proteins (Goberdhan, 1999). This second approach will be examined in detail, since it foreshadows future approaches that will be taken to analyze Drosophila genes based on genomics. Previously, two Drosophila genes had been identified in the vicinity of the gene that was later to be identified as Drosophila Pten: Dror encodes a neural-specific receptor tyrosine kinase, and basket encodes the homolog of c-Jun amino-terminal kinase. These two genes map adjacent to each other at 31B/C on the second chromosome. Several deficiency chromosomes have been characterized that uncover one or both of these genes and also affect the adjacent gene chico, which codes for a homolog of mammalian insulin receptor substrates, IRS1-4 (Böhni, 1999). One of these deficiencies, Df(2L)170B, is of particular interest because it deletes sequences proximal to Dror and produces an overgrowth phenotype in homozygous clones. This effect was not observed with deficiencies [Df(2L)41C and Df(2L)147F] that only delete Dror, DJNK, and chico. Clones generated with these smaller deficiencies contain reduced numbers of small cells attributable to the loss of chico function. During a chemical mutagenesis screen using the Df(2L)170B chromosome, a new lethal complementation group has been identified that maps proximal to Dror and affects tissue growth. Sequence analysis of genomic and cDNA clones reveals a novel gene at this locus encoding the Drosophila PTEN homolog, Pten (Goberdhan, 1999 and references therein).

Drosophila Pten regulates cell number and size and affects assembly of specific cytoskeleton-dependent structures. Because animals transheterozygous for strong Pten alleles die with no obvious phenotypes, the functions of this gene have been elucidated further by generating homozygous mutant clones in heterozygous animals using the FLP/FRT system. Two Pten alleles, DPTEN¹ and DPTEN³, produce growth phenotypes slightly more severe than those generated by Df(2L)170B, the chromosomal deficiency deleting Pten, Dror, DJNK, and chico. DPTEN¹ and DPTEN³ also behave in a manner similar to the deficiency in combination with a weak, nonlethal Pten mutation; this suggests that they are strong or null Pten alleles (Goberdhan, 1999).

Overexpression of Pten also produces enlargement of wing cells. Wild-type Pten cDNA is overexpressed in particular areas of the wing using the GAL4-UAS system. Initially flies carrying a dpp-GAL4 construct were used. This drives gene expression in cells that will normally populate the region between the third and fourth longitudinal wing veins (LIII and LIV). Overexpression of Pten reduces the size of these regions by nearly 25% compared with wild type. This is not a consequence of a general reduction in wing size in overexpressing flies, since an adjacent area of the wing between LIV and LV is essentially unaffected. The effect on wing area is similar to that produced by overexpression of Dp110^D954A, a dominant-negative, kinase-dead version of Pi3K92E. The reduction is caused by both a decrease in cell size and cell number and is opposite of the effect of overexpressing an activated, membrane-associated form of Pi3K92E, Dp110-CAAX, in the same region. To test whether Pten's growth regulatory functions are primarily mediated by its effects on the insulin receptor-Pi3K92E signaling pathway and not by an independent signaling cascade, the genetic effects of Pten alleles were sought using mutant phenotypes associated with chico and Pi3K92E (Goberdhan, 1999).

The recent characterization of chico, a Drosophila IRS1-4 homolog, has shown that chico, Pi3K92E and Insulin receptor(Inr) act as positive elements in a Drosophila insulin signaling pathway to regulate cell proliferation and cell size. Consistent with the role of Pten as a negative regulator in this insulin pathway, removal of one copy of the chico gene genetically enhances the eye/Pten eye phenotype. Overexpression of Inr (eye/Inr) causes lethality at 25°C. At room temperature, few animals survive with overproliferated eyes. Strikingly, co-overexpression of Pten completely rescues lethality and the overproliferation phenotype. This suggests that all signals from the insulin receptor can be antagonized by Pten function. Together with the previous findings that mammalian and C. elegans PTEN molecules interact with components of the insulin pathway, these genetic data argue that Pten functions as a major conserved negative regulator in the insulin signaling pathway (Huang, 1999).

The importance of Drosophila Pten in negatively regulating the growth-promoting effects of insulin signaling in vivo, however, is best illustrated in homozygous clones mutant for both chico and Pten. In these clones, the reduced growth phenotype normally seen in chico mutant cells is masked completely by the overgrowth phenotype associated with loss of Pten function, suggesting that Pten normally has a critical role downstream of Chico in maintaining growth-promoting signals at nonhyperplastic levels (Goberdhan, 1999).

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 (RPS6-p70-protein kinase), 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, 2000).

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, 2000).

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, 2000).

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, 2000 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, 2000 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, 2000 and references therein).

Understanding how stem-cell proliferation is controlled to maintain adult tissues is of fundamental importance. Drosophila oogenesis provides an attractive system to study this issue since cell production in the ovary depends on small populations of observable germ-line and somatic stem cells. By controlling the amount of protein-rich nutrients in the diet, conditions have been established under which the rate of egg production varies 60-fold. Using a cell-lineage labeling system, it was found that both germ-line and somatic stem cells, as well as their progeny, adjust their proliferation rates in response to nutrition. However, the number of active stem cells does not appear to change. Proliferation rates varied fourfold; the remaining 15-fold difference in egg production results from different frequencies of cell death at two precise developmental points: (1) the region 2a/2b transition within the germarium, and (2) stage 8 egg chambers that are entering vitellogenesis. To initiate a genetic analysis of these changes in cell proliferation and apoptosis, it has been shown that ovarian cells require an intact insulin pathway to fully upregulate their rate of cycling in response to a protein-rich diet and to enter vitellogenesis (Drummond-Barbosa, 2001).

Insulin is an important mediator of energy metabolism in vertebrates and is known to be required for the growth of ovarian follicles in mammals. chico encodes an IRS-like protein that functions in the Drosophila insulin pathway, and chico¹ mutant females are sterile. To investigate the role of chico under different nutritional conditions, newly eclosed chico¹ homozygous females that contain the beta-gal marking system were cultured on either rich or poor food. Following a heat-shock to induce marked clones, the size of beta-gal-positive follicle cell clones was measured over the next 4 days. chico¹ mutation partially impairs the ability of ovarian follicle cells to proliferate faster in the presence of abundant nutrients. On poor food, follicle cells divide at similar rates in females heterozygous or homozygous for chico¹. Follicle cells divide faster in both genotypes on rich food, but heterozygotes increase their doubling rate significantly more than chico¹ homozygotes. Clone sizes were comparable all along the ovariole and within the germarium, suggesting that the response is uniform from the stem cells throughout the somatic lineage. Thus, chico-mediated insulin receptor signaling appears to play a small but detectable role in controlling the rate of follicle cell proliferation in response to rich food. In addition, the chico¹ mutation causes a large effect on egg chamber progression into vitellogenesis. Egg chambers did not develop beyond vitellogenic stages in chico homozygotes, despite the presence of abundant food. Among 40 chico¹ ovarioles analyzed, none had vitellogenic stages, while 33 out of 33 heterozygous ovarioles had vitellogenic stages. Thus, chico function is needed for vitellogenesis and to accelerate the rate of follicle cell proliferation within the entire lineage, in response to rich food (Drummond-Barbosa, 2001).

The Drosophila gene chico encodes an insulin receptor substrate that functions in an insulin/insulin-like growth factor (IGF) signaling pathway. In the nematode C. elegans, insulin/IGF signaling regulates adult longevity. Mutation of chico extends fruit fly median life-span by up to 48% in homozygotes and 36% in heterozygotes. Extension of life-span is not a result of impaired oogenesis in chico females, nor is it consistently correlated with increased stress resistance. The dwarf phenotype of chico homozygotes was also unnecessary for extension of life-span. The role of insulin/IGF signaling in regulating animal aging is therefore evolutionarily conserved (Clancy, 2001).

Mutations that extend life-span illuminate the molecular mechanisms underlying aging and longevity. In Caenorhabditis elegans, mutation of the genes daf-2 and age-1, which encode components of an insulin/IGF signaling (IIS) pathway, enhances stress resistance and increases adult life-span by up to 200%. This pathway also controls the formation of dauer larvae, which are developmentally arrested, stress resistant, long-lived, and produced in response to crowding and reduced food. Potentially, insulin/IGF mutants could be long-lived by virtue of expression of dauer longevity in the adult, in which case the extension of adult life-span by these mutations could be a peculiarity of C. elegans (Clancy, 2001).

In Drosophila, the insulin/IGF receptor INR, the insulin receptor substrate Chico, the phosphatidylinositol 3-kinase (PI3K) Dp110/p60, and the PI3K target protein kinase B (PKB, also known as DAkt1) form a signaling pathway that regulates growth and size. The effects on aging of hypomorphic mutations in Inr (equivalent to daf-2) and PKB, and null mutations in chico and the catalytic (Dp110, equivalent to age-1) and adapter (p60) PI3K subunits were examined. All mutants were tested as heterozygotes. chico¹ and PKB³ homozygotes and Inr^GC25/Inr^E19 transheterozygotes, which form viable dwarf adults, were also examined. The remaining mutations were homozygous lethal (Clancy, 2001).

Most mutants tested had normal or significantly decreased life-span. For example, PKB³ homozygotes and Inr^GC25/Inr^E19 flies are short-lived. By contrast, chico¹ extends life-span. Homozygous chico¹ females exhibit an increase of median and maximum life-span of up to 48% and 41%, respectively. chico¹ heterozygotes also exhibit increases in median life-span of up to 36% and 13% in females and males, respectively. Homozygous males, however, are slightly short-lived (Clancy, 2001).

To confirm that chico¹ itself extends life-span, the effect on life-span of pCSR4-chico, a P element containing chico(+) was examined. This construct fully rescues the dwarf phenotype of chico¹. chico¹ was crossed to two stocks containing independent pCSR4-chico insertions (pCSR4-chico 1.1 and 2.3). As a control, chico¹ was also crossed to the base stock in which the P element insertions were made. Progeny with either two copies (chico¹ heterozygotes with one chico transgene) or one copy (chico¹ heterozygotes alone) of chico(+) were compared. The rescue construct significantly reduces life-span relative to the +/chico¹ control. The median female life-span of 54 days in +/chico¹ was reduced to 46 days in +/chico¹, +/pCSR4-chico 1.1 flies and 52 days in +/chico¹, +/pCSR4-chico 2.3 flies. Similar effects were observed in males. Thus, mutation of chico itself increases life-span. Because chico¹ is a null allele, its effect on life-span indicates that the wild-type chico gene acts to accelerate aging (Clancy, 2001).

Of the mutations tested, only chico¹ increases life-span. This may be because the effect of reduced IIS on life-span depends on the degree to which signaling is reduced. Unlike the other null mutations in IIS genes tested, chico¹ is not homozygous lethal, presumably because the INR receptor can signal to PI3K directly, as well as indirectly via Chico. Thus, chico¹ mutants may be long-lived because of the relatively mild reduction in pathway activity that they bring about. Notably, severe IIS mutations in C. elegans can cause premature mortality in some adults, although the maximum life-span of populations is invariably increased. This is probably why Inr^GC25/Inr^E19 flies are short-lived: Demographic analysis indicates that a reduction in the age-specific mortality rate acceleration occurs, whose effect on survival is masked by an elevated rate of age-independent mortality. Furthermore, a different heteroallelic Drosophila Inr mutant to that tested here exhibits an 85% increase in female life-span. By contrast, in short-lived PKB³ populations, no reduction in mortality rate acceleration is seen. This raises the possibility that a second pathway downstream of chico might regulate aging in Drosophila. Interestingly, Chico contains potential binding sites for the Drk/Grb2 docking protein, consistent with signaling via Ras/mitogen-activated protein kinase (Clancy, 2001).

Tests were made of whether extension of life-span by chico¹ is mediated by processes previously shown to affect aging. A reduction in fecundity extends life-span in Drosophila females; chico¹ heterozygous females have reduced fecundity, and the homozygotes are almost sterile. To test whether the increased life-span of chico¹ females was due to reduced fecundity, the interactions between chico¹ and the dominant, female-sterile mutant ovo^D1 were examined. This mutation blocks oogenesis at stage 4, before vitellogenesis commences, and extends female life-span. If chico¹ extends female life-span by the exact same mechanism as ovo^D1, then the three sterile genotypes (chico¹, +/ovo^D1, and +/ovo^D1 +/chico¹) should have similar life-spans and live longer than the subfertile chico¹ heterozygotes. In fact, the chico homozygotes live significantly longer than all other genotypes. In addition, the partially fertile chico¹ heterozygotes live as long as the sterile flies that are heterozygous for both ovo^D1 and chico¹ and live significantly longer than the sterile ovo^D1 heterozygotes. The effect of chico¹ on female life-span is therefore not a consequence of the same mechanism of reduced fecundity as is produced by ovo^D1. If chico¹ does extend female life-span through an effect on reproductive effort, the interaction must occur through some process other than oogenesis (for instance yolk protein synthesis) or before stage 4 in oogenesis because ovo^D1 flies are blocked at that stage (Clancy, 2001).

In C. elegans, long-lived IIS mutants are stress resistant and overexpress the antioxidant enzyme superoxide dismutase (SOD). The resistance of chico¹ flies to three stressors was examined, but only one shows any correspondence with life-span. No resistance to heat stress (37°C) was seen. Slight resistance to oxidative stress (methyl viologen) is observed in chico¹ heterozygotes but not in homozygotes. However, some correspondence between starvation resistance and life-span is seen. Increased SOD levels are seen in chico¹ homozygotes but not in heterozygotes. Thus, modulation by IIS of longevity, and of SOD levels, has evidently been conserved between C. elegans and Drosophila. Furthermore, effects of this pathway on fertility are widespread. However, effects on stress resistance are not well conserved, nor do any of the above associated affects appear to be causal in extending life-span (Clancy, 2001).

These results raise the question of whether IIS regulates aging in mammals. Whereas both the C. elegans and Drosophila genomes contain a single insulin/IGF receptor, mammals possess distinct receptors for insulin and IGF-I, plus a third insulin receptor-like receptor of unknown function. Potentially, any or all of these receptors may play a role in regulating aging. Caloric restriction (CR), which increases life-span in rodents, and possibly primates, reduces circulating levels of both insulin and IGF-I. In the case of IGF-I, there is further evidence for a role in the control of longevity. Growth hormone (GH) acts via IGF-I to control mammalian body size, and circulating IGF-I levels correlate with body size in mice, dogs, and humans. Furthermore, CR can reduce body size. In mice and dogs (and possibly humans), there is a marked negative correlation between body size and longevity. In addition, long-lived Ames hypopituitary mouse dwarves are deficient in GH and other pituitary hormones and have reduced circulating IGF-I. Mutation of the human equivalent of the Ames dwarf gene, Prop-1, also causes dwarfism and, possibly, delayed aging. The Laron dwarf mouse, which has no GH receptor and very low IGF-I levels, exhibits life-span increases of up to 55% (Clancy, 2001 and references therein).

Whereas the effects of chico¹ on development that result in reduced body size are recessive, its effects on life-span are semidominant. This may reflect the noncatalytic and dosage-dependent nature of the function of Chico as a docking protein. It has been proposed that reduced body size per se increases life-span in mammals. Alternatively, the same genes may independently regulate growth during the preadult period and regulate survival during the adult period. These data support the latter interpretation because chico¹ heterozygotes are long-lived, yet of normal size. Likewise, the effect of CR on aging may be observed in the absence of its effects on body size. Together, these results with fruit flies and recent findings with nematodes and mice suggest that the role of IIS (perhaps IGF-I in mammals) in regulating longevity is evolutionarily conserved throughout the animal kingdom (Clancy, 2001).

Analysis of Drosophila Foxo indicates that it is a critical PKB target, but that it mediates only one aspect of PKB function. Several lines of evidence support this model. (1) The effects of ectopic overexpression of Foxo and the human homolog hFOXO3a in the developing Drosophila eye are altered by Dp110 and PKB signaling as well as by nutrient levels. Under conditions of lowered insulin signaling, the phenotypes resulting from expression of foxo and hFOXO3a are dramatically enhanced. This situation was mimicked by expressing a PKB-insensitive phosphorylation mutant, suggesting that endogenous PKB signaling is required to mitigate the effects of ectopically expressed Foxo and hFOXO3a. (2) The physiological relevance of Foxo in PKB signaling is most vividly demonstrated by the observation that the larval lethality associated with the complete loss of PKB is rescued by foxo mutations to the extent that some flies develop to pharate adults. The lethality associated with loss of PKB function is therefore to a large extent due to the hyperactivation of Foxo. (3) Loss of Foxo function suppresses the effects of insulin-signaling mutations only partially; Foxo mediates a reduction in cell number but not in cell size in response to reduced insulin signaling (Jünger, 2003).

Genetic analysis of the control of body size in Drosophila has revealed two classes of mutations. Flies carrying mutations in chico or viable allelic combinations of Inr, Dp110, and PKB are reduced in body size by up to 50% owing to a reduction in both cell size and cell number. Conversely, flies mutant for S6K exhibit a more moderate reduction in body size, caused almost exclusively by a reduction in cell size. This suggests that the pathways controlling cell number and cell size bifurcate at or below PKB. Although foxo single mutants have no obvious size phenotype, loss of foxo substantially suppresses the cell-number reduction observed in insulin-signaling mutants. It appears that Foxo mediates the repression of proliferation in flies mutant for Inr, chico, Dp110, and PKB without being required for the reduction in cell size. Chico-Foxo double mutant flies even have slightly smaller cells than chico mutants, suggesting that removal of Foxo permits cell-cycle acceleration under conditions of impaired insulin signaling. The pathway controlling body size in response to insulin therefore bifurcates at the level of PKB: PKB controls cell number by inhibiting Foxo function and PKB controls cell size, at least under some conditions, by regulating S6K activity by phosphorylation of TSC2 (Jünger, 2003).

The signaling systems controlling cell size and cell number are tightly interconnected. Genetic and biochemical analyses have revealed five different links between the TSC-TOR-S6K pathway and the Inr-PKB-Foxo pathway. (1) Under conditions of unnaturally high insulin-signaling activity (that is, following the oncogenic activation of PKB) PKB phosphorylates and inactivates TSC2, resulting in increased activation of S6K. Under normal culture conditions this regulation does not seem critical, however, loss of dPKB function does not lower dS6K activity in larval extracts. (2) Under physiological conditions, PDK1 regulates PKB as well as S6K. (3) S6K itself downregulates dPKB activity in a negative feedback loop. (4) Under severe starvation conditions, nuclear Foxo presumably activates target genes that reduce cell proliferation. One of these target genes is 4E-BP, which encodes an inhibitor of translation initiation. When conditions improve, the insulin and TOR signaling pathways can stimulate translation by disrupting the 4E-BP/eIF4E complex via phosphorylation of 4E-BP, and in parallel by repressing FOXO-dependent 4E-BP expression. (5) Under even more severe starvation or stress conditions, full activation of Foxo upregulates expression of the insulin receptor itself, thus rendering the cell hypersensitive to low insulin levels. These multiple positive and negative interactions ensure a continuous fine adjustment of the growth rate to changing environmental conditions (Jünger, 2003).

Insulin-IGF receptor (InR) signaling has a conserved role in regulating lifespan, but little is known about the genetic control of declining organ function. This study describes progressive changes of heart function in aging fruit flies: from one to seven weeks of a fly's age, the resting heart rate decreases and the rate of stress-induced heart failure increases. These age-related changes are minimized or absent in long-lived flies when systemic levels of insulin-like peptides are reduced and by mutations of the only receptor, InR, or its substrate, Chico. Moreover, interfering with InR signaling exclusively in the heart, by overexpressing the phosphatase PTEN or the forkhead transcription factor FOXO, prevents the decline in cardiac performance with age. Thus, insulin-IGF signaling influences age-dependent organ physiology and senescence directly and autonomously, in addition to its systemic effect on lifespan. The aging fly heart is a model for studying the genetics of age-sensitive organ-specific pathology (Wessells, 2004).

Abe, H., et al. (1998). Hypertension, hypertriglyceridemia, and impaired endothelium-dependent vascular relaxation in mice lacking insulin receptor substrate-1. J. Clin. Invest. 101: 1784-1788.

Anai M., et al. (1998). Different subcellular distribution and regulation of expression of insulin receptor substrate (IRS)-3 from those of IRS-1 and IRS-2. J. Biol. Chem. 273(45): 29686-92.

Araki, E., et al. (1994). Alternative pathway of insulin signalling in mice with targeted disruption of the IRS-1 gene. Nature 372(6502): 186-90.

Berg, C.A., and Spradling, A.C. (1991). Studies on the rate and site-specificity of P-element transposition. Genetics 127: 515-524.

Bohni, R., et al. (1999). Autonomous control of cell and organ size by CHICO, a Drosophila homolog of vertebrate IRS1-4. Cell 97(7): 865-75.

Britton, J.S., and Edgar, B.A. (1998). Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms. Development 125: 2149-2158

Chaika, O. V., et al. (1999). Mutation of tyrosine 960 within the insulin receptor juxtamembrane domain impairs glucose transport but does not inhibit ligand-mediated phosphorylation of insulin receptor substrate-2 in 3T3-L1 adipocytes. J. Biol. Chem. 274(17): 12075-80.

Chen, C., Jack, J. and Garofalo, R. S. (1996). The Drosophila insulin receptor is required for normal growth. Endocrinology 137(3):8 46-56.

Clancy, D. J., et al. (2001). Extension of life-span by loss of Chico, a Drosophila Insulin receptor substrate protein. Science 292: 104-6. 11292874

Clark, S. F., et al. (1998). Intracellular localization of phosphatidylinositide 3-kinase and insulin receptor substrate-1 in adipocytes: potential involvement of a membrane skeleton. J. Cell Biol. 140(5): 1211-25.

Drummond-Barbosa, D. and Spradling, A. C. (2001). Stem cells and their progeny respond to nutritional changes during Drosophila oogenesis. Dev. Bio. 231: 265-278. 11180967

Egawa, K., et al. (1999). Membrane-targeted phosphatidylinositol 3-kinase mimics insulin actions and induces a state of cellular insulin resistance. J. Biol. Chem. 274(20): 14306-14.

Farooq, A., et al. (1999). Phosphotyrosine binding domains of Shc and insulin receptor substrate 1 recognize the NPXpY motif in a thermodynamically distinct manner. J. Biol. Chem. 274(10): 6114-21.

Fernandez, R., et al. (1995). The Drosophila insulin receptor homolog: a gene essential for embryonic development encodes two receptor isoforms with different signaling potential. EMBO J. 14(14): 3373-84.

Gallant, P., Shiio, Y., Cheng, P. F., Parkhurst, S. M., and Eisenman, R. N. (1996). Myc and Max homologs in Drosophila. Science 274: 1523-1527.

Goberdhan, D. C., et al. (1999). Drosophila tumor suppressor PTEN controls cell size and number by antagonizing the Chico/PI3-kinase signaling pathway. Genes Dev. 13(24): 3244-58.

Heller-Harrison, R. A., Morin, M. and Czech, M. P. (1995). Insulin regulation of membrane-associated insulin receptor substrate 1. J. Biol. Chem. 270(41): 24442-50.

Huang, H., et al. (1999). PTEN affects cell size, cell proliferation and apoptosis during Drosophila eye development. Development 126(23): 5365-72.

Jhala, U. S., et al. (2003). cAMP promotes pancreatic ß-cell survival via CREB-mediated induction of IRS2. Genes Dev. 17: 1575-1580. 12842910

Jünger, M. A., Rintelen, F., Stocker, H., Wasserman, J. D., Vegh, M., Radimerski, T., Greenberg, M. E. and Hafen E. (2003). The Drosophila Forkhead transcription factor FOXO mediates the reduction in cell number associated with reduced insulin signaling. J. Biol. 2: 20. 12908874

Kawamura, K., Shibata, T., Saget, O., Peel, D., and Bryant, P.J. (1999). A new family of growth factors produced by the fat body and active on Drosophila imaginal disc cells. Development 126: 211-219.

Kimura, K.D., Tissenbaum, H.A., Liu, Y., and Ruvkun, G. (1997). daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans. Science 277, 942-946.

Laustsen, P. G., et al. (2002). Lipoatrophic diabetes in Irs1^-/-/Irs3^-/- double knockout mice. Genes Dev. 16(24): 3213-22. 12502742

Lee, A. V., et al. (1999). Enhancement of insulin-like growth factor signaling in human breast cancer: estrogen regulation of insulin receptor substrate-1 expression in vitro and in vivo. Mol. Endocrinol. 13(5): 787-96.

Lee, Y. H., et al. (2003). c-Jun N-terminal kinase (JNK) mediates feedback inhibition of the insulin signaling cascade. J. Biol. Chem. 278(5): 2896-2902. 12417588

Leevers, S.J., Weinkove, D., MacDougall, L.K., Hafen, E., and Waterfield, M.D. (1996). The Drosophila phosphoinositide 3-kinase Dp110 promotes cell growth. EMBO J. 15: 6584-6594.

Li, J., DeFea, K. and Roth, R. A. (1999). Modulation of insulin receptor substrate-1 tyrosine phosphorylation by an Akt/phosphatidylinositol 3-kinase pathway. J. Biol. Chem. 274(14): 9351-6.

Liang, L., et al. (1999). Insulin receptor substrate-1 enhances growth hormone-induced proliferation. Endocrinology 140(5): 1972-83.

Marin-Hincapie, M. and Garofalo, R. S. (1999). The carboxyl terminal extension of the Drosophila insulin receptor homologue binds IRS-1 and influences cell survival. J. Biol. Chem. 274(35): 24987-94.

Miele, C., et al. (1999). Differential role of insulin receptor substrate (IRS)-1 and IRS-2 in L6 skeletal muscle cells expressing the Arg1152 --> Gln insulin receptor. J. Biol. Chem. 274(5): 3094-102.

Miki, H., et al. (2001). Essential role of Insulin receptor substrate 1 (IRS-1) and IRS-2 in adipocyte differentiation. Mol. Cell. Bio. 21: 2521-2532. 11259600

Mussig, K., et al. (2005). Shp2 is required for protein kinase C-dependent phosphorylation of serine 307 in insulin receptor substrate-1. J. Biol. Chem. 280(38): 32693-9. 16055440

Oldham, S., et al. (2000). Genetic and biochemical characterization of dTOR, the Drosophila homolog of the target of rapamycin. Genes Dev. 14: 2689-2694. 0523807

Oldham, S., et al. (2002). The Drosophila insulin/IGF receptor controls growth and size by modulating PtdInsP₃ levels. Development 129: 4103-4109. 12163412

Patti, M. E., et al. (1995). 4PS/insulin receptor substrate (IRS)-2 is the alternative substrate of the insulin receptor in IRS-1-deficient mice. J. Biol. Chem. 270(42): 24670-3.

Poltilove, R. M. K., et al. (2000). Characterization of Drosophila insulin receptor substrate. J. Biol. Chem. 275(30): 23346-23354. 10801879

Ridderstrale, M., Degerman, E. and Tornqvist, H.(1996). Growth hormone stimulates the tyrosine phosphorylation of the insulin receptor substrate-1 and its association with phosphatidylinositol 3-kinase in primary adipocytes. J. Biol. Chem. 270(8): 3471-4.

Rother, K. I., et al. (1998). Evidence that IRS-2 phosphorylation is required for insulin action in hepatocytes. J. Biol. Chem. 273(28): 17491-7.

Rui, L., et al. (2002). SOCS-1 and SOCS-3 block insulin signaling by ubiquitin-mediated degradation of IRS1 and IRS2. J. Biol. Chem. 277(44): 42394-8. 12228220

Stewart, C.E., and Rotwein, P. (1996). Growth, differentiation, and survival: multiple physiological functions for insulin-like growth factors. Physiol. Rev. 76: 1005-26.

Skolnik EY., et al. (1993). The SH2/SH3 domain-containing protein GRB2 interacts with tyrosine-phosphorylated IRS1 and Shc: implications for insulin control of ras signalling. EMBO J. 12(5): 1929-36.

Staubs, P. A., et al. (1998). Platelet-derived growth factor inhibits insulin stimulation of insulin receptor substrate-1-associated phosphatidylinositol 3-kinase in 3T3-L1 adipocytes without affecting glucose transport. J. Biol. Chem. 273(39): 25139-47.

Staveley, B.E., Ruel, L., Jin, J., Stambolic, V., Mastronardi, F.G., Heitzler, P., Woodgett, J.R., and Manoukian, A.S. (1998). Genetic analysis of protein kinase B (AKT) in Drosophila. Curr. Biol. 8: 599-602

Summers, S. A., et al. (1999). The role of glycogen synthase kinase 3beta in insulin-stimulated glucose metabolism. J. Biol. Chem. 274(25): 17934-40.

Sun, X. J., et al. (1991). Structure of the insulin receptor substrate IRS-1 defines a unique signal transduction protein. Nature 352(6330): 73-7.

Terauchi, Y., et al. (1997). Development of non-insulin-dependent diabetes mellitus in the double knockout mice with disruption of insulin receptor substrate-1 and beta cell glucokinase genes. Genetic reconstitution of diabetes as a polygenic disease. J. Clin. Invest. 99(5): 861-6.

Valverde, A. M., et al. (1998). Insulin receptor substrate (IRS) proteins IRS-1 and IRS-2 differential signaling in the insulin/insulin-like growth factor-I pathways in fetal brown adipocytes. Mol. Endocrinol. 12(5): 688-97.

Valverde, A. M., et al. (2001). Association of Insulin receptor substrate 1 (IRS-1) Y895 with Grb-2 mediates the insulin signaling involved in IRS-1-deficient brown adipocyte mitogenesis. Mol. Cell. Bio. 21: 2269-2280. 11259577

Vassen, L., Wegrzyn, W. and Klein-Hitpass, L. (1998). Human insulin receptor substrate-2 (IRS-2) is a primary progesterone response gene. Mol. Endocrinol. 13(3): 485-94.

Ward, C. W., et al. (1996). Systematic mapping of potential binding sites for Shc and Grb2 SH2 domains on insulin receptor substrate-1 and the receptors for insulin, epidermal growth factor, platelet-derived growth factor, and fibroblast growth factor. J. Biol. Chem. 271(10): 5603-9.

Wessells, R. J., Fitzgerald, E., Cypser, J. R., Tatar, M. and Bodmer, R. (2004). Insulin regulation of heart function in aging fruit flies. Nat. Genet. 36(12): 1275-81. 15565107

Xu, G. G., et al. (1999). Insulin receptor substrate 1-induced inhibition of endoplasmic reticulum Ca2+ uptake in beta-cells. Autocrine regulation of intracellular ca2+ homeostasis and insulin secretion. J. Biol. Chem. 274(25): 18067-74.

Yamamoto-Honda, R., et al. (1996). Mutant of insulin receptor substrate-1 incapable of activating phosphatidylinositol 3-kinase did not mediate insulin-stimulated maturation of Xenopus laevis oocytes. J. Biol. Chem. 271(45): 28677-81.

Yamaguchi, T., Fernandez, R. and Roth, R. A. (1995). Comparison of the signaling abilities of the Drosophila and human insulin receptors in mammalian cells. Biochemistry 34(15): 4962-8.

Yamauchi, T., Tobe, K. and Tamemoto, H., et al. (1996). Insulin signalling and insulin actions in the muscles and livers of insulin-resistant, insulin receptor substrate 1-deficient mice. Mol. Cell. Biol. 16(6): 3074-84.

Yamauchi, T., et al. (1998). Growth hormone and prolactin stimulate tyrosine phosphorylation of insulin receptor substrate-1, -2, and -3, their association with p85 phosphatidylinositol 3-kinase (PI3-kinase), and concomitantly PI3-kinase activation via JAK2 kinase. J. Biol. Chem. 273(25): 15719-26.

Yenush, L., et al. (1996a). The Drosophila insulin receptor activates multiple signaling pathways but requires insulin receptor substrate proteins for DNA synthesis. Mol. Cell. Biol. 16(5): 2509-17.

Yenush, L., et al. (1996b). The pleckstrin homology domain is the principal link between the insulin receptor and IRS-1. J. Biol. Chem. 271(39): 24300-6.

Yenush, L., and White, M. F. (1997). The IRS-signaling system during insulin and cytokine action. Bioessays 19: 491-500.

Zhou L., et al. (1997). Insulin receptor substrate-2 (IRS-2) can mediate the action of insulin to stimulate translocation of GLUT4 to the cell surface in rat adipose cells. J. Biol. Chem. 272(47): 29829-33.

Zhou, L., et al. (1999). Action of insulin receptor substrate-3 (IRS-3) and IRS-4 to stimulate translocation of GLUT4 in rat adipose cells. Mol. Endocrinol. 13(3): 505-14.

date revised: 1 March 2004

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D

The Interactive Fly resides on the
Society for Developmental Biology's Web server.