Drosophila PI3K Pathway
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Drosophila PI3K Pathway

The PI3K (Phosphatidylinositde-3 Kinase) family of enzymes is recruited upon growth factor receptor activation and produces 3 phosphoinositide lipids. The lipid products of PI3K act as second messengers by binding to and activating diverse cellular target proteins. Therefore, PI3Ks play a central role in many cellular functions. In mammals, the PI3Ks have a p110 catalytic subunit associated with a p85, p55 or p50 regulatory subunit (Ref.1). In Drosophila, the dPI3K (Phosphatidylinositde-3 Kinase) regulatory subunit dp60 (p60 Regulatory Subunit of PI3K) lacks the SH3 and BH (Bcr homology) domains like that of the mammals showing that these additional domains have been added during evolution to confer additional functions or modes of regulation to the PI3K signaling pathways. The dPI3K along with dp60 also contain dp110 (PI3K Class-IA) as the catalytic subunit. dp60 is an SH2 domain-containing adaptor molecule that binds to the dp110. dp60 contains two SH2 domains that recognize phosphorylated tyrosine residues within the consensus Tyr-X-X-Met and an inter-SH2 domain region through which it interacts with dp110. dp60 shares sequence homology with mammalian p85Alpha and p85Beta. dp110 is homologous to mammalian p110Alpha, p110Beta and p110Delta. dp110 contains an N-terminal domain which binds to the SH2 domain of dp60, a dRas (Ras Oncogene)-binding domain, a C2 domain, an Alpha-helical PIK domain and a catalytic domain (Ref.2). dp110 phosphorylates dPtdIns (Phosphatidylinositol) to generate dPtdIns(3)P (Phosphatidylinositol 3-Phosphate) and ultimately dPtdIns(3,4)P2 (Phosphatidylinositol 3,4-bisphosphate) and dPIP3 (Phosphatidylinositol 3,4,5-trisphosphate). In addition, dp110 possess intrinsic protein kinase activity and auto-phosphorylates itself. dp60 and dp110 are required for the normal growth of Drosophila imaginal discs, epithelial organs that give rise to the epidermal organs of the adult fly (Ref.3 & 4).

dPI3Ks phosphorylate inositol lipids in the cell membrane, generating a range of second messengers including dPIP3. dPIP3 recruits and activates downstream effectors such as dAkt (v-Akt Murine Thymoma Viral Oncogene Homolog-1)/dPKB (Protein Kinase-B) by binding to their PH (Pleckstrin Homology) domains. dRas signaling plays a major role in cell fate specification in Drosophila and C. elegans (Caenorhabiditis elegans) (Ref.5). In contrast, PI3K signaling does not regulate cell fate in C. elegans. The ability of dRas to regulate both patterning and growth explains that dRas regulate growth via Dm (Diminutive)/dMyc (v-Myc Avian Myelocytomatosis Viral Oncogene Homolog) and/or dPI3K. dPI3K signaling is regulated by a family of secreted dILPs (Insulin-Like Peptides) that bind and activate dInR (Insulin-Like Receptor). dILPs are structurally similar to pre-pro insulin, with a signal peptide, B-chain, C-peptide and A-chain. The dInR is an essential gene that is necessary for formation of the embryonic epidermis and nervous system. The dInR encodes a pro-receptor that is cleaved to produce a ligand-binding Alpha-subunit and a Beta-subunit containing a tyrosine kinase domain and a novel C-terminal extension. The dRas GTPase links extracellular signals to intracellular mechanisms that control cell growth, the cell cycle, and cell identity. An activated form of dRas promotes these processes in the developing wing, but the effector pathways involved are unclear. dRas promotes cell growth and G1 to S progression by increasing Dm/dMyc protein levels and activating dPI3K signaling, and that it does so via separate effector pathways. Endogenous dRas is required to maintain normal levels of Dm/dMyc, but not dPI3K signaling during wing development (Ref.6). Induction of Dm/dMyc and regulation of cell identity are separable effects of dRaf (Raf Oncogene)-Rl (Rolled)/dmMAPK (MAPK (Mitogen-Activated Protein Kinase)) signaling. dRas is a membrane-associated guanine nucleotide-binding protein that is normally activated in response to the binding of extracellular signals, such as growth factors, to receptor tyrosine kinases. These receptors include the dEGFR (Epidermal Growth Factor Receptor), Sevenless and Torso. Extracellular signals enhance binding of dEGFRL (Epidermal Growth Factor Receptor Ligand) to dEGFR. In the developing Drosophila wing dRas, Dm/dMyc and dPI3K regulate rates of cellular growth and progression through the G1 to S transition of the cell cycle without affecting overall rates of cell division. Thus dRas generates a more robust and balanced growth response than activation of either Dm/dMyc or dPI3K alone. Furthermore, the ability of dRas to de-regulate cell identity and adhesion underlie the strong synergy between dRas and other growth-promoting oncogenes in vivo (Ref.5 & 7).

In Drosophila, the dInR/dPI3K/dPTEN (Phosphatase and Tensin Homolog) pathway combines both metabolism and growth control into one pathway that later diverged into two separate, yet interacting systems in mammals. The dPI3K is negatively regulated by dPTEN, a dPIP3 lipid phosphatase. dPTEN is the Drosophila homolog of the human tumor suppressor gene, PTEN (Phosphatase and Tensin Homolog), which antagonizes Class-Ia PI3K signaling by removing the D3 phosphate group from dPtdIns(3)P, dPtdIns(3,4)P2 and dPIP3. dPTEN activity blocks the recruitment and activation of two key downstream effectors, the dPDK1 (Phosphoinositide-Dependent Protein Kinase-1) and dAkt/dPKB (Ref.8 & 9). Thus dPTEN acts as a negative effector of dPI3K in the dInR growth response. dPDK1 contains an N-terminal serine/threonine kinase domain and C-terminal PH domain. The PH domain binds specifically to the lipid products of dPI3K, dPtdIns(3,4)P2 and dPIP3, and thereby localizes dPDK1 to the plasma membrane. dPDK1 carries out an analogous activating phosphorylation reaction that contributes to the activation of dAkt/dPKB and dS6K (S6 Kinase). The dAkt/dPKB is a central regulator of cellular migration and one that angiogenic tubulogenesis requires. dAkt/dPKB has an N-terminal PH domain and a C-terminal serine/threonine kinase domain. The PH domain of dAkt/dPKB binds to the lipid products of dPI3K: dPtdIns(3,4)P2 or dPIP3. This binding localizes dAkt/dPKB to the plasma membrane where it is phosphorylated on Thr423 (Threonine-423) in the activation loop of the catalytic domain by dPDK1. dAkt/dPKB is required for cell survival during early embryogenesis and is essential for tracheal cell fate determination. The molecular mechanisms controlled by dPI3K and dAkt/dPKB are complex and involve multiple players (Ref.10).

Since dS6K requires direct phosphorylation by dPDK1, it is susceptible to variations in its levels. On the other hand, Thor/d4EBP (Eukaryotic Translation Initiation Factor-4E-Binding Protein), which relies on a signal relayed by dAkt/dPKB, is less affected by variations in dPDK1. The co-expression of dS6K with dPI3K does not cause additive cellular overgrowth, unlike co-expression of dAkt/dPKB and dPI3K. Thor/d4EBP requires higher dAkt/dPKB activity than dS6K in order to be phosphorylated (Ref.11). In Drosophila, dS6K and a single Thor/d4EBP (Eukaryotic Translation Initiation Factor-4E-Binding Protein) are phosphorylated via the insulin and target dTOR (Target of Rapamycin) signaling pathways. The phosphorylation of Thr46 (Threonine-46) is the major phosphorylation event that regulates Thor/d4EBP activity. There are three 4E-BPs (Eukaryotic Translation Initiation Factor-4E-Binding Protein) in mammals but only one in Drosophila melanogaster. Thor/d4EBP is a downstream target of the dPI3K-dAkt/dPKB-dTSC (Tuberous Sclerosis)-dTOR signaling cascade. The dInR-Chico/dIRS (Insulin Receptor Substrate)-dPI3K-dPTEN signaling module coordinates cellular metabolism with the nutritional state. The primary outcome of its activation is the modulation of the dPI3K/dPTEN cycle and consequent dPIP3 production. The increase in dPIP3 facilitates the recruitment of PH domain-containing proteins, such as dAkt/dPKB and dPDK1, to the plasma membrane. Chico/dIRS is the Drosophila homolog of mammalian IRS (Insulin Receptor Substrate) proteins, contains an N-terminal PH domain, a PTB domain and a C-terminal region containing multiple potential tyrosine phosphorylation sites. The PTB domain binds to phosphotyrosine residues in dInR, whereas phosphorylated C-terminal tyrosine residues within the consensus Tyr-X-X-Met bind to the dp60. Chico/dIRS controls fecundity and body size develpoment. When dTSC2 subunit of dTSC is phosphorylated by dAkt/dPKB, it results in inhibition of the complex by causing its dissociation or by blocking its interaction with other proteins. dTSC inhibits dS6K, Thor/d4EBP and dTOR (Ref.12 & 13).

In Drosophila cells amino acids alone do not stimulate dPI3K. Amino acids stimulate dS6K by a dPI3K-independent pathway, which is mediated through dTOR. Interestingly, in the presence of insulin and amino acids, dS6K is activated 5-fold higher levels than in the presence of amino acids alone. This enhanced dS6K activation, is mediated through the phosphorylation of dTSC2 by dAkt/dPKB, which results in a greater activation of dTOR than by amino acids alone. dPI3K and dAkt/dPKB, as well as dTOR, are required for insulin-stimulated dS6K activation in Drosophila. The growth suppressing role of dTSC1 and dTSC2 acting as a complex occurs in Drosophila (Ref.14). The dTSC1 and dTSC2 act together to restrict growth in a cell-autonomous manner. The dTSC complex acts in a dominant fashion in the dInR pathway upstream of dS6K or on a parallel pathway to that of the dInR pathway. dS6K is involved in the translational regulation of mRNAs (Messenger RNAs) encoding components of the protein synthetic apparatus. Furthermore, the dTOR, a direct upstream regulator of dS6K is implicated in the control of rRNA (Ribosomal RNA) transcription. Because dTSC1/2-controls cell growth and is involved in the regulation of vesicle trafficking, it is also that dTSC1/2 controls a step involved in ribosome biogenesis, an event which in itself is controlled through dS6K or a common target of dS6K function. A direct affect of the dInR signaling pathway is to drive ribosome biogenesis, modulated through dTSC1/2. Although dS6K activation is controlled by dTOR, the latter reside on a parallel nutrient-sensing pathway to that of Chico/dIRS. The dTSC1/2 negatively regulates cell growth downstream of dPI3K and dAkt/dPKB, but upstream or parallel to dS6K, Dm/dMyc and CycD (Cyclin-D)-dmCDK4 (Cyclin-Dependent Kinase-4). Despite the potential link between dS6K and CycE (Cyclin-E), it is intriguing that over-expression of CycE suppresses the growth defect induced by dTSC1/2 over-expression, because CycE is implicated in cell proliferation, rather than increasing cell mass. The dTSC1/2 functions to restrict the G1 to S transition. Although modulating dTSC1/2 levels acts on cell growth, affecting cell size and cell proliferation, the direct targets and the molecular mode of action of dTSC1/2 remains to be established (Ref.15).