InteractiveFly: GeneBrief

chico : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

In higher vertebrates, hormones and growth factors play an important role in the control of overall growth because they orchestrate cell growth, cell cycle, and cell survival. Reducing or increasing levels of growth hormone or of the growth hormone mediators, IGF1 and its receptor (IGFR), dramatically influences body and organ size (for review see Stewart, 1996).

Overall growth is affected by the availability of nutrients, as is cell size, in some cases. Many organisms have developed special survival strategies for growth during periods of low nutrition. Under inadequate nutritional conditions, yeast cells, for example, reduce growth and divide at a smaller size, whereas nematodes like C. elegans enter a diapause called the dauer stage. Raising Drosophila under adverse food conditions also results in the production of small flies with fewer and smaller cells. Still, little is known in higher organisms about how growth is controlled at the cellular level: what are the genes involved in the regulation of cell growth, and what determines the critical size at which cells undergo mitosis? In Drosophila, a class of mutations known as Minutes (M) dominantly delay development and in some cases reduce body size. Some of the M genes encode ribosomal proteins and are thought to slow down growth by reducing protein synthesis. Partial loss-of-function mutations in the Drosophila myc gene diminutive cause a reduction in overall body size (Gallant, 1996). However, it is not yet known how Drosophila myc controls growth (Böhni, 1999 and references).

The Drosophila insulin receptor (INR) pathway, and in particular the adaptor protein Chico, which is homologous to vertebrate insulin receptor substrates (IRS), plays a critical role in the control of cell proliferation, cell size, and overall body growth. In Spanish, chico means 'small boy'. chico mutant flies are smaller in size, owing to a reduction in cell size and cell number. The effect of chico mutations on cell size and cell growth is strictly cell autonomous. In addition to its overall effect on growth, Chico also controls cellular metabolism; even though chico flies are only half the size of normal flies, they show an almost 2-fold increase in lipid levels, when compared with their heterozygous siblings. These results provide evidence for a cell-autonomous requirement of the INR signaling pathway in the control of cell size and overall growth (Böhni, 1999).

Many aspects of the insulin system appear to be conserved in both flies and mammals. In mammalian cells, activation of either the insulin receptor or the IGF1 (insulin-like growth factor 1) receptor by insulin and IGF1, respectively, results in the recruitment of IRS1 or IRS2 to the receptor via interaction of the IRS phosphotyrosine binding domains, with a phosphotyrosine motif (NPXY) in the juxtamembrane region of the receptors. Phosphorylation of the multiple tyrosine residues of IRS1 triggers the activation of various signaling pathways, including the RAS/MAP kinase pathway via the SH2/SH3 adaptor GRB2 and the PI3K/PKB pathway via the p85 SH2 adaptor subunit of p110 PI3K (Yenush, 1997). The Drosophila INR shares many structural features with its human homologs, including its heterotetrameric structure and a conserved PTB consensus binding site in the juxtamembrane region. However, the Drosophila INR contains a 400-amino acid C-terminal extension not found in any of the vertebrate receptors. This C-terminal tail contains three YXXM consensus binding sites for the SH2 domain of the p60 subunit of PI3K (see Phosphotidylinositol 3 kinase 92E) and four additional NPXY consensus PTB-binding sites. The C-terminal domain is functional, since expression of a chimeric receptor consisting of the extracellular domain of the human INR and the intracellular domain of the Drosophila INR in murine 32D cells lacking endogenous IRS1 can partially activate mammalian PI3K and S6K. In contrast, the ability of the human INR to activate PI3K in this system is strictly dependent on the coexpression of IRS1 (Yenush, 1996a). These findings and the identification of Chico suggest that in Drosophila, INR couples to the downstream effector PI3K in two different ways, one using docking sites in the INR C-terminal tail and the other connecting through docking sites in Chico (Böhni, 1999).

The homology of Chico with mammalian IRS1, IRS2, IRS3 and IRS4 (collectively referred to as IRS1-4) prompted a test for genetic interactions with other components involved in signaling via IRS proteins, such as the insulin receptor and the p110 catalytic subunit of PI3 kinase. Loss-of-function mutations in Inr are lethal, but certain heteroallelic combinations survive to adulthood. Such Inr mutant flies are reduced in size (Chen, 1996). As in chico mutants, cell size is reduced by 28% in Inr³¹³/Inr³²⁷ flies. Furthermore, targeted expression of a dominant-negative variant of Drosophila p110 PI3K in the developing eye or wing causes a reduction in cell size in the eye, and in both cell size and cell number in the wing. Conversely, overexpression of a constitutively active, membrane-targeted version of PI3K increases cell size and cell number (Leevers, 1996). In flies that are homozygous for chico, heterozygosity for a hypomorphic Inr allele leads to a further reduction in cell number in the wing and the eye. Thus, in the absence of chico function, a reduction of the receptor level potentiates the growth reduction. This Chico independent signaling of INR is likely to be mediated by PI3K-binding sites in the C-terminal tail of the INR (Yenush, 1996). Similarly, expressing a catalytically inactive version of PI3K in chico homozygous wing discs leads to a further reduction in wing size by 48%. Thus, the chico mutant phenotype is modified by mutations in Inr and PI3K. This is consistent with the notion that INR, Chico, and PI3K form a conserved signaling pathway involved in the cell-autonomous control of growth and cell size in Drosophila (Böhni, 1999).

Given the role of the insulin signaling pathway in the control of cellular metabolism in vertebrates and in C. elegans, a test was performed to see whether energy stores are altered in chico mutant flies. The amount of lipid, protein, and glycogen per unit of fresh weight was determined. While there was no significant difference in levels of proteins and glycogen, lipid levels are increased significantly in chico males. In fact, despite their smaller size, chico males have almost twice as much lipids as wild-type males per milligram of fresh weight. The dramatic increase in lipids in chico mutant males is reminiscent of hypertriglyceridemia in IRS1-deficient mice (Abe, 1998) and of fat accumulation observed in C. elegans containing a mutation in the daf-2 gene, which encodes the insulin receptor homolog (Kimura, 1997). Thus, it appears that the INR signaling pathway controls cellular metabolism in vertebrates, nematodes, and insects (Böhni, 1999).

Given the fact that in vertebrates, the insulin receptor pathway plays a critical role in regulating cellular metabolism and growth, it is interesting to speculate that the insulin signaling pathway in flies is part of a nutritional sensing system for each individual cell. Growth is dependent on the availability of nutrients. The reduced body size of chico flies is similar to that of flies reared under poor nutritional conditions. Poorly fed flies also eclose later and possess fewer and smaller cells. In multicellular organisms a system of overall growth coordination is required, since nutrient conditions are unlikely to be the same for all cells in an organism. Under abundant food conditions, an insulin-like peptide and/or other growth factors may be produced and secreted into the open circulatory system to activate the INR pathway in each cell. As a result, cells grow and divide when a critical cell size has been reached. However, when the level of these growth factors drops because of adverse food conditions, the INR pathway activity is reduced. As a consequence, cells slow down cell cycle progression and cell growth, resulting in fewer and smaller cells. The increased accumulation of lipids in chico adults suggests that in addition to its growth-regulating function, Chico may also alter cellular metabolism, resulting in the accumulation of lipids. Interestingly, in homozygous chico mutant larvae no significant difference in lipid levels is observed. This may suggest that during larval stages chico function is required for cell growth and only during pupal development and in the adult Chico controls metabolism. Thus, at the end of development the INR pathway may control the physiological response required to endure periods of low nutrient availability (Böhni, 1999).

Evidence from other studies points to global hormonal regulation of cell growth in Drosophila. Although in Drosophila amino acid withdrawal prevents imaginal disc cells and larval neuroblasts from entering the cell cycle, culturing brains of starved larvae in amino acid-rich medium is not sufficient to induce cell cycle entry of quiescent neuroblasts (Britton and Edgar, 1998). When such brains were cocultured with fat body from fed larvae, however, the neuroblasts started to divide. This suggests that growth control is mediated by growth factors secreted from the larval fat body (Britton, 1998). Recently, Kawamura (1999) characterized imaginal disc growth factors (IDGFs), which are expressed primarily in yolk cells and fat body. Moreover, IDGFs have been shown to cooperate with insulin to stimulate proliferation, and it was speculated that the IDGFs, as chitin-specific lectins, could interact with the INR (Kawamura, 1999). Under abundant food conditions, an insulin-like peptide and/or other growth factors, like IDGFs, may be produced and secreted by the larval fat body into the open circulatory system to activate the INR pathway in each cell. As a result, cells grow and divide when a critical cell size has been reached. However, when the level of these growth factors drops because of adverse food conditions, the INR pathway activity is reduced. As a consequence, cells slow down cell cycle progression and cell growth, resulting in fewer and smaller cells (Böhni, 1999).

The effects on growth and cell size of chico mutants are remarkably similar to the phenotypes of mutations in genes encoding other components of the INR pathway in Drosophila. Although loss-of-function mutations in the Drosophila INR gene are lethal, certain heteroallelic combinations are viable and show delayed development, reduced body size, and decreased cell number (Chen, 1996) and cell size. Expression of dominant-negative or constitutively active variants of p110 PI3K in the developing wing and eye reduces or increases cell number and cell size, respectively (Leevers, 1996). Furthermore, viable mutations in the gene encoding Drosophila protein kinase B (Staveley, 1998), a downstream effector of PI3K, cause a reduction in cell number and cell size. The striking similarities between the phenotypes of chico and mutations in the genes encoding INR and DPKB, as well as the genetic interactions between mutations in Inr, chico, and PI3K, emphasize the specific role of the INR pathway in control of cell growth and cell number as a process independent of pattern formation (Böhni, 1999).

Chico controls growth at three different levels: it regulates the size of individual cells, of organs, and of the entire organism. The similarities observed in the phenotypes of loss-of-function mutations in the INR pathway in Drosophila extend to the phenotypes caused by defects in insulin/IGF signaling pathways in humans and mice. For example, severe insulin resistance in humans causes intrauterine growth retardation and low birth weight. Mice lacking IGF1, IGF1 receptor, IRS1, or IRS2 function are also delayed in development and have a reduced body size. Therefore, it appears that the INR/IGFR signaling pathway is conserved from vertebrates to Drosophila, not only in regard to its structure but also to its function (Böhni, 1999 and references).

The Drosophila SH2B family adaptor Lnk acts in parallel to chico in the insulin signaling pathway

Insulin/insulin-like growth factor signaling (IIS) plays a pivotal role in the regulation of growth at the cellular and the organismal level during animal development. Flies with impaired IIS are developmentally delayed and small due to fewer and smaller cells. In the search for new growth-promoting genes, mutations were identified in the gene encoding Lnk, the single fly member of the SH2B family of adaptor molecules. Flies lacking lnk function are viable but severely reduced in size. Furthermore, lnk mutants display phenotypes reminiscent of reduced IIS, such as developmental delay, female sterility, and accumulation of lipids. Genetic epistasis analysis places lnk downstream of the insulin receptor (InR) and upstream of phosphoinositide 3-kinase (PI3K) in the IIS cascade, at the same level as chico (encoding the single fly insulin receptor substrate [IRS] homolog). Both chico and lnk mutant larvae display a similar reduction in IIS activity as judged by the localization of a PIP₃ reporter and the phosphorylation of protein kinase B (PKB). Furthermore, chico; lnk double mutants are synthetically lethal, suggesting that Chico and Lnk fulfill independent but partially redundant functions in the activation of PI3K upon InR stimulation (Werz, 2009).

The core components of the Drosophila IIS pathway include Chico, the homolog of the insulin receptor substrates (IRS), the lipid kinase phosphoinositide 3-kinase (PI3K), the lipid phosphatase PTEN, and the serine-threonine kinase PKB. Chico gets phosphorylated upon IIS pathway activation, providing binding sites for the Src Homology 2 (SH2) domain of p60, the regulatory subunit of PI3K. Increased PI3K activity leads to the accumulation of phosphatidylinositol-(3,4,5)-trisphosphate(PIP₃) at the plasma membrane, which recruits PKB to the membrane via its pleckstrin homology (PH) domain. PKB takes a central position in the regulation of multiple cellular processes such as cellular growth, proliferation, apoptosis, transcription and cell motility (Werz, 2009).

In Drosophila, mutations in IIS components result in reduced cell, organ and body size with little effect on cell fate and differentiation. For example, hypomorphic mutants of essential IIS components and, in particular, homozygous null mutants of chico are viable but only approximately half the size of wild-type flies, due to smaller and fewer cells. Furthermore, characteristic defects caused by reduced IIS activity include female sterility, an increase in total lipid levels of adults, and a severe developmental delay (Werz, 2009).

chico encodes an adaptor protein, a group of proteins without catalytic activity usually carrying domains mediating specific interactions with other proteins such as an SH2 domain, a PH domain, or a phosphotyrosine-binding (PTB) domain. Adaptor proteins play an important role in the formation of protein-protein interactions and thus in the formation of protein networks. The various interaction domains within adaptor proteins and the specificity of those domains provide adaptor molecules with the ability to elicit characteristic responses to a particular signal (Werz, 2009).

Recently, a novel family of adaptor proteins, the SH2B family, has been identified in mammals. It consists of three members -- SH2B1 (SH2B/PSM), SH2B2 (APS) and SH2B3 (Lnk) -- that share a common protein structure with an N-terminal proline-rich stretch, a PH domain, an SH2 domain and a highly conserved C-terminal Cbl recognition motif (Huang, 1995; Riedel, 1997; Yokouchi, 1997). They have been shown to regulate signal transduction by receptor tyrosine kinases such as the InR, IGF-I receptor and receptors for nerve growth factor, hepatocyte growth factor, platelet-derived growth factor and fibroblast growth factor, as well as by the JAK family of tyrosine kinases (Riedel, 1997; Wakioka, 1999; Rui, 1997). Whereas SH2B3 (Lnk) has been described to function exclusively by negatively regulating receptor kinases that are specialized in the development of a subset of immune and hematopoietic cells, the picture for the other two family members is not as clear yet (Werz, 2009).

Although both SH2B1 and SH2B2 have been shown to be directly involved in the regulation of JAK tyrosine kinases and of IIS, their specificities and physiological functions are complex and remain largely elusive. For example, depletion of SH2B1 in mice leads to severe obesity, leptin and insulin resistance as well as female infertility. However, a number of studies suggest that SH2B1 exerts its function predominantly in the association with JAK2 and regulation of related signaling cascades. For example, binding of SH2B1 to JAK2 results in an enhancement of JAK2 activation and JAK2-mediated growth hormone signaling, and depletion of SH2B1 leads to decreased leptin-stimulated JAK2 activation and reduced phosphorylation of its substrates (Werz, 2009 and refereces therein).

SH2B2 is also able to bind to JAK2 and to the InR but recent research has mainly focused on the mechanisms related to the connection of SH2B2 and c-Cbl. Phosphorylation of Tyr618 in SH2B2 stimulates binding of c-Cbl and thus mediates GLUT4 translocation and inhibition of erythropoietin-dependent activation of Stat5. However, the general impact of SH2B2 on receptor tyrosine kinase signaling remains controversial. Whereas one study showed that SH2B2 overexpression delayed InR and IRS dephosphorylation and enhanced PKB activation, several other studies (e.g., on SH2B2 knockout mice) have suggested a negative regulatory role for SH2B2 in IIS, which might also be mediated via c-Cbl dependent ubiquitination and subsequent degradation of target kinases (Werz, 2009 and references therein).

Although interactions with the IIS pathway and the InR have been described for SH2B1 and SH2B2, the physiological significance of these connections in mammals appears to be the regulation of metabolism and energy homeostasis rather than the control of cell growth and proliferation (Werz, 2009 and references therein).

In contrast to the mammalian situation, the Drosophila genome encodes a single adaptor protein that shares a common domain structure with the SH2B family, termed Lnk. This study shows that Drosophila lnk predominantly regulates cellular and organismal growth in a cell-autonomous way. Loss of lnk function leads to a reduction in cell size and cell number, reminiscent of decreased IIS activity. A thorough genetic analysis placed Lnk as a positive regulator of IIS at the level of IRS/Chico (Werz, 2009).

lnk was identified in an unbiased screen for growth-regulating genes based on the eyFLP/FRT technique in Drosophila. In principle, mutations in growth-promoting genes led to flies with smaller heads (the so-called pinheads), whereas negative regulators of tissue growth resulted in larger heads (referred to as bighead mutants). Among others, four mutations were identified causing a pinhead phenotype that fell into a single complementation group on the right arm of the third chromosome. The complementation group mapped close to the lnk locus (CG 17367) at the cytological position 96F. Subsequent sequencing revealed EMS-induced mutations in the lnk coding region for each allele (Werz, 2009).

Flies homozygous mutant for lnk are small but do not show any obvious patterning defects. Homozygous mutant pupae are also small, indicating that lnk is essential for proper organismal growth throughout development. lnk mutant flies are severely reduced in dry weight, as shown for male and female flies . This defect is fully rescued by introducing a genomic rescue construct comprising the entire lnk locus, proving that the mutations in lnk are responsible for the growth phenotype (Werz, 2009).

The most closely related group of proteins to Drosophila Lnk in vertebrates is the SH2B family of adaptor proteins sharing a common protein structure. Alignment of Drosophila Lnk with its human homologs (SH2B1, SH2B2 and SH2B3) shows high sequence identity in particular in the conserved PH and SH2 domains. The four lnk alleles recovered in the screen (7K1, 4Q3, 6S2, 4H2) contain a single point mutation in either of these two highly conserved protein domains resulting in a premature stop (4Q3, 6S2) or an amino acid exchange in conserved residues (7K1, 4H2). Since hemizygous and heteroallelic lnk mutant animals display identical phenotypes, all lnk alleles are genetically null, suggesting an essential role of both the PH and the SH2 domain for Lnk function (Werz, 2009).

SH2B1 and SH2B2, two members of the mammalian family of Lnk-related adaptor proteins, have been shown to associate with several signaling molecules including JAK2 and the InR. However, the different proteins seem to have distinct impacts on the respective pathways, regulating them either in a positive or negative manner. Using the new mutations in the single member of the SH2B family in Drosophila allowed determination of whether lnk plays an essential role in either of these pathways (Werz, 2009).

Although the tyrosines in JAK2 and JAK3 mediating their interaction with the SH2B family proteins in mammals are not conserved in the Drosophila homolog, it was wondered whether Lnk has a function in the regulation of Drosophila JAK. Misregulation of JAK/Stat signaling in Drosophila results in formation of melanotic tumors and proliferative defects in larval blood cells, held out wings and rough or disrupted eye phenotypes as well as male sterility and fused egg chambers in the vitellarium due to the absence of stalk cells. In the characterization of homozygous lnk mutant animals none of the phenotypes that are characteristic for impaired JAK/Stat signaling are observed. Moreover, genetic interaction experiments of lnk with any of the core JAK/Stat pathway components did not reveal a connection of Lnk to JAK/Stat signaling. These results suggest that in Drosophila, Lnk is not involved in the regulation of signaling activity downstream of JAK (Werz, 2009).

The initial observation that lnk mutations reduced organ and body size pointed at a role of Lnk in the IIS pathway. The growth phenotype of lnk mutants was characterized further by quantifying ommatidia number and generating tangential sections of mosaic eyes to study the impact of lnk on cell number and cell size. SEM pictures of heads of lnk mutant adults compared to wild type and quantification of ommatidia number revealed that mutations in lnk caused a reduction in cell number by about 30%. Induction of lnk mutant clones in the eye resulted in a cell-autonomous reduction of cell size in photoreceptor cells and rhabdomeres, as shown by tangential eye sections and subsequent quantification of photoreceptor cell and rhabdomere area in lnk mutant tissue compared to wild type. Therefore, lnk function is important to ensure proper regulation of cell number and cell size, similar to IIS components (Werz, 2009).

It has previously been shown that IIS is required in oogenesis beyond the last previtellogenic stage; a reduction in IIS activity leads to an arrest in oogenesis and female sterility. Female flies lacking lnk function are also sterile and have small ovaries. These ovaries only contain oocytes that developed until the last previtellogenic stage and resemble ovaries of females mutant for chico (Werz, 2009).

A further characteristic phenotype of impaired IIS is the accumulation of lipids in adult flies. The lipid levels in three-day old male chico flies are more than twice the level than in the control despite their smaller body size. Homozygous lnk mutant flies reach the same lipid levels as chico mutants. Taken together, these results strongly indicate a role of Lnk in the IIS pathway (Werz, 2009).

The phenotypes of homozygous lnk mutants suggest that Lnk regulates cellular growth exclusively via IIS. However, the protein sequence of Lnk contains two putative Drk/Grb2 YXN binding sites. In addition, all SH2B family members, except for the beta, gamma and delta isoform of SH2B1, carry a highly conserved consensus site for binding of Cbl. The functionality of this Cbl binding site has only been demonstrated in SH2B2 so far. In order to test the functional significance of the individual binding motifs, rescue constructs consisting of the genomic lnk locus but carrying specific mutations that result in amino acid exchanges in the core tyrosine of the respective motifs were generated. These constructs fully rescued the reduction in dry weight in lnk mutants, suggesting that neither binding of Drk to the YXN site nor an interaction of Lnk with Cbl through the C-terminal binding motif is important in the regulation of growth. In contrast, both the PH and the SH2 domains of Lnk are essential for its function because the lnk alleles disrupting either domain behave genetically as null mutations (Werz, 2009).

In order to study the consequences of the loss of lnk function on cell growth, a clonal analysis in larval wing discs was performed using the 4Q3 allele. The hsFLP/FRT system was used to induce mitotic recombination, thus to generate homozygous lnk mutant cell clones (marked by the absence of GFP) adjacent to clones that consist of wild-type cells (marked by two copies of GFP). All mutant clones were smaller than their wild-type sister clones, and they contained fewer cells. Although a clear tendency to a cell size reduction of lnk mutant cells, as determined by the ratio of clone area to cell number, was apparent, the relative reduction was not significant in larval wing discs. It is thus speculated that the influence of lnk on cell size is rather subtle in early stages of development (Werz, 2009).

Molecular readouts of IIS activity were used to investigate the consequences of the loss of lnk function. Stimulation of the InR activates PI3K, which increases the levels of phosphatidylinositol-(3,4,5)-trisphosphate(PIP₃) at the plasma membrane. Previously, a reporter containing a PH domain fused to GFP (tGPH) that localizes to the plasma membrane as a result of PI3K activity had been described. Using this reporter, PIP₃ levels were monitored in wild-type and lnk mutant fat body cells as well as in clones of lnk mutant cells in the fat body. Whereas the tGPH reporter localized to the membrane in wild-type cells, the GFP signal was predominantly observed in the cytoplasm in lnk mutant cells, indicating that the loss of lnk function causes a reduction of PI3K signaling activity. The impact of lnk on tGPH localization is comparable to the effects observed in chico mutant cells (Werz, 2009).

As another molecular readout of IIS activity, the phosphorylation levels of PKB, a downstream kinase of IIS, were measured. Lysates of homozygous lnk and chico mutant larvae were subjected to Western analysis and compared to wild-type controls. Whereas the PKB protein levels were comparable in all genotypes, the amount of phosphorylated PKB was reduced in both lnk and chico mutant larvae. Thus, Lnk and Chico contribute similarly to the activity of PI3K (Werz, 2009).

In order to establish where lnk acts in the IIS cascade, genetic epistasis experiments were performed. The ability of lnk to suppress the overgrowth phenotype caused by overexpression of InR during eye development was measured. In this sensitized background loss of lnk function reduced the eye size almost to wild-type size, suggesting that Lnk modulates the IIS pathway downstream of the receptor. In contrast, homozygosity for lnk was not sufficient to suppress the overgrowth caused by a membrane-tethered form of PI3K. Thus, Lnk acts between the InR and the lipid kinase PI3K in the IIS pathway (Werz, 2009).

The phenotypic similarities between lnk and chico mutants are striking. Both genes encode adaptor proteins with a PH domain and a phosphotyrosine-binding motif (an SH2 domain in the case of Lnk and a PTB domain in the case of Chico, respectively), and both act between the InR and PI3K. Thus, it is conceivable that Lnk is required for proper Chico function, for example by stabilizing the phosphorylated InR and thereby allowing a stable InR-Chico interaction. Attempts were made to genetically test whether Lnk acts via Chico. If this were the case, chico; lnk double mutants would be expected to display similar phenotypes as the single mutants. However, chico; lnk double mutants were lethal. Removing one copy of PTEN (encoding the lipid phosphatase that antagonizes PI3K) restored viability of the chico; lnk double mutants, suggesting that the chico; lnk double mutants suffer from reduced IIS activity and thus insufficient levels of the second messenger PIP₃. Reducing the amount of PTEN, the negative regulator of PIP₃ production, allows for PIP₃ levels above a critical threshold for survival but still insufficient to ensure normal growth. These results imply that Chico and Lnk independently act downstream of the InR, and that both adaptors are required for the full activation of PI3K upon InR stimulation. Consistently, it was found that the levels of phospho-PKB were further reduced in chico; lnk double mutant larvae as compared to single mutants (Werz, 2009).

These data clearly indicate that both Lnk and Chico are required for the full activity of PI3K, with each adaptor being sufficient for a partial stimulation of PI3K activity. This might explain why chico and lnk are among the few non-essential genes in the IIS cascade. How does Lnk contribute to the activation of PI3K? Probably, Lnk does not exert its function in the same way as Chico. In contrast to Chico, Lnk lacks an YXXM consensus binding site for the SH2 domain of the regulatory subunit of PI3K. Upon activation of the InR, Lnk might connect the signal from the InR with Chico in order to enhance PI3K activation. Interestingly, such a mechanism has been proposed in vertebrates, where SH2B1 promotes IRS1 and IRS2-mediated activation of the PI3K pathway in response to Leptin. However, a model is favored in which Lnk promotes the membrane localization of PI3K by recruiting another binding partner of PI3K or by counteracting a negative regulator of PI3K localization. It will thus be important to identify physical interactors of Lnk (Werz, 2009).

Hyperinsulinemia drives epithelial tumorigenesis by abrogating cell competition

Metabolic diseases such as type 2 diabetes are associated with increased cancer incidence. This study shows that hyperinsulinemia promotes epithelial tumorigenesis by abrogating cell competition. In Drosophila eye imaginal epithelium, oncogenic scribble (scrib) mutant cells are eliminated by cell competition when surrounded by wild-type cells. Through a genetic screen, this study found that flies heterozygous for the insulin receptor substrate chico allow scrib cells to evade cell competition and develop into tumors. Intriguingly, chico is required in the brain's insulin-producing cells (IPCs) to execute cell competition remotely. Mechanistically, chico downregulation in IPCs causes hyperinsulinemia by upregulating a Drosophila insulin Dilp2, which activates insulin-mTOR signaling and thus boosts protein synthesis in scrib cells. A diet-induced increase in insulin levels also triggers scrib tumorigenesis, and pharmacological repression of protein synthesis prevents hyperinsulinemia-induced scrib overgrowth. These findings provide an in vivo mechanistic link between metabolic disease and cancer risk via systemic regulation of cell competition (Sanaki, 2020).

Metabolic diseases such as type 2 diabetes and obesity are often accompanied by hyperinsulinemia, which is characterized by high levels of circulating insulin. In epidemiology, hyperinsulinemia has been implicated in increased cancer incidence. For instance, the risk of liver, pancreas, endometrium, kidney, and bladder cancers increases 1.5- to 2-fold in people with hyperinsulinemia. Although previous studies in Drosophila and rodents unveiled some aspects of the mechanism by which hyperinsulinemia promotes tumor growth and malignancy, the underlying mechanisms are still largely unknown (Sanaki, 2020).

Most cancers originate from epithelial cells that frequently lose apicobasal polarity during tumor progression. In Drosophila imaginal epithelium, loss-of-function mutations in evolutionarily conserved apicobasal polarity genes, such as scrib or discs large (dlg), disrupt epithelial integrity and result in tumorous overgrowth. Intriguingly, such oncogenic polarity-deficient cells do not overproliferate but are eliminated from the tissue when surrounded by wild-type cells, a phenomenon called tumor-suppressive cell competition. Previous work found multiple mechanisms that drive this cell elimination via cell-cell interaction between scrib and wild-type cells, which include Sas-PTP10D ligand-receptor interaction, Slit-Robo2-Ena/VASP-mediated scrib cell extrusion, and engulfment of scrib cells by wild-type cells. Through a genetic screen in Drosophila, this study found an unexpected new regulatory mechanism whereby hyperinsulinemia systemically abrogates tumor-suppressive cell competition and thus causes tumorigenesis in the epithelium. These data could provide a mechanistic explanation for the epidemiological evidence that links hyperinsulinemia and cancer incidence, thus contributing to a better understanding of cancer biology in vivo (Sanaki, 2020).

This study found that hyperinsulinemia in flies systemically suppresses cell competition in the eye epithelium, leading to tumorous overproliferation of polarity-deficient cells that are normally eliminated when surrounded by wild-type cells. It has been reported that high-sugar diet promotes tumor growth and metastasis of fly tumors with elevated Ras and Src signaling, providing a model of how abnormal physiology promotes tumor progression. In addition, studies in mice have shown that high-fat diet-induced obesity suppresses extrusion of oncogenic RasV12-expressing cells from mice intestine and that endogenous hyperinsulinemia contributes to pancreatic ductal adenocarcinoma. Thus, abnormal physiology, especially hyperinsulinemia, has a promotive effect on tumor development and progression, yet the mechanism by which hyperinsulinemia controls the initial step of tumorigenesis has been unclear. The current observations indicate that chico heterozygous mutant or IPCs-specific chico-knockdown larvae can be used as a Drosophila model of hyperinsulinemia. Consistently, although chico homozygous mutant flies drastically decrease their body weight, chico heterozygous mutant flies show increased body weight, implying a phenotypic outcome of hyperinsulinemia (Sanaki, 2020).

The findings that hyperinsulinemia systemically abrogates tumor-suppressive cell competition by boosting InR-TOR-mediated protein synthesis in pre-malignant cells may provide an in vivo mechanistic link between metabolic diseases and cancer risk. Previous work has shown that Sas-PTP10D signaling in scrib cells promotes their elimination by repressing epidermal growth factor receptor (EGFR) signaling. Defects in Sas-PTP10D signaling attenuates scrib cell elimination via cooperation between EGFR-Ras and TNF-JNK signaling, which leads to activation of the Hippo pathway effector Yorkie (Yki). On the other hand, this study found that hyperinsulinemia attenuates scrib cell elimination by fueling insulin-mTor signaling. Given that these two signaling pathways are independent, there would be no direct cross talk between Sas-PTP10D signaling and hyperinsulinemia-driven tumorigenesis. Rather, it is possible that both Sas-PTP10D inactivation (Yki activation) and insulin signaling activation (Tor activation) lead to the same biological outcome, namely, elevation of protein synthesis, which could explain how insulin signaling overrides Sas-PTP10D signaling (Sanaki, 2020).

Notably, differential levels of protein synthesis between cells has long been implicated in regulating classical Minute cell competition, which is a competitive elimination of cells with a heterozygous mutation for a ribosomal protein gene. In addition, recent work has found that losers of cell competition triggered by different mutations such as Minute, Myc, Mahjong, and Hel25E commonly show lower protein synthesis levels than that neighboring winners do (Nagata, 2019). Moreover, insulin-TOR signaling has been shown to control cell competition during mouse embryonic development. These observations suggest that differential levels of insulin-TOR signaling and protein synthesis between cells are the key for cell competition. Supporting this notion, scrib-induced cell competition can be compromised either by introducing Minute mutation in wild-type winners or by overexpressing Myc in scrib losers. These data show that scrib cells are insensitive to environmental insulin and thus are lower in insulin-TOR signaling and protein synthesis levels compared with that of the neighbors, and hyperinsulinemia reverses this balance and causes scrib tumorigenesis. Given that a drug treatment targeting cellular metabolism could prevent hyperinsulinemia-driven tumorigenesis, cancer risk risen by metabolic diseases may become controllable in the future (Sanaki, 2020).

REGULATION

Protein Interactions

Chimeric receptors encoding either the whole or a portion of the cytoplasmic domain of the Drosophila Insulin-like receptor (InR) with the extracellular domain of the human insulin receptor (IR) were expressed either transiently in COS cells or stably in Chinese hamster ovary cells and compared with the wild-type human IR. All three receptors bind insulin equally and exhibit an insulin-activated tyrosine kinase activity. The ability of the Drosophila cytoplasmic domain to mediate the tyrosine phosphorylation of insulin receptor substrate 1, stimulate cell proliferation, and activate MAP kinase is indistinguishable from that of the human IR. The chimeric Drosophila receptors do not bind more phosphatidylinositol 3-kinase (see Phosphotidylinositol 3 kinase 92E) than the human IR, despite containing a C-terminal extension with potential tyrosine phosphorylation sites in the motif recognized by the SH2 domain of this enzyme. Thus, the essential signal-transducing abilities of the IR appear to have been conserved from invertebrates to mammals, despite the considerable differences in the sequences of these receptors (Yamaguchi, 1995).

Drosophila contain an insulin receptor homolog, encoded by the InR gene located at position 93E4-5 on the third chromosome. The receptor protein is strikingly homologous to the human receptor, exhibiting the same alpha2beta2 subunit structure and containing a ligand-activated tyrosine kinase in its cytoplasmic domain. Chemical mutagenesis was used to induce mutations in the inr gene. Six independent mutations that lead to a loss of expression or function of the receptor protein were identified. These mutations are recessive, embryonic, or early larval lethals, but some alleles exhibit heteroallelic complementation to yield adults with a severe developmental delay (10 days), growth-deficiency, female-sterile phenotype. Interestingly, the severity of the mutant phenotype correlates with biochemical measures of loss of function of the receptor tyrosine kinase. The growth deficiency appears to be due to a reduction in cell number, suggesting a role for InR in regulation of cell proliferation during development. The phenotype is reminiscent of those seen in syndromes of insulin-resistance or IGF-I and IGF-I receptor deficiencies in higher organisms, suggesting a conserved function for this growth factor family in the regulation of growth and body size (Chen, 1996).

The cloning and primary structure of the Drosophila insulin receptor gene (InR) is reported, along with functional expression of the predicted polypeptide, and the isolation of mutations in the InR locus. The structure and processing of the Drosophila insulin proreceptor are somewhat different from those of the mammalian insulin and IGF 1 receptor precursors. The InR proreceptor (M(r) 280 kDa) is processed proteolytically to generate an insulin-binding alpha subunit (M(r) 120 kDa) and a beta subunit (M(r) 170 kDa) with protein tyrosine kinase domain. The InR beta 170 subunit contains a novel domain at the carboxyterminal side of the tyrosine kinase, in the form of a 60 kDa extension that contains multiple potential tyrosine autophosphorylation sites. This 60 kDa C-terminal domain undergoes cell-specific proteolytic cleavage that leads to the generation of a total of four polypeptides (alpha 120, beta 170, beta 90, and a free 60 kDa C-terminus) from the inr gene. These subunits assemble into mature InR receptors with the structures alpha 2(beta 170)2 or alpha 2(beta 90)2. Mammalian insulin stimulates tyrosine phosphorylation for both types of beta subunits; in turn, the phosphorylation allows the beta 170, but not the beta 90 subunit, to bind directly to p85 SH2 domains of PI-3 kinase. It is likely that the two different isoforms of InR have different signaling potentials. Loss of function mutations in the InR gene, induced by either a P-element insertion occurring within the predicted ORF, or by ethylmethane sulfonate treatment, renders pleiotropic recessive phenotypes that lead to embryonic lethality. The activity of InR appears to be required in the embryonic epidermis and nervous system among organ systems, since development of the cuticle, as well as the peripheral and central nervous systems are affected by InR mutations (Fernandez, 1995).

The Drosophila insulin receptor (InR) contains a 368-amino-acid COOH-terminal extension that contains several tyrosine phosphorylation sites in YXXM motifs. This extension is absent from the human insulin receptor but resembles a region in insulin receptor substrate (IRS) proteins that binds to the phosphatidylinositol (PI) 3-kinase and mediates mitogenesis. The function of a chimeric InR containing the human insulin receptor binding domain (hDIR) was investigated in 32D cells, which contain few insulin receptors and no IRS proteins. Insulin stimulated tyrosine autophosphorylation of the human insulin receptor and hDIR, and both receptors mediate tyrosine phosphorylation of Shc and activate mitogen-activated protein kinase. IRS-1 is required by the human insulin receptor to activate PI 3-kinase and p70s6k (see Drosophila RPS6-p70-protein kinase), whereas hDIR associate with PI 3-kinase and activates p70s6k without IRS-1. However, both receptors required IRS-1 to mediate insulin-stimulated mitogenesis. These data demonstrate that the InR possesses additional signaling capabilities when compared with its mammalian counterpart but still requires IRS-1 for the complete insulin response in mammalian cells (Yenush, 1996a).

Like the mammalian insulin receptor, the Drosophila insulin receptor (INR)¹ is a tetramer formed by two alpha subunits and two beta subunits. INR alpha and beta subunits are synthesized together as a proreceptor precursor, proteolytically processed, and linked together by disulfide bonds. The alpha subunits, with a molecular mass of 110-120 kDa, are extracellular and contain the ligand binding domains that are capable of binding mammalian insulin with a K_d of 15 nM. The beta subunits traverse the plasma membrane and have an insulin-stimulated tyrosine kinase in the cytoplasmic portion. DNA sequence analysis and expression of the INR beta subunit in mammalian and Drosophila cells indicate that the INR beta subunit is larger than its mammalian homolog and exhibits an apparent molecular mass of ~180 kDa. The increased mass is due to the presence of a 400-amino acid carboxyl-terminal extension. However, the majority of INR beta subunits are processed to 92/102-kDa forms in Drosophila embyros and some cell lines, the difference being due to proteolytic cleavage of the carboxyl-terminal extension. Both truncated and full-length beta subunits are autophosphorylated on tyrosine residues in response to insulin binding (Marin-Hincapie, 1999 and references therein).

The 400-amino acid carboxyl-terminal extension of the beta INR contains clusters of motifs known to be involved in the interaction with SH2 and PTB domain-containing proteins, suggesting a role for this domain in signaling through interaction with other signaling molecules. Interestingly, four tyrosines are found in 'hybrid' amino acid motifs in which residues amino-terminal to each tyrosine form the motif NP X Y, resembling known PTB domain binding sites, and residues carboxyl-terminal to the same tyrosines form the motifs YXXM, YMXM, or YXLLD -- all known to be involved in binding to SH2 domains. Thus, tyrosines 1993 and 2030 appear in the motif SXNPXYXX M; tyrosine 2009 is part of S X NPXYMXM, and tyrosine 1969 appears in the sequence SDNPXYRLLD. Whether these motifs serve to bind SH2 or PTB domain-containing proteins upon tyrosine phosphorylation and whether one is preferred over the other is not clear. The cytoplasmic domain of the INR expressed in cells lacking IRS-1 has been shown to bind PI3-kinase. However, a similar construct expressed in Chinese hamster ovary cells that contain IRS-1 fails to do so. Since a significant percentage of the INR beta subunit undergoes tissue- or stage-specific proteolytic processing in Drosophila embryos to remove the carboxyl-terminal extension and once it is removed it appears not to be phosphorylated, its role in signal transduction by the INR is not clear. Therefore, the signaling capacity conferred by the beta INR carboxyl-terminal extension has been explored by expressing either full-length or truncated INR beta subunit forms in mammalian cells and determining the effect on protein-protein interactions and cell growth (Marin-Hincapie, 1999 and references therein).

In order to explore the role of the 400 AA extention in INR function, mammalian expression vectors encoding either the complete INR beta subunit (beta-Myc) or the INR beta subunit without the carboxyl-terminal extension (betaDelta) were constructed, and the membrane-bound beta subunits were expressed in 293 and Madin-Darby canine kidney (MDCK) cells in the absence of the ligand-binding alpha subunits. beta-Myc and betaDelta proteins are constitutively active tyrosine kinases of 180 and 102 kDa, respectively. INR beta-Myc co-immunoprecipitates a phosphoprotein of 170 kDa identified as insulin receptor substrate-1 (IRS-1, Flipper or Chico), whereas INR betaDelta does not, suggesting that the site of interaction is within the carboxyl-terminal extension. IRS-1 is phosphorylated on tyrosine to a much greater extent in cells expressing INR beta-Myc than in parental or INR betaDelta cells. Despite this, a variety of PTB or SH2 domain-containing signaling proteins, including IRS-2, mSos-1, Shc, p85 subunit of phosphatidylinositol 3-kinase, SHP-2, Raf-1, and JAK2, are not associated with the INR beta-Myc.IRS-1 complex. Overexpression of INR beta-Myc and betaDelta kinases confers an equivalent increase in cell proliferation in both 293 and Madin-Darby canine kidney cells, indicating that this growth response is independent of the carboxyl-terminal extension. However, INR beta-Myc-expressing cells exhibit enhanced survival, relative to parental and betaDelta cells, suggesting that the carboxyl-terminal extension, through its interaction with IRS-1, plays a role in the regulation of cell death (Marin-Hincapie, 1999).

Thus, overexpression of constitutively active INR beta and betaDelta receptors in 293 and MDCK cells promotes cell proliferation, indicating that the INR can engage the mammalian proliferation pathways. The equivalent proliferative responses induced by INR beta-Myc and betaDelta kinases suggests that the growth-promoting function of the INR in these cells is independent of the carboxyl-terminal extension. In contrast, cells expressing the full-length INR beta subunit exhibit significantly enhanced survival as compared with cells expressing the betaDelta INR. Relative to the parental 293 and MDCK cells, the INR beta-Myc and betaDelta proteins confer somewhat different behavior; beta-Myc clearly promotes survival in 293 cells, whereas betaDelta more dramatically accelerates cell death in MDCK cells. Nonetheless, a clear difference in the behavior of cells expressing the full-length or truncated INR beta subunits is evident in both backgrounds. Despite the presence of a juxtamembrane NPXY motif predicted to interact with IRS-1 in both beta-Myc and betaDelta proteins, IRS-1 is not highly phosphorylated in betaDelta cells. This suggests that the carboxyl-terminal extension of the INR beta subunit is required for sustained association and phosphorylation of IRS-1. This persistent IRS-1 phosphorylation distinguishes beta-Myc from betaDelta cells and may be of primary importance in promoting cell survival. Without this sustained interaction, cell death may actually be accelerated, as observed in MDCK cells transfected with the INR betaDelta kinase (Marin-Hincapie, 1999).

IRS-1 that is bound to the INR beta subunit is phosphorylated on tyrosine; however, no evidence has been found for increased association of PI3-kinase or other candidate signaling molecules with this complex. Therefore, the mechanism whereby this association leads to increased cell survival is unclear at present. Interestingly, a recent report demonstrates that expression of a truncated IRS-1 containing only the pleckstrin homology and phosphotyrosine binding domains, without any tyrosine phosphorylation sites, mediates PI3-kinase and phosphotyrosine-independent signals that contribute to the regulation of cell survival and apoptosis. IRS-1 that is bound to the carboxyl-terminal extension of INR in 293 and MDCK cells may have similarly activated pathways that promote cell survival in the absence of PI3-kinase activation (Marin-Hincapie, 1999 and references therein).

Thus, two isoforms of an activated INR beta subunit have been expressed in mammalian cells, and a functional difference between them has been demonstrated. The data presented here indicate that the stimulation of cell proliferation by INR is mediated by the kinase domain independent of the carboxyl-terminal extension. In contrast, the carboxyl-terminal extension mediates an interaction with IRS-1 and influences cell survival. Since an IRS homolog is present in Drosophila, this may reflect an inherent function of the INR which, in flies, is modulated by tissue- or stage-specific processing of the receptor. These data also suggest that in mammalian cells, persistent localization of IRS-1 to membranes via the interaction of IRS-1 with receptors and/or persistent tyrosine phosphorylation generates signals independent of association with PI3-kinase (Marin-Hincapie, 1999 and references therein).

Insulin receptor substrate (IRS) proteins are phosphorylated by multiple tyrosine kinases, including the insulin receptor. Phosphorylated IRS proteins bind to SH2 domain-containing proteins, thereby triggering downstream signaling pathways. The Drosophila insulin receptor (InR) C-terminal extension contains potential binding sites for signaling molecules, suggesting that InR might not require an IRS protein to accomplish its signaling functions. However, a cDNA encoding Drosophila IRS (Chico, but referred to in this study as dIRS) has been obtained and one for Chico in a Drosophila cell line has also been demonstrated. Like mammalian IRS proteins, the N-terminal portion of Chico contains a pleckstrin homology domain and a phosphotyrosine binding domain that binds to phosphotyrosine residues in both human and Drosophila insulin receptors. When coexpressed with Chico in COS-7 cells, a chimeric receptor (the extracellular domain of human IR fused to the cytoplasmic domain of InR) mediates the insulin-stimulated tyrosine phosphorylation of Chico. Mutating the juxtamembrane NPXY motif markedly reduces the ability of the receptor to phosphorylate Chico. In contrast, the NPXY motifs in the C-terminal extension of InR are required for stable association with Chico. Coimmunoprecipitation experiments demonstrate insulin-dependent binding of Chico to phosphatidylinositol 3-kinase and SHP2. However, interactions with Grb2, SHC, or phospholipase C-gamma were not detected. Taken together with published genetic studies, these biochemical data support the hypothesis that Chico functions directly downstream from the insulin receptor in Drosophila (Poltilove, 2000).

Quantitative proteomic analysis of protein complexes: concurrent identification of interactors and their state of phosphorylation

Protein complexes have largely been studied by immunoaffinity purification and (mass spectrometric) analysis. Although this approach has been widely and successfully used it is limited because it has difficulties reliably discriminating true from false protein complex components, identifying post-translational modifications, and detecting quantitative changes in complex composition or state of modification of complex components. A protocol has been developed that enables determination, in a single LC-MALDI-TOF/TOF analysis, the true protein constituents of a complex, to detect changes in the complex composition, and to localize phosphorylation sites and estimate their respective stoichiometry. The method is based on the combination of fourplex iTRAQ (isobaric tags for relative and absolute quantification) isobaric labeling and protein phosphatase treatment of substrates. It was evaluated on model peptides and proteins and on the complex Ccl1-Kin28-Tfb3 isolated by tandem affinity purification from yeast cells. The two known phosphosites in Kin28 and Tfb3 could be reproducibly shown to be fully modified. The protocol was then applied to the analysis of samples immunopurified from Drosophila melanogaster cells expressing an epitope-tagged form of the insulin receptor substrate homologue Chico. These experiments allowed identification 14-3-3ε;, 14-3-3zeta, and the insulin receptor as specific Chico interactors. In a further experiment, the immunopurified materials obtained from tagged Chico-expressing cells that were either treated with insulin or left unstimulated were cmpared. This analysis showed that hormone stimulation increases the association of 14-3-3 proteins with Chico and modulates several phosphorylation sites of the bait, some of which are located within predicted recognition motives of 14-3-3 proteins (Pflieger, 2008: Full text of article).

The two 14-3-3 proteins ε and zeta were identified as interactors of Chico, and their association appeared to increase upon insulin stimulation of cells. The mammalian homologues of Chico, IRS-1 as well as IRS-2 and IRS-4, were also shown to bind to 14-3-3 proteins. IRS-1 was proven to interact with 14-3-3β in 3T3L1 adipocytes, and this binding was shown to increase with insulin treatment. In contrast, another study did not observe a significant change of interaction between 14-3-3ε and IRS-1 upon hormonal stimulation in HepG2 cells; nevertheless this observation relied on Western blotting, which provides less accurate quantitative data than MS-based approaches and may not have been able to detect changes at or below 2-fold, such as those observed here using mass spectrometry techniques. In NIH-3T3 cells, 14-3-3ε was shown to interact with IRS-1 and protein kinase C-α, thus modulating insulin signaling and degradation. This study also observed an increased association of Chico and IR after a 7-min insulin treatment, which reflects activation of the insulin pathway involving tyrosine phosphorylation of Chico by IR (Pflieger, 2008).

Kc cells were stimulated with an insulin concentration and within a time window previously established to give a robust induction of the whole pathway. As a result, several insulin-dependent phosphosites, mainly phosphoserines, were identified, in Chico. The roles of phosphoserines/phosphothreonines in the mammalian homologue IRS-1 have been studied with regard to the regulation of the insulin pathway. Some serine residues, when phosphorylated, participate in the negative control of insulin signaling, whereas others appear to have a positive regulatory function. The homology of the Chico sequence to the mammalian IRS homologues is too weak to allow precise comparison of phosphosites. Nonetheless it is worth mentioning that some serine residues were shown previously to become partially or fully phosphorylated in rat and mouse IRS-1 after 5-min stimulation with 80-100 nM insulin, which is in agreement with the current observations. Among the phosphorylated residues identified in Chico, several appear to correlate with insulin stimulation either positively or negatively. Most interestingly, five sequences overlap with predicted recognition motives of 14-3-3 proteins. All but one of them were shown to become more highly phosphorylated upon stimulation, which correlates well with an enhanced association of the two 14-3-3 proteins with Chico. The differences of phosphorylation levels measured in samples Chico3 and Chico4 may be, at least in part, due to the different cell densities reached before induction. Despite differences in absolute phosphorylation levels, similar variations of the phosphorylation states (increase or decrease) were observed in the two samples upon insulin stimulation (Pflieger, 2008).

Phosphorylations on tyrosine residues were also expected at least upon insulin treatment. The presence of phosphotyrosine-containing peptides could not be conclusively established by the MS data. Nonetheless the intact protein Chico could be shown to contain phosphorylated tyrosines: a fraction of the samples Chico3 and Chico4 was analyzed by Western blot using an anti-phosphotyrosine antibody, and signal was detected in both insulin conditions with increased signal in the +INS case as expected (Pflieger, 2008).

SH2B regulation of growth, metabolism, and longevity in both insects and mammals

SH2B1 is a key regulator of body weight in mammals. This study identified dSH2B (Lnk) as the Drosophila homolog of SH2B1. dSH2B binds to Chico and directly promotes insulin-like signaling. Disruption of dSH2B decreases insulin-like signaling and somatic growth in flies. dSH2B deficiency also increases hemolymph carbohydrate levels, whole-body lipid levels, life span, and resistance to starvation and oxidative stress. Systemic overexpression of dSH2B results in opposite phenotypes. dSH2B overexpression in fat body decreases lipid and glucose levels, whereas neuron-specific overexpression of dSH2B decreases oxidative resistance and life span. Genetic deletion of SH2B1 also results in growth retardation, obesity, and type 2 diabetes in mice; surprisingly, life span and oxidative resistance are reduced in SH2B1 null mice. These data suggest that dSH2B regulation of insulin-like signaling, growth, and metabolism is conserved in SH2B1, whereas dSH2B regulation of oxidative stress and longevity may be conserved in other SH2B family members (Song, 2010).

SH2B1 has been a component of the IIS pathway in mice. The SH2B family members (SH2B1, 2, and 3) contain characteristic PH and SH2 domains; SH2B1 is believed to serve as an adaptor in cell signaling. It has been shown that genetic disruption of SH2B1 results in obesity and type 2 diabetes in mice. Neuron-specific restoration of SH2B1 fully rescues obesity and type 2 diabetes in SH2B1 null mice. Neuronal SH2B1 controls appetite, energy balance, and body weight at least in part by enhancing leptin sensitivity in the brain. Importantly, mutations in the SH2B1 loci link to obesity in humans. A chromosomal deletion of the SH2B1 loci cosegregates with early-onset severe obesity and insulin resistance in humans (Song, 2010).

The Drosophila genome contains a single dSH2B gene. This gene has evolved into three distinct genes (SH2B1, 2, and 3) in mammals. It is hypothesized that the core functions of dSH2B (e.g., growth, reproduction, and metabolism) are evolutionarily conserved; however, they are not equally distributed among the three SH2B family members. SH2B1, 2 and/or 3 may also evolve new functions in mammals (Song, 2010).

It has been reported that in mammals, SH2B1 binds to both the insulin receptor and IRS proteins. SH2B1 directly enhances insulin signaling by promoting insulin receptor phosphorylation of IRS proteins and by preventing dephosphorylation of IRS proteins. Genetic deletion of SH2B1 results in insulin resistance and type 2 diabetes in mice. This study shows that dSH2B binds to Chico and promoted insulin-stimulated phosphorylation of Chico, dAkt, and dFOXO. Disruption of dSH2B increases dILP resistance and hemolymph glucose in flies; conversely, dSH2B overexpression decreases dILP resistance and hemolymph glucose. dSH2B null flies are dwarf, and females were sterile. SH2B1 null mice also exhibit growth retardation. These data suggest that SH2B regulation of the IIS pathway, growth, glucose metabolism, and reproduction is largely conserved in SH2B1. Consistent with this idea, deletion of SH2B2 or SH2B3 does not alter growth and glucose metabolism in mice, (Song, 2010).

Werz has reported similar dwarf phenotypes in dSH2B null flies (Werz, 2009). It was proposed that dSH2B (dLnk) acts in parallel to Chico, because simultaneous disruption of both dSH2B and Chico are lethal. This study also observed a reduced survival rate, but not completely synthetic lethality, of Chico^C/C;dSH2B^D/D double mutant flies. Chico^C/C flies had the Chico hypomorphic but not null alleles, which may explain the discrepancy between these two studies. The Chico/dSH2B synthetic lethality is rescued by PTEN haploinsufficiency; dSH2B deficiency does not further inhibit growth, as revealed by similar body sizes between Chico and Chico/dSH2B double null animals. These results are consistent with the proposal that dSH2B and Chico may act in the same pathway(s) downstream of dInR. However, the results do not exclude the possibility that dSH2B may activate additional Chico-independent pathways (Song, 2010).

It was observed that disruption of dSH2B increased lipid levels and energy conservation in flies; conversely, dSH2B overexpression decreased energy conservation. Moreover, dSH2B overexpression in fat bodies but not neuronal tissues decreased lipid levels, hemolymph glucose, and energy conservation. These observations indicate that in insects, dSH2B in fat body plays a key role in regulating lipid metabolism and energy homeostasis (Song, 2010).

Deletion of SH2B1 but not the other SH2B family members results in obesity and type 2 diabetes in mice, suggesting that the metabolic functions of dSH2B are largely conserved in SH2B1. Moreover, mutations in the SH2B1 loci are genetically linked to obesity in humans. A rare chromosomal deletion of the SH2B1 loci cosegregates with early-onset severe obesity and insulin resistance in humans (Bochukova, 2010). Neuronal restoration of SH2B1 fully rescues the obesity and type 2 diabetes phenotypes in SH2B1 null mice, suggesting that SH2B1 in the central nervous system plays a dominant role in controlling energy homeostasis (Ren, 2007). Neuronal SH2B1 controls energy metabolism and body weight at least in part by promoting the anorexigenic response to leptin in the brain (Song, 2010).

The IIS system is conserved in Caenorhabditis elegans, Drosophila melanogaster, and mammals to regulate longevity. Given the fact that dSH2B promotes the activation of the IIS pathway, it is not surprising that disruption of dSH2B increased both oxidative resistance and life span in flies. Conversely, ubiquitous overexpression of dSH2B decreased oxidative resistance and longevity. In agreement with these observations, Slack have independently reported that dSH2B deficiency increases stress resistance and life span. dFOXO is a critical component of the IIS system. Loss of dFOXO reduces life span; conversely, dFOXO activation in the adult head fat body increases oxidative resistance and life span. However, neuronal dFOXO appears not to be involved in regulating longevity. It was observed in Drosophila that neuron-specific but not fat-body-specific overexpression of dSH2B decreased life span and oxidative resistance. These data suggest that dFOXO is unlikely to mediate dSH2B regulation of oxidative resistance and longevity. Moreover, dSH2B may also regulate life span by an additional IIS-independent mechanism (Song, 2010).

In contrast, deletion of SH2B1 reduced longevity and oxidative resistance in female mice in the absence of type 2 diabetes. The shortened life span cannot be explained by obesity and insulin resistance, because brain-specific deletion of IRS2 extends life span in the presence of life-long obesity and insulin resistance. A simple interpretation of these observations is that dSH2B regulation of oxidative resistance and longevity is conserved in other SH2B family members. SH2B3 is unlikely to regulate longevity because its expression is restricted to the immune system. SH2B2, which is expressed in multiple tissues, may act as dSH2B to regulate longevity in mammals. However, the possibility cannot be excluded that SH2B1 may regulate life span in a cell type-specific manner similar to dSH2B; however, systemic deletion of SH2B1 may cause an unknown pathological alteration that shortens the life span independently of aging in the mouse models. In agreement with this idea, systemic deletion of the insulin receptor results in neonatal death, whereas fat-specific deletion of the insulin receptor extends life span in mice (Song, 2010).

In summary, key functions of dSH2B (e.g., its regulation of the IIS pathway, growth, glucose metabolism, energy homeostasis, and reproduction) are conserved in SH2B1. While dSH2B in fat body plays a key role in regulating energy metabolism in flies, neuronal SH2B1 has evolved a more prominent role in controlling energy homeostasis and body weight in mammals. dSH2B, particularly neuronal dSH2B, negatively regulates longevity in flies; in contrast, SH2B1 deficiency shortens life span in mice. The other SH2B family members may regulate oxidative response and longevity in mammals (Song, 2010).

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

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).

Regulation of lifespan, metabolism, and stress responses by the Drosophila SH2B protein, Lnk

Drosophila Lnk is the single ancestral orthologue of a highly conserved family of structurally-related intracellular adaptor proteins, the SH2B proteins. As adaptors, they lack catalytic activity but contain several protein-protein interaction domains, thus playing a critical role in signal transduction from receptor tyrosine kinases to form protein networks. Physiological studies of SH2B function in mammals have produced conflicting data. However, a recent study in Drosophila has shown that Lnk is an important regulator of the insulin/insulin-like growth factor (IGF)-1 signaling (IIS) pathway during growth, functioning in parallel to the insulin receptor substrate, Chico. Since this pathway also has an evolutionary conserved role in the determination of organism lifespan, whether Lnk is required for normal lifespan in Drosophila was investigated. Phenotypic analysis of mutants for Lnk revealed that loss of Lnk function results in increased lifespan and improved survival under conditions of oxidative stress and starvation. Starvation resistance was found to be associated with increased metabolic stores of carbohydrates and lipids indicative of impaired metabolism. Biochemical and genetic data suggest that Lnk functions in both the IIS and Ras/Mitogen activated protein Kinase (MapK) signaling pathways. Microarray studies support this model, showing transcriptional feedback onto genes in both pathways as well as indicating global changes in both lipid and carbohydrate metabolism. Finally, these data also suggest that Lnk itself may be a direct target of the IIS responsive transcription factor, dFoxo, and that dFoxo may repress Lnk expression. Therefore this study describes novel functions for a member of the SH2B protein family and provide the first evidence for potential mechanisms of SH2B regulation. These findings suggest that IIS signaling in Drosophila may require the activity of a second intracellular adaptor, thereby yielding fundamental new insights into the functioning and role of the IIS pathway in ageing and metabolism.

Understanding of the physiological roles of the SH2B family of intracellular adaptors has been complicated by the presence of multiple family members in mammals. Furthermore, phenotypic analysis of genetic knockouts in mice has produced contradictory results. Recent genetic evidence has described a role for the single ancestral SH2B protein in Drosophila (Lnk) during IIS-mediated growth control. This study has characterised a critical role for Lnk in the regulation of lifespan, stress responses and cellular metabolism. The results support a model in which Lnk functions as an intracellular adaptor for transduction of the IIS and Ras/MapK signaling cascades to mediate these physiological processes (Slack, 2010).

The precise mechanisms whereby mammalian SH2B proteins transduce intracellular signaling from the insulin receptor remain unclear although like the IRS proteins, they have been shown to bind to multiple downstream mediators such as PI3K and Grb2. However, Drosophila Lnk lacks a consensus binding site for PI3K which is present in Chico so it is unlikely that they regulate similar downstream mechanisms (Slack, 2010).

The IIS pathway has an evolutionary conserved role in the determination of adult lifespan mediated by the Chico/PI3K/dFoxo branch of the IIS cascade. Previous studies have shown that flies either homozygous or heterozygous for chico¹, a strong loss-of-function allele of chico, show increased lifespan. This study has shown that Lnk homozygotes also show increased lifespan although no obvious effects on lifespan were observed in heterozygous animals. Interestingly, the effects of Lnk mutation on lifespan extension were similar in both males and females, which is uncommon in Drosophila, even for IIS mutants. This data therefore suggests that as during growth regulation, signaling via the activated dInR during lifespan determination may require a second intracellular adaptor in addition to the insulin receptor substrate, Chico, and provides the first evidence of a role for SH2B proteins in lifespan determination (Slack, 2010).

Lifespan extension in females was associated with reduced fecundity as a result of an arrest in oogenesis. However, there were no visible effects of Lnk mutation on male fertility as measured by offspring production. As male homozygous mutants were also long-lived, this suggests that the extended lifespan of Lnk mutant females is not simply due to reduced fecundity. Genetic knockouts of SH2B1 in mice also show infertility due to impaired signal transduction from the IGF-1 receptor resulting in poor gonad development. The sex-specific differences on fertility observed in Lnk mutants are probably due to sex-specific differences in Lnk transcript expression; microarray analyses of Drosophila gene expression has shown that Lnk transcripts are enriched within the female ovary but not in the male testis or accessory glands (Slack, 2010).

A comparison of the transcriptomes of Lnk mutant flies to controls revealed a number of gene expression changes associated with genes that encode components of the Drosophila IIS pathway. Hence, upregulation was observed of a number of factors that potentiate IIS such as the insulin-like ligands dilp2, dilp3, dilp5 and dilp6, as well as the insulin receptor substrate chico, the Drosophila class I PI3K, Dp110, phosphoinositide-dependent protein kinase PDK-1 and dAkt. In contrast, the expression of negative regulators of IIS such as the IGFBP-like ImpL2 and the PI3kinase inhibitor susi were downregulated. Several of these changes in expression were confirmed by qRT-PCR analysis and these data suggest that IIS transduction is affected by Lnk mutation, further strengthening the genetic evidence that Lnk is a component of the IIS pathway in flies. Transcriptional regulation downstream of IIS is in part mediated by the dFoxo transcription factor which is activated in response to low IIS by dAkt-mediated phosphorylation. While no differences were observed in dFoxo mRNA or protein levels in Lnk mutants compared to controls, a number of dFoxo target genes did show changes in expression. Thus, split-ends (CG18497), ches-1-like (CG12690), eIF-4E (CG4035) and CG9009 all showed upregulated expression in the microarray data set. Increased expression of two well-characterised dFoxo target genes, 4eBP and dInR, was observed by quantitative RT-PCR. Taken together, these data suggest that dFoxo activity may be increased in Lnk mutant animals (Slack, 2010).

Interestingly, a marked difference was observed in the magnitude of increased expression of both 4eBP and dInR between different body parts. Thus, for 4eBP a 1.1-fold increase was observed in expression in head RNA extracts compared to a 3.8-fold increase in RNA extracts from bodies. Similarly, for dInR, a 1.5-fold increase in expression was observed in head RNA extracts compared to a 2.6-fold increase in body RNA extracts. These data suggest that different tissues may exhibit differences in the magnitude of the transcriptional response to Lnk loss of function. Since the microarray experiments were performed on RNA isolated from adult heads only, this may explain why 4eBP and dInR were not identified in the microarray data set; microarray analysis of gene expression is generally regarded as less sensitive than qRT-PCR especially when changes in expression are small (Slack, 2010).

The observations that upstream components of the IIS pathway show transcriptional upregulation in response to Lnk loss of function suggest that transcriptional feedback back onto multiple components of the pathway may play an important regulatory role in IIS signal transduction. Previous studies have shown that dInR is itself a direct target of dFoxo so that when IIS levels are low, activated dFoxo increases dInR expression. In this study, it was shown that dFoxo also binds to the Lnk promoter in vivo suggesting that Lnk itself may be a direct target of dFoxo. dFoxo activity may also regulate transcription of IIS genes under basal conditions. Previous studies have shown that dFoxo is required for the basal expression of the dilp3 ligand. In the current study, it was found that in the absence of dFoxo, Lnk transcript expression increases suggesting that dFoxo activity is normally required for Lnk repression. Thus, regulation by dFoxo may involve both positive and negative effects on gene expression (Slack, 2010).

The microarray data set also contained a number of differentially expressed genes that function within the Ras/MapK signal transduction pathway. Previous studies have shown that the Ras binding domain of Drosophila PI3K is required for maximal PI3K activity during growth and female egg laying linking Ras/MapK and IIS during growth and development in Drosophila. Furthermore, this study has shown that RNAi-mediated knockdown of Lnk inhibits insulin-stimulated Erk phosphorylation in insect cells. The possibility cannot be excluded that Lnk may play an adaptor function for Ras signaling downstream of other RTKs in addition to the insulin receptor. However, it should be noted that Lnk RNAi knockdown has no effect on Spitz-stimulated Erk phosphorylation via activation of the Drosophila EGF receptor (Slack, 2010).

Despite their small body size, Lnk mutants contain elevated levels of both lipid and carbohydrate stores. Consistent with their increased metabolic stores, Lnk mutants also showed increased survival under starvation conditions. Transcriptome analysis revealed gene expression changes in a number of components of metabolic regulation in Lnk mutants compared to controls. Thus, reduced expression was observed of several enzymes that function in the glycolytic pathway and along with upregulation of genes that function in glycogen synthesis. In addition, several genes in the mitochondrial β-oxidation pathway were downregulated whereas genes involved in the regulation of lipid storage showed increased expression. Taken together, these changes in gene expression are consistent with an overall inhibition of catabolic processes and upregulation of pathways that regulate the synthesis and storage of carbohydrates and lipids (Slack, 2010).

Studies on the metabolic defects of SH2B knockouts in mice have proved inconsistent. One group has shown that genetic deletion of SH2B1 impairs adipogenesis by downregulating adipogenic gene expression including PPARγ resulting in mice with decreased fat mass. A Drosophila PPAR homolog has yet to identified but the closest Drosophila relative is the orphan receptor, E75. This gene was not among the differentially expressed gene list from the microarray data. Other studies have shown that SH2B1 null mice actually increase their body mass and develop obesity as a result of hyperphagia. In mammals, feeding is regulated by hypothalmic leptin signaling. Binding of leptin to its receptor results in receptor activation which in turn interacts with the non-receptor Janus kinase (Jak) stimulating downstream signaling events. Leptin stimulation of Jak is strongly potentiated by SH2B1 binding and so SH2B1 deletion impairs leptin signaling via Jak. This study did not observe any obvious differences in the feeding behaviour of Lnk mutant flies and there is no evidence to date that a leptin-like hormone exists in Drosophila. A functional Jak has been identified encoded by the hopscotch (hop) gene that has a well characterised role in hematopoesis in flies. No obvious hematopoetic defects were observed in Lnk mutants, and Lnk was not found to genetically interact with any of the core JAK/STAT pathway components. The data therefore suggests that the increased adiposity in Lnk mutant flies is unlikely to be mediated by increased feeding or by defects in Jak signaling. In fact, the data suggest that the ancestral function of Lnk in Drosophila is to regulate carbohydrate and fat storage by regulating gene expression of several key metabolic regulatory pathways (Slack, 2010).

In mammalian cells, SH2B proteins have been shown to have dual functions during insulin signaling transduction by both activating and inhibiting downstream intracellular signaling events. Phosphorylation of SH2B2 by the activated insulin receptor creates a binding site for the proto-oncogene product c-Cbl. This promotes the ubiquitination of tyrosine kinase receptors by functioning as a RING-type E2-dependent ubiquitin protein ligase facilitating either endocytosis or proteasomal degradation of the receptor. The c-Cbl binding motif is conserved in Drosophila Lnk and so it will be of interest to determine whether the interaction with c-Cbl is important for Lnk function especially during lifespan regulation (Slack, 2010).

Tissue-specific coupling between insulin/IGF and TORC1 signaling via PRAS40 in Drosophila

PRAS40 has recently been identified as a protein that couples insulin/IGF signaling (IIS) to TORC1 activation in cell culture; however, the physiological function of PRAS40 is not known. This study investigate flies lacking PRAS40 (FlyBase name: Lobe). Surprisingly, it was found, both biochemically and genetically, that PRAS40 couples IIS to TORC1 activation in a tissue-specific manner, regulating TORC1 activity in ovaries but not in other tissues of the animal. PRAS40 thereby regulates fertility but not growth of the fly, allowing distinct physiological functions of TORC1 to be uncoupled. The main function of PRAS40 in vivo is to regulate TORC1 activity, and not to act as a downstream target and effector of TORC1. Finally, this work sheds some light on the question of whether TORC1 activity is coupled to IIS in vivo (Pallares-Cartes, 2012).

PRAS40 has been proposed to link IIS to TORC1 in cell culture. Two reports showed that PRAS40 binds the TORC1 complex thereby inhibiting its activity, and that phosphorylation of PRAS40 by Akt relieves this inhibition (Nascimento, 2010; Sancak, 2007; Vander Haar, 2007). Other studies, however, identified PRAS40 as a TORC1 substrate, suggesting that the apparent inhibitory effects of PRAS40 on the canonical TORC1 substrates 4EBP and S6K may reflect competition for substrate binding. This would place PRAS40 downstream, rather than upstream of TORC1. Indeed, as these studies point out, PRAS40 might function concomitantly as a TORC1 substrate and a TORC1 regulator, regulating mTORC1 activity via direct inhibition of substrate binding. These studies have led to several open questions: (1) does PRAS40 regulate TORC1 activity in vivo, as it does in cell culture? (2) does PRAS40 link IIS to TOR activation in vivo? and (3) is the main function of PRAS40 to act as a TOR substrate or as a TOR regulator? These two options can be distinguished in an animal context. If the main function of PRAS40 is to regulate TORC1 activity (i.e., it is genetically upstream of TORC1), then PRAS40 mutant phenotypes should be rescued by reducing activity of TORC1 or of a TORC1 target other than PRAS40. If, instead, PRAS40 functions mainly as a TOR substrate downstream of TORC1, then loss of PRAS40 cannot be rescued by manipulating TORC1. No animal models for PRAS40 loss of function have yet been reported to address these questions (Pallares-Cartes, 2012).

One physiological function of IIS and TORC1 of particular relevance to this present study is the regulation of fertility. In Drosophila, insulin-like peptides (DILPs) secreted by neurosecretory cells regulate the rate of germline stem cell division in the ovary. This links metabolic status to fertility, so that rich nutrient conditions cause high DILP secretion, leading to increased egg production. If IIS is abrogated in the ovary, as in the case of chico or InR mutants, egg production is completely blocked and the animals are sterile. The defect in chico mutant ovaries is ovary-autonomous because transplantation of chico mutant ovaries into wild-type hosts, containing normal levels of DILPS, does not rescue their defects. At the cellular level, IIS and TORC1 regulate almost all aspects of oogenesis including the rate of proliferation of ovarian somatic and germline cells, germline stem cell maintenance, vitellogenesis, and oocyte loss. Interestingly, the roles of IIS and TORC1 in regulating fertility are highly conserved throughout evolution, regulating similar processes in Caenorhabditis elegans and in mammals. As in flies, reduction of IIS via knockout of IGF-1 or IRS-2 causes infertility in mice. As in flies, normal TORC1 in mice prevents oocyte loss (Thomson, 2010) and hyperactivation of IIS or TORC1 leads to premature activation of all primordial follicles, resulting in premature follicular depletion (Reddy, 2010; Sun, 2010). In sum, IIS and TORC1 play critical roles in regulating fertility in an evolutionarily conserved manner (Pallares-Cartes, 2012).

This study presents a PRAS40 loss-of-function animal model. By generating PRAS40 knockout Drosophila, the in vivo function of PRAS40, as well as the connection between IIS and TORC1, were studied. PRAS40 is shown function to link IIS to TORC1 in the animal. Unexpectedly, however, it does so in a tissue-specific manner, influencing TORC1 activity predominantly in the fly ovary, but not in other tissues of the animal. As a result, PRAS40 regulates development of the ovary, but not growth or proliferation of somatic tissues, thereby influencing animal fertility but not animal growth. Because PRAS40 is present in all tissues of the fly, this indicates PRAS40 is a link between IIS and TORC1 that can be switched on and off in a tissue-specific manner. Furthermore, PRAS40 knockout phenotypes can be rescued by inhibiting TORC1 or by reducing S6K gene dosage, indicating that PRAS40 functions mainly as a TORC1 inhibitor in vivo. Finally, this work sheds light on the conundrum whether the IIS and TORC1 signaling pathways are linked under normal physiological conditions, showing that they are indeed linked, but only in particular tissues (Pallares-Cartes, 2012).

Both biochemically and genetically this study found that PRAS40 and IIS do not affect TORC1 activity in most tissues during growth of the fly. Removal of PRAS40 does not cause elevated TORC1 activity in larvae and, in agreement with previous studies, removal of chico does not lead to reduced TORC1 activity in the adult body or in larvae. Removal of PRAS40 does not cause any size abnormalities in the fly, which is a very sensitive readout for TORC1 activity during development. It was surprising to find, however, that in ovaries both IIS and PRAS40 do affect TORC1 activity. TORC1 activity drops in ovaries of chico⁻ animals, and increases in ovaries of PRAS40⁻ animals. Furthermore, in ovaries, PRAS40 links IIS to TORC1 in that removal of both chico and PRAS40 leads to renormalized TORC1 activity. These biochemical data are reflected by genetic epistasis data. Chico mutant flies are completely infertile, laying no eggs, and this phenotype is rescued by removal of PRAS40. These data indicate that under normal physiological conditions, IIS activates TORC1 in a tissue-specific manner (Pallares-Cartes, 2012).

Does PRAS40 also link IIS to TORC1 in the male germline? The fact that mutation of PRAS40 rescues the infertility of PDK1^4/5 mutant males, and that PRAS40 mutant testes are larger than control testes suggests that it does. PRAS40⁻, chico⁻ mutant testes also appear mildly increased in size compared to chico⁻ mutant testes, however, the result is not as clear cut as with ovaries, because chico mutant females are completely sterile whereas chico mutant males have only mildly reduced fertility. Further work will be required to look at this carefully (Pallares-Cartes, 2012).

All these data, indicating an ovary-specific link between IIS and TORC1 result from manipulations within physiological range. In contrast, overexpression of PRAS40 does cause reduced tissue growth as well as reduced TORC1 activity, indicating that PRAS40 can inhibit TORC1 in most tissues when overexpressed. Furthermore, in contrast to the tissue-specific link between IIS and TORC1 under normal physiological conditions, it was also observed that hyper-stimulation of IIS above physiological range does activate TORC1 in most tissues, for instance in tissue explants treated with insulin, or in animals overexpressing activated PI3K (Dp110-CAAX). This mechanism might be relevant for pathophysiological conditions with elevated IIS, such as in cancer cells. This may occur via elevated ATP production in the cell, inhibiting AMPK, because this activation was also observed in tissues simultaneously lacking PRAS40 and all Akt phosphorylation sites on Tsc1 and Tsc2 (Pallares-Cartes, 2012).

One open question is whether the main function of PRAS40 is to regulate TORC1 activity or whether it functions mainly as a downstream target and effector of TORC1. The data suggest the former is the case. If PRAS40 had effector functions downstream of TORC1, these functions would not be rescued by additional removal of other TORC1 substrates such as S6K. Instead, it was found that the elevated fertility of PRAS40 mutants is rescued by removal of one copy of S6K, suggesting that the phenotype found in PRAS40 mutants is due to elevated S6K activity (Pallares-Cartes, 2012).

Why does PRAS40 regulate TORC1 activity in ovaries but not in other tissues of the animal? PRAS40 is expressed in all tissues that were tested. Therefore, the fact that removal of PRAS40 from larval tissues, for instance, has no effect on TORC1 activity must mean that larval PRAS40 protein is inactive. Data is presented suggesting that the state of phosphorylation of PRAS40 may be different in larval tissues compared to ovaries, providing a possible explanation for this inactivation. To date, a number of phosphorylations on PRAS40 have been reported, all of which are inhibitory in terms of TORC1 binding. These include phosphorylations by Akt, TORC1 itself, PIM1, and PKA. Intriguingly, this correlates with the observation that PRAS40 is highly phosphorylated in many cancers and that PRAS40 phosphorylation correlates with bad prognosis. The possibility is favored that PRAS40 phosphorylation on an inhibitory site could be regulated by a kinase that is absent in ovaries, or a phosphatase that is enriched in ovaries compared to other tissues. Future studies will shed light on this issue (Pallares-Cartes, 2012).

TORC1 has multiple physiological roles in various tissues. In Drosophila, TORC1 in the growing larva regulates both growth and metabolism of the animal whereas in the adult fly, it regulates mainly metabolic parameters. TORC1 in ovaries regulates fertility of the animal, whereas in the nervous system it regulates dendritic tiling. Therefore, unless TORC1 activity can be differentially regulated in various tissues, all these physiological functions would have to be controlled in a correlated fashion. Tissue-specific differential regulation of PRAS40 presents a mechanism that allows TORC1 activity to be uncoupled in a tissue-specific manner (Pallares-Cartes, 2012).

Insulin signaling mediates sexual attractiveness in Drosophila

Sexually attractive characteristics are often thought to reflect an individual's condition or reproductive potential, but the underlying molecular mechanisms through which they do so are generally unknown. Insulin/insulin-like growth factor signaling (IIS) is known to modulate aging, reproduction, and stress resistance in several species and to contribute to variability of these traits in natural populations. This study shows that IIS determines sexual attractiveness in Drosophila through transcriptional regulation of genes involved in the production of cuticular hydrocarbons (CHC), many of which function as pheromones. Using traditional gas chromatography/mass spectrometry (GC/MS) together with newly introduced laser desorption/ionization orthogonal time-of-flight mass spectrometry (LDI-MS), it was established that CHC profiles are significantly affected by genetic manipulations that target IIS. Manipulations that reduce IIS also reduce attractiveness, while females with increased IIS are significantly more attractive than wild-type animals. IIS effects on attractiveness are mediated by changes in CHC profiles. Insulin signaling influences CHC through pathways that are likely independent of dFOXO and that may involve the nutrient-sensing Target of Rapamycin (TOR) pathway. These results suggest that the activity of conserved molecular regulators of longevity and reproductive output may manifest in different species as external characteristics that are perceived as honest indicators of fitness potential (Kuo, 2012).

The Ras-Erk-ETS-signaling pathway is a drug target for longevity

Identifying the molecular mechanisms that underlie aging and their pharmacological manipulation are key aims for improving lifelong human health. This study has identified a critical role for Ras-Erk-ETS signaling in aging in Drosophila. Inhibition of Ras was shown to be sufficient for lifespan extension downstream of reduced insulin/IGF-1 (IIS) signaling. Moreover, direct reduction of Ras or Erk activity leads to increased lifespan. ETS transcriptional repressor Anterior open (Aop) was identified as central to lifespan extension caused by reduced IIS or Ras attenuation. Importantly, it was demonstrates that adult-onset administration of the drug trametinib, a highly specific inhibitor of Ras-Erk-ETS signaling, can extend lifespan. This discovery of the Ras-Erk-ETS pathway as a pharmacological target for animal aging, together with the high degree of evolutionary conservation of the pathway, suggests that inhibition of Ras-Erk-ETS signaling may provide an effective target for anti-aging interventions in mammals (Slack, 2015).

The key role of IIS in determining animal lifespan has been well appreciated for more than two decades and shows strong evolutionary conservation. Alleles of genes encoding components of this pathway have also been linked to longevity in humans. Multiple studies have demonstrated the importance of the PI3K-Akt-Foxo branch of IIS, while this study has identified an equally important role for Ras-Erk-ETS signaling in IIS-dependent lifespan extension (Slack, 2015).

Downstream of chico, preventing the activation of either Ras or PI3K is sufficient to extend lifespan. Ras can interact directly with the catalytic subunit of PI3K, which is required for maximal PI3K activation during growth. Thus, inhibition of Ras could increase lifespan via inactivation of PI3K. However, several lines of evidence indicate that the Erk-ETS pathway must also, if not solely, be involved. In this study and elsewhere, it has been demonstrated that direct inhibition of the Ras-dependent kinase, Erk, or activation of the Aop transcription factor, a negative effector of the Ras-Erk pathway, is sufficient to extend lifespan. Importantly, this study shows that Ras-Erk-ETS signaling is genetically linked to chico because activation of Aop is required for lifespan extension due to chico loss of function. Furthermore, altering the ability of Chico to activate Ras or PI3K does not result in equivalent phenotypes: it has been shown that mutation of the Grb2/Drk docking site in Chico is dispensable for multiple developmental phenotypes associated with chico mutation, while disruption of the Chico-PI3K interaction is not. Overall, the observations strongly suggest that lifespan extension downstream of chicomutation involves inhibition of the Ras-Erk-ETS-signaling pathway (Slack, 2015).

A simple model integrates the role of Ras-Erk-ETS signaling with the PI3K-Akt-Foxo branch in extension of lifespan by reduced IIS. It is proposed that, downstream of Chico, the IIS pathway bifurcates into branches delineated by Erk and Akt, with inhibition of either sufficient to extend lifespan, as is activation of either responsive TF, Aop or Foxo. The two branches are not redundant, because mutation of chico or the loss of its ability to activate either branch results in the same magnitude of lifespan extension. Furthermore, Aop and Foxo are each individually required downstream of chico mutation for lifespan extension. At the same time, the effects of the two branches are not additive, as simultaneous activation of Aop and Foxo does not extend lifespan more than activation of either TF alone. Taken together, these data suggest that the two pathways re-join for transcriptional regulation, where Aop and Foxo co-operatively regulate genes required for lifespan extension. The model is corroborated by a previous finding that, in the adult gut and fat body, some 60% of genomic locations bound by Foxo overlap with regions of activated-Aop binding (Alic, 2014; Slack, 2015).

It is proposed that functional interactions of Aop and Foxo at these sites may be such that each factor is both necessary and sufficient to achieve the beneficial changes in target gene expression upon reduced IIS. It remains to be determined how promoter-based Foxo and Aop interactions produce such physiologically relevant, transcriptional changes. It is, however, curious that activation of either TF alone promotes longevity when one is known as a transcriptional activator (Foxo) and the other as a transcriptional repressor (Aop). A subset of Foxo-bound genes, albeit a minority, has been consistently observed that are transcriptionally repressed when Foxo is activated (Alic, 2014). Furthermore, the Foxo target gene myc is downregulated in larval muscle when Foxo is active under low insulin conditions, while deletion of foxo or its binding site within the myc promoter results in de-repression of myc expression in adipose of fed larvae (Teleman, 2008). Thus, on some promoters under certain conditions, Drosophila Foxo appears to act as a transcriptional repressor. Mammalian Foxo3a may also directly repress some genes. It will therefore be important to test whether the lifespan-relevant interactions between Foxo and Aop occur on promoters where Foxo acts as a repressor with Foxo possibly acting as a cofactor for Aop or vice versa (Slack, 2015).

In mediating the effects of IIS on lifespan, the Ras-Erk-ETS- and PI3K-Akt-Foxo-signaling pathways both appear to inhibit Aop/Foxo. To understand why signaling might be so wired, it is important to consider that the two pathways are also regulated by other stimuli, such as other growth factors, stress signals, and nutritional cues. The re-joining of the two branches at the transcriptional level would therefore allow for their outputs to be integrated, producing a concerted transcriptional response, a feature that is also seen in other contexts. For example, stability of the Myc transcription factor is differentially regulated in response to Erk and PI3K signals, allowing it to integrate signals from the two kinases. Transcriptional integration in response to RTK signaling also confers specificity during cell differentiation, with combinatorial effects of multiple transcriptional modulators inducing tissue-specific responses to inductive Ras signals. Similar integrated responses of lifespan could be orchestrated by transcriptional coordination of Aop and Foxo (Slack, 2015).

Direct inhibition of Ras in Drosophila can extend lifespan, suggesting that the role of Ras in aging is evolutionarily conserved. In budding yeast, deletion of RAS1 extends replicative lifespan, and deletion of RAS2 increases chronological lifespan by altering signaling through cyclic-AMP/protein kinase A (cAMP/PKA), downregulation of which is sufficient to extend both replicative and chronological lifespan. This role of cAMP/PKA in aging may be conserved in mammals, as disruption of adenylyl cyclase 5' and PKA function extend murine lifespan. However, cAMP/PKA are not generally considered mediators of Ras function in metazoa. Instead, the data suggest that signaling through Erk and the ETS TFs mediates the longevity response to Ras. Interestingly, fibroblasts isolated from long-lived mutant strains of mice and long-lived species of mammals and birds show altered dynamics of Erk phosphorylation in response to stress, further suggesting a link between Erk activity and longevity. Importantly, the ETS TFs are conserved mediators of Ras-Erk signaling in mammals. Investigation of the effects of Ras inhibition on mammalian lifespan and the role of the mammalian Aop ortholog Etv6 are now warranted (Slack, 2015).

A role for Ras-Erk-ETS signaling in lifespan offers multiple potential targets for small-molecule inhibitors that could function as anti-aging interventions. Importantly, due to the key role of this pathway in cancer, multiple such inhibitors exist or are in development (Slack, 2015).

This study has shown that trametinib, a highly specific allosteric inhibitor of the Mek kinase, prolongs Drosophila lifespan, thus validating the Ras-Erk-ETS pathway as a pharmacological target for anti-aging therapeutics. Trametinib joins a very exclusive list of FDA-approved drugs that promote longevity in animals, the most convincing other example being rapamycin (Slack, 2015).

Rapamycin not only increases lifespan in multiple organisms, including mammals, but also improves several indices of function during aging (Ehninger, 2014; Lamming, 2013). While rapamycin can protect against tumor growth, the effects on longevity appear to be independent of cancer prevention, as rapamcyin-treated animals still develop tumors and rapamycin can increase lifespan in tumor-free species. Furthermore, increased activity of certain tumor suppressors such as lnk4a/Arf and PTEN as well as the RasGrf1 deficiency all increase lifespan independently of anti-tumor activity. The findings that trametinib can increase lifespan inDrosophila, which are mainly post-mitotic in adulthood, and that doses of trametinib that increase lifespan do not alter proliferation rates of ISCs inDrosophila suggest that the anti-aging effects of trametinib are separable from its anti-cancer activity (Slack, 2015).

Finally, due to the high degree of evolutionary conservation in the Ras-Erk-ETS pathway, this study suggests the intriguing possibility that pharmacological inhibition of Ras-Erk-ETS may also increase lifespan in mammal (Slack, 2015).

Drosophila longevity assurance conferred by reduced insulin receptor substrate Chico partially requires d4eBP

Mutations of the insulin/IGF signaling (IIS) pathway extend Drosophila lifespan. Based on genetic epistasis analyses, this longevity assurance is attributed to downstream effects of the FOXO transcription factor. However, as reported FOXO accounts for only a portion of the observed longevity benefit. One candidate is target of rapamycin complex 1 (TORC1). Reduced TORC1 activity is reported to slow aging, whereas reduced IIS is reported to repress TORC1 activity. The eukaryotic translation initiation factor 4E binding protein (4E-BP) is repressed by TORC1, and activated 4E-BP is reported to increase Drosophila lifespan. This study uses genetic epistasis analyses to test whether longevity assurance mutants of chico, the Drosophila insulin receptor substrate homolog, require Drosophila d4eBP to slow aging. In chico heterozygotes, which are robustly long-lived, d4eBP is required but not sufficient to slow aging. Remarkably, d4eBP is not required or sufficient for chico homozygotes to extend longevity. Likewise, chico heterozygote females partially require d4eBP to preserve age-dependent locomotion, and both chico genotypes require d4eBP to improve stress-resistance. It thus appears that altered IRS function within the IIS pathway of Drosophila appears to have partial, conditional capacity to regulate aging through an unconventional interaction with 4E-BP (Bai, 2015).

EVOLUTIONARY HOMOLOGS

The IRS-signaling system during insulin and cytokine action

The discovery of the first intracellular substrate for insulin, IRS-1, has redirected the field of diabetes research and led to many important advances in the understanding of insulin action. Detailed analysis of IRS-1 demonstrates structure/function relationships for this modular docking molecule, including mechanisms of substrate recognition and signal propagation. Recent work has also identified other structurally similar molecules, including IRS-2, the Drosophila protein DOS, and the Grb2-binding protein Gab1, suggesting that this intracellular signaling strategy is conserved evolutionarily and is utilized by an expanding number of receptor systems. In fact, IRS-1 itself has been shown to be important in other growth factor and cytokine signaling systems, including growth hormone and several interleukins. Analysis of mice lacking IRS-1 confirms an important physiological role for this protein in glucose metabolism and general cell growth in the intact animal. Disregulation of the signaling pathways integrated by the IRS proteins may contribute to the pathophysiology of non-insulin-dependent diabetes mellitus or other diseases (Yenush, 1997).

Structure of the insulin receptor substrate IRS-1 defines a unique signal transduction protein

Since the discovery of insulin in 1921, there has been no problem more fundamental to diabetes research than understanding how insulin works at the cellular level. Insulin binds to the alpha subunit of the insulin receptor, which activates the tyrosine kinase in the beta subunit, but the molecular events linking the receptor kinase to insulin-sensitive enzymes and transport processes are unknown. The discovery that insulin stimulates tyrosine phosphorylation of a protein with a relative molecular mass between 165,000 and 185,000 kDa, collectively called pp185, shows that the insulin receptor kinase has specific cellular substrates. The pp185 is a minor cytoplasmic phosphoprotein found in most cells and tissues; its phosphorylation is decreased in cells expressing mutant receptors defective in signaling. IRS-1, which encodes a component of the pp185 band, has now been cloned. IRS-1 contains over ten potential tyrosine phosphorylation sites, six of which are in Tyr-Met-X-Met motifs. During insulin stimulation, the IRS-1 protein undergoes tyrosine phosphorylation and binds phosphatidylinositol 3-kinase, suggesting that IRS-1 acts as a multisite 'docking' protein to bind signal-transducing molecules containing Src-homology 2 and Src-homology-3 domains. Thus IRS-1 may link the insulin receptor kinase and enzymes regulating cellular growth and metabolism (Sun, 1991).

4PS/insulin receptor substrate (IRS)-2 is the alternative substrate of the insulin receptor in IRS-1-deficient mice

Insulin receptor substrate-1 (IRS-1) is the major cytoplasmic substrate of the insulin and insulin-like growth factor (IGF)-1 receptors. Transgenic mice lacking IRS-1 are resistant to insulin and IGF-1, but exhibit significant residual insulin action that corresponds to the presence of an alternative high molecular weight substrate in liver and muscle. 4PS, the major substrate of the IL-4 receptor-associated tyrosine kinase in myeloid cells, has significant structural similarity to IRS-1. To determine if 4PS is the alternative substrate of the insulin receptor in IRS-1-deficient mice, immunoprecipitation, immunoblotting, and phosphatidylinositol (PI) 3-kinase assays were performed using specific antibodies to 4PS. Following insulin stimulation, 4PS is rapidly phosphorylated in liver and muscle; it binds to the p85 subunit of PI 3-kinase, and activates the enzyme. Insulin stimulation also results in the association of 4PS with Grb 2 in both liver and muscle. In IRS-1-deficient mice, both the phosphorylation of 4PS and associated PI 3-kinase activity are enhanced, without an increase in protein expression. Immunodepletion of 4PS from liver and muscle homogenates removes most of the phosphotyrosine-associated PI 3-kinase activity in IRS-1-deficient mice. Thus, 4PS is the primary alternative substrate, i.e. IRS-2, which plays a major role in physiologic insulin signal transduction via both PI 3-kinase activation and Grb 2/Sos association. In IRS-1-deficient mice, 4PS/IRS-2 provides signal transduction to these two major pathways of insulin signaling (Patti, 1995).

Phosphotyrosine binding domains of Shc and insulin receptor substrate 1 recognize the NPXpY motif in a thermodynamically distinct manner

Phosphotyrosine binding (PTB) domains of the adaptor protein Shc and insulin receptor substrate (IRS-1) interact with a distinct set of activated and tyrosine-phosphorylated cytokine and growth factor receptors and play important roles in mediating mitogenic signal transduction. By using the technique of isothermal titration calorimetry, the thermodynamics of binding of the Shc and IRS-1 PTB domains to tyrosine-phosphorylated NPXY-containing peptides derived from known receptor binding sites were determined. The results show that relative contributions of enthalpy and entropy to the free energy of binding are dependent on specific phosphopeptides. Binding of the Shc PTB domain to tyrosine-phosphorylated peptides from TrkA, epidermal growth factor, ErbB3, and insulin receptors is achieved via an overall entropy-driven reaction. In contrast, the recognition of the phosphopeptides of insulin and interleukin-4 receptors by the IRS-1 PTB domain is predominantly an enthalpy-driven process. Mutagenesis and amino acid substitution experiments show that in addition to the tyrosine-phosphorylated NPXY motif, the PTB domains of Shc and IRS-1 prefer a large hydrophobic residue at pY-5 and a small hydrophobic residue at pY-1, respectively (where pY is phosphotyrosine). These results agree with the calculated solvent accessibility of these two key peptide residues in the PTB domain/peptide structures and support the notion that the PTB domains of Shc and IRS-1 employ functionally distinct mechanisms to recognize tyrosine-phosphorylated receptors (Farooq, 1999).

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

Multipin peptide synthesis has been employed to produce biotinylated 11-mer phosphopeptides that account for every tyrosine residue in insulin receptor substrate-1 (IRS-1) and the cytoplasmic domains of the insulin-, epidermal growth factor-, platelet-derived growth factor- and basic fibroblast growth factor receptors. These phosphopeptides have been screened for their capacity to bind to the SH2 domains of Shc and Grb in a solution phase enzyme-linked immunosorbent assay. The data reveal new potential Grb2 binding sites at Tyr-1114 [epidermal growth factor receptor (EGFR) C-tail]; Tyr-743 [platelet-derived growth factor receptor (PDGFR) insert region], Tyr-1110 from the E-helix of the catalytic domain of insulin receptor (IR), and Tyr-47, Tyr-939, and Tyr-727 in IRS-1. None of the phosphopeptides from the juxtamembrane or C-tail regions of IR bind Grb2 significantly, and only one phosphopeptide from the basic fibroblast growth factor receptor (Tyr-556) binds Grb2 but with medium strength. Tyr-1068 and -1086 from the C-tail of EGFR, Tyr-684 from the kinase insert region of PDGFR, and Tyr-895 from IRS-1 were confirmed as major binding sites for the Grb2 SH2 domain. With regard to Shc binding, the data reveal new potential binding sites at Tyr-703 and Tyr-789 from the catalytic domain of EGFR and at Tyr-557 in the juxtamembrane region of PDGFR. The data also identify new potential Shc binding sites at Tyr-764, in the C-tail of basic fibroblast growth factor receptor, and Tyr-960, in the juxtamembrane of IR, a residue previously known to be required for Shc phosphorylation in response to insulin. The study confirms the previous identification of Tyr-992 and Tyr-1173 in the C-tail of EGFR and several phosphopeptides from the PDGFR as medium strength binding sites for the SH2 domain of Shc. None of the 34 phosphopeptides from IRS-1 bind Shc strongly, although Tyr-690 shows medium strength binding. The specificity characteristics of the SH2 domains of Grb2 and Shc are discussed. This systematic peptide mapping strategy provides a way of rapidly scanning candidate proteins for potential SH2 binding sites as a first step to establishing their involvement in kinase-mediated signaling pathways (Ward, 1996).

The pleckstrin homology domain is the principal link between the insulin receptor and IRS-1

Interaction domains located in the NH2 terminus of IRS-1 mediate its recognition by the insulin receptor. Alignment of IRS-1 and IRS-2 reveals two homology regions: the IH1(PH) contains a pleckstrin homology (PH) domain, and the IH2(PTB) contains a phosphotyrosine binding (PTB) domain. A third region in IRS-1 called SAIN has been proposed to contain another functional PTB domain. Peptide competition experiments demonstrate that the IH2(PTB) in IRS-2, like the corresponding domain in IRS-1, binds directly to peptides containing NPXY motifs. In contrast, these peptides do not bind to IH1(PH) or the SAIN regions. In 32D cells the IH1(PH) is essential for insulin-stimulated tyrosine phosphorylation of IRS-1 and insulin-stimulated phosphatidylinositol 3-kinase activity and p70(s6k: see Drosophila RPS6-p70-protein kinase) phosphorylation. In contrast, the IH2(PTB) and the SAIN regions are not required for these insulin actions; however, the IH2(PTB) improves the coupling between IRS-1 and the insulin receptor. Overexpression of the insulin receptor in 32DIR cells increases IRS-1 tyrosine phosphorylation and mediates insulin-stimulated DNA synthesis. The sensitivity of these responses is partially reduced by deletion of either the IH1(PH) or the IH2(PTB) and significantly reduced when both regions are deleted. Thus, the PH and PTB domains equally couple IRS-1 to high levels of insulin receptor normally expressed in most cells, whereas at low levels of insulin receptors the PTB domain is inefficient and the PH domain is essential for a productive interaction (Yenush, 1996b).

Different subcellular distribution and regulation of expression of insulin receptor substrate (IRS)-3 from those of IRS-1 and IRS-2

Adipocytes contain three major substrate proteins of the insulin receptor, termed IRS-1, IRS-2, and IRS-3. IRS-1 and IRS-2 are located mainly in the low density microsome (LDM) fraction and are tyrosine phosphorylated in response to insulin stimulation, leading to phosphatidylinositol (PI) 3-kinase activation. In contrast, IRS-3 is located mainly in the plasma membrane (PM) fraction and contributes to PI 3-kinase activation in the PM fraction. The different cellular localizations of IRS proteins may account for the mechanism of insulin resistance induced by a high fat diet, considering that PI 3-kinase activation in the LDM fraction is reportedly essential for the translocation of GLUT4 in adipocytes. High fat feeding in rats increases both protein and mRNA levels of IRS-3 but decreases those of IRS-1 and IRS-2 in epididymal adipocytes. As a result, selective impairment of insulin-induced PI 3-kinase activation is observed in the LDM fraction, whereas PI 3-kinase activation is conserved in the PM fraction. This is the first report showing that different IRS proteins function in different subcellular compartments, which may contribute to determining the insulin sensitivity in adipocytes (Anai, 1998).

Insulin regulation of membrane-associated insulin receptor substrate 1

Insulin stimulation of 3T3-L1 adipocytes results in rapid activation of the insulin receptor tyrosine kinase followed by autophosphorylation of the receptor and phosphorylation of insulin receptor substrate 1 (IRS-1), its major substrate. The insulin receptor resides mostly at the cell surface of 3T3-L1 adipocytes under basal conditions, while about two-thirds of IRS-1 fractionates with intracellular membranes and one-third fractionates with cytosol. To test whether insulin receptor internalization is required for optimal tyrosine phosphorylation of IRS-1, 3T3-L1 adipocytes and CHO-T cells were incubated at 4 degrees C: this inhibits receptor endocytosis but not its tyrosine kinase activity. Under these conditions, tyrosine phosphorylation of IRS-1 in the low density microsome fraction in response to insulin is as intense as that observed at 37 degrees C, indicating that endocytosis of insulin receptors is not necessary for tyrosine phosphorylation of IRS-1 to occur. Surprisingly, at 37 degrees C, insulin action on 3T3-L1 adipocytes progressively decreases the amount of IRS-1 protein associated with the low density microsome fraction and increases that in the cytosol. This redistribution of IRS-1 from the low density microsome fraction to the cytosol in response to insulin is accompanied by decreased electrophoretic mobility of IRS-1 on SDS-polyacrylamide gel electrophoresis. Incubation of adipocytes at 4 degrees C blocks the appearance of tyrosine-phosphorylated IRS-1 in the cytosol. Taken together, these data indicate that insulin receptors phosphorylate IRS-1 at the cell surface, perhaps in coated pits that are included in the low density microsome fraction. The results also suggest a desensitization mechanism in which the tyrosine-phosphorylated membrane-bound IRS-1, associated with signaling molecules such as phosphatidylinositol 3-kinase, is released into the cytoplasm in concert with its serine/threonine phosphorylation (Heller-Harrison, 1995).

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

CSF-1 is equipotent with insulin in its ability to stimulate 2-[3H]deoxyglucose uptake in 3T3-L1 adipocytes expressing the colony stimulating factor-1 receptor/insulin receptor chimera (CSF1R/IR). However, CSF-1-stimulated glucose uptake and glycogen synthesis is reduced by 50% in comparison to insulin in 3T3-L1 cells expressing a CSF1R/IR mutated at Tyr960 (CSF1R/IRA960). CSF-1-treated adipocytes expressing the CSF1R/IRA960 are impaired in their ability to phosphorylate insulin receptor substrate 1 (IRS-1) but not in their ability to phosphorylate IRS-2. Immunoprecipitation of IRS proteins followed by Western blotting reveals that the intact CSF1R/IR co-precipitates with IRS-2 from CSF-1-treated cells. In contrast, the CSF1R/IRA960 co-precipitates poorly with IRS-2. These observations suggest that Tyr960 is important for interaction of the insulin receptor cytoplasmic domain with IRS-2, but it is not essential to the ability of the insulin receptor tyrosine kinase to use IRS-2 as a substrate. These observations also suggest that in 3T3-L1 adipocytes, tyrosine phosphorylation of IRS-2 by the insulin receptor tyrosine kinase is not sufficient for maximal stimulation of receptor-regulated glucose transport or glycogen synthesis (Chaika, 1999).

Differential role of insulin receptor substrate (IRS)-1 and IRS-2 in L6 skeletal muscle cells expressing the Arg1152 --> Gln insulin receptor

In L6 muscle cells expressing the Arg1152 --> Gln insulin receptor (Mut), basal tyrosine phosphorylation of insulin receptor substrate (IRS)-1 is increased by 35% compared with wild-type cells (WT). Upon exposure to insulin, IRS-1 phosphorylation increases by 12-fold in both Mut and WT cells. IRS-2 is constitutively phosphorylated in Mut cells and not further phosphorylated by insulin. The maximal phosphorylation of IRS-2 in basal Mut cells is paralleled by a 4-fold increased binding of the kinase regulatory loop binding domain of IRS-2 to the Arg1152 --> Gln receptor. Grb2 and phosphatidylinositol 3-kinase association to IRS-1 and IRS-2 reflects the phosphorylation levels of the two IRSs. Mitogen-activated protein kinase activation and [3H]thymidine incorporation closely correlates with IRS-1 phosphorylation in Mut and WT cells, while glycogen synthesis and synthase activity correlates with IRS-2 phosphorylation. The Arg1152 --> Gln mutant does not signal Shc phosphorylation or Shc-Grb2 association in intact L6 cells, while binding Shc in a yeast two-hybrid system and phosphorylating Shc in vitro. Thus, IRS-2 appears to mediate insulin regulation of glucose storage in Mut cells, while insulin-stimulated mitogenesis correlates with the activation of the IRS-1/mitogen-activated protein kinase pathway in these cells. IRS-1 and Shc-mediated mitogenesis may be redundant in muscle cells (Miele, 1999).

Association of Insulin receptor substrate 1 (IRS-1) Y895 with Grb-2 mediates the insulin signaling involved in IRS-1-deficient brown adipocyte mitogenesis

Immortalized fetal brown adipocyte cell lines have been generated from insulin receptor substrate 1 (IRS-1) knockout mice and an impairment in insulin-induced lipid synthesis has been demonstrated. The consequences of IRS-1 deficiency on mitogenesis in response to insulin have been investigated. The lack of IRS-1 results in the inability of insulin-stimulated IRS-1-deficient brown adipocytes to increase DNA synthesis and enter into S/G2/M phases of the cell cycle. These cells show a severe impairment in activating mitogen-activated protein kinase kinase (MEK1/2) and p42-p44 mitogen-activated protein kinase (MAPK) upon insulin stimulation. IRS-1-deficient cells also lack tyrosine phosphorylation of SHC and show no SHC-Grb-2 association in response to insulin. The mitogenic response to insulin can be partially restored by enhancing IRS-2 tyrosine phosphorylation and its association with Grb-2 by inhibition of phosphatidylinositol 3-kinase activity through a feedback mechanism. Reconstitution of IRS-1-deficient brown adipocytes with wild-type IRS-1 restores insulin-induced IRS-1 and SHC tyrosine phosphorylation and IRS-1-Grb-2, IRS-1-SHC, and SHC-Grb-2 associations, leading to the activation of MAPK and enhancement of DNA synthesis. Reconstitution of IRS-1-deficient brown adipocytes with the IRS-1 mutant Tyr895Phe, which lacks IRS-1-Grb-2 binding, restores SHC-IRS-1 association and SHC-Grb-2 association. However, the lack of IRS-1-Grb-2 association impairs MAPK activation and DNA synthesis in insulin-stimulated mutant cells. These data provide strong evidence for an essential role of IRS-1 and its direct association with Grb-2 in the insulin signaling pathway leading to MAPK activation and mitogenesis in brown adipocytes (Valverde, 2001).

The SH2/SH3 domain-containing protein GRB2 interacts with tyrosine-phosphorylated IRS1 and Shc: implications for insulin control of ras signalling

GRB2, a small protein comprising one SH2 domain and two SH3 domains, represents the human homolog of the Caenorhabditis elegans protein, sem-5. Both GRB2 and sem-5 have been implicated in a highly conserved mechanism that regulates p21ras signaling by receptor tyrosine kinases. In response to insulin, GRB2 forms a stable complex with two tyrosine-phosphorylated proteins. One protein is the major insulin receptor substrate IRS-1 and the second is the SH2 domain-containing oncogenic protein, Shc. The interactions between GRB2 and these two proteins require ligand activation of the insulin receptor and are mediated by the binding of the SH2 domain of GRB2 to phosphotyrosines on both IRS-1 and Shc. Although GRB2 associates with IRS-1 and Shc, it is not tyrosine-phosphorylated after insulin stimulation, implying that GRB2 is not a substrate for the insulin receptor. Furthermore, a short sequence motif (YV/IN) present in IRS-1, EGFR and Shc, has been identified that specifically binds the SH2 domain of GRB2 with high affinity. Interestingly, both GRB2 and phosphatidylinositol-3 (PI-3) kinase can simultaneously bind distinct tyrosine phosphorylated regions on the same IRS-1 molecule, suggesting a mechanism whereby IRS-1 could provide the core for a large signaling complex. A model is proposed whereby insulin stimulation leads to formation of multiple protein-protein interactions between GRB2 and the two targets IRS-1 and Shc. These interactions may play a crucial role in activation of p21ras and the control of downstream effector molecules (Skolnik, 1993).

Shp2 is required for protein kinase C-dependent phosphorylation of insulin receptor substrate

The function of insulin receptor substrate-1 (IRS-1), a key molecule of insulin signaling, is modulated by phosphorylation at multiple serine/threonine residues. Phorbol ester stimulation of cells induces phosphorylation of two inhibitory serine residues in IRS-1, i.e. Ser-307 and Ser-318, suggesting that both sites may be targets of protein kinase C (PKC) isoforms. However, in an in vitro system using a broad spectrum of PKC isoforms (alpha, beta1, beta2, delta, epsilon, eta, mu), only Ser-318, but not Ser-307 phosphorylation was detected, suggesting that phorbol ester-induced phosphorylation of this site in intact cells requires additional signaling elements and serine kinases that link PKC activation to Ser-307 phosphorylation. Since the tyrosine phosphatase Shp2, a negative regulator of insulin signaling, is a substrate of PKC, the role of Shp2 in this context was examined. Phorbol ester-induced Ser-307 phosphorylation is reduced markedly in Shp2-deficient mouse embryonic fibroblasts (Shp2-/-) whereas Ser-318 phosphorylation is unaltered. The Ser-307 phosphorylation was rescued by transfection of mouse embryonic fibroblasts with wild-type Shp2 or with a phosphatase-inactive Shp2 mutant, respectively. In this cell model, tumor necrosis factor-alpha-induced Ser-307 phosphorylation as well depends on the presence of Shp2. Furthermore, Shp2-dependent phorbol ester effects on Ser-307 are blocked by wortmannin, rapamycin, and the c-Jun NH2-terminal kinase (JNK) inhibitor SP600125. This suggests an involvement of the phosphatidylinositol 3-kinase/mammalian target of rapamycin cascade and of JNK in this signaling pathway resulting in IRS-1 Ser-307 phosphorylation. Because the activation of these kinases does not depend on Shp2, it is concluded that the function of Shp2 is to direct these activated kinases to IRS-1 (Mussig, 2005).

Intracellular localization of phosphatidylinositide 3-kinase and insulin receptor substrate-1 in adipocytes: potential involvement of a membrane skeleton

Phosphatidylinositide (PI) 3-kinase binds to tyrosyl-phosphorylated insulin receptor substrate-1 (IRS-1) in insulin-treated adipocytes, and this step plays a central role in the regulated movement of the glucose transporter, GLUT4, from intracellular vesicles to the cell surface. PDGF, which also activates PI 3-kinase in adipocytes, has no significant effect on GLUT4 trafficking in these cells. It is proposed that this specificity may be mediated by differential localization of PI 3-kinase in response to insulin versus PDGF activation. Using subcellular fractionation in 3T3-L1 adipocytes, it has been shown that insulin- and PDGF-stimulated PI 3-kinase activities are located in an intracellular high speed pellet (HSP) and in the plasma membrane (PM), respectively. The HSP is also enriched in IRS-1, insulin-stimulated tyrosyl-phosphorylated IRS-1 and intracellular GLUT4-containing vesicles. Using sucrose density gradient sedimentation, the HSP could be separated into two separate subfractions: one enriched in IRS-1, tyrosyl-phosphorylated IRS-1, PI 3-kinase as well as cytoskeletal elements, and another enriched in membranes, including intracellular GLUT4 vesicles. Treatment of the HSP with nonionic detergent, liberates all membrane constituents, whereas IRS-1 and PI 3-kinase remain insoluble. Conversely, at high ionic strength, membranes remain intact, whereas IRS-1 and PI 3-kinase become freely soluble. This IRS-1-PI 3-kinase complex exists in CHO cells overexpressing IRS-1 and, in these cells, the cytosolic pool of IRS-1 and PI 3-kinase is released subsequent to permeabilization with Streptolysin-O, whereas the particulate fraction of these proteins is retained. These data suggest that IRS-1, PI 3-kinase, as well as other signaling intermediates, may form preassembled complexes that may be associated with the actin cytoskeleton. This complex must be in close apposition to the cell surface, enabling access to the insulin receptor and presumably other signaling molecules that somehow confer the absolute specificity of insulin signaling in these cells (Clark, 1998).

Membrane-targeted phosphatidylinositol 3-kinase mimics insulin actions and induces a state of cellular insulin resistance

Phosphatidylinositol (PI) 3-kinase plays an important role in various insulin-stimulated biological responses, including glucose transport, glycogen synthesis, and protein synthesis. However, the molecular link between PI 3-kinase and these biological responses is still unclear. Is targeting of the catalytic p110 subunit of PI 3-kinase to cellular membranes both sufficient and necessary to induce PI 3-kinase dependent signaling responses, as is characteristic of insulin action? Myc-tagged, membrane-targeted p110 [p110(CAAX)], and wild-type p110 [p110(WT)] in 3T3-L1 adipocytes were engineered by adenovirus-mediated gene transfer. Overexpressed p110(CAAX) exhibits approximately 2-fold increase in basal kinase activity in p110 immunoprecipitates; this further increases to approximately 4-fold with insulin. Even at this submaximal PI 3-kinase activity, p110(CAAX) fully stimulates p70 S6 kinase, Akt, 2-deoxyglucose uptake, and Ras, whereas, p110(WT) has little or no effect on these downstream effects. Interestingly, p110(CAAX) does not activate MAP kinase, despite its stimulation of p21(ras). Surprisingly, p110(CAAX) does not increase basal glycogen synthase activity, and inhibits insulin stimulated activity, indicative of cellular resistance to this action of insulin. p110(CAAX) also inhibits insulin stimulated, but not platelet-derived growth factor-stimulated mitogen-activated protein kinase phosphorylation, demonstrating that the p110(CAAX) induced inhibition of mitogen-activated protein kinase and insulin signaling is specific, and not due to some toxic or nonspecific effect on the cells. Moreover, p110(CAAX) stimulates IRS-1 Ser/Thr phosphorylation, and inhibits IRS-1 associated PI 3-kinase activity, without affecting insulin receptor tyrosine phosphorylation, suggesting that it may play an important role as a negative regulator for insulin signaling. In conclusion, these studies show that membrane-targeted PI 3-kinase can mimic a number of biologic effects normally induced by insulin. In addition, the persistent activation of PI 3-kinase induced by p110(CAAX) expression leads to desensitization of specific signaling pathways. Interestingly, the state of cellular insulin resistance is not global, in that some of insulin's actions are inhibited, whereas others are intact (Egawa, 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

Serine/threonine phosphorylation of insulin receptor substrate 1 (IRS-1) has been implicated as a negative regulator of insulin signaling. Prior studies have indicated that this negative regulation by protein kinase C involves the mitogen-activated protein kinase and phosphorylation of serine 612 in IRS-1. In the present studies, the negative regulation by platelet-derived growth factor (PDGF) was compared with that induced by endothelin-1, an activator of protein kinase C. In contrast to endothelin-1, the inhibitory effects of PDGF does not require mitogen-activated protein kinase or the phosphorylation of serine 612. Instead, three other serines in the phosphorylation domain of IRS-1 (serines 632, 662, and 731) are required for the negative regulation by PDGF. In addition, the PDGF-activated serine/threonine kinase called Akt is found to inhibit insulin signaling. Moreover, this inhibition requires the same IRS-1 serine residues as the inhibition by PDGF. Finally, the negative regulatory effects of PDGF and Akt are inhibited by rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), one of the downstream targets of Akt. These studies implicate the phosphatidylinositol 3-kinase/Akt kinase cascade as an additional negative regulatory pathway for the insulin signaling cascade (Li, 1999).

Platelet-derived growth factor inhibits insulin stimulation of insulin receptor substrate-1-associated phosphatidylinositol 3-kinase in 3T3-L1 adipocytes without affecting glucose transport

Phosphatidylinositol 3-kinase (PI3K) activation is necessary for insulin-responsive glucose transporter (GLUT4) translocation and glucose transport. Insulin and platelet-derived growth factor (PDGF) stimulate PI3K activity in 3T3-L1 adipocytes, but only insulin is capable of stimulating GLUT4 translocation and glucose transport. PDGF causes serine/threonine phosphorylation of insulin receptor substrate 1 (IRS-1) in 3T3-L1 cells, measured by altered mobility on SDS-polyacrylamide gel, and this leads to a decrease in insulin-stimulated tyrosine phosphorylation of IRS-1. The PI3K inhibitors wortmannin and LY294002 inhibit the PDGF-induced phosphorylation of IRS-1, whereas the MEK inhibitor PD98059 is without a major effect. PDGF pretreatment for 60-90 min leads to a marked 80%-90% reduction in insulin stimulatable phosphotyrosine and IRS-1-associated PI3K activity. The functional consequences of this decrease in IRS-1-associated PI3K activity were examined. Interestingly, insulin stimulation of GLUT4 translocation and glucose transport is unaffected by 60-90 min of PDGF preincubation. Furthermore, insulin activation of Akt and p70(s6kinase: see Drosophila RPS6-p70-protein kinase), kinases downstream of PI3K, is unaffected by PDGF pretreatment. Wortmannin is capable of blocking these insulin actions following PDGF pretreatment, suggesting that PI3K was still necessary for these effects. In conclusion, (1) PDGF causes serine/threonine phosphorylation of IRS-1, and PI3K, or a kinase downstream of PI3K, mediates this phosphorylation. (2) This PDGF-induced phosphorylation of IRS-1 leads to a significant decrease in insulin-stimulated PI3K activity. (3) PDGF has no effect on insulin stimulation of Akt, p70(s6kinase), GLUT4 translocation, or glucose transport. (4) This suggests the existence of an IRS-1-independent pathway leading to the activation of PI3K, Akt, and p70(s6kinase), and of GLUT4 translocation and glucose transport (Staubs, 1998).

Growth hormone stimulates the tyrosine phosphorylation of the insulin receptor substrate-1 and its association with phosphatidylinositol 3-kinase in primary adipocytes

Insulin receptor substrate-1 (IRS-1) is tyrosine-phosphorylated in response to insulin resulting in association with and activation of phosphatidylinositol 3-kinase (PI 3-kinase), thereby initiating some of the effects of insulin. The insulin-like effects of growth hormone (GH) in adipocytes can be inhibited by the selective PI 3-kinase inhibitor wortmannin, suggesting a similar role for PI 3-kinase in GH action. IRS-1 is tyrosine-phosphorylated in a time- and dose-dependent manner in response to GH in primary rat adipocytes. This phosphorylation coincides with the extent of interaction between IRS-1 and the 85-kDa subunit of PI 3-kinase as evidenced by coimmunoprecipitation. Stimulation with 23 nM GH increases the PI 3-kinase activity associated with IRS1 4-fold. These data suggest that GH-induced tyrosine phosphorylation of IRS-1 and the subsequent docking of PI 3-kinase are important postreceptor events in GH action. The mechanism for the phosphorylation of IRS-1 induced by GH is unknown, but involvement of JAK2, the only known GH receptor-associated tyrosine kinase, seems possible (Ridderstrale, 1996).

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

Growth hormone (GH) and prolactin (PRL) binding to their receptors, which belong to the cytokine receptor superfamily, activate Janus kinase (JAK) 2 tyrosine kinase, thereby leading to their biological actions. GH mainly stimulates tyrosine phosphorylation of epidermal growth factor receptor and its association with Grb2, and concomitantly stimulates mitogen-activated protein kinase activity in liver, a major target tissue. Using specific antibodies, it has been shown that GH is also able to induce tyrosine phosphorylation of IRS-1/IRS-2 in liver. In addition, the major tyrosine-phosphorylated protein in anti-p85 phosphatidylinositol 3-kinase (PI3-kinase) immunoprecipitate from liver of wild-type mice is IRS-1, and IRS-2 in IRS-1 deficient mice, but not epidermal growth factor receptor. These data suggest that tyrosine phosphorylation of IRS-1 may be a major mechanism for GH-induced PI3-kinase activation in liver -- the physiological target organ of GH. PRL was able to induce tyrosine phosphorylation of both IRS-1 and IRS-2 in COS cells transiently transfected with PRLR and in CHO-PRLR cells. Moreover, tyrosine phosphorylation of IRS-3 is induced by both GH and PRL in COS cells transiently transfected with IRS-3 and their cognate receptors. By using the JAK2-deficient cell lines or by expressing a dominant negative JAK2 mutant, it has been shown that JAK2 is required for the GH- and PRL-dependent tyrosine phosphorylation of IRS-1, -2, and -3. Finally, a specific PI3-kinase inhibitor, wortmannin, completely blocks the anti-lipolytic effect of GH in 3T3 L1 adipocytes. Taken together, the role of IRS-1, -2, and -3 in GH and PRL signalings appears to be phosphorylated by JAK2, thereby providing docking sites for p85 PI3-kinase and activating PI3-kinase and its downstream biological effects (Yamauchi, 1998).

Insulin receptor substrate-1 enhances growth hormone-induced proliferation

GH exerts a variety of metabolic and growth-promoting effects. GH induces activation of the GH receptor (GHR)-associated cytoplasmic tyrosine kinase, JAK2, resulting in tyrosine phosphorylation of the GHR and activation of STAT (signal transducer and activator of transcription), Ras-mitogen-activated protein kinase, and phosphoinositol 3-kinase signaling pathways, among others. GH-stimulated tyrosine phosphorylation of insulin receptor substrate (IRS) proteins has been demonstrated in vitro and in vivo. IRS-1 is a multiply phosphorylated cytoplasmic docking protein involved in metabolic and proliferative signaling by insulin, IL-4, and other cytokines, but the physiological role of IRS-1 in GH signaling is unknown. In murine 3T3-F442A pre-adipocytes, GH-dependent tyrosine phosphorylation of IRS-1 is observed as is specific GH-induced coimmunoprecipitation of IRS-1 with JAK2. This interaction was examined by in vitro affinity precipitation experiments with glutathione-S-transferase fusion proteins incorporating regions of rat IRS-1 and, as a source of JAK2, extracts of 3T3-F442A cells. Fusion proteins containing amino-terminal regions of IRS-1 that include the pleckstrin homology, phosphotyrosine-binding, and Shc and IRS-1 NPXY-binding domains, but not those containing other IRS-1 regions or glutathione-S-transferase alone, bind JAK2 from cell extracts. Tyrosine-phosphorylated JAK2 resulting from GH stimulation is included in the amino-terminal IRS-1 fusion precipitates; however, neither tyrosine phosphorylation of JAK2 nor treatment of cells with GH before extraction is necessary for the specific JAK2-IRS-1 interaction to be detected. In contrast, in this assay, specific insulin receptor association with the IRS-1 phosphotyrosine-binding, and Shc and IRS-1 NPXY-binding domains is insulin and phosphotyrosine dependent. To test for significance of IRS-1 with regard to GH signaling, IRS- and GHR-deficient 32D cells were stably reconstituted with the rabbit (r) GHR, either alone (32D-rGHR) or with IRS-1 (32D-rGHR-IRS-1). As assayed by three independent methods, GH induces proliferation in 32D-rGHR cells, even in the absence of transfected IRS-1. Notably, however, GH-induced proliferation is markedly enhanced in cells expressing IRS-1. Similarly, GH-induced mitogen-activated protein kinase activation is significantly augmented in IRS-1-expressing cells relative to that in cells harboring no IRS-1. These results indicate that IRS-1 enhances GH-induced proliferative signaling (Liang, 1999).

Evidence that IRS-2 phosphorylation is required for insulin action in hepatocytes

Insulin receptor substrates (IRSs) are tyrosine-phosphorylated following stimulation with insulin, insulin-like growth factors (IGFs), and interleukins. A key question is whether different IRSs play different roles to mediate insulin's metabolic and growth-promoting effects. In a novel system of insulin receptor-deficient hepatocytes, insulin fails to (1) stimulate glucose phosphorylation; (2) enhance glycogen synthesis; (3) suppress glucose production, and (4) promote mitogenesis. However, insulin's ability to induce IRS-1 and gab-1 phosphorylation and binding to phosphatidylinositol (PI) 3-kinase is unaffected, by virtue of the compensatory actions of IGF-1 receptors. In contrast, phosphorylation of IRS-2 and generation of IRS-2/PI 3-kinase complexes are markedly reduced. Thus, absence of insulin receptors selectively reduces IRS-2, but not IRS-1 phosphorylation, and the impairment of IRS-2 activation is associated with lack of insulin effects. To address whether phosphorylation of additional IRSs is also affected, phosphotyrosine-containing proteins were analyzed in PI 3-kinase immunoprecipitates from insulin-treated cells. However, these experiments indicate that IRS-1 and IRS-2 are the main PI 3-kinase-bound proteins in hepatocytes. These data identify IRS-2 as the main effector of both the metabolic and growth-promoting actions of insulin through PI 3-kinase in hepatocytes, and IRS-1 as the main substrate mediating the mitogenic actions of IGF-1 receptors (Rother, 1998).

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

Insulin receptor substrates-1 and -2 (IRS-1 and -2) are important substrates of the insulin receptor tyrosine kinase. IRS-2 can mediate translocation of the insulin responsive glucose transporter GLUT4 in a physiologically relevant target cell for insulin action. Co-immunoprecipitation experiments performed on cell lysates derived from freshly isolated rat adipose cells incubated in the presence or absence of insulin indicate that twice as much phosphatidylinositol 3-kinase is associated with endogenous IRS-1 as with IRS-2 after insulin stimulation. When rat adipose cells in primary culture are transfected with expression vectors for IRS-1 or IRS-2, 40-fold overexpression of human IRS-1 or murine IRS-2 is observed. In addition, anti-phosphotyrosine immunoblotting experiments confirm that the recombinant substrates are phosphorylated in response to insulin stimulation. To examine the role of IRS-2 in insulin-stimulated translocation of GLUT4, the effects of overexpression of IRS-1 and -2 on translocation of a co-transfected epitope-tagged GLUT4 (GLUT4-HA) were studied. Overexpression of IRS-1 or IRS-2 in adipose cells results in a significant increase in the basal level of cell surface GLUT4 (in the absence of insulin). Interestingly, at maximally effective concentrations of insulin (60 nM), the level of cell surface GLUT4 in cells overexpressing IRS-1 or -2 significantly exceeds the maximal recruitment observed in the control cells (160% and 135% of control, respectively; p < 0.003). These data directly demonstrate that IRS-2, like IRS-1, is capable of participating in insulin signal transduction pathways leading to the recruitment of GLUT4. Thus, IRS-2 may provide an alternative pathway for critical metabolic actions of insulin (Zhou, 1997).

Action of insulin receptor substrate-3 (IRS-3) and IRS-4 to stimulate translocation of GLUT4 in rat adipose cells

The insulin receptor initiates insulin action by phosphorylating multiple intracellular substrates. Insulin receptor substrates (IRS)-1 and -2 can mediate insulin's action to promote translocation of GLUT4 glucose transporters to the cell surface in rat adipose cells. Although IRS-1, -2, and -4 are similar in overall structure, IRS-3 is approximately 50% shorter and differs with respect to sites of tyrosine phosphorylation. Nevertheless both IRS-3 and IRS-4 can also stimulate translocation of GLUT4. Rat adipose cells were cotransfected with expression vectors for hemagglutinin (HA) epitope-tagged GLUT4 (GLUT4-HA) and human IRS-1, murine IRS-3, or human IRS-4. Overexpression of IRS-1 leads to a 2-fold increase in cell surface GLUT4-HA in cells incubated in the absence of insulin; overexpression of either IRS-3 or IRS-4 elicits a larger increase in cell surface GLUT4-HA. Indeed, the effect of IRS-3 in the absence of insulin is approximately 40% greater than the effect of a maximally stimulating concentration of insulin in cells not overexpressing IRS proteins. Because phosphatidylinositol (PI) 3-kinase is essential for insulin-stimulated translocation of GLUT4, a mutant IRS-3 molecule (IRS-3-F4) was studied in which Phe was substituted for Tyr in all four YXXM motifs (the phosphorylation sites predicted to bind to and activate PI 3-kinase). Interestingly, overexpression of IRS-3-F4 does not promote translocation of GLUT4-HA, but actually inhibits the ability of insulin to stimulate translocation of GLUT4-HA to the cell surface. These data suggest that IRS-3 and IRS-4 are capable of mediating PI 3-kinase-dependent metabolic actions of insulin in adipose cells, and that IRS proteins play a physiological role in mediating translocation of GLUT4 (Zhou, 1999).

Insulin receptor substrate 1-induced inhibition of endoplasmic reticulum Ca2+ uptake in beta-cells. Autocrine regulation of intracellular ca2+ homeostasis and insulin secretion

To understand the role of the insulin receptor pathway in beta-cell function, stable beta-cells (betaIRS1-A) were created that overexpress by 2-fold the insulin receptor substrate-1 (IRS-1) and they were compared to vector-expressing controls. IRS-1 overexpression dramatically increases basal cytosolic Ca2+ levels from 81 to 278 nM, but it does not affect Ca2+ response to glucose. Overexpression of the insulin receptor also causes an increase in cytosolic Ca2+. Increased cytosolic Ca2+ is due to inhibition of Ca2+ uptake by the endoplasmic reticulum, because endoplasmic reticulum Ca2+ uptake and content are reduced in betaIRS1-A cells. Fractional insulin secretion is significantly increased 2-fold, and there was a decrease in betaIRS1-A insulin content and insulin biosynthesis. Steady-state insulin mRNA levels and glucose-stimulated ATP are unchanged. High IRS-1 levels also reduce beta-cell proliferation. These data demonstrate a direct link between the insulin receptor signaling pathway and the Ca2+-dependent pathways regulating insulin secretion of beta-cells. It is postulated that during regulated insulin secretion, released insulin binds the beta-cell insulin receptor and activates IRS-1, thus further increasing cytosolic Ca2+ by reducing Ca2+ uptake. The existence of a novel pathway of autocrine regulation of intracellular Ca2+ homeostasis and insulin secretion in the beta-cell of the endocrine pancreas is suggested (Xu, 1999).

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

The contribution of the insulin receptor substrate proteins (IRS-1 and IRS-2) to insulin/insulin like growth factor I (IGF-I)-signaling pathways was investigated in fetal rat brown adipocytes, a model that expresses both insulin and IGF-I receptors. Insulin/IGF-I rapidly stimulates IRS-1 and IRS-2 tyrosine phosphorylation, their association with p85alpha, and IRS-1- and IRS-2-associated phosphatidylinositol (PI) 3-kinase activation to the same extent, the effect of insulin being stronger than the effect of IGF-I at the same physiological dose (10 nM). Furthermore, insulin/IGF-I stimulates IRS-1-associated Grb-2 phosphorylation. However, IRS-2-associated Grb-2 phosphorylation is barely detected. Pull-down experiments with glutathione-S-transferase-fusion proteins containing SH2-domains of p85alpha reveal a strong association between IRS-1 and IRS-2 with p85alpha in response to insulin/IGF-I, the insulin effect being stronger than IGF-I. However, the Grb-2-SH2 domain shows functional differences. While a strong association between IRS-1/Grb-2 is found, IRS-2/Grb-2 association is virtually absent in response to insulin/IGF-I, as also demonstrated in competition studies with a phosphopeptide containing the phosphotyrosine 895 residue within the putative Grb-2-binding domain. Finally, insulin/IGF-I stimulates tyrosine phosphorylation of the three SHC proteins (46, 52, and 66 kDa). Moreover, insulin/IGF-I markedly increases the amount of Grb-2-associated SHC proteins by the same extent. These results suggest that both IRS-1 and IRS-2 are required for phosphatidylinositol 3-kinase activation, which leads to adipogenic and thermogenic differentiation of fetal brown adipose tissue; meanwhile, IRS-1 and SHC, but not IRS-2, associate with Grb-2, leading to the ras-mitogen-activated protein kinase-signaling pathway required for fetal brown adipocyte proliferation (Valverde, 1998).

Essential role of Insulin receptor substrate 1 (IRS-1) and IRS-2 in adipocyte differentiation

To investigate the role of insulin receptor substrate 1 (IRS-1) and IRS-2, the two ubiquitously expressed IRS proteins, in adipocyte differentiation, embryonic fibroblast cells were established with four different genotypes, i.e., wild-type, IRS-1 deficient (IRS-1^-/-), IRS-2 deficient (IRS-2^-/-), and IRS-1 IRS-2 double deficient (IRS-1^-/- IRS-2^-/-), from mouse embryos of the corresponding genotypes. The abilities of IRS-1^-/- cells and IRS-2^-/- cells to differentiate into adipocytes are approximately 60% and 15%, respectively, lower than that of wild-type cells, at day 8 after induction and, surprisingly, IRS-1^-/- IRS-2^-/- cells have no ability to differentiate into adipocytes. The expression of CCAAT/enhancer binding protein alpha (C/EBPalpha) and peroxisome proliferator-activated receptor gamma (PPARgamma) is severely decreased in IRS-1^-/- IRS-2^-/- cells at both the mRNA and the protein level, and the mRNAs of lipoprotein lipase and adipocyte fatty acid binding protein are severely decreased in IRS-1^-/- IRS-2^-/- cells. Phosphatidylinositol 3-kinase (PI 3-kinase) activity that increases during adipocyte differentiation is almost completely abolished in IRS-1^-/- IRS-2^-/- cells. Treatment of wild-type cells with a PI 3-kinase inhibitor, LY294002, markedly decreases the expression of C/EBPalpha and PPARgamma, a result that is associated with a complete block of adipocyte differentiation. Moreover, histologic analysis of IRS-1^-/- IRS-2^-/- double-knockout mice 8 h after birth reveals severe reduction in white adipose tissue mass. These results suggest that IRS-1 and IRS-2 play a crucial role in the upregulation of the C/EBPalpha and PPARgamma expression and adipocyte differentiation (Miki, 2001).

The role of glycogen synthase kinase 3beta in insulin-stimulated glucose metabolism

To characterize the contribution of glycogen synthase kinase 3beta (GSK3beta) inactivation to insulin-stimulated glucose metabolism, wild-type (WT-GSK), catalytically inactive (KM-GSK), and uninhibitable (S9A-GSK) forms of GSK3beta were expressed in insulin-responsive 3T3-L1 adipocytes using adenovirus technology. WT-GSK, but not KM-GSK, reduces basal and insulin-stimulated glycogen synthase activity without affecting the stimulation of the enzyme by insulin. S9A-GSK similarly decreases cellular glycogen synthase activity, but also partially blocks insulin stimulation of the enzyme. S9A-GSK expression also markedly inhibits insulin stimulation of IRS-1-associated phosphatidylinositol 3-kinase activity, but only weakly inhibits insulin-stimulated Akt/PKB phosphorylation and glucose uptake, with no effect on GLUT4 translocation. To further evaluate the role of GSK3beta in insulin signaling, the GSK3beta inhibitor lithium was used to mimic the consequences of insulin-stimulated GSK3beta inactivation. Although lithium stimulates the incorporation of glucose into glycogen and glycogen synthase enzyme activity, the inhibitor is without effect on GLUT4 translocation and pp70 S6 kinase. Lithium stimulation of glycogen synthesis is insensitive to wortmannin, which is consistent with its acting directly on GSK3beta downstream of phosphatidylinositol 3-kinase. These data support the hypothesis that GSK3beta contributes to insulin regulation of glycogen synthesis, but is not responsible for the increase in glucose transport (Summers, 1999).

SOCS-1 and SOCS-3 block insulin signaling by ubiquitin-mediated degradation of IRS1 and IRS2

Inflammation associates with peripheral insulin resistance, which dysregulates nutrient homeostasis and leads to diabetes. Inflammation induces the expression of suppressors of cytokine signaling (SOCS) proteins. SOCS1 and SOCS3 target IRS1 and IRS2, two critical signaling molecules for insulin action, for ubiquitin-mediated degradation. SOCS1 or SOCS3 bind both recombinant and endogenous IRS1 and IRS2 and promote their ubiquitination and subsequent degradation in multiple cell types. Mutations in the conserved SOCS box of SOCS1 abrogate its interaction with the elongin BC ubiquitin-ligase complex without affecting its binding to IRS1 or IRS2. The SOCS1 mutants also fail to promote the ubiquitination and degradation of either IRS1 or IRS2. Adenoviral-mediated expression of SOCS1 in mouse liver dramatically reduce hepatic IRS1 and IRS2 protein levels and cause glucose intolerance; by contrast, expression of the SOCS1 mutants has no effect. Thus, SOCS-mediated degradation of IRS proteins, presumably via the elongin BC ubiquitin-ligase, might be a general mechanism of inflammation-induced insulin resistance, providing a target for therapy (Rui, 2002).

c-Jun N-terminal kinase (JNK) mediates feedback inhibition of the insulin signaling cascade

Activation of the c-Jun N-terminal kinase (JNK) by proinflammatory cytokines inhibits insulin signaling, at least in part, by stimulating phosphorylation of rat/mouse insulin receptor substrate 1 (Irs1) at Ser(307) [Ser(312) in human IRS1]. JNK mediated feedback inhibition of the insulin signal has been demonstrated in mouse embryo fibroblasts, 3T3-L1 adipocytes, and 32D(IR) cells. Insulin stimulation of JNK activity requires phosphatidylinositol 3-kinase and Grb2 signaling. Moreover, activation of JNK by insulin is inhibited by a cell-permeable peptide that disrupts the interaction of JNK with cellular proteins. However, the direct binding of JNK to Irs1 is not required for its activation by insulin, whereas direct binding is required for Ser(307) phosphorylation of Irs1. Insulin-stimulated Ser(307) phosphorylation was reduced 80% in cells lacking JNK1 and JNK2 or in cells expressing a mutant Irs1 protein lacking the JNK binding site. Reduced Ser(307) phosphorylation is directly related to increased insulin-stimulated tyrosine phosphorylation, Akt phosphorylation, and glucose uptake. These results support the hypothesis that JNK is a negative feedback regulator of insulin action by phosphorylating Ser(307) in Irs1 (Lee, 2003).

Phenotypic effects of IRS gene mutation

Alternative pathway of insulin signalling in mice with targeted disruption of the IRS-1 gene

The principal substrate for the insulin and insulin-like growth factor-1 (IGF-1) receptors is the cytoplasmic protein insulin-receptor substrate-1 (IRS-1/pp185). After tyrosine phosphorylation at several sites, IRS-1 binds to and activates phosphatidylinositol-3'-OH kinase (PI(3)K) and several other proteins containing SH2 (Src-homology 2) domains. To elucidate the role of IRS-1 in insulin/IGF-1 action, IRS-1-deficient mice were created by targeted gene mutation. These mice have no IRS-1 and show no evidence of IRS-1 phosphorylation or IRS-1-associated PI(3)K activity. They also have a 50% reduction in intrauterine growth, impaired glucose tolerance, and a decrease in insulin/IGF-1-stimulated glucose uptake in vivo and in vitro. The residual insulin/IGF-1 action correlates with the appearance of a new tyrosine-phosphorylated protein (IRS-2) which binds to PI(3)K, but is slightly larger than and immunologically distinct from IRS-1. These results provide evidence for IRS-1-dependent and IRS-1-independent pathways of insulin/IGF-1 signaling and for the existence of an alternative substrate of these receptor kinases (Araki, 1994).

Insulin signalling and insulin actions in the muscles and livers of insulin-resistant, insulin receptor substrate 1-deficient mice

Mice with a targeted disruption of the insulin receptor substrate 1 (IRS-1) gene exhibit growth retardation and have resistance to the glucose-lowering effect of insulin. Insulin initiates its biological effects by activating at least two major signaling pathways, one involving phosphatidylinositol 3-kinase (PI3-kinase) and the other involving a ras/mitogen-activated protein kinase (MAP kinase) cascade. In this study the roles of IRS-1 and IRS-2 in the biological action in the physiological target organs of insulin was investigated by comparing the effects of insulin in wild-type and IRS-1-deficient mice. In muscles from IRS-1-deficient mice, the responses to insulin-induced PI3-kinase activation, glucose transport, p70 S6 kinase and MAP kinase activation, mRNA translation, and protein synthesis are significantly impaired compared with those in wild-type mice. Insulin-induced protein synthesis is wortmannin insensitive in IRS-1 deficient mice. However, in another target organ, the liver, the responses to insulin-induced PI3-kinase and MAP kinase activation are not significantly reduced. The amount of tyrosine-phosphorylated IRS-2 (in IRS-1-deficient mice) is roughly equal to that of IRS-1 (in wild-type mice) in the liver, whereas it only 20% to 30% of that of IRS-1 in the muscles. In conclusion, (1) IRS-1 plays central roles in two major biological actions of insulin in muscles, glucose transport and protein synthesis; (2) the insulin resistance of IRS-1-deficient mice is mainly due to resistance in the muscles, and (3) the degree of compensation for IRS-1 deficiency appears to be correlated with the amount of tyrosine-phosphorylated IRS-2 (in IRS-1-deficient mice) relative to that of IRS-1 (in wild-type mice) (Yamauchi, 1996).

Hypertension, hypertriglyceridemia, and impaired endothelium-dependent vascular relaxation in mice lacking insulin receptor substrate-1

Insulin resistance is often associated with atherosclerotic diseases in subjects with obesity and impaired glucose tolerance. This study examined the effects of insulin resistance on coronary risk factors in IRS-1 deficient mice, a nonobese animal model of insulin resistance. Blood pressure and plasma triglyceride levels are significantly higher in IRS-1 deficient mice than in normal mice. Impaired endothelium-dependent vascular relaxation is also observed in IRS-1 deficient mice. Furthermore, lipoprotein lipase activity is lower than in normal mice, suggesting impaired lipolysis to be involved in the increase in plasma triglyceride levels under insulin-resistant conditions. Thus, insulin resistance plays an important role in the clustering of coronary risk factors which may accelerate the progression of atherosclerosis in subjects with insulin resistance (Abe, 1998).

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

Non-insulin-dependent diabetes mellitus (NIDDM) is considered a polygenic disorder in which insulin resistance and insulin secretory defect are the major etiologic factors. Homozygous mice with insulin receptor substrate-1 (IRS-1) gene knockout show normal glucose tolerance associated with insulin resistance and compensatory hyperinsulinemia. Heterozygous mice with beta cell glucokinase (GK) gene knockout show impaired glucose tolerance due to decreased insulin secretion to glucose. To elucidate the interplay between insulin resistance and insulin secretory defect for the development of NIDDM, double knockout mice were generated with disruption of IRS-1 and beta cell GK genes by crossing the mice with each of the single gene knockout. The double knockout mice develope overt diabetes. Blood glucose levels 120 min after intraperitoneal glucose load (1.5 mg/g body wt) are 108 +/- 24 (wild type), 95 +/- 26 (IRS-1 knockout), 159 +/- 68 (GK knockout), and 210 +/- 38 (double knockout) mg/dl (mean +/- SD) (double versus wild type, IRS-1, or GK; P < 0.01). The double knockout mice show fasting hyperinsulinemia and selective hyperplasia of the beta cells as the IRS-1 knockout mice (fasting insulin levels: 0.38 +/- 0.30 [double knockout], 0.35 +/- 0.27 [IRS-1 knockout] versus 0.25 +/- 0.12 [wild type] ng/ml) (proportion of areas of insulin-positive cells to the pancreas: 1.18 +/- 0.68%; P < 0.01 [double knockout], 1.20 +/- 0.93%; P < 0.05 [IRS-1 knockout] versus 0.54 +/- 0.26% [wild type]), but impaired insulin secretion to glucose (the ratio of increment of insulin to that of glucose during the first 30 min after load: 31 [double knockout] versus 163 [wild type] or 183 [IRS-1 knockout] ng insulin/mg glucose x 10³). In conclusion, the genetic abnormalities, each of which is nondiabetogenic by itself, cause overt diabetes if they coexist. This report provides the first genetic reconstitution of NIDDM as a polygenic disorder in mice (Terauchi, 1997).

Mutant of insulin receptor substrate-1 incapable of activating phosphatidylinositol 3-kinase did not mediate insulin-stimulated maturation of Xenopus laevis oocytes

Insulin receptor substrate-1 (IRS-1) is rapidly phosphorylated on multiple tyrosine residues in response to insulin and binds several Src homology 2 domain-containing proteins, thereby initiating downstream signaling. To assess the tyrosine phosphorylation sites that mediate relevant downstream signaling and biological effects, site-directed mutants of IRS-1 were created and they were overexpressed in the Xenopus laevis oocyte. In oocytes overexpressing IRS-1 or IRS-1-895F (Tyr-895 replaced with phenylalanine), insulin activatea phosphatidylinositol (PI) 3-kinase, p70 S6 kinase, and mitogen-activated protein kinase and induces oocyte maturation. In contrast, in oocytes overexpressing IRS-1-4F (Tyr-460, Tyr-608, Tyr-939, and Tyr-987 of IRS-1 replaced with phenylalanine), insulin does not activate PI 3-kinase, p70 S6 kinase, and mitogen-activated protein kinase and fails to induce oocyte maturation. These observations indicate that in X. laevis oocytes overexpressing IRS-1, the association of PI 3-kinase rather than Grb2 (growth factor-bound protein 2) with IRS-1 plays a major role in insulin-induced oocyte maturation. Activation of PI 3-kinase may lie upstream of mitogen-activated protein kinase activation and p70 S6 kinase activation in response to insulin (Yamamoto-Honda, 1996).

Lipoatrophic diabetes in Irs1^-/-/Irs3^-/- double knockout mice

Based on the phenotypes of knockout mice and cell lines, as well as pathway-specific analysis, the insulin receptor substrates IRS-1, IRS-2, IRS-3, and IRS-4 have been shown to play unique roles in insulin signal transduction. To investigate possible functional complementarity within the IRS family, mice with double knockout of the genes for IRS-1/IRS-3 and IRS-1/IRS-4 were generated. Mice with a combined deficiency of IRS-1 and IRS-4 showed no differences from Irs1^-/- mice with respect to growth and glucose homeostasis. In contrast, mice with a combined deficiency of IRS-1 and IRS-3 developed early-onset severe lipoatrophy associated with marked hyperglycemia, hyperinsulinemia, and insulin resistance. However, in contrast to other models of lipoatrophic diabetes, there was no accumulation of fat in liver or muscle. Furthermore, plasma leptin levels were markedly decreased, and adenovirus-mediated expression of leptin in liver reversed the hyperglycemia and hyperinsulinemia. The results indicate that IRS-1 and IRS-3 play important complementary roles in adipogenesis and establish the Irs1^-/-/Irs3^-/- double knockout mouse as a novel model of lipoatrophic diabetes (Laustsen, 2002).

Human insulin receptor substrate-2 (IRS-2) is a primary progesterone response gene

Elevated cAMP has been shown to unmask agonist activity of antiprogestin/antiglucocorticoid RU486. In a search for cellular target genes induced through this cross-talk mechanism, human insulin receptor substrate-2 (IRS-2) was identified. IRS-2 is a cytoplasmic signaling molecule that mediates effects of insulin, insulin-like growth factor-1 (IGF-I), and other cytokines by acting as a molecular adaptor between diverse receptor tyrosine kinases and downstream effectors. Analysis of the regulation of IRS-2 in HeLa cell models shows that synergistic induction of IRS-2 by cAMP and RU486 can be mediated by progesterone receptors (PR) and glucocorticoid receptors (GR) and occurs through a relative slow mechanism that requires ongoing protein synthesis. IRS-2 mRNA is also inducible by progesterone, while glucocorticoid effects are only observed in the presence of cAMP. Up-regulation of IRS-2 by progesterone depends strictly on the presence of PR and occurs through a rapid mechanism, suggesting that it represents a primary transcriptional response. Expression of IRS-1, which also binds to receptors of insulin, IGF-I, and cytokines, is unaffected by progesterone. Thus, these results demonstrate that progesterone alters the ratio of IRS-1 and IRS-2 in PR-positive cells and implicate a mechanism through which progesterone can modulate the effects of insulin, IGF-I, and cytokines on cell proliferation, differentiation, and homeostasis (Vassen, 1998).

Enhancement of insulin-like growth factor signaling in human breast cancer: estrogen regulation of insulin receptor substrate-1 expression in vitro and in vivo

Cross-talk between insulin-like growth factor (IGF)- and estrogen receptor (ER)-signaling pathways results in synergistic growth. Estrogen enhances IGF signaling by inducing expression of three key IGF-regulatory molecules, the type 1 IGF receptor (IGFR1) and its downstream signaling molecules, insulin receptor substrate (IRS)-1 and IRS-2. Estrogen induction of IGFR1 and IRS expression result in enhanced tyrosine phosphorylation of IRS-1 after IGF-I stimulation, followed by enhanced mitogen-activated protein kinase activation. To examine whether these pathways are similarly activated in vivo, MCF-7 cells grown as xenografts in athymic mice were examined. IRS-1 is expressed at high levels in estrogen-dependent growth of MCF-7 xenografts, but withdrawal of estrogen, which decreases tumor growth, results in a dramatic decrease in IRS-1 expression. High IRS-1 expression is an indicator of early disease recurrence in ER-positive human primary breast tumors. Taken together, these data not only reinforce the concept of cross-talk between IGF- and ER-signaling pathways, but indicate that IGF molecules may be critical regulators of estrogen-mediated growth and breast cancer pathogenesis (Lee, 1999).

cAMP promotes pancreatic ß-cell survival via CREB-mediated induction of IRS2

The incretin hormone GLP1 promotes islet-cell survival via the second messenger cAMP. Mice deficient in the activity of CREB, caused by expression of a dominant-negative A-CREB transgene in pancreatic ß-cells, develop diabetes secondary to ß-cell apoptosis. Remarkably, A-CREB severely disrupts expression of IRS2, an insulin signaling pathway component that is shown in this study to be a direct target for CREB action in vivo. Since induction of IRS2 by cAMP enhances activation of the survival kinase Akt in response to insulin and IGF-1, these results demonstrate a novel mechanism by which opposing pathways cooperate in promoting cell survival (Jhala, 2003).

REFERENCES

Search PubMed for articles about Drosophila chico

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. PubMed Citation: 9541510

Alic, N., Giannakou, M. E., Papatheodorou, I., Hoddinott, M. P., Andrews, T. D., Bolukbasi, E. and Partridge, L. (2014). Interplay of dFOXO and two ETS-family transcription factors determines lifespan in Drosophila melanogaster. PLoS Genet 10: e1004619. PubMed ID: 25232726

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. PubMed Citation: 9792680

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. PubMed Citation: 7526222

Bai, H., Post, S. Kang, P. and Tatar, M. (2015). Drosophila longevity assurance conferred by reduced insulin receptor substrate Chico partially requires d4eBP. PLoS One 10: e0134415. PubMed ID: 26252766

Berg, C.A., and Spradling, A.C. (1991). Studies on the rate and site-specificity of P-element transposition. Genetics 127: 515-524. PubMed Citation: 1849859

Bochukova, E. G., et al. (2010) Large, rare chromosomal deletions associated with severe early-onset obesity. Nature 463: 666-670. PubMed Citation: 19966786

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. PubMed Citation: 10399915

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. PubMed Citation: 9570778

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. PubMed Citation: 10207032

Chen, C., Jack, J. and Garofalo, R. S. (1996). The Drosophila insulin receptor is required for normal growth. Endocrinology 137(3):846-56. PubMed Citation: 8603594

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. PubMed Citation: 9490733

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. PubMed Citation: 10318852

Ehninger, D., Neff, F. and Xie, K. (2014). Longevity, aging and rapamycin. Cell Mol Life Sci 71: 4325-4346. PubMed ID: 25015322

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. PubMed Citation: 10037694

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. PubMed Citation: 7628438

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. PubMed Citation: 8929412

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. PubMed Citation: 10617573

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. PubMed Citation: 7592659

Huang, H., et al. (1999). PTEN affects cell size, cell proliferation and apoptosis during Drosophila eye development. Development 126(23): 5365-72. PubMed Citation: 10556061

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

Kuo, T. H., Fedina, T. Y., Hansen, I., Dreisewerd, K., Dierick, H. A., Yew, J. Y. and Pletcher, S. D. (2012). Insulin signaling mediates sexual attractiveness in Drosophila. PLoS Genet 8: e1002684. Pubmed: 22570625

Lamming, D. W., Ye, L., Sabatini, D. M. and Baur, J. A. (2013). Rapalogs and mTOR inhibitors as anti-aging therapeutics. J Clin Invest 123: 980-989. PubMed ID: 23454761

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

Nagata, R., Nakamura, M., Sanaki, Y. and Igaki, T. (2019). Cell competition is driven by autophagy. Dev Cell 51(1): 99-112. PubMed ID: 31543447

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

Pallares-Cartes, C., Cakan-Akdogan, G. and Teleman, A. A. (2012). Tissue-specific coupling between insulin/IGF and TORC1 signaling via PRAS40 in Drosophila. Dev. Cell 22(1): 172-82. PubMed Citation: 22264732

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

Pflieger, D., et al. (2008). Quantitative proteomic analysis of protein complexes: concurrent identification of interactors and their state of phosphorylation. Mol. Cell Proteomics. 7(2): 326-46. PubMed Citation: 17956857

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

Reddy, P., Zheng, W. and Liu, K. (2010). Mechanisms maintaining the dormancy and survival of mammalian primordial follicles. Trends Endocrinol. Metab. 21: 96-103. PubMed Citation: 19913438

Ren, E., et al. (2007). Neuronal SH2B1 is essential for controlling energy and glucose homeostasis. J. Clin. Invest. 117: 97-406. PubMed Citation: 17235396

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

Sanaki, Y., Nagata, R., Kizawa, D., Leopold, P. and Igaki, T. (2020). Hyperinsulinemia drives epithelial tumorigenesis by abrogating cell competition. Dev Cell 53(4): 379-389 PubMed ID: 32386602

Sancak, Y., et al. (2007). PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase Mol. Cell 25: 903-915. PubMed Citation: 17386266

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

Slack, C., et al. (2010). Regulation of lifespan, metabolism, and stress responses by the Drosophila SH2B protein, Lnk. PLoS Genet. 6(3): e1000881. PubMed Citation: 20333234

Slack, C., Alic, N., Foley, A., Cabecinha, M., Hoddinott, M.P. and Partridge, L. (2015). The Ras-Erk-ETS-signaling pathway is a drug target for longevity. Cell 162(1):72-83.. PubMed ID: 26119340

Song, W., et al. (2010). SH2B regulation of growth, metabolism, and longevity in both insects and mammals. Cell Metab. 11(5): 427-37. PubMed Citation: 20417156

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

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

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, P., et al. (2010). TSC1/2 tumour suppressor complex maintains Drosophila germline stem cells by preventing differentiation. Development 137: 2461-2469. PubMed Citation: 20573703

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

Teleman, A. A., Hietakangas, V., Sayadian, A. C. and Cohen, S. M. (2008). Nutritional control of protein biosynthetic capacity by insulin via Myc in Drosophila. Cell Metab 7: 21-32. PubMed ID: 18177722

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

Thomson, T. C., Fitzpatrick, K. E. and Johnson, J. (2010). Intrinsic and extrinsic mechanisms of oocyte loss. Mol. Hum. Reprod. 16: 916-927. PubMed Citation: 20651035

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

Vander Haar, R., et al. (2007). Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40. Nat. Cell Biol. 9: 316-323. PubMed Citation: 17277771

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

Werz, C., Köhler, K., Hafen, E. and Stocker, H. (2009). The Drosophila SH2B family adaptor Lnk acts in parallel to chico in the insulin signaling pathway. PLoS Genet. 5(8): e1000596. PubMed Citation: 19680438

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