chico
Chimeric receptors encoding either the whole or a portion of the cytoplasmic domain of the Drosophila Insulin-like receptor (InR) with the extracellular domain of the human insulin receptor (IR) were expressed either transiently in COS cells or stably in Chinese hamster ovary cells and compared with the wild-type human IR. All three receptors bind insulin equally and exhibit an insulin-activated tyrosine kinase activity. The ability of the Drosophila cytoplasmic domain to mediate the tyrosine phosphorylation of insulin receptor substrate 1, stimulate cell proliferation, and activate MAP kinase is indistinguishable from that of the human IR. The chimeric Drosophila receptors do not bind more phosphatidylinositol 3-kinase (see Phosphotidylinositol 3 kinase 92E) than the human IR, despite containing a C-terminal extension with potential tyrosine phosphorylation sites in the motif recognized by the SH2 domain of this enzyme. Thus, the essential signal-transducing abilities of the IR appear to have been conserved from invertebrates to mammals, despite the considerable differences in the sequences of these receptors (Yamaguchi, 1995).
Drosophila contain an insulin receptor homolog, encoded by the InR gene located at position 93E4-5 on the third chromosome. The receptor protein is strikingly homologous to the human receptor, exhibiting the same alpha2beta2 subunit structure and containing a ligand-activated tyrosine kinase in its cytoplasmic domain. Chemical mutagenesis was used to induce mutations in the inr gene. Six independent mutations that lead to a loss of expression or function of the receptor protein were identified. These mutations are recessive, embryonic, or early larval lethals, but some alleles exhibit heteroallelic complementation to yield adults with a severe developmental delay (10 days), growth-deficiency, female-sterile phenotype. Interestingly, the severity of the mutant phenotype correlates with biochemical measures of loss of function of the receptor tyrosine kinase. The growth deficiency appears to be due to a reduction in cell number, suggesting a role for InR in regulation of cell proliferation during development. The phenotype is reminiscent of those seen in syndromes of insulin-resistance or IGF-I and IGF-I receptor deficiencies in higher organisms, suggesting a conserved function for this growth factor family in the regulation of growth and body size (Chen, 1996).
The cloning and primary structure of the Drosophila insulin receptor gene (InR) is reported, along with functional expression of the predicted polypeptide, and the isolation of mutations in the InR locus. The structure and processing of the Drosophila insulin proreceptor are somewhat different from those of the mammalian insulin and IGF 1 receptor precursors. The InR proreceptor (M(r) 280 kDa) is processed proteolytically to generate an insulin-binding alpha subunit (M(r) 120 kDa) and a beta subunit (M(r) 170 kDa) with protein tyrosine kinase domain. The InR beta 170 subunit contains a novel domain at the carboxyterminal side of the tyrosine kinase, in the form of a 60 kDa extension that contains multiple potential tyrosine autophosphorylation sites. This 60 kDa C-terminal domain undergoes cell-specific proteolytic cleavage that leads to the generation of a total of four polypeptides (alpha 120, beta 170, beta 90, and a free 60 kDa C-terminus) from the inr gene. These subunits assemble into mature InR receptors with the structures alpha 2(beta 170)2 or alpha 2(beta 90)2. Mammalian insulin stimulates tyrosine phosphorylation for both types of beta subunits; in turn, the phosphorylation allows the beta 170, but not the beta 90 subunit, to bind directly to p85 SH2 domains of PI-3 kinase. It is likely that the two different isoforms of InR have different signaling potentials. Loss of function mutations in the InR gene, induced by either a P-element insertion occurring within the predicted ORF, or by ethylmethane sulfonate treatment, renders pleiotropic recessive phenotypes that lead to embryonic lethality. The activity of InR appears to be required in the embryonic epidermis and nervous system among organ systems, since development of the cuticle, as well as the peripheral and central nervous systems are affected by InR mutations (Fernandez, 1995).
The Drosophila insulin receptor (InR) contains a 368-amino-acid COOH-terminal extension that contains several tyrosine phosphorylation sites in YXXM motifs. This extension is absent from the human insulin receptor but resembles a region in insulin receptor substrate (IRS) proteins that binds to the phosphatidylinositol (PI) 3-kinase and mediates mitogenesis. The function of a chimeric InR containing the human insulin receptor binding domain (hDIR) was investigated in 32D cells, which contain few insulin receptors and no IRS proteins. Insulin stimulated tyrosine autophosphorylation of the human insulin receptor and hDIR, and both receptors mediate tyrosine phosphorylation of Shc and activate mitogen-activated protein kinase. IRS-1 is required by the human insulin receptor to activate PI 3-kinase and p70s6k (see Drosophila RPS6-p70-protein kinase), whereas hDIR associate with PI 3-kinase and activates p70s6k without IRS-1. However, both receptors required IRS-1 to mediate insulin-stimulated mitogenesis. These data demonstrate that the InR possesses additional signaling capabilities when compared with its mammalian counterpart but still requires IRS-1 for the complete insulin response in mammalian cells (Yenush, 1996a).
Like the mammalian insulin receptor, the Drosophila insulin receptor (INR)1 is a
tetramer formed by two alpha subunits and two beta subunits. INR alpha and
beta subunits are synthesized together as a proreceptor precursor, proteolytically processed, and linked together by disulfide bonds. The alpha subunits, with a molecular mass of 110-120 kDa, are
extracellular and contain the ligand binding domains that are capable
of binding mammalian insulin with a Kd of 15 nM. The beta subunits traverse the plasma membrane and have an insulin-stimulated tyrosine kinase in the cytoplasmic portion. DNA sequence analysis and expression of the INR beta subunit
in mammalian and Drosophila cells indicate that the
INR beta subunit is larger than its mammalian homolog and exhibits an
apparent molecular mass of ~180 kDa. The increased mass is due to the
presence of a 400-amino acid carboxyl-terminal extension. However,
the majority of INR beta subunits are processed to 92/102-kDa forms in Drosophila embyros and some cell lines, the difference being
due to proteolytic cleavage of the carboxyl-terminal extension.
Both truncated and full-length beta subunits are autophosphorylated on
tyrosine residues in response to insulin binding (Marin-Hincapie, 1999 and references therein).
The 400-amino acid carboxyl-terminal extension of the beta INR contains
clusters of motifs known to be involved in the interaction with SH2 and
PTB domain-containing proteins, suggesting a role for this domain
in signaling through interaction with other signaling molecules.
Interestingly, four tyrosines are found in 'hybrid' amino acid
motifs in which residues amino-terminal to each tyrosine form the motif
NP X Y, resembling known PTB domain binding sites, and
residues carboxyl-terminal to the same tyrosines form the motifs
YXXM, YMXM, or YXLLD -- all known to be
involved in binding to SH2 domains. Thus, tyrosines 1993 and 2030 appear in the motif
SXNPXYXX M;
tyrosine 2009 is part of
S X NPXYMXM, and tyrosine
1969 appears in the sequence SDNPXYRLLD.
Whether these motifs serve to bind SH2 or PTB domain-containing
proteins upon tyrosine phosphorylation and whether one is preferred
over the other is not clear. The cytoplasmic domain of the INR
expressed in cells lacking IRS-1 has been shown to bind PI3-kinase.
However, a similar construct expressed in Chinese hamster ovary cells
that contain IRS-1 fails to do so. Since a significant percentage
of the INR beta subunit undergoes tissue- or stage-specific proteolytic
processing in Drosophila embryos to remove the
carboxyl-terminal extension and once it is removed it appears not
to be phosphorylated, its role in signal transduction by the INR is
not clear. Therefore, the signaling capacity conferred by the beta INR
carboxyl-terminal extension has been explored by
expressing either full-length or truncated INR beta subunit forms in
mammalian cells and determining the effect on protein-protein
interactions and cell growth (Marin-Hincapie, 1999 and references therein).
In order to explore the role of the 400 AA extention in INR function, mammalian
expression vectors encoding either the complete INR beta subunit (beta-Myc) or the INR beta subunit without the carboxyl-terminal extension (betaDelta) were
constructed, and the membrane-bound beta subunits were expressed in 293 and Madin-Darby canine kidney (MDCK) cells in the absence of the ligand-binding alpha
subunits. beta-Myc and betaDelta proteins are constitutively active tyrosine kinases of 180 and 102 kDa, respectively. INR beta-Myc co-immunoprecipitates a
phosphoprotein of 170 kDa identified as insulin receptor substrate-1 (IRS-1, Flipper or Chico), whereas INR betaDelta does not, suggesting that the site of interaction is within the
carboxyl-terminal extension. IRS-1 is phosphorylated on tyrosine to a much greater extent in cells expressing INR beta-Myc than in parental or INR betaDelta
cells. Despite this, a variety of PTB or SH2 domain-containing signaling proteins, including IRS-2, mSos-1, Shc, p85 subunit of phosphatidylinositol 3-kinase,
SHP-2, Raf-1, and JAK2, are not associated with the INR beta-Myc.IRS-1 complex. Overexpression of INR beta-Myc and betaDelta kinases confers an
equivalent increase in cell proliferation in both 293 and Madin-Darby canine kidney cells, indicating that this growth response is independent of the carboxyl-terminal
extension. However, INR beta-Myc-expressing cells exhibit enhanced survival, relative to parental and betaDelta cells, suggesting that the carboxyl-terminal
extension, through its interaction with IRS-1, plays a role in the regulation of cell death (Marin-Hincapie, 1999).
Thus, overexpression of constitutively active INR beta and betaDelta receptors in
293 and MDCK cells promotes cell proliferation, indicating that the INR
can engage the mammalian proliferation pathways. The equivalent
proliferative responses induced by INR beta-Myc and betaDelta kinases
suggests that the growth-promoting function of the INR in these cells
is independent of the carboxyl-terminal extension. In contrast, cells
expressing the full-length INR beta subunit exhibit significantly
enhanced survival as compared with cells expressing the betaDelta INR.
Relative to the parental 293 and MDCK cells, the INR beta-Myc and betaDelta
proteins confer somewhat different behavior; beta-Myc clearly
promotes survival in 293 cells, whereas betaDelta more dramatically accelerates cell death in MDCK cells. Nonetheless, a clear difference in the behavior of cells expressing the full-length or truncated INR
beta subunits is evident in both backgrounds. Despite the presence of a
juxtamembrane NPXY motif predicted to interact with IRS-1 in
both beta-Myc and betaDelta proteins, IRS-1 is not highly phosphorylated in
betaDelta cells. This suggests that the
carboxyl-terminal extension of the INR beta subunit is required for
sustained association and phosphorylation of IRS-1. This persistent
IRS-1 phosphorylation distinguishes beta-Myc from betaDelta cells and may
be of primary importance in promoting cell survival. Without this
sustained interaction, cell death may actually be accelerated, as
observed in MDCK cells transfected with the INR betaDelta kinase (Marin-Hincapie, 1999).
IRS-1 that is bound to the INR beta subunit is phosphorylated on
tyrosine; however, no evidence has been found for increased association of
PI3-kinase or other candidate signaling molecules with this complex.
Therefore, the mechanism whereby this association leads to increased cell
survival is unclear at present. Interestingly, a recent report
demonstrates that expression of a truncated IRS-1 containing only the
pleckstrin homology and phosphotyrosine binding domains, without any
tyrosine phosphorylation sites, mediates PI3-kinase and
phosphotyrosine-independent signals that contribute to the regulation
of cell survival and apoptosis. IRS-1 that is bound to the
carboxyl-terminal extension of INR in 293 and MDCK cells may have
similarly activated pathways that promote cell survival in the absence
of PI3-kinase activation (Marin-Hincapie, 1999 and references therein).
Thus, two isoforms of an activated INR beta subunit have been expressed
in mammalian cells, and a functional difference between them has been
demonstrated. The data presented here indicate that the stimulation of
cell proliferation by INR is mediated by the kinase domain independent
of the carboxyl-terminal extension. In contrast, the carboxyl-terminal
extension mediates an interaction with IRS-1 and influences cell
survival. Since an IRS homolog is present in Drosophila, this may reflect an inherent function of the INR which, in flies,
is modulated by tissue- or stage-specific processing of the receptor.
These data also suggest that in mammalian cells, persistent localization of IRS-1 to membranes via the interaction of IRS-1 with receptors and/or persistent tyrosine phosphorylation generates signals
independent of association with PI3-kinase (Marin-Hincapie, 1999 and references therein).
Insulin receptor substrate (IRS) proteins are phosphorylated by multiple
tyrosine kinases, including the insulin receptor. Phosphorylated IRS proteins
bind to SH2 domain-containing proteins, thereby triggering downstream signaling
pathways. The Drosophila insulin receptor (InR) C-terminal extension contains
potential binding sites for signaling molecules, suggesting that InR might not
require an IRS protein to accomplish its signaling functions. However, a cDNA encoding Drosophila IRS (Chico, but referred to in this study as dIRS) has been obtained and one for Chico in a Drosophila cell line has also been demonstrated. Like mammalian IRS proteins, the N-terminal
portion of Chico contains a pleckstrin homology domain and a phosphotyrosine
binding domain that binds to phosphotyrosine residues in both human and
Drosophila insulin receptors. When coexpressed with Chico in COS-7 cells, a
chimeric receptor (the extracellular domain of human IR fused to the cytoplasmic
domain of InR) mediates the insulin-stimulated tyrosine phosphorylation of Chico.
Mutating the juxtamembrane NPXY motif markedly reduces the ability of the
receptor to phosphorylate Chico. In contrast, the NPXY motifs in the C-terminal
extension of InR are required for stable association with Chico.
Coimmunoprecipitation experiments demonstrate insulin-dependent binding of Chico
to phosphatidylinositol 3-kinase and SHP2. However,
interactions with Grb2, SHC, or phospholipase C-gamma were not detected. Taken together with
published genetic studies, these biochemical data support the hypothesis that
Chico functions directly downstream from the insulin receptor in Drosophila (Poltilove, 2000).
Protein complexes have largely been studied by immunoaffinity purification and (mass spectrometric) analysis. Although this approach has been widely and successfully used it is limited because it has difficulties reliably discriminating true from false protein complex components, identifying post-translational modifications, and detecting quantitative changes in complex composition or state of modification of complex components. A protocol has been developed that enables determination, in a single LC-MALDI-TOF/TOF analysis, the true protein constituents of a complex, to detect changes in the complex composition, and to localize phosphorylation sites and estimate their respective stoichiometry. The method is based on the combination of fourplex iTRAQ (isobaric tags for relative and absolute quantification) isobaric labeling and protein phosphatase treatment of substrates. It was evaluated on model peptides and proteins and on the complex Ccl1-Kin28-Tfb3 isolated by tandem affinity purification from yeast cells. The two known phosphosites in Kin28 and Tfb3 could be reproducibly shown to be fully modified. The protocol was then applied to the analysis of samples immunopurified from Drosophila melanogaster cells expressing an epitope-tagged form of the insulin receptor substrate homologue Chico. These experiments allowed identification 14-3-3ε;, 14-3-3zeta, and the insulin receptor as specific Chico interactors. In a further experiment, the immunopurified materials obtained from tagged Chico-expressing cells that were either treated with insulin or left unstimulated were cmpared. This analysis showed that hormone stimulation increases the association of 14-3-3 proteins with Chico and modulates several phosphorylation sites of the bait, some of which are located within predicted recognition motives of 14-3-3 proteins (Pflieger, 2008: Full text of article).
The two 14-3-3 proteins ε and zeta were identified as interactors of Chico, and their association appeared to increase upon insulin stimulation of cells. The mammalian homologues of Chico, IRS-1 as well as IRS-2 and IRS-4, were also shown to bind to 14-3-3 proteins. IRS-1 was proven to interact with 14-3-3β in 3T3L1 adipocytes, and this binding was shown to increase with insulin treatment. In contrast, another study did not observe a significant change of interaction between 14-3-3ε and IRS-1 upon hormonal stimulation in HepG2 cells; nevertheless this observation relied on Western blotting, which provides less accurate quantitative data than MS-based approaches and may not have been able to detect changes at or below 2-fold, such as those observed here using mass spectrometry techniques. In NIH-3T3 cells, 14-3-3ε was shown to interact with IRS-1 and protein kinase C-α, thus modulating insulin signaling and degradation. This study also observed an increased association of Chico and IR after a 7-min insulin treatment, which reflects activation of the insulin pathway involving tyrosine phosphorylation of Chico by IR (Pflieger, 2008).
Kc cells were stimulated with an insulin concentration and within a time window previously established to give a robust induction of the whole pathway. As a result, several insulin-dependent phosphosites, mainly phosphoserines, were identified, in Chico. The roles of phosphoserines/phosphothreonines in the mammalian homologue IRS-1 have been studied with regard to the regulation of the insulin pathway. Some serine residues, when phosphorylated, participate in the negative control of insulin signaling, whereas others appear to have a positive regulatory function. The homology of the Chico sequence to the mammalian IRS homologues is too weak to allow precise comparison of phosphosites. Nonetheless it is worth mentioning that some serine residues were shown previously to become partially or fully phosphorylated in rat and mouse IRS-1 after 5-min stimulation with 80-100 nM insulin, which is in agreement with the current observations. Among the phosphorylated residues identified in Chico, several appear to correlate with insulin stimulation either positively or negatively. Most interestingly, five sequences overlap with predicted recognition motives of 14-3-3 proteins. All but one of them were shown to become more highly phosphorylated upon stimulation, which correlates well with an enhanced association of the two 14-3-3 proteins with Chico. The differences of phosphorylation levels measured in samples Chico3 and Chico4 may be, at least in part, due to the different cell densities reached before induction. Despite differences in absolute phosphorylation levels, similar variations of the phosphorylation states (increase or decrease) were observed in the two samples upon insulin stimulation (Pflieger, 2008).
Phosphorylations on tyrosine residues were also expected at least upon insulin treatment. The presence of phosphotyrosine-containing peptides could not be conclusively established by the MS data. Nonetheless the intact protein Chico could be shown to contain phosphorylated tyrosines: a fraction of the samples Chico3 and Chico4 was analyzed by Western blot using an anti-phosphotyrosine antibody, and signal was detected in both insulin conditions with increased signal in the +INS case as expected (Pflieger, 2008).
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).
SH2B1 is a key regulator of body weight in mammals. This study identified dSH2B (Lnk) as the Drosophila homolog of SH2B1. dSH2B binds to Chico and directly promotes insulin-like signaling. Disruption of dSH2B decreases insulin-like signaling and somatic growth in flies. dSH2B deficiency also increases hemolymph carbohydrate levels, whole-body lipid levels, life span, and resistance to starvation and oxidative stress. Systemic overexpression of dSH2B results in opposite phenotypes. dSH2B overexpression in fat body decreases lipid and glucose levels, whereas neuron-specific overexpression of dSH2B decreases oxidative resistance and life span. Genetic deletion of SH2B1 also results in growth retardation, obesity, and type 2 diabetes in mice; surprisingly, life span and oxidative resistance are reduced in SH2B1 null mice. These data suggest that dSH2B regulation of insulin-like signaling, growth, and metabolism is conserved in SH2B1, whereas dSH2B regulation of oxidative stress and longevity may be conserved in other SH2B family members (Song, 2010).
SH2B1 has been a component of the IIS pathway in mice. The SH2B family members (SH2B1, 2, and 3) contain characteristic PH and SH2 domains; SH2B1 is believed to serve as an adaptor in cell signaling. It has been shown that genetic disruption of SH2B1 results in obesity and type 2 diabetes in mice. Neuron-specific restoration of SH2B1 fully rescues obesity and type 2 diabetes in SH2B1 null mice. Neuronal SH2B1 controls appetite, energy balance, and body weight at least in part by enhancing leptin sensitivity in the brain. Importantly, mutations in the SH2B1 loci link to obesity in humans. A chromosomal deletion of the SH2B1 loci cosegregates with early-onset severe obesity and insulin resistance in humans (Song, 2010).
The Drosophila genome contains a single dSH2B gene. This gene has evolved into three distinct genes (SH2B1, 2, and 3) in mammals. It is hypothesized that the core functions of dSH2B (e.g., growth, reproduction, and metabolism) are evolutionarily conserved; however, they are not equally distributed among the three SH2B family members. SH2B1, 2 and/or 3 may also evolve new functions in mammals (Song, 2010).
It has been reported that in mammals, SH2B1 binds to both the insulin receptor and IRS proteins. SH2B1 directly enhances insulin signaling by promoting insulin receptor phosphorylation of IRS proteins and by preventing dephosphorylation of IRS
proteins. Genetic deletion of SH2B1 results in insulin resistance and type 2 diabetes in mice. This study shows that dSH2B binds to Chico and promoted insulin-stimulated phosphorylation of Chico, dAkt, and dFOXO. Disruption of dSH2B increases dILP resistance and hemolymph glucose in flies; conversely, dSH2B overexpression
decreases dILP resistance and hemolymph glucose. dSH2B null flies are dwarf, and females were sterile. SH2B1 null mice also exhibit growth retardation. These data suggest that SH2B regulation of the IIS pathway, growth, glucose metabolism, and reproduction is largely conserved in SH2B1. Consistent with this idea, deletion of SH2B2 or SH2B3 does not alter growth and glucose metabolism in mice, (Song, 2010).
Werz has reported similar dwarf phenotypes in dSH2B null flies (Werz, 2009). It was proposed that dSH2B (dLnk) acts in parallel to Chico, because
simultaneous disruption of both dSH2B and Chico are lethal. This study also observed a reduced survival rate, but not completely synthetic lethality, of
ChicoC/C;dSH2BD/D double mutant flies.
ChicoC/C flies had the Chico hypomorphic but not null alleles, which may explain the discrepancy between these two studies. The
Chico/dSH2B synthetic lethality is rescued by PTEN haploinsufficiency; dSH2B deficiency does not further inhibit growth, as revealed by similar body sizes between Chico and Chico/dSH2B double null animals. These results are consistent with the proposal that dSH2B and Chico may act in the same pathway(s)
downstream of dInR. However, the results do not exclude the possibility that dSH2B may activate additional Chico-independent pathways (Song, 2010).
It was observed that disruption of dSH2B increased lipid levels and energy conservation in flies; conversely, dSH2B overexpression decreased energy conservation. Moreover, dSH2B overexpression in fat bodies but not neuronal tissues decreased lipid levels, hemolymph
glucose, and energy conservation. These observations indicate that in insects, dSH2B in fat body plays a key role in regulating lipid metabolism and energy homeostasis (Song, 2010).
Deletion of SH2B1 but not the other SH2B family members results in obesity and type 2 diabetes in mice, suggesting that the metabolic functions of dSH2B are largely conserved in SH2B1. Moreover, mutations in the SH2B1 loci are genetically linked to obesity in humans. A rare chromosomal deletion of the
SH2B1 loci cosegregates with early-onset severe obesity and insulin resistance in humans (Bochukova, 2010). Neuronal restoration of SH2B1 fully rescues the obesity and type 2 diabetes phenotypes in SH2B1 null mice, suggesting that SH2B1 in the central nervous system plays a dominant role in controlling energy homeostasis (Ren, 2007). Neuronal SH2B1 controls energy metabolism and body weight at least in part by promoting the anorexigenic response to leptin in the brain (Song, 2010).
The IIS system is conserved in Caenorhabditis elegans, Drosophila melanogaster, and mammals to regulate longevity. Given the fact that dSH2B promotes the activation of the IIS pathway, it is not surprising that disruption of dSH2B increased both oxidative resistance
and life span in flies. Conversely, ubiquitous overexpression of dSH2B decreased oxidative resistance and longevity. In agreement with these observations, Slack have independently reported that dSH2B deficiency increases stress resistance and life span. dFOXO is a critical component of the IIS system. Loss of dFOXO reduces life span; conversely, dFOXO activation in the adult head fat body increases oxidative resistance and life span. However, neuronal dFOXO appears not to be involved in regulating longevity. It was observed in Drosophila that neuron-specific but not fat-body-specific overexpression of dSH2B decreased life span and oxidative resistance. These data suggest that dFOXO is unlikely to mediate dSH2B regulation of oxidative resistance and longevity. Moreover, dSH2B may also regulate life span by an additional IIS-independent mechanism (Song, 2010).
In contrast, deletion of SH2B1 reduced longevity and oxidative resistance in female mice in the absence of type 2 diabetes. The shortened life span cannot be explained by obesity and insulin resistance, because brain-specific deletion of IRS2 extends life span in the presence of life-long obesity and insulin resistance. A simple interpretation of these observations is that dSH2B regulation of oxidative
resistance and longevity is conserved in other SH2B family members. SH2B3 is unlikely to regulate longevity because its expression is restricted to the immune system. SH2B2, which is expressed in multiple tissues, may act as dSH2B to regulate longevity in mammals. However,
the possibility cannot be excluded that SH2B1 may regulate life span in a cell type-specific manner similar to dSH2B; however, systemic deletion of SH2B1 may cause an unknown pathological alteration that shortens the life span independently of aging in the mouse models. In agreement with this idea, systemic deletion of the insulin receptor results in neonatal death, whereas fat-specific deletion of the insulin receptor extends life span in mice (Song, 2010).
In summary, key functions of dSH2B (e.g., its regulation of the IIS pathway, growth, glucose metabolism, energy homeostasis, and reproduction) are conserved in SH2B1. While dSH2B in fat body plays a key role in regulating energy metabolism
in flies, neuronal SH2B1 has evolved a more prominent role in controlling energy homeostasis and body weight in mammals. dSH2B, particularly neuronal dSH2B, negatively regulates longevity in flies; in contrast, SH2B1 deficiency shortens life span in mice. The other SH2B family members may regulate oxidative response and longevity in mammals (Song, 2010).
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