Gene name - Phosphotidylinositol 3 kinase 92E
Synonyms - Dp110
Cytological map position - 92E12--13
Function - signal transduction
Keywords - cell proliferation, growth response, insulin signaling pathway
Symbol - Pi3K92E
FlyBase ID: FBgn0015279
Genetic map position -
Classification - Phosphatidylinositol 3-kinase
Cellular location - cytoplasmic
|Recent literature||Ejeskar, K., Vickes, O., Kuchipudi, A., Wettergren, Y., Uv, A. and Rotter Sopasakis, V. (2015). The unique non-catalytic C-Terminus of P37delta-PI3K adds proliferative properties in vitro and in vivo. PLoS One 10: e0127497. PubMed ID: 26024481
The PI3K/Akt pathway is central for numerous cellular functions and is frequently deregulated in human cancers. The fruit fly, Drosophila melanogaster, is a highly suitable system to investigate PI3K signaling, expressing one catalytic, Dp110, and one regulatory subunit, Dp60, and both show strong homology with the human PI3K proteins p110 and p85. p37delta, an alternatively spliced product of human PI3K p110delta, has been shown to display strong proliferation-promoting properties despite lacking the catalytic domain completely. This study functionally evaluated the different domains of human p37delta in Drosophila. The N-terminal region of Dp110 alone promotes cell proliferation, and the unique C-terminal region of human p37delta further enhances these proliferative properties, both when expressed in Drosophila, and in human HEK-293 cells. Surprisingly, although the N-terminal region of Dp110 and the C-terminal region of p37delta both display proliferative effects, over-expression of full length Dp110 or the N-terminal part of Dp110 decreases survival in Drosophila, whereas the unique C-terminal region of p37delta prevents this effect. Furthermore, it was found that the N-terminal region of the catalytic subunit of PI3K p110, including only the Dp60 (p85)-binding domain and a minor part of the Ras binding domain, rescues phenotypes with severely impaired development caused by Dp60 over-expression in Drosophila, possibly by regulating the levels of Dp60, and also by increasing the levels of phosphorylated Akt. These results indicate a novel kinase-independent function of the PI3K catalytic subunit.
|Chang, Y. J., Zhou, L., Binari, R., Manoukian, A., Mak, T., McNeill, H. and Stambolic, V. (2016). The Rho guanine nucleotide exchange factor DRhoGEF2 is a genetic modifier of the PI3K pathway in Drosophila. PLoS One 11: e0152259. PubMed ID: 27015411
The insulin/IGF-1 signaling pathway mediates various physiological processes associated with human health. Components of this pathway are highly conserved throughout eukaryotic evolution. In Drosophila, the PTEN ortholog and its mammalian counterpart downregulate insulin/IGF signaling by antagonizing the PI3-kinase function. From a dominant loss-of-function genetic screen, this study discovered that mutations of a Dbl-family member, the guanine nucleotide exchange factor DRhoGEF2 (DRhoGEF22(l)04291), suppressed the PTEN-overexpression eye phenotype. dAkt/dPKB phosphorylation, a measure of PI3K signaling pathway activation, increased in the eye discs from the heterozygous DRhoGEF2 wandering third instar larvae. Overexpression of DRhoGEF2, and it's functional mammalian ortholog PDZ-RhoGEF (ArhGEF11), at various stages of eye development, resulted in both dPKB/Akt-dependent and -independent phenotypes, reflecting the complexity in the crosstalk between PI3K and Rho signaling in Drosophila.
|Cinnamon, E., Makki, R., Sawala, A., Wickenberg, L.P., Blomquist, G.J., Tittiger, C., Paroush, Z. and Gould, A.P. (2016). Drosophila Spidey/Kar regulates oenocyte growth via PI3-kinase signaling. PLoS Genet 12: e1006154. PubMed ID: 27500738
Cell growth and proliferation depend upon many different aspects of lipid metabolism. One key signaling pathway that is utilized in many different anabolic contexts involves Phosphatidylinositide 3-kinase (PI3K) and its membrane lipid products, the Phosphatidylinositol (3,4,5)-trisphosphates. It remains unclear, however, which other branches of lipid metabolism interact with the PI3K signaling pathway. This study focused on specialized fat metabolizing cells in Drosophila called larval oenocytes. In the presence of dietary nutrients, oenocytes undergo PI3K-dependent cell growth and contain very few lipid droplets. In contrast, during starvation, oenocytes decrease PI3K signaling, shut down cell growth and accumulate abundant lipid droplets. It was shown that PI3K in larval oenocytes, but not in fat body cells, functions to suppress lipid droplet accumulation. Several enzymes of fatty acid, triglyceride and hydrocarbon metabolism are required in oenocytes primarily for lipid droplet induction rather than for cell growth. In contrast, a very long chain fatty-acyl-CoA reductase (FarO) and a putative lipid dehydrogenase/reductase (Spidey, also known as Kar) not only promote lipid droplet induction but also inhibit oenocyte growth. In the case of Spidey/Kar, it was found that the growth suppression mechanism involves inhibition of the PI3K signaling pathway upstream of Akt activity. Together, these findings show how Spidey/Kar and FarO regulate the balance between the cell growth and lipid storage of larval oenocytes.
|Arnes, M., Romero, N., Casas-Tinto, S., Acebes, A. and Ferrus, A. (2019). PI3K activation prevents Abeta42-induced synapse loss and favors insoluble amyloid deposits formation. Mol Biol Cell: mbcE19050303. PubMed ID: 31877058
Excess of Abeta42 peptide is considered a hallmark of the disease. This study expressed the human Abeta42 peptide to assay the neuroprotective effects of PI3K in adult Drosophila melanogaster. The neuronal expression of the human peptide elicits progressive toxicity in the adult fly. The pathological traits include reduced axonal transport, synapse loss, defective climbing ability and olfactory perception, as well as lifespan reduction. The Abeta42-dependent synapse decay does not involve transcriptional changes in the core synaptic protein encoding genes: bruchpilot, liprin and synaptobrevin. All toxicity features, however, are suppressed by the co-expression of PI3K. Moreover, PI3K activation induces a significant increase of 6E10 and Thioflavin-positive amyloid deposits. Mechanistically, it is suggested that Abeta42-Ser26 could be a candidate residue for direct or indirect phosphorylation by PI3K. Along with these in vivo experiments this study further analyzed Abeta42 toxicity and its suppression by PI3K activation in in vitro assays with SH-SY5Y human neuroblastoma cell cultures, where Abeta42 aggregation into large insoluble deposits is reproduced. Finally, it was shown that the Abeta42 toxicity syndrome includes the transcriptional shut down of PI3K expression. Taken together, these results uncover a potential novel pharmacological strategy against this disease through the restoration of PI3K activity.
|Xu, L., Qiu, Y., Wang, X., Shang, W., Bai, J., Shi, K., Liu, H., Liu, J. P., Wang, L. and Tong, C. (2022). ER-mitochondrial contact protein Miga regulates autophagy through Atg14 and Uvrag. Cell Rep 41(5): 111583. PubMed ID: 36323251
Mitochondrial malfunction and autophagy defects are often concurrent phenomena associated with neurodegeneration. This study shows that Miga, a mitochondrial outer-membrane protein that regulates endoplasmic reticulum-mitochondrial contact sites (ERMCSs), is required for autophagy. Loss of Miga results in an accumulation of autophagy markers and substrates, whereas PI3P and Syx17 levels are reduced. Further experiments indicated that the fusion between autophagosomes and lysosomes is defective in Miga mutants. Miga binds to Atg14 and Uvrag; concordantly, Miga overexpression results in Atg14 and Uvrag recruitment to mitochondria. The heightened PI3K activity induced by Miga requires Uvrag, whereas Miga-mediated stabilization of Syx17 is dependent on Atg14. Miga-regulated ERMCSs are critical for PI3P formation but are not essential for the stabilization of Syx17. In summary, this study identified a mitochondrial protein that regulates autophagy by recruiting two alternative components of the PI3K complex present at the ERMCSs.
Phosphoinositide 3-kinases (PI3Ks) have been identified in an evolutionarily diverse range of organisms, including mammals, Drosophila, yeast, plants and Dictyostelium. They are activated by a multitude of extracellular signals and are implicated in mitogenesis, differentiation and cell survival, as well as in the control of the cytoskeleton and cell shape. Inositol is a sugar moiety that is found attached to cell membrane lipids. One type of PI3K, exemplified by Phosphotidylinositol 3 kinase 92E, the protein described in this essay, phosphorylates inositol bearing lipids (phosphatidylinositols or PtdIns) at the D3 position of the inositol ring. Substrates of PI3Ks include PtdIns, PtdIns (4)P, and PtdIns (4,5)P2: these are phosphatidylinositols with phosphates attached to different hydroxide residues of the inositol ring, generating (respectively) PtdIns (3P), PtdIns (3,4)P2, and PtdIns (3.4,5)P3. These phosphoinositide residues created by PI3K phosphorylation have been proposed to act as second messengers, functioning to recruit regulatory proteins to the plasma membrane. The recuited regulatory proteins act in turn to trigger downstream signaling events in multiple regulatory pathways.
Drosophila has three genes coding for PI3Ks. The class I gene, Phosphotidylinositol 3 kinase 92E, informally termed Drosophila p110 or Dp110, the subject of this overview, codes for a protein that phosphorylates PtdIns, PtdIns (4)P, and PtdIns (4,5)P2 in vitro, though the major substrates of these PI3Ks are though to be PtdIns (4)P and PtdIns (4,5)P2. This class includes enzymes that mose closely resemble the prototypical p110 catalytic subunit and which associate with a regulatory subunit [e.g. p85 alpha, p85 beta and p55 PIK]. The regulatory subunits contain Sh2 domains that bind to specific phosphotyrosine residues and recruit p110/p85 heterodimers to activated receptor tyrosine kinases, thereby facilitating PI3K activation.
Interest in the Drosophila PI3K pathway has recently be rekindled by the cloning of chico, the Drosophila homolog of vertebrate insulin receptor substrates (IRSs). Many aspects of the insulin system appear to be conserved in flies and mammals. In mammalian cells, activation of the insulin or IGF1 (insulin-like growth factor 1) receptors by insulin and IGF1, respectively, results in the recruitment of IRS1 or IRS2 to the receptor via interaction of the IRS phosphotyrosine-binding domains with a phosphotyrosine motif (NPXY) in the juxtamembrane region of the receptors. Phosphorylation of multiple tyrosine residues of IRS1 triggers the activation of various signaling pathways, including the RAS/MAP kinase pathway via the SH2/SH3 adaptor GRB2 and the PI3K pathway via the p85 SH2 adaptor subunit of p110 PI3K (Yenush, 1997). The Drosophila Insulin-like receptor (InR) shares many structural features with its human homologs, including its heterotetrameric structure and a conserved PTB consensus binding site in the juxtamembrane region. However, Drosophila InR contains a 400-amino acid C-terminal extension not found in any of the vertebrate receptors. This C-terminal tail contains three YXXM consensus binding sites for the SH2 domain of the p60 subunit of PI3K and four additional NPXY consensus PTB-binding sites. The C-terminal domain is functional, since expression of a chimeric receptor consisting of the extracellular domain of the human INR and the intracellular domain of the Drosophila InR in murine 32D cells lacking endogenous IRS1 can partially activate mammalian PI3K and S6K. In contrast, the ability of the human INR to activate PI3K in this system is strictly dependent on the coexpression of IRS1 (Yenush, 1996). These findings and the identification of Chico suggest that in Drosophila, InR couples to the downstream effector PI3K in two different ways, one using docking sites in the InR C-terminal tail and the other connecting through docking sites in Chico (Böhni, 1999).
Ectopic expression of Dp110 affects wing growth. The adult wing consists of two layers of ectodermal cells (dorsal and ventral) that secrete cuticular structures (wing hairs and veins) and contact sensory organs (campaniform sensilla on the wing surface and sensory bristles along the anterior wing margin). The effect of ectopic Dp110 expression on development during larval stages was examined. At this time the wing imaginal disc is a monolayer epithelium divided by anterior/posterior (A/P) an dorsal/ventral (D/V) compartment boundaries. The expression of WT-Dp110 or Dp110-CAAX (membrane targeted Dp110) in different regions of the wing imaginal disc results in expansion of the corresponding regions in the adult wing blade. Conversely, Dp110D954A (Dp110 with the mutation in the putative ATP binding site) expression reduces the size of these regions. For example, ectopic expression of WT-Dp110 or Dp110-CAAX in the prospective dorsal surface of the wing blade results in wings that are bigger than wild-type and curve downwards. In contrast Dp110D954A wings are smaller and curve upwards. Similarly when Dp110 is expressed along, and immediately anterior to, the A/P boundary of the wing disc, the corresponding region of the adult wing (visualized by assessing the distance between longitudinal veins III and IV) expand or contract. Interestingly, the proximal to distal alignment of the non-sensory wing hairs that are present on the surface of the wing blade, is often disrupted by the expression of Wt-Dp110 or Dp110-CAAX (Leevers, 1996).
To further examine the effect of Dp110 on wing growth, wing discs from third instar larvae were examined in which GAL4 was used to drive the co-expression of nuclear beta-galactosidase and different forms of Dp110 at high levels in the dorsal wing pouch and at lower levels in the ventral wing pouch. The region of Gal4 expression was revealed by immunostaining for beta-galactosidase expression, and ectopic Dp110 expression was detected by a monoclonal antibody directed against a myc peptide epitope engineered into the N-terminus of the Dp110 expression constructs. Consistent with the effects observed in adult wings, the Gal4 expression domain is expanded by WT-Dp110 and Dp110-CAAX, and contracted by Dp110D954A. Similar differences are observed in discs expressing Dp110 along the A/P boundary, even in regions that give rise to non-wing blade adult structures, such as the notum and hinge. Significantly, the same effects are also observed in leg and haltere discs expressing Dp110 along the A/P boundary, indicating that the effect of Dp110 on the growth of imaginal discs is not specific to wing discs (Leevers, 1996).
An examination was made of whether the observed differences in wing size result from alterations in cell size or cell number. The number of cells per unit area was assessed by counting wing hairs, single apical extensions found on the surface of each wing blade cell. Wings generated by transgene expression along the A/P boundary were analyzed by examining an area of fixed size on the wing blade, and by looking at the wing margin between veins III and IV. Surprisingly, it was found that Dp110 and Dp110-CAAX expression increases both the overall number of cells and their size. In contrast, Dp110D954A decreases both cell size and cell number (Leevers, 1996).
To further analyze the role that Dp110 plays in the growth of imaginal discs, effects of the ectopic expression during eye development were examined. The Drosophila compound eye is a repetitive and highly organized structure generated by the stepwise recruitment of cells to ommatidial clusters behind an indentation in the eye imaginal disc known as the morphogenetic furrow. These clusters grow and differentiate during larval and pupal development and ultimately give rise to the adult retina. The Dp110 transgenes are expressed in cells posterior to the morphogenetic furrow using a GMR-Gal4 line (Leevers, 1996).
The expression of wild type or membrane targeted Dp110 generates enlarged and bulging, roughened eyes with fused ommatidia and misplaced or duplicated bristles, whereas Dp110D954A eyes are smaller than normal and flatter. The hexagonal lenses or facets that form the surface of each ommatidium are larger (ectopic WT-Dp110) or smaller (Dp110D954A). The small eyes contain the wild-type number of facets, whereas the enlarged eyes actually contained fewer ommatidia. To investigate the internal changes resulting in these differences, radial and tangential sections through the eyes were examined. The radial sections reflect differences seen from the outside: the adult retina is either increased or decreased, in both size and thickness. In addition, in eyes expressing ectopic WT-Dp110, an apical region above the photoreceptor rhabdomeres and immediately behind the lens was filled with cells. This is particularly evident in apical tangential section, where the space occupied by the secreted pseudocone in control eyes has been found to contain many swollen cell bodies in WT-Dp110 eyes. In more basal transverse sections the normally regular array of photoreceptors is disrupted by WT-Dp110 expression. The rhabdomeres appeared twisted and are disorganized with respect to one another and neighboring ommatidia: the number of photoreceptor rhabdomeres is often reduced. In Dp110D954A eyes the lattice is also disrupted though to a lesser degree, and the orientation of photoreceptors relative to those in neighboring ommatidia is at least partially maintained (Leevers, 1996).
These adult eye phenotypes indicate that Dp110 transgenes can also modulate growth during eye development. Therefore, differences in cell size and/or cell number were sought by examining confocal images in fluorescently labeled larval and pupal discs. The process of photoreceptor determination, as judged by the pattern of expression of the neuronal marker Elav is unaffected by ectopic WT-Dp110 or Dp110D954A expression. Interestingly though, the nuclei of WT-Dp110 discs are more widely spaced. Similarly, the distance between the centers of the developing ommatidial clusters revealed by immunostaining larval and pupal discs with anti-Armadillo, is affected by Dp110 transgene expression. Armadillo, the Drosophila homolog of the vertebrate beta-catenin, localizes to the adherens junctions just below the apical membranes of the developing photoreceptors, where the cells are constricted in close contact. The apical clustering of the photoreceptor membranes is disrupted in Dp110D954A pupal discs, where a 'ring' of Armadillo staining can be seen that persists more basally than in control discs (Leevers, 1996).
Immunostaining of the membranes of pupal cells with anti-alpha-Spectrin indicates that the Dp110 transgenes affect the size and not the number of cells in the eye disc. The pigment, cone, and photoreceptor cell bodies are swollen in ectopic WT-Dp110 discs and smaller than normal in the Dp110D954A discs. Notably, and in spite of these differences in cell size, the specific arrangement of the different cell types within each ommatidial cluster remains undisturbed. Consistent with this, cell proliferation and apoptosis are not detectably affected in the developing eye discs. Presumably the roughness and degeneration of the ommatidial pattern seen in adult WT-Dp110 eyes must arise during the late pupal stages of eye development (Leevers, 1996).
These results implicate Dp110 in the control imaginal disc growth, since the Dp110 transgenes affect both the size of individual cells and (in the wing disc) the overall number of cells. Currently, it cannot be determined how closely linked these two effects are, though it is conceivable that the differences seen in cell number might arise as a direct consequence of the effects on cell size. An alternative possibility is that the differences in the wing cell number result from a more direct effect on the rate of cell division or cell death in the developing discs. Studies in a mammalian system have implicated PI3K in the control of both processes. Interestingly, the simultaneous disruption of two of the three genes encoding class I PI3Ks in Dictyostelium also affects cell growth; double knock-out strains grow slowly and produce cells of reduced size (Leevers, 1996 and references).
Since the effect of the Dp110 transgenes on cell size might arise from alterations in biosynthesis or cytoskeletal architecture, it is noteworthy that likely downstream targets of class I PI3Ks identified in mammalian cells include P70S6K and the small GTP binding protein Rac. P70S6K is a serine/threonine kinase implicated in the up-regulation of translation and cell growth. Furthermore, studies in the class of Drosophila mutants termed 'Minutes' have indicated that protein synthesis is the rate limiting step in growth and development. Heterozygote Minute mutations, many of which correspond to or have been genetically mapped to the vicinity of ribosomal genes, delay development and often result in flies with reduced body size. Studies in mammalian cells have also implied a role for class I PI3Ks in the organization of actin cytoskeleton and membrane ruffling via Rac. Thus it is possible that effects on the architecture of the cytoskeleton or membrane composition and organization contribute to the differences in cell size that are observed. Furthermore, the disrupted adherens junctions in Dp110D954A pupal eye discs, the degeneration seen in adult eyes, and the loss of wing hair polarity observed in the wing blade might all be mediated via effects on actin organization (Leevers, 1996 and references).
The phenotypes generated by ectopic Dp110 expression suggest a possible role downstream of the Drosophila homolog of the EGF receptor or the insulin receptor. One of the multiple phenotypes generated by mutation in Egfr and downstream components of the Ras/MAP kinase pathway is a decrease in wing cell size. Thus, both the Ras/MAP kinase pathway and a signal mediated by Dp110 might cooperate in the control of wing cell size. Although Egfr contains no YXXM motifs (the motif to which the PI3K regulatory subunit binds), the receptor tyrosine kinase substrate Dos, which is also required for correct wing cell size, contains a YXXM motif. Another possible upstream regulator of Dp110 is the Insulin receptor and the Drosophila Insulin receptor substrate protein Chico (Bohni, 1999). Not only does the Drosophila Insulin receptor contain three pYXXM motifs and interact in vitro with the SH2 domains of mammalian p85 (the PI3K regulatory subunit), but flies heteroallelic for mutations in inr, which codes for the Drosophila Insulin receptor, grow slowly and are significantly reduced in size (Fernandez, 1995 and Chen, 1996). Furthermore, these growth defects arise as a result of prolonged larval instars during which the imaginal discs fail to grow to their wild type size. Flies defective in chico exhibit a similar phenotype (Bohni, 1999). Mice deficient in IRS1 are also retarded in growth and have a reduced final body size (Araki, 1994; Tamemoto, 1994 and Leevers, 1996).
Although this study implies a general role for Dp110 in the control of cell growth, many aspects of endogenous Dp110 function may not have been revealed by ectopic expression. Thus it will be informative in the future to examine phenotypes generated by mutations in the gene encoding Dp110. It is of note that when similar experiments were performed to express the Drosophila class II and class III PI3Ks, no general effects on growth are observed. PI3K_59F (class III) expression gives no detectable phenotype, whereas PI3K_68D (class II) expression produces phenotypes that imply a role in differentiation as opposed to growth. It is hoped that further analyses utilizing Drosophila as a model system will enable the characterization of different functions for different PI3Ks during Drosophila development and the performance of genetic screens to identify key downstream targets. The identification of such targets should help to further elucidate the mode of activation of class I PI3Ks in both Drosophila and mammals (Leevers, 1996).
In metazoans, the insulin signalling pathway has a key function in regulating energy metabolism and organismal growth. Its activation stimulates a highly conserved downstream kinase cascade that includes phosphatidylinositol-3-OH kinase (PI(3)K) and the serine-threonine protein kinase Akt. This study identifies a new component of insulin signalling in Drosophila, the steppke gene (step). step encodes a member of the cytohesin family of guanine nucleotide exchange factors (GEFs), which have been characterized as activators for ADP-ribosylation factor (ARF) GTPases. In step mutant animals both cell size and cell number are reduced, resulting in decreased body size and body weight in larvae, pupae and adults. step acts upstream of PI(3)K and is required for the proper regulation of Akt and the transcription factor FOXO. Temporally controlled interference with the GEF activity of the Step protein by feeding the chemical inhibitor SecinH3 causes a block of insulin signalling and a phenocopy of the step mutant growth defect. Step represses its own expression and the synthesis of growth inhibitors such as the translational repressor 4E-BP. These findings indicate a crucial role of an ARF-GEF in insulin signalling that has implications for understanding insulin-related disorders, such as diabetes and obesity (Fuss, 2006).
All animals coordinate growth to reach their final size and shape. The insulin–insulin-like growth factor signalling pathway, which is genetically conserved from flies to humans, has been identified as a key regulator of cell growth in response to extrinsic signals such as growth factors and nutrient availability. In mammals, loss of the ability to respond to insulin, a phenomenon known as insulin resistance, is associated with pathological manifestations such as type 2 diabetes. In Drosophila, activation of a unique insulin-like receptor (InR) stimulates a conserved downstream cascade that includes PI(3)K and Akt. This signalling cascade controls organismal growth directly by regulating cell size and cell number (Fuss, 2006).
In a search for genes controlling larval growth in Drosophila, a genetic locus was identified that was named steppke (step). Molecular analysis and genetic rescue experiments show that the lethality of the P element alleles is linked to the step gene function. The step gene encodes a protein that belongs to the highly conserved cytohesin protein family of GEFs that consists of four family members in humans and one family member in invertebrates such as the nematode, mosquito and fly. GEFs mediate the exchange of GDP for GTP on the ARFs, which belong to the Ras superfamily of small GTPases. Like other Ras-related GTP-binding proteins, the ARF proteins cycle between their active GTP-bound and inactive GDP-bound conformations. In concert with ARFs, cytohesin proteins regulate vesicle trafficking, cell adhesion, migration and structural organization at the cell surface (Fuss, 2006).
Cytohesin proteins contain two characteristic motifs: a Sec7 domain responsible for the GEF activity, and a pleckstrin homology domain (PH) required for plasma membrane recruitment as a result of specific binding to phosphatidylinositol-3,4,5-trisphosphate, the second messenger generated by class I PI(3)Ks. The Sec7 and PH domains of Step are highly conserved compared with the corresponding protein domains of mammalian cytohesins (Fuss, 2006).
Phenotypic analysis of homozygous stepk08110 and stepSH0323 mutants and transheterozygous allelic combinations indicate an essential role of step in regulating growth and body size at all stages of the Drosophila life cycle. Both males and females of stepk08110/stepSH0323 transheterozygous adults are significantly smaller than control animals; however, the body proportions of these animals are not changed. Consistently, larval and pupal development are also slowed down in step mutants and body size is reduced. The observed growth defects mimic a starvation phenotype that is not caused by a failure of food intake, as verified by feeding coloured yeast and by the analysis of a metabolic marker gene (Fuss, 2006).
It is known that larval growth is largely based on an increase in cell size in all terminally differentiated tissues that is accomplished by endoreplication, a modified cell cycle, consisting of successive rounds of DNA synthesis without intervening mitoses. To examine the cause for the growth defects of step mutant larvae, cell cycle activity was investigated in the midgut and the salivary glands, which are representative endoreplicating tissues in the larval stage. A general decrease in endoreplication activity was found, indicated by a slowing down of the S phase of the cell cycle. Both the size and the total number of salivary gland cells are decreased, resulting in a smaller organ (Fuss, 2006).
Because embryonic lethality was observed in a small proportion of the homozygous stepk08110 mutants, it was important to exclude the possibility that the growth defects observed in the mutant larvae derive from a defect laid down during embryogenesis. For this purpose an assay was established to analyse step function exclusively in the larval stage, in which the growth rate is maximal. Use was made of the small molecule SecinH3, which was recently identified as an inhibitor of the Drosophila Step protein and the vertebrate cytohesin family members. SecinH3 binds to the Sec7 domain of Step, thereby inhibiting the guanine nucleotide exchange of interacting ARF proteins. Feeding SecinH3 to wild-type larvae induced a phenocopy of the growth defects observed in step mutants and led to a marked decrease in body size. It is concluded from the phenotypic analysis of step mutants and from the experiments inhibiting Step protein function directly by using the chemical inhibitor SecinH3 that Step is essential for organismal growth of Drosophila larvae, pupae and adults (Fuss, 2006).
In step mutants, organismal growth is strongly reduced and development is delayed, which is also a hallmark of mutants affecting the insulin signalling pathway. To investigate whether step has a function in insulin signalling, the expression of two known target genes of the pathway was analysed in step mutants, namely 4E-BP, encoding a translational repressor, and InR, encoding the insulin receptor, by using quantitative reverse-transcriptase-mediated polymerase chain reaction (RT–PCR); both 4E-BP and InR transcription are upregulated in response to repressed insulin signalling. Lipase3 (Lip3) expression was used as a starvation marker in these experiments. In step mutant larvae and also in wild-type larvae treated with the Step inhibitor SecinH3, 4E-BP and InR transcription is activated, whereas Lip3 expression is unaffected. This indicates that the growth phenotype observed in step mutant larvae is not caused by a complete block of nutrition but is associated with a specific downregulation of insulin signalling activity. Similarly, interfering with Step function by feeding SecinH3 to transheterozygous step mutant flies or applying SecinH3 in S2 tissue culture cells also results in an activation of 4E-BP and InR transcription (Fuss, 2006).
It has been shown previously that 4E-BP and InR are target genes of the transcription factor FOXO (forkhead box, sub-group ‘O’). In Drosophila cells, insulin receptor signalling results in a high activity of PI(3)K and phosphorylation of Akt. Akt phosphorylates FOXO and causes cytoplasmic retention of FOXO, whereas low activities of PI(3)K and Akt allow FOXO to enter the nucleus, where it promotes the expression of factors such as 4E-BP that retard cell growth and proliferation. In step mutant larvae or in S2 tissue culture cells in which Step protein function is inhibited with SecinH3, a nuclear localization of FOXO was found, indicating that step is required for insulin-signalling-dependent cytoplasmic localization of FOXO. Because this is regulated by phosphorylation by means of Akt, whether step is necessary for Akt phosphorylation was tested, and it was found that under conditions in which the step function is affected, the amount of phosphorylated Akt protein is significantly decreased (Fuss, 2006).
It has been shown that activation of Akt during growth in Drosophila is regulated by the class I PI(3)K Dp110. Overexpression of Dp110-CAAX, a constitutively active form of PI(3)K, in wing or eye imaginal discs enhances cellular growth, resulting in enlarged cells and organs, whereas mutations in Dp110 are lethal and result in a larval growth arrest in the third instar. It has been shown previously that Dp110 interacts with key components of the insulin signalling pathway including Chico, PTEN and Akt to control insulin-signalling-dependent cell and organ growth in Drosophila. To test whether step acts together with PI(3)K in a common pathway involved in Akt and FOXO regulation and, if so, to address whether step is genetically upstream or downstream of PI(3)K in the insulin pathway, Dp110-CAAX was expressed in heterozygous and transheterozygous step mutant animals (Fuss, 2006).
step mutant adults are greatly decreased in size and weight in comparison with wild-type animals. In control flies in which Dp110-CAAX has been overexpressed, body size and weight are greatly increased in comparison with wild-type flies. If step were positioned downstream of PI(3)K, the oversize phenotype induced by the expression of Dp110-CAAX should be suppressed or at least strongly reduced, whereas if step were positioned upstream of PI(3)K, Dp110-CAAX expression would rescue the growth phenotype of step mutants. The latter was found, providing in vivo evidence that the cytohesin family member step is upstream of PI(3)K (Fuss, 2006).
Tight regulation of insulin signalling activity has been shown to be crucial for cell and organ growth in Drosophila and for numerous growth-related and homeostasis-related diseases such as cancer and type 2 diabetes in humans. It is known from recent studies in Drosophila that InR represses its own synthesis by a feedback mechanism directed by the transcription factor FOXO. To test whether step is also part of a negative feedback control mechanism, step transcription was analysed at different levels of insulin signalling activity in vivo by using quantitative RT–PCR experiments. Similarly to the 4E-BP and InR genes, step transcription was found to be upregulated under conditions promoting FOXO activity such as starvation or in mutants of the insulin signalling pathway, such as chico mutants. Consistently, step transcription is induced 24-fold in response to a brief pulse of ectopic FOXO expression during larval development. These results indicate a FOXO-dependent transcription of step, which may be direct, presumably through several FOXO consensus binding motifs present in the step promoter, or indirect (Fuss, 2006).
It is therefore proposed that Step is a previously unrecognized and essential component of the insulin signalling cascade in Drosophila that regulates organismal growth. These results are consistent with the findings of a parallel study on the role of mammalian cytohesins. Both papers provide independent evidence for the central involvement of cytohesins in the insulin pathway upstream of PI(3)K and show a functional conservation of these proteins for at least 900 million years (Fuss, 2006).
A full-length Dp110 cDNA was isolated and found to encode a protein homologous throughout its length to the class I mammalian PI3Ks p110alpha and p110beta. Dp110 can be divided into domains of different predicted function. The most conserved regions, found at the C-terminus, are the catalytic core (HR1) and PIK domain (HR2), a domain that is also found in PI4-kinases. The N-terminus contains regions known to be both necessary and sufficient for mammalian p110alpha to associate with the regulatory subunit p85. The central portion of the protein contains two further PI3K homology regions: HR3, which is of unknown function, and a stretch of basic amino acids followed by a short leucine zipper, reminiscent of the dimerization domains of bZIP transcription factors and which therefore might play a role in intra- or inter-molecular interactions (Leevers, 1996).
date revised: 10 June 2023
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