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

Gene name - chico

Synonyms - flipper

Cytological map position - 31B1

Function - signaling protein

Keywords - growth response, insulin signaling pathway

Symbol - chico

FlyBase ID: FBgn0024248

Genetic map position -

Classification - insulin receptor substrate family

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW


Recent literature

Ismail, M.Z., Hodges, M.D., Boylan, M., Achall, R., Shirras, A. and Broughton, S.J. (2015). The Drosophila insulin receptor independently modulates lifespan and locomotor senescence. PLoS One 10: e0125312. PubMed ID: 26020640
Summary:
The Insulin/IGF-like signalling (IIS) pathway plays an evolutionarily conserved role in ageing. In model organisms reduced IIS extends lifespan and ameliorates some forms of functional senescence. However, little is known about IIS in nervous system ageing and behavioural senescence. To investigate this role in Drosophila melanogaster, the effect of reduced IIS on senescence of two locomotor behaviours, negative geotaxis and exploratory walking, was measured in this study. Two long-lived fly models with systemic IIS reductions (daGAL4/UAS-InRDN (ubiquitous expression of a dominant negative insulin receptor) and d2GAL/UAS-rpr (ablation of insulin-like peptide producing cells)) showed an amelioration of negative geotaxis senescence similar to that previously reported for the long-lived IIS mutant chico. In contrast, exploratory walking in daGAL4/UAS-InRDN and d2GAL/UAS-rpr flies declined with age similarly to controls. To determine the contribution of IIS in the nervous system to these altered senescence patterns and lifespan, the InRDN was targeted to neurons (elavGAL4/UAS-InRDN), which resulted in extension of lifespan in females, normal negative geotaxis senescence in males and females, and detrimental effects on age-specific exploratory walking behaviour in males and females. These data indicate that the Drosophila insulin receptor independently modulates lifespan and age-specific function of different types of locomotor behaviour. The data suggest that ameliorated negative geotaxis senescence of long-lived flies with systemic IIS reductions is due to ageing related effects of reduced IIS outside the nervous system. The lifespan extension and coincident detrimental or neutral effects on locomotor function with a neuron specific reduction (elavGAL4/UAS-InRDN) indicates that reduced IIS is not beneficial to the neural circuitry underlying the behaviours despite increasing lifespan

Egenriether, S. M., Chow, E. S., Krauth, N. and Giebultowicz, J. M. (2015). Accelerated food source location in aging Drosophila. Aging Cell [Epub ahead of print] PubMed ID: 26102220
Summary:
Adequate energy stores are essential for survival, and sophisticated neuroendocrine mechanisms evolved to stimulate foraging in response to nutrient deprivation. Food search behavior is usually investigated in young animals, and it is not known how aging alters this behavior. To address this question in Drosophila melanogaster, the ability to locate food by olfaction was investigated in young and old flies using a food-filled trap. As aging is associated with a decline in motor functions, learning, and memory, it was expected that aged flies would take longer to enter the food trap than their young counterparts. Surprisingly, old flies located food with significantly shorter latency than young ones. Robust food search behavior was associated with significantly lower fat reserves and lower starvation resistance in old flies. Food-finding latency (FFL) was shortened in young wild-type flies that were starved until their fat was depleted but also in heterozygous chico mutants with reduced insulin receptor activity and higher fat deposits. Conversely, food trap entry was delayed in old flies with increased insulin signaling. These results suggest that the difference in FFL between young and old flies is linked to age-dependent differences in metabolic status and may be mediated by reduced insulin signaling.

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
Summary:
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, suggesting there are additional outputs of IIS to mediate aging. 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 use 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. Reproduction and most measures of growth affected by either chico genotype are always independent of d4eBP. In females, chico heterozygotes paradoxically produce more rather than less phosphorylated 4E-BP (p4E-BP). 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.

Naganos, S., Ueno, K., Horiuchi, J. and Saitoe, M. (2016). Learning defects in Drosophila growth restricted chico mutants are caused by attenuated adenylyl cyclase activity. Mol Brain 9: 37. PubMed ID: 27048332
Summary:
Reduced insulin/insulin-like growth factor signaling (IIS) is a major cause of symmetrical intrauterine growth retardation (IUGR), an impairment in cell proliferation during prenatal development that results in global growth defects and mental retardation. In Drosophila, chico encodes the only insulin receptor substrate. The physiological and molecular bases of learning defects caused by chico mutation are not clear. This study found that chico mutations impair memory-associated synaptic plasticity in the mushroom bodies (MBs), neural centers for olfactory learning. Mutations in chico reduce expression of the rutabaga-type adenylyl cyclase (rut), leading to decreased cAMP synthesis in the MBs. Expressing a rut + transgene in the MBs restores memory-associated plasticity and olfactory associative learning in chico mutants, without affecting growth. Thus chico mutations disrupt olfactory learning, at least in part, by reducing cAMP signaling in the MBs. These results suggest that some cognitive defects associated with reduced IIS may occur, independently of developmental defects, from acute reductions in cAMP signaling.

McCormack, S., Yadav, S., Shokal, U., Kenney, E., Cooper, D. and Eleftherianos, I. (2016). The insulin receptor substrate Chico regulates antibacterial immune function in Drosophila. Immun Ageing 13: 15. PubMed ID: 27134635
Summary:
Molecular and genetic studies in model organisms have recently revealed a dynamic interplay between immunity and ageing mechanisms. In Drosophila, inhibition of the insulin signaling pathway prolongs lifespan, and mutations in the insulin receptor substrate Chico extend the survival of mutant flies against certain bacterial pathogens. This study investigated the immune function of chico mutant adult flies against the virulent insect pathogen Photorhabdus luminescens as well as to non-pathogenic E. coli. chico loss-of-function mutant flies were equally able to survive infection by P. luminescens or E. coli compared to their background controls, but they contain fewer numbers of bacterial cells at most time-points after the infection. Analysis of immune signaling activation in flies infected with either bacteria shows reduced transcript levels of antimicrobial peptide genes in the chico mutants than in controls. Evaluation of immune function in infected flies reveals increased phenoloxidase activity and melanization response to P. luminescens and E. coli together with reduced phagocytosis of bacteria in the chico mutants. Changes in the antibacterial immune function in the chico mutants is not due to altered metabolic activity. These results indicate a novel role for chico in the regulation of the antibacterial immune function in D. melanogaster.

McCormack, S., Yadav, S., Shokal, U., Kenney, E., Cooper, D. and Eleftherianos, I. (2016). The insulin receptor substrate Chico regulates antibacterial immune function in Drosophila. Immun Ageing 13: 15. PubMed ID: 27134635
Summary:
Molecular and genetic studies in model organisms have recently revealed a dynamic interplay between immunity and ageing mechanisms. In Drosophila, inhibition of the insulin signaling pathway prolongs lifespan, and mutations in the insulin receptor substrate Chico extend the survival of mutant flies against certain bacterial pathogens. This study investigated the immune function of chico mutant adult flies against the virulent insect pathogen Photorhabdus luminescens as well as to non-pathogenic E. coli. chico loss-of-function mutant flies were equally able to survive infection by P. luminescens or E. coli compared to their background controls, but they contain fewer numbers of bacterial cells at most time-points after the infection. Analysis of immune signaling activation in flies infected with either bacteria shows reduced transcript levels of antimicrobial peptide genes in the chico mutants than in controls. Evaluation of immune function in infected flies reveals increased phenoloxidase activity and melanization response to P. luminescens and E. coli together with reduced phagocytosis of bacteria in the chico mutants. Changes in the antibacterial immune function in the chico mutants is not due to altered metabolic activity. These results indicate a novel role for chico in the regulation of the antibacterial immune function in D. melanogaster.

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).


GENE STRUCTURE

The putative transcriptional start site of chico lies 221 bp 3' from the end of basket encoding DJNK. ME31B, immediately terminating at the 3' end of chico, encodes a DEAD box RNA helicase (Böhni, 1999).

Open reading frame - 3.6 kb

Exons - 9


PROTEIN STRUCTURE

Amino Acids - 967

Structural Domains

The Chico amino acid sequence exhibits the strongest similarity with members of a family of vertebrate insulin receptor substrate proteins known as IRS1-4. Vertebrate IRS family members are characterized by an N-terminal pleckstrin homology (PH) domain, a phosphotyrosine-binding (PTB) domain, and by a number of phosphotyrosine motifs that can serve as docking sites for SH2-containing proteins (for review, see Yenush, 1997). Sequence similarity between Chico and IRS1, IRS2, IRS3 and IRS4 (collectively called IRS1-4) is confined to the N-terminal region including the PH domain and the PTB domain. The amino acid identity is 41% in the PH domain and 38% in the PTB domain. Although there is no significant overall homology within the C-terminal domain, the Chico protein contains several putative SH2-binding motifs characteristic of IRS family members. Two motifs at positions 411 and 641 fit the consensus binding site (YXXM) for the p85/p60 adaptor subunit of P110 PI(3)K, and one (at position 243) corresponds to the consensus (YXN) for GRB2/DRK binding (Böhni, 1999).


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

date revised: 3 August 99

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