InteractiveFly: GeneBrief

Lnk: Biological Overview | References

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 (Ren, 2005). 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 (Wakioka, 1999; Moodie, 1999) 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 (Wakioka, 1999; Liu, 2002). 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 (Riedel, 1997; Maures, 2007; Li, 2007). 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 (Britton, 2002). 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 (Duan, 2004a). 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).

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 (Ohtsuka, 2002). 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 (Duan, 2004a; Li, 2006). 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).

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 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).

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).

In mammals, SH2B1 binds to both the insulin receptor and IRS proteins (Duan, 2004a; Morris, 2009). SH2B1 directly enhances insulin signaling by promoting insulin receptor phosphorylation of IRS proteins and by preventing dephosphorylation of IRS proteins (Morris, 2009). Genetic deletion of SH2B1 results in insulin resistance and type 2 diabetes in mice (Duan, 2004b; Morris, 2009). Deletion of SH2B1 also impairs reproduction (Ohtsuka, 2002). This study shows that dSH2B binds to Chico and promotes 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 are 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).

Similar dwarf phenotypes have been reported in dSH2B null flies (Werz, 2009). The Werz study proposed that dSH2B (dLnk) acts in parallel to Chico, because simultaneous disruption of both dSH2B and Chico are lethal (Werz, 2009). 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 (Werz, 2009). These results are consistent with the proposal that dSH2B and Chico may act in the same pathway(s) downstream of dInR. However, the current results do not exclude the possibility that dSH2B may activate additional Chico-independent pathways (Song, 2010).

It was observed that disruption of dSH2B increases lipid levels and energy conservation in flies; conversely, dSH2B overexpression decreases energy conservation. Moreover, dSH2B overexpression in fat bodies but not neuronal tissues decreases 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 (Jamshidi, 2007; Thorleifsson, 2009; Willer, 2009). 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 decreases oxidative resistance and longevity. In agreement with these observations, Slack have independently reported that dSH2B deficiency increases stress resistance and life span (Slack, 2010). 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. This study observed that neuron-specific but not fat-body-specific overexpression of dSH2B decreases 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 reduces 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 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, this study reports that 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).

Insulin signalling regulates remating in female Drosophila

Mating rate is a major determinant of female lifespan and fitness, and is predicted to optimize at an intermediate level, beyond which superfluous matings are costly. In female Drosophila melanogaster, nutrition is a key regulator of mating rate but the underlying mechanism is unknown. The evolutionarily conserved insulin/insulin-like growth factor-like signalling (IIS) pathway is responsive to nutrition, and regulates development, metabolism, stress resistance, fecundity and lifespan. This study shows that inhibition of IIS, by ablation of Drosophila insulin-like peptide (DILP)-producing median neurosecretory cells, knockout of dilp2, dilp3 or dilp5 genes, expression of a dominant-negative DILP-receptor (InR) transgene or knockout of Lnk, results in reduced female remating rates. IIS-mediated regulation of female remating can occur independent of virgin receptivity, developmental defects, reduced body size or fecundity, and the receipt of the female receptivity-inhibiting male sex peptide. These results provide a likely mechanism by which females match remating rates to the perceived nutritional environment. The findings suggest that longevity-mediating genes could often have pleiotropic effects on remating rate. However, overexpression of the IIS-regulated transcription factor dFOXO in the fat body-which extends lifespan-does not affect remating rate. Thus, long life and reduced remating are not obligatorily coupled (Wigby, 2011).

The effects of IIS on female remating can - at least to some extent - act independently of SP, the major male-derived molecular effector of female receptivity. This finding is consistent with the lack of interaction effects between nutrition and SP on female mating rate found by Fricke (2010). These two major regulators of female remating, IIS and SP, are likely to signal the normal requirement for remating in response to factors that limit female reproduction, namely nutrients required to produce eggs and sperm required for fertilization. This dual mechanism for controlling remating, via IIS and SP, may enable female mating rate to most effectively match reproductive opportunities while avoiding costly superfluous matings (Wigby, 2011).

Females may benefit unconditionally from their first mating as they need to obtain sperm to fertilize eggs. Thus, the lack of effect of IIS on virgin receptivity may be because sexually mature females gain from a rapid first mating - and there is no benefit to delaying mating -- whatever may be the nutritional conditions. However, in D. melanogaster, as in many insects, a single mating fails to provide sufficient sperm to fertilize all the eggs produced over a lifetime, meaning that females must remate to replenish sperm stores. A tighter calibration of nutrition with remating rate may be beneficial following the first mating, because nutrition affects female fecundity and the rate of sperm use such that, under poor nutritional conditions, females will need to replenish stored sperm (i.e. mate) less frequently. Hence, the regulation of female remating receptivity in response to nutritional status is likely to be key for female fitness (Wigby, 2011).

The sexual behaviour of IIS mutant females broadly mimics that of females on a poor diet, which is consistent with the hypothesis that reduced IIS partly (though not wholly) mimics dietary restriction. Like reduced IIS, restriction of dietary nutrients can result in increased lifespan and decreased mating rates. Manipulating components of the IIS pathway, as performed in this study, could generate a mismatch between the perceived and real nutritional environment, resulting in potentially sub-optimal mating rates for a given rate of egg-laying. However, it is clear that there is no obligatory link between egg-laying and mating rate, because females that lack the ability to produce eggs display normal mating and remating behaviours. Moreover, this study shows that females can possess normal fecundity but show reduced mating rates under IIS suppression (Wigby, 2011)

Lifespan can be extended by genetic manipulations that reduce IIS, including several mutants used in this study (MNC-ablated; dilp2 and dilp2-3; InRDN; Lnk). However, lifespan can also be extended by reducing mating frequency. The results therefore highlight the importance of controlling mating rates in studies that investigate the genetics of ageing, to avoid confounding effects of differential sexual activity on lifespan. The discovery that several IIS manipulations that increase lifespan also increase the inter-mating interval raises an important potential confound regarding the conclusions of ageing studies in which flies are maintained in mixed sex groups. Reduced mating rates in experimental mutant lines could potentially confound ageing studies because females might live longer owing to reduced mating rates rather than as a direct effect of the genetic manipulations themselves. The solution to this potential confound is to control mating rates in lifespan studies in order to test for direct effects on lifespan. However, the results from the dFOXO experiment show that it is also possible to uncouple the regulation of female sexual behaviour and the regulation of lifespan, in accordance with the uncoupling of lifespan and fecundity. Thus, both behavioural and physiological aspects of reproduction can be uncoupled from lifespan extension under certain conditions (Wigby, 2011).

The effects of single dilp mutants on remating were, surprisingly, only marginally weaker than the effects of MNC ablation or dilp2-3 double mutants, despite the apparently weaker genetic intervention. However, ablation of the MNCs is incomplete, and DILP levels are reduced rather than abolished in the flies that were used. Moreover, there is compensation and synergism between DILPs such that knockouts of single dilp genes can affect the expression of one or more of the other dilps. For example, dilp2 and dilp2-3 mutant flies exhibit increased expression of dilp5, while dilp3 mutants exhibit reduced levels of dilp2 and dilp5 expression. Such effects could explain the relatively strong phenotypes of the single dilp knockouts in comparison with the dilp2-3 knockout and MNC-ablated females (Wigby, 2011).

The extracellular DILPs, the InR and the intracellular IIS component, Lnk, all regulate female remating rate, but it is currently unclear which downstream molecules are involved. A major downstream target of the IIS pathway is the transcription factor dFOXO, but no effect of fat body dFOXO expression was found on female mating. One possibility is that dFOXO mediates the effect of reduced IIS on remating rates in tissues other than the fat body. Another possibility is that the effect of IIS on remating rate occurs via the target of rapamycin (TOR) pathway. The TOR pathway senses amino acids and runs parallel to, and interacts with, IIS. The IIS and TOR pathways interact to control growth, and TOR signalling, like IIS, has been shown to regulate lifespan. Moreover, recent work shows that the TOR pathway is involved in mating-induced changes in diet choice, supporting the idea that TOR functions in the coordination of behavioural responses to mating and the nutritional environment. It will be important to investigate the mating behaviour of TOR-pathway mutants to determine whether this pathway is involved in the regulation of mating and whether the effects of IIS on female remating are mediated through TOR signalling. It will also be important to determine through which tissues IIS regulates remating (Wigby, 2011).

This work shows that components of the IIS pathway modulate sexual behaviour by significantly altering the receptivity of mated female D. melanogaster. Thus, a likely molecular basis is provided for the link between nutrition and sexual behaviour in insects, which is an important step in understanding the mechanisms underlying life-history traits and trade-offs. Reproduction and nutrition are linked across a broad range of taxa, including mammals, and many of the effects of IIS (e.g. on lifespan and fecundity) are highly evolutionarily conserved. It is concluded that the regulation of mating behaviour via IIS could be common among animals (Wigby, 2011).

Modulation of gurken translation by insulin and TOR signaling in Drosophila

Localized Gurken (Grk) translation specifies the anterior-posterior and dorsal-ventral axes of the developing Drosophila oocyte; spindle-class females lay ventralized eggs resulting from inefficient grk translation. This phenotype is thought to result from inhibition of the Vasa RNA helicase. In a screen for modifiers of the eggshell phenotype in spn-B flies, a mutation was identified in the lnk gene. lnk mutations restore Grk expression but do not suppress the persistence of double-strand breaks nor other spn-B phenotypes. This suppression does not affect Egfr directly, but rather overcomes the translational block of grk messages seen in spindle mutants. Lnk was recently identified as a component of the insulin/insulin-like growth factor signaling (IIS) and TOR pathway. Interestingly, direct inhibition of TOR with rapamycin in spn-B or vas mutant mothers can also suppress the ventralized eggshell phenotype. When dietary protein is inadequate, reduced IIS-TOR activity inhibits cap-dependent translation by promoting the activity of the translation inhibitor eIF4E-binding protein (4EBP). It is hypothesized that reduced TOR activity promotes grk translation independent of the canonical Vasa- and cap-dependent mechanism. This model might explain how flies can maintain the translation of developmentally important transcripts during periods of nutrient limitation when bulk cap-dependent translation is repressed (Ferguson, 2012).

Reproduction represents a substantial energy investment for an organism. Many studies have shown that ovarian physiology is exquisitely sensitive to nutritional status. Limitation of dietary protein intake results in a dramatic slowing of egg chamber maturation via developmental arrest, programmed cell death, or loss of germline stem cells. Several signaling pathways are integrated to bring about this response including 20-hydroxyecdysone, Juvenile Hormone (JH), and insulin/insulin-like signaling (IIS). IIS is stimulated by protein feeding and is required for oogenesis to progress. The IIS pathway integrates nutritional signals at two distinct points during oogenesis. The first is in region 2A of the germarium where developing germline cysts undergo apoptosis in the absence of a source of maternal dietary protein. The second point of nutritional control is at stage 8 of oogenesis during the onset of vitellogenesis. In the absence of food, egg chambers develop to stage 8, where they are arrested until a favorable food source is located. These two checkpoints represent points at which the energetically expensive process of oogenesis can be halted if insufficient resources are available (Ferguson, 2012 and references therein).

The IIS pathway elicits its effect on Drosophila physiology through several effector pathways, namely the dFOXO transcription factor and the Target of Rapamycin kinase (TOR). IIS inhibits dFOXO activity by promoting its phosphorylation by PKB/Akt and subsequent exclusion from the nucleus. Starvation or mutations in the insulin pathway allow dFOXO to translocate to the nucleus where it directs the transcription of genes that promote longevity, stress resistance, fat storage, and growth attenuation. TOR activity is stimulated by both IIS through the dRheb GTPase and by amino acids via Rag GTPases. When nutrients are plentiful, high TOR activity stimulates the translation of mRNA by phosphorylating S6K which in turn phosphorylates eIF4B and promotes its interaction with eIF3. These steps are critical for recruiting the translation preinitiation complex (PIC) to the m7G cap at the 5’ end of the mRNA. Once bound, the PIC recruits the small ribosomal subunit and proceeds to scan the transcript for an initiating AUG codon. This process requires the activity of the eIF4A RNA helicase. TOR also phosphorylates and inactivates the inhibitory eIF4E binding protein, 4EBP. Starvation inhibits cap- dependent translation through reduced TOR activity. When nutrients are limiting and TOR activity is low, eIF4B is not phosphorylated and can no longer participate in PIC assembly, furthermore 4EBP inhibition is lifted and it proceeds to inhibit cap-recognition by eIF4E. Both activities have the effect of strongly blocking cap-dependent translation initiation when nutrients are scarce. A select few transcripts escape this translational block by upregulating the utilization of an alternative mechanism that relies on an Internal Ribosomal Entry Site (IRES) that obviates the requirement for cap recognition and start codon scanning. The list of transcripts that contain IRES sequences is growing and includes numerous growth factors such as VEGF-A , PDGF2, and IGF-II. A prominent example of IRES-mediated nutritional adaptation is the Drosophila insulin receptor dInR, the translation of which is upregulated in response to starvation as a way to sensitize the cell to insulin when nutrients become available (Ferguson, 2012 and references therein).

Control of translation is vitally important to developmental patterning. The transcripts of many morphogens, including nanos, oskar, and gurken, are co-transcriptionally packaged into silencing particles and transported in a translationally quiescent form. Once localized, this repression is alleviated and translation proceeds in the developmentally appropriate locale. Gurken (Grk) is a TGF-α related ligand for the Drosophila Egfr. Localized translation of the spatially restricted grk transcript results in signaling by germline-derived Grk to the Egfr in the overlying follicle cells. This signal is required to specify the posterior fate in early oogenesis and the dorsal fate during mid oogenesis. Mutations that reduce grk translation are female sterile due to an inability to correctly pattern the developing oocyte and result in concomitant patterning defects in the embryo. grk translation requires the eIF4A-related DEAD-box helicase Vasa (Vas). Mutations in vas are female sterile owing to a failure to specify dorsal structures in the egg shell or posterior structures in the embryo (Ferguson, 2012 and references therein).

Spindle class genes are responsible for repairing DNA double strand breaks (DSBs) that are induced during homologous recombination in Drosophila oogenesis. In wild type females, DSBs are induced in germ line cells entering pachytene in region 2A of the germarium. This process is initiated by the Spo11 homologue Mei-W68 and Mei-P22, a protein that aids in break site selection. These breaks are then repaired by homologous recombination, a process that requires the RAD-51 homologue spindle-B (spn-B). Mutations in spn-B result in an accumulation of unrepaired DSBs that lead to activation of a meiotic checkpoint. The checkpoint is comprised of the ATR homologue mei-41 and the downstream kinase chk-2. Persistent DSBs in spn-B^BU females activate the checkpoint that requires the Mei-41 and Chk2 kinases and leads to inefficient grk translation and ventralized eggshell phenotypes. Checkpoint activation also results in phosphorylation of Vasa, a modification that is thought to inhibit its function. Early in oogenesis, the oocyte nucleus becomes arrested in pachytene and forms a compact structure called the karyosome. The formation of the karyosome is disrupted in spindle-class mutants where the chromatin appears fractured or ellipsoid. Weak grk translation and an inability to properly form the karyosome are both spindle phenotypes that are consistent with reduced Vasa activity (Ferguson, 2012).

This study has identified the SH2B family adaptor gene lnk in a genetic screen for modifiers of the ventralized eggshell phenotype seen in spn-B^BU mutant flies. SH2B proteins are known to regulate intracellular signaling by membrane bound receptor tyrosine kinases (RTKs). SH2Bs can promote signaling by scaffolding downstream effectors to the RTK or mediate proteosomal receptor destruction by recruiting the Cbl ubiquitin ligase. Lnk was recently identified as a positive regulator of the Insulin/Insulin-like Signaling (IIS) pathway that functions at the level of the insulin receptor substrate Chico. This study shows that lnk mutations can promote grk translation and suppress the ventralized eggshell phenotype in a spn-B^BU mutant background. This suppression occurs independent of Vasa activity and does not suppress the karyosome phenotype. No genetic interactions were found with a weak grk allele nor downstream targets of Egfr suggesting that lnk-mediated suppression of spindle phenotypes does not occur by directly modulating Egfr activity. The data suggest that lnk mutations promote grk translation by inhibiting TOR activity as Rapamycin feeding experiments can also suppress the eggshell phenotype of spn-B and vas mutant flies. A model is proposed in which reduced IIS/TOR signaling inhibits cap-dependent translation and promotes utilization of an alternative translation initiation mechanism of the grk mRNA. This mechanism enables flies to faithfully pattern their oocytes when nutrients are scarce (Ferguson, 2012).

This study demonstrates a novel interaction between a meiotic checkpoint, the insulin/insulin- like signaling pathway, and translation of gurken mRNA in Drosophila oogenesis. Mutations in meiotic DNA repair enzymes such as spn-B result in persistent DSBs in early oogenesis that activate an ATR- Chk2-dependent meiotic checkpoint. Checkpoint activation results in phosphorylation of the eIF4A-like RNA helicase Vasa, the activity of which is important for grk translation. In these mutants, low levels of Grk protein are synthesized which is insufficient to pattern the eggshell correctly and results in ventralized eggs. Using forward genetics, an allele was isolated of the insulin receptor adapter, lnk. This mutation can suppress the weak grk translation phenotype and restore normal patterning to eggs laid by spn-B^BU flies. Clonal analysis has shown that lnk mutations reduce IIS in a cell-autonomous manner in the ovary. As in mammals, Drosophila IIS controls the rate of cap-dependent translation initiation in the cell by regulating the activity of the TOR kinase. Rapamycin inhibits TOR activity, and feeding rapamycin can suppress the ventralized eggshell phenotype not only in spn-B^BU females, but also in vasa^PH165 / vas^aRG53 flies. These data suggest an alternative translation initiation mechanism for the grk mRNA by which flies can maintain D/V axis patterning in times of moderate nutrient limitation (Ferguson, 2012).

The discovery that mutations in lnk, a positive regulator of IIS, can suppress the patterning defects in spn-B flies was initially surprising. The eggshell phenotypes of the different genotypes were assessed after keeping the flies on apple or grape juice agar plates on which abundant amounts of yeast paste had been added thus allowing the females to eat a very protein rich diet. A protein rich diet stimulates the activity of the TOR kinase via two mechanisms. Insulin-like peptides (dilps) are secreted into the hemolymph by neuroendocrine cells in response to nutrient availability. This in turn activates the IIS cascade comprised of Chico/Lnk, PI3K, Akt, Tsc1/2, and Rheb which promotes TOR-C1 activity. The second mechanism acts more directly through the levels of intracellular amino acids that are imported in part by the slimfast and pathetic transporters. Both of these mechanisms stimulate TOR-C1 activity which has been shown to promote cap-dependent translation by inhibiting 4EBP sequestration of eIF4E. Therefore, reducing TOR activity either by a mutation in lnk or by addition of rapamycin, would be expected to interfere with cap-dependent translation and therefore further enhance the mutant phenotype. However, in spn-B mutant flies, cap-dependent translation is already inhibited by the activity of the checkpoint, presumably acting via Vasa modification. The fact that a suppression of the ventralized phenotype was observed in lnk mutants indicates that reduction in TOR signaling must activate a second mode of translation that allows Gurken protein to be produced independently of the block in cap-dependent translation (Ferguson, 2012).

Several ovarian phenotypes are shared between mutations in spindle genes and vas mutants, including failure to form a compact karyosome, very weak grk translation, and ventralized eggs. Combined with the reproducible phosphorylation of Vas protein in spindle-class mutants, these phenotypes are consistent with a defect in Vas activity. While the specific effect of this phosphorylation is unknown, Vas serves several functions in cap-dependent translation initiation of grk mRNA. Vasa has been shown to interact with eIF5B and mutations that interfere with this interaction inhibit grk translation. This interaction is thought to facilitate assembly of the 60S ribosomal subunit at the AUG start codon. Furthermore, as a DEAD-box RNA helicase, Vasa may permit the pre-initiation complex to scan the 5’ UTR of grk and negotiate secondary structures that may impede the progress of this complex. IRES sequences adopt strong secondary structures in the 5’ UTR of RNAs that they regulate. If it can be demonstrated in the future that grk possess an IRES sequence, this may explain the requirement for Vasa helicase activity to unwind this structure when translation is initiated from the 5'cap during conditions of adequate nutrient availability. Whether the checkpoint dependent phosphorylation of Vas affects its stability, RNA helicase activity, or its eIF5B interaction, the expected result is a block in cap-dependent translation initiation of grk mRNA and concomitant D/V patterning defects. The observation that grk translation can be induced to occur in spn-B^BU and in vasa^PH165 / vas^aRG53 flies indicates that an alternative mechanism for supporting translation initiation is taking place. Because reduced IIS and TOR activity both block bulk cap-dependent translation initiation through sequestration of eIF4E by 4EBP, yet stimulate IRES activity, it is proposed that the latter may provide an explanation for the results (Ferguson, 2012).

Grk plays a central role in shaping the development of the egg and subsequent embryo. Mutations that disrupt Grk / Egfr signaling during oogenesis result in female sterility. Blocking the translation of this essential morphogen in spindle class mutants that are unable to repair DNA damage is an effective mechanism to prevent the transmission of mutations to the progeny. This reproductive checkpoint is effective when nutrients are abundant, however as this study has demonstrated, the strategy breaks down when IIS/TOR activity is low. Under these conditions, grk can be translated and result in eggs that are patterned correctly, even though the DNA damage and karyosome malformation phenotypes persist. It is proposed that this difference occurs because the DNA-damage checkpoint can only impinge on one of the two mechanisms by which grk translation can be initiated (Ferguson, 2012).

One mechanism by which suppression of the D/V patterning defects of spn-B^BU may occur is through the effects of the additional time that lnk^CR642 egg chambers spend completing oogenesis. While Grk production is reduced in spn-B^BU flies, it is not completely blocked and some Grk protein is made. If the reduced rate of Grk production is integrated over the extended time spent during mid oogenesis, sufficient Grk levels could accumulate and support normal D/V patterning. However, this model is inconsistent with the inability of lnk^CR642 to suppress the ventralized eggs laid by grk^ED22 females. These flies do retain some Grk activity as is evident by the single appendage that is specified, however if the mechanism of suppression were via accumulation, then grk^ED22 should be suppressed by lnk mutations. Therefore, the IRES-dependent model proposed in this study is favored (Ferguson, 2012).

The selective pressure that may have driven the evolution of this bi-modal translation mechanism for grk can be best understood by considering that in wild populations of Drosophila, females feed and oviposit at locations where yeast is abundant. This behavior ensures adequate nutrition to support oogenesis in the female as well as for the developing larvae. If however nutrients become scarce, females adjust the rate of oogenesis to match nutrient availability. In response to complete starvation, egg chambers undergo apoptosis and are reabsorbed, however moderate reductions in IIS slow the rate of oogenesis until an abundant protein source is found. The conserved response to dietary restriction is to repress cap- dependent translation of most cellular transcripts while a select population of RNAs that are essential for survival escape this repression by utilizing a cap-independent IRES mechanism. It is posited that grk may be one such transcript. Oocytes that are in mid development when nutrients are scarce must still be patterned appropriately so that the resulting eggs are fertile. IRES activity may facilitate Grk expression to maintain normal D/V patterning in times of lean whereas when nutrients are abundant, cap-dependent translation predominates (Ferguson, 2012).

Search PubMed for articles about Drosophila Lnk

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

Britton, J. S., Lockwood, W. K., Li, L., Cohen, S. M. and Edgar, B. A. (2002). Drosophila's insulin/PI3-kinase pathway coordinates cellular metabolism with nutritional conditions. Dev. Cell 2: 239-249. PubMed ID: 11832249

Duan, C., Li, M. and Rui, L. (2004a). SH2-B promotes insulin receptor substrate 1 (IRS1)- and IRS2-mediated activation of the phosphatidylinositol 3-kinase pathway in response to leptin. J. Biol. Chem. 279: 43684-43691. PubMed ID: 15316008

Duan, c., et al. (2004b). Disruption of the SH2-B gene causes age-dependent insulin resistance and glucose intolerance. Mol. Cell. Biol. 24: 7435-7443. PubMed ID: 15314154

Fricke C., Bretman A. and Chapman T. (2010). Female nutritional status determines the magnitude and sign of responses to a male ejaculate signal in Drosophila melanogaster. J. Evol. Biol. 23: 157-165. PubMed ID: 19888937

Ferguson, S. B., Blundon, M. A., Klovstad, M. S. and Schüpbach, T. (2012). Modulation of gurken translation by insulin and TOR signaling in Drosophila. J. Cell Sci. 125(Pt 6): 1407-19. PubMed ID: 22328499

Huang, X., et al. (1995). Cloning and characterization of Lnk, a signal transduction protein that links T-cell receptor activation signal to phospholipase C gamma 1, Grb2, and phosphatidylinositol 3-kinase. Proc. Natl. Acad. Sci. 92: 11618-11622. PubMed ID: 8524815

Jamshidi, Y., et al. (2007). The SH2B gene is associated with serum leptin and body fat in normal female twins. Obesity (Silver Spring) 15: 5-9. PubMed ID: 17228025

Li, M., Li, Z., Morris, D. L. and Rui, L. (2007) Identification of SH2B2beta as an inhibitor for SH2B1- and SH2B2alpha-promoted Janus kinase-2 activation and insulin signaling. Endocrinology 148: 1615-1621. PubMed ID: 17204555

Liu, J., Kimura, A., Baumann, C. A. and Saltiel, A. R. (2002). APS facilitates c-Cbl tyrosine phosphorylation and GLUT4 translocation in response to insulin in 3T3-L1 adipocytes. Mol. Cell. Biol. 22: 3599-3609. PubMed ID: 11997497

Maures, T. J., Kurzer, J. H. and Carter-Su, C. (2007). SH2B1 (SH2-B) and JAK2: a multifunctional adaptor protein and kinase made for each other. Trends Endocrinol. Metab. 18: 38-45. PubMed ID: 17140804

Moodie, S. A., Alleman-Sposeto, J. and Gustafson, T. A. (1999). Identification of the APS protein as a novel insulin receptor substrate. J. Biol. Chem. 274: 11186-11193. PubMed ID: 10196204

Morris, D. L., et al. (2009). SH2B1 enhances insulin sensitivity by both stimulating the insulin receptor and inhibiting tyrosine dephosphorylation of insulin receptor substrate proteins. Diabetes 58: 2039-2047. PubMed ID: 19542202

Ohtsuka, S., et al. (2002). SH2-B is required for both male and female reproduction. Mol. Cell. Biol. 22: 3066-3077. PubMed ID: 11940664

Ren, D., Li, M., Duan, C. and Rui, L. (2005). Identification of SH2-B as a key regulator of leptin sensitivity, energy balance, and body weight in mice. Cell Metab. 2: 95-104. PubMed ID: 16098827

Riedel, H., Wang, J., Hansen, H. and Yousaf, N. (1997). PSM, an insulin-dependent, pro-rich, PH, SH2 domain containing partner of the insulin receptor. J. Biochem. 122: 1105-1113. PubMed ID: 9498552

Rui, L., et al. (1997). Identification of SH2-Bbeta as a substrate of the tyrosine kinase JAK2 involved in growth hormone signaling. Mol. Cell. Biol. 17: 6633-6644. PubMed ID: 9343427

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

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

Thorleifsson, G., et al. (2009) Genome-wide association yields new sequence variants at seven loci that associate with measures of obesity. Nat. Genet. 41: 18-24. PubMed ID: 19079260

Wakioka, T., et al. (1999). APS, an adaptor protein containing Pleckstrin homology (PH) and Src homology-2 (SH2) domains inhibits the JAK-STAT pathway in collaboration with c-Cbl. Leukemia 13: 760-767. PubMed ID: 10374881

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 ID: 19680438

Wigby, S., et al. (2011). Insulin signalling regulates remating in female Drosophila. Proc. Biol. Sci. 278(1704): 424-31. PubMed ID: 20739318

Willer, C. J., et al. (2009). Six new loci associated with body mass index highlight a neuronal influence on body weight regulation. Nat. Genet. 41: 25-34. PubMed ID: 19079261

Yokouchi, M., et al. (1997). Cloning and characterization of APS, an adaptor molecule containing PH and SH2 domains that is tyrosine phosphorylated upon B-cell receptor stimulation. Oncogene 15: 7-15. PubMed ID: 9233773

date revised: 20 August 2012

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