BIOLOGICAL OVERVIEW

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).

PROTEIN STRUCTURE

Amino Acids - 1088

Structural Domains

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: 14 August 99

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