Rheb functions downstream of PTEN

Insulin signalling is a potent inhibitor of cell growth and has been proposed to function, at least in part, through the conserved protein kinase TOR (target of rapamycin). Recent studies suggest that the tuberous sclerosis complex Tsc1-Tsc2 may couple insulin signalling to Tor activity. However, the regulatory mechanism involved remains unclear, and additional components are most probably involved. In a screen for novel regulators of growth, Rheb (Ras homolog enriched in brain), a member of the Ras superfamily of GTP-binding proteins, was identified. Increased levels of Rheb in Drosophila promote cell growth and alter cell cycle kinetics in multiple tissues. In mitotic tissues, overexpression of Rheb accelerates passage through G1-S phase without affecting rates of cell division, whereas in endoreplicating tissues, Rheb increases DNA ploidy. Mutation of Rheb suspends larval growth and prevents progression from first to second instar. Genetic and biochemical tests indicate that Rheb functions in the insulin signalling pathway downstream of Tsc1-Tsc2 and upstream of TOR. Levels of rheb mRNA are rapidly induced in response to protein starvation, and overexpressed Rheb can drive cell growth in starved animals, suggesting a role for Rheb in the nutritional control of cell growth (Saucedo, 2003).

Because the growth and cell cycle phenotypes after Rheb overexpression are reminiscent of those caused by hyperactivation of insulin/phosphatidylinositol-3-OH kinase [PI(3)K] signalling, the potential role of Rheb in this network was tested. Using a pleckstrin homology (PH) domain-green fluorescent protein (GFP) fusion protein as a reporter of PI(3)K activity, it was found that Rheb dies not stimulate PI(3)K function, indicating that if Rheb has a role in insulin/PI(3)K signalling, it functions further downstream. The lipid phosphatase PTEN (phosphatase and tensin homolog deleted in chromosome 10) directly antagonizes the kinase function of PI(3)K and suppresses growth when overexpressed. Co-overexpression of Rheb bypasses PTEN-mediated growth inhibition in the adult eye, providing further evidence that Rheb functions downstream of PI(3)K activity. Whether PI(3)K signalling occurs in the absence of Rheb was tested. Animals overexpressing PI(3)K are sensitive to starvation, most probably because of inappropriate anabolic metabolism. Removal of one or both copies of rheb suppresses this hypersensitivity, suggesting that Rheb is required for PI(3)K signalling (Saucedo, 2003).

Molecular networks linked by Moesin drive remodeling of the cell cortex during mitosis

The cortical mechanisms that drive the series of mitotic cell shape transformations remain elusive. This paper identifies two novel networks that collectively control the dynamic reorganization of the mitotic cortex. Moesin, an actin/membrane linker, integrates these two networks to synergize the cortical forces that drive mitotic cell shape transformations. The Pp1-87B/Slik phosphatase restricts high Moesin activity to early mitosis and down-regulates Moesin at the polar cortex, after anaphase onset. Overactivation of Moesin at the polar cortex impairs cell elongation and thus cytokinesis, whereas a transient recruitment of Moesin is required to retract polar blebs that allow cortical relaxation and dissipation of intracellular pressure. This fine balance of Moesin activity is further adjusted by Skittles and Pten, two enzymes that locally produce phosphoinositol 4,5-bisphosphate and thereby, regulate Moesin cortical association. These complementary pathways provide a spatiotemporal framework to explain how the cell cortex is remodeled throughout cell division (Roubinet, 2011).

These findings unravel how, by integrating two regulatory networks, Moe activity provides a spatiotemporal framework to control cell shape transformations during division (see Model of the spatiotemporal regulation of Moe activity throughout the successive steps of the cell cycle). The increase in cortical rigidity that drives cell shape remodeling at the interphase/mitosis transition involves a Pp1-87B/Slik molecular switch that timely regulates Moe phosphorylation (Roubinet, 2011).

PI(4,5)P2 was further identified as a spatial cue that controls Moe distribution at the cortex. This latter aspect coordinates the spatial balance in cortical stiffness/contractility that is required for anaphase cell elongation and cytokinesis. It is proposed that the concerted action of these two regulatory networks ensures the proper series of mitotic cell shape transformations required for the fidelity of cell division (Roubinet, 2011).

A global increase in cortical actomyosin forces generate cell rounding at mitosis entry. These forces are transmitted to the plasma membrane through the activation of ERM proteins. At mitosis exit, both cortical contractions and ERM activity must be down-regulated to allow cells to go back to their interphase shape. In Drosophila cells, the Slik kinase was shown to activate Moe at mitosis entry (Carreno, 2008; Kunda, 2008). This study identifies the Pp1-87B phosphatase as essential for Moe inactivation after cytokinesis and in interphase (Roubinet, 2011).

Although Slik homogenously associates with the cell cortex in both interphase and early mitosis, Pp1-87B is cytoplasmic in interphase and relocalizes to the spindle in pro/metaphase. An attractive model would be that together with a 'constitutive' cortical association of the Slik activator in interphase and pro/metaphase, intracellular redistribution of the Pp1-87B inhibitor represents an efficient way to restrict high levels of Moe phosphorylation to mitosis entry. During anaphase, Pp1-87B concentrates near the chromosomes migrating toward the polar cortex, whereas Slik accumulates at the cleavage furrow. In this model, redistribution of both Pp1-87B and Slik after the anaphase onset contributes to enrich Moe at the equator and to decrease it at poles. Finally, relocalization of Pp1-87B in the cytoplasm after cytokinesis would contribute to relax the cortex for the next interphase by maintaining low Moe activity. A growing number of evidence supports that Pp1 phosphatases play important roles in the temporal control of cell division. Pp1-87B being required for mitotic spindle morphogenesis, this phosphatase could contribute to synchronize cell shape control operated through Moe regulation to chromosome segregation. Although additional investigations will be required to unravel how the activity and distribution of Pp1-87B and Slik are regulated, these results indicate that the Slik/Pp1-87B switch represents an important control of Moe activity during the cell cycle (Roubinet, 2011).

The results show that local levels of PI(4,5)P2 provide an additional mechanism to regulate Moe function at the cortex of dividing cells. Several studies have established a role of PI(4,5)P2 in the localization of ERM proteins in polarized processes of differentiated cells. This study provides evidence that during mitosis, PI(4,5)P2-rich membrane domains act as a spatial cue that regulates both Moe distribution and activation at the cortex (Roubinet, 2011).

The distribution of PI(4,5)P2 at the plasma membrane is tightly regulated during mitosis. As in mammalian cells, it was found that PI(4,5)P2 is actively enriched at the equator of anaphase Drosophila S2 cells, suggesting that equatorial accumulation of PI(4,5)P2 is a feature shared by most animal cells. Although a previous study did not detect PI(4,5)P2 enrichment at the cleavage furrow of Drosophila spermatocytes, whether this is caused by an intrinsic difference between mitosis and meiosis or by experimental limitations in vivo remains to be established. However, how this dynamic localization is regulated remained unknown. This study shows that the equatorial enrichment of PI(4,5)P2 relies, at least in part, on the enzymatic activity of Skittles and Pten. During cytokinesis, the equatorial accumulation of PI(4,5)P2 plays a role in cleavage furrow formation and ingression, through controlling the activity and/or recruitment of several components of the contractile ring. PI(4,5)P2 hydrolysis is also necessary for maintaining cleavage furrow stability and efficient cytokinesis. The current findings extend the functional repertoire of PI(4,5)P2 during mitosis to the control of local properties of the mitotic cortex, which are required for polar relaxation and cell elongation. Through functional screenings, novel regulators of cell division were identified among the entire set of enzymes implicated in phosphoinositide biosynthesis. Two main pathways regulate PI(4,5)P2 levels in mitotic cells, and their alterations provoke similar cortical disorganization. The first pathway involves the Pten tumor suppressor, a PI(3,4,5)P3 3-phosphatase. Pten was shown to accumulate at the septum of dividing yeast cells, as well as at the cleavage furrow in Dictyostelium discoideum. The results of living Drosophila cells show a progressive delocalization of Pten from the polar cortex to the equator after anaphase onset, suggesting that Pten dynamics rely on mechanisms conserved throughout evolution. Furthermore, depletion of Pten leads to a significant enrichment of PI(3,4,5)P3 at the cortex, especially at the cleavage furrow. These results show that Pten uses PI(3,4,5)P3 to spatially control PI(4,5)P2 levels at the mitotic cortex (Roubinet, 2011).

The second pathway relies on Skittles, a PI(4)P 5-kinase that plays a major role in regulating the levels and localization of PI(4,5)P2 during mitosis. Skittles switches from an isotropic cortical distribution in pro/metaphase to equatorial enrichment after the anaphase onset. Depletion of Skittles results in a phenotype similar to the mitotic cortical defects observed after inducible PI(4,5)P2 hydrolysis. It was also found that CG10260, a phosphoinositide 4-kinase, contributes to the organization of the mitotic cortex. Genetics screens have identified a role for phosphoinositide 4-kinases in the division of budding and fission yeast as well as for cytokinesis of male spermatocytes in flies. CG10260 is involved in PI(4)P synthesis, the major substrate of Skittles to produce PI(4,5)P2. Together, these data show that Skittles acts as a key regulator of PI(4,5)P2 levels and Moe activation at the mitotic cortex. Interestingly, Skittles is required for Moe activation in Drosophila oocytes, suggesting that this enzyme plays a broad role in the regulation of ERM proteins (Roubinet, 2011).

An important question is how Skittles and Pten are enriched at the equator in anaphase. It has been reported that activated RhoA stimulates a PI(4)P 5-kinase activity and promotes PI(4,5)P2 synthesis in mammalian cells. During anaphase, activated RhoA localizes at the equatorial cortex, where it could recruit and/or activates Skittles to promote PI(4,5)P2 production. This anisotropy in PI(4,5)P2 distribution might be in turn reinforced by the localized activity of Pten, whose membrane association is itself dependent on PI(4,5)P2. Together, the activity of Skittles and Pten could therefore provide a feed-forward regulatory loop of local PI(4,5)P2 levels at the cortex of dividing cells (Roubinet, 2011).

The metaphase/anaphase transition is characterized by a break in cortical symmetry, with concomitant relaxation of the polar cortex and contraction of the equator. The anisotropic distribution of Moe participates in coordinating this differential in cortical tension. Overactivation of Moe impairs cell elongation and causes cytokinesis failure, suggesting that the polar cortex is too rigid for cell division. Accumulation of F-actin at the cleavage furrow can be attributed, at least in part, to a cortical flow of F-actin filaments from polar regions to the equator. Overactivation of Moe at the poles could block this actin cortical flow, through an excessive bridging of the actin cytoskeleton with the plasma membrane, leading to an abnormal stiffness of the polar cortex. Therefore, redistribution of activated Moe from the polar cortex to the equator participates in polar relaxation, anaphase cell elongation, and cytokinesis fidelity (Roubinet, 2011).

Contraction of the equatorial actomyosin ring increases the cytoplasmic pressure exerted on the plasma membrane. Relaxation of the polar cortex is thus required to dissipate this extra pressure by increasing the cellular volume, a process that was proposed to involve short-lived polar blebs. These polar blebs were recently found to play important roles during cell division. Perturbation of their dynamics triggers anaphase spindle rocking and destabilization of cleavage furrow positioning. Although recent studies have addressed how cortical blebs are regulated in interphase, understanding of the signalization that controls dynamics of cortical blebs in mitosis has poorly progressed since pioneering studies. The results show that a transient recruitment of Moe at the mitotic bleb membrane is required for efficient polar bleb retraction, as are the functions of the Moe positive regulators Slik, Skittles, and Pten. Active Moe contributes to cortical bleb organization because alteration of Moe function (or regulation) disrupts actin organization and efficient bleb retraction. This leads to disorganization of the mitotic cortex, characterized by giant blebs that continue growing in an unregulated manner. Therefore, although a global decrease in Moe activity at the polar cortex contributes to cell elongation and cytokinesis, transient and local association of Moe at the rim of polar blebs is important for their retraction. If the binding of Moe to PI(4,5)P2 is required at both the equator and bleb membrane, the influence of the Slik kinase on Moe activation appears different between these two regions of the anaphase cortex. Although Slik depletion abolishes Moe recruitment to polar blebs, remnants of cortical Moe are still visible at the equator, likely as a result of high PI(4,5)P2 levels at the furrow (Roubinet, 2011).

Although these mechanisms synergistically contribute to the cortical contractility at the equator, they also allow cortical relaxation at the polar cortex through control of transient anaphase blebs. It is proposed that this dual mechanism of Moe regulation is exploited by animal cells to ensure proper cell division (Roubinet, 2011).

Protein Interactions

Direct association of Bazooka/PAR-3 with the lipid phosphatase PTEN reveals a link between the PAR/aPKC complex and phosphoinositide signaling

Cell polarity in Drosophila epithelia, oocytes and neuroblasts is controlled by the evolutionarily conserved PAR/aPKC complex, which consists of the serine-threonine protein kinase aPKC and the PDZ-domain proteins Bazooka (Baz) and PAR-6. The PAR/aPKC complex is required for the separation of apical and basolateral plasma membrane domains, for the asymmetric localization of cell fate determinants and for the proper orientation of the mitotic spindle. How the complex exerts these different functions is not known. The lipid phosphatase PTEN directly binds to Baz in vitro and in vivo, and colocalizes with Baz in the apical cortex of epithelia and neuroblasts. PTEN is an important regulator of phosphoinositide turnover that antagonizes the activity of PI3-kinase. Pten mutant ovaries and embryos lacking maternal and zygotic Pten function display phenotypes consistent with a function for PTEN in the organization of the actin cytoskeleton. In freshly laid eggs, the germ plasm determinants, oskar mRNA and Vasa, are not localized properly to the posterior cytocortex and pole cells do not form. In addition, the actin-dependent posterior movement of nuclei during early cleavage divisions does not occur and the synchrony of nuclear divisions at syncytial blastoderm stages is lost. Pten mutant embryos also show severe defects during cellularization. The data provide evidence for a link between the PAR/aPKC complex, the actin cytoskeleton and PI3-kinase signaling mediated by PTEN (von Stein, 2005).

In order to find molecules that bind to Baz/PAR-3 and that may provide a link between the PAR/aPKC complex and the cortical cytoskeleton, a yeast two-hybrid screen was performed using the three PDZ domains of Baz as bait. Three independent clones of the lipid phosphatase PTEN were isolated that specifically bind to Baz. PTEN catalyzes the dephosphorylation of phosphoinositide lipids at the D3 position of the inositol ring, One substrate of particular importance is the lipid phosphatidylinositol (3,4,5) trisphosphate [PtdIns(3,4,5)P3], which is converted by the activity of PTEN to phosphatidylinositol (4,5) bisphosphate [PtdIns(4,5)P2]. PtdIns(3,4,5)P3 is produced by activation of phosphatidylinositol 3-kinase (PI3-kinase) in response to stimulation by a multitude of growth factors and cytokines. Interestingly, PtdIns(3,4,5)P3 locally activates Cdc42 by recruitment of guanine nucleotide exchange factors (GEFs) that promote the exchange of GDP for GTP specifically on Cdc42. Moreover, in mammalian cells PtdIns(3,4,5)P3 recruits phosphoinositide dependent kinase 1 (PDK1; Pk61C – FlyBase) which activates aPKC by direct phosphorylation of a conserved threonine residue in the activation loop of the kinase. Thus, PtdIns(3,4,5)P3 is likely to activate two components of the PAR/aPKC complex, Cdc42 and aPKC. Because PTEN is predicted to antagonize the activation of both Cdc42 and aPKC by lowering the level of PtdIns(3,4,5)P3 in the plasma membrane, the association of PTEN with Baz may have a significant impact on the activity of these two key components of the PAR/aPKC complex (von Stein, 2005 and references therein).

PI3-kinase signaling and PTEN have been implicated in the polarization of Dictyostelium amoebae in response to a source of chemoattractant. PI3-kinase and PTEN are localized to the leading edge and uropod, respectively, in a very dynamic fashion. PI3-kinase signaling also appears to be required for directed migration of leukocytes. In both cases, PI3-kinase and PTEN are thought to participate in a self-sustaining loop that intracellularly amplifies the shallow concentration gradient of the chemoattractant. PI3-kinase and PTEN also affect the polarization of hippocampal neurons in culture and, more specifically, the localization of PAR-3 and aPKC to the tip of the neurite that is going to become the axon. Thus, there is increasing evidence that PTEN and the PAR/aPKC complex may cooperate in the control of cell polarity (von Stein, 2005 and references therein).

In Drosophila, the function of PTEN has mainly been studied with respect to its role in the regulation of growth and proliferation in larval and adult tissues. Pten mutant cells have elevated PtdIns(3,4,5)P3 levels and are larger than wild-type cells owing to increased growth. Clones of Pten mutant cells in imaginal discs also show subtle defects in the organization the actin cytoskeleton. Pten interacts genetically with components of the insulin signaling pathway including the insulin receptor, the insulin receptor substrate IRS-1/Chico, PI3-kinase and protein kinase B (PKB). These findings provided solid evidence for an antagonistic relationship between PTEN and PI3-kinase and show that the regulation of phosphoinositide levels is the main vital function of PTEN (von Stein, 2005).

This study shows that PTEN directly binds to Baz/PAR-3 and colocalizes with Baz in the apical cortex of epithelia and neuroblasts. Pten mutant embryos lacking maternal and zygotic Pten function show defects during early embryonic development that point to a function for Pten in the organization of the actin cytoskeleton. Removal of Pten function from the germline in ovaries causes abnormal actin organization in nurse cells and in the oocyte. It is proposed that recruitment of PTEN by Baz may contribute to the polarization of the actin cytoskeleton, most likely by creating local differences in the balance between PtdIns(3,4,5)P3 and PtdIns(4,5)P2 in the plasma membrane. Moreover, PTEN may affect the activity of two key components of the PAR/aPKC complex, aPKC and Cdc42. The binding of PTEN to Baz provides the first molecular link between the PAR/aPKC complex, the actin cytoskeleton and phosphoinositide signaling (von Stein, 2005).

Therefore, Baz/PAR-3 and PTEN directly bind to each other and colocalize in the apical cortex of neuroblasts and epithelia. What could be the physiological meaning of this interaction? Evidence for a functional link between the PAR/aPKC complex and PI3-kinase signaling comes from a recent study that showed that both pathways are required for polarization of cultured hippocampal neurons. In this system, the PAR/aPKC complex localizes to the tip of the outgrowing axon and its localization is abolished upon overexpression of PTEN. However, no information is available on the mechanism of how PTEN interacts with the PAR/aPKC complex in this system (von Stein, 2005).

Mammalian PTEN contains a canonical PDZ-binding motif at its C terminus, and this motif has been reported to interact with the multi-PDZ proteins MAGI-2 and MAGI-3. Both PDZ proteins localize to tight junctions in mammalian epithelia and cooperate with PTEN to control the activity of the downstream kinase PKB/Akt, indicating that subcellular targeting of PTEN may be important for its biological activity. This hypothesis is supported by studies of a deletion mutant of PTEN lacking the PDZ-binding motif. Although this mutant retains lipid phosphatase activity, its activity differed from the full-length wild-type form of PTEN in several biological assays. Together, these observations demonstrate that targeting of PTEN to a specific subcellular location may be essential for its proper function in the control of cell polarity. The data show that PTEN is specifically recruited to the apical plasma membrane of epithelia and neuroblasts by direct association with Baz/PAR-3, a key regulator of cell polarity (von Stein, 2005).

In order to address the issue of whether Pten activity is required for the control of cell polarity in Drosophila, the phenotype of mutant ovaries and embryos lacking maternal and zygotic Pten activity was analyzed. The organization of the actin cytoskeleton in nurse cells and in the oocyte of Pten germ-line clones becomes abnormal from stage 9 onwards, resulting in the production of smaller, misshapen eggs. Ptenmat,zyg embryos show defects in the axial expansion of nuclei during nuclear division cycles 4-7 and fail to synchronize the cell cycle in syncytial blastoderm nuclei. In addition, pole cells are strongly reduced in number or are missing altogether, which is accompanied by the failure to maintain oskar mRNA and Vasa protein localization at the posterior pole. Very similar phenotypes have been reported for embryos treated with the actin depolymerizing drug cytochalasin D and for mutants in genes that are required for the organization of the actin cytoskeleton. Mutations in the gene shackleton also show defects in axial expansion and lack pole cells, but the posterior localization of oskar mRNA is normal, indicating that defects in axial expansion alone are sufficient to cause the lack of pole cells. Interestingly, although germ plasm determinants were mislocalized or absent in early Ptenmat,zyg embryos, they were still localized normally during oogenesis, pointing to a function for Pten in maintenance, rather than establishment, of germ plasm determinant localization. Studies on ovaries and embryos mutant for the actin-binding protein tropomyosin II give essentially the same results. Thus, all of the phenotypes of Ptenmat,zyg mutant ovaries and embryos described here can be related to a function for PTEN in actin-dependent processes (von Stein, 2005).

The links between PTEN and actin are obviously the substrate and the product of the enzymatic activity of PTEN, PtdIns(3,4,5)P3 and PtdIns(4,5)P2. Both phosphoinositide lipids are important regulators of the actin cytoskeleton. PtdIns(4,5)P2 acts mostly by direct binding to actin-associated proteins that link the actin cytoskeleton to the plasma membrane or by binding to proteins that are involved in the initiation of de novo actin polymerization, e.g., profilin and WASP. PtdIns(3,4,5)P3 in turn acts on the actin cytoskeleton via recruitment of guanine nucleotide exchange factors (GEFs) for the small GTPases Rac1, Rho and Cdc42, which can activate WASP proteins and the Arp2/3 complex. Because the subcellular localization of endogenous PTEN is not known, it is not possible to predict at present how exactly PTEN may affect the organization of the actin cytoskeleton during early embryonic development. However, the fact that overexpressed PTEN2 colocalizes with PtdIns(4,5)P2 to the junctional region of epithelial cells indicates that PTEN may locally alter the balance between PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in the plasma membrane, leading to a modification of the actin cytoskeleton in defined regions of the cytocortex. Studies of PTEN knockout cells and Pten mutants in Drosophila have indeed shown that loss of Pten leads to a significant increase in the amount of PtdIns(3,4,5)P3 in the plasma membrane (von Stein, 2005).

Surprisingly, PTEN does not appear to be required for the control of apicobasal polarity of neuroblasts and epithelia, despite its apical colocalization with Baz in these two cell types. The asymmetric localization of cell fate determinants to the basal cortex of mitotic neuroblasts requires both an intact actin cytoskeleton and the activity of the PAR/aPKC complex. Thus, the PAR/aPKC complex must be communicating with the actin cytoskeleton, but how this occurs is unknown. The finding that mutations in Pten lead to severe defects in several actin dependent processes during oogenesis and early embryonic development support the hypothesis that PTEN may provide a link between the PAR/aPKC complex and the actin cytoskeleton in neuroblasts and epithelia. However, this link may not be essential in these cell types because of functional redundancy in the system that controls the levels of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 at the plasma membrane. Functional redundancy has recently been uncovered for the pathways that control the different cell size of neuroblasts and ganglion mother cells during asymmetric neuroblast division. During this division, the activity of either the PAR/aPKC complex or the Pins/Gαi complex alone is sufficient to generate two daughter cells of different size. Only the simultaneous inactivation of both complexes leads to loss of cell size asymmetry. Alternatively, even if the balance between PtdIns(4,5)P2 and PtdIns(3,4,5)P3 at the plasma membrane were altered in neuroblasts and epithelia of Ptenmat,zyg embryos, alterations in the biological activity of downstream components of the system may compensate for this imbalance. In support of this interpretation, the loss of Pten function that are described in this study affects only a subset of actin-dependent processes in oogenesis and early embryogenesis, while a participation of PTEN in other actin-dependent processes may be masked by the redundant activities of additional actin effectors (von Stein, 2005).

Besides its function in the regulation of actin, PTEN may regulate the catalytic activity of aPKC, a core component of the PAR/aPKC complex that directly binds to Baz. The mammalian homologs of aPKC, the atypical PKC isoforms l and zeta, require phosphorylation by the upstream kinase PDK1 in order to become fully active. PDK1 is recruited to the plasma membrane by direct binding of its pleckstrin homology (PH) domain to PtdIns(3,4,5)P3. PDK1, PTEN and several downstream effectors of the PI3-kinase signaling pathway in Drosophila show strong genetic interactions and are crucial for the regulation of cell growth and proliferation. Biochemical evidence has been obtained that aPKC is a substrate for PDK1 and it is proposed that aPKC is phosphorylated in response to elevated PtdIns(3,4,5)P3 levels. According to this hypothesis, PTEN would be a negative regulator of the kinase activity of aPKC (von Stein, 2005).

In addition to PDK1, PtdIns(3,4,5)P3 recruits GEFs that activate the small GTPases Cdc42 and Rac1. Intriguingly, active GTP-bound Cdc42 is also a component of the PAR/aPKC complex in mammalian cells and in Drosophila. GTP-bound Cdc42 binds directly to the CRIB domain of PAR-6 and this interaction might elevate the kinase activity of aPKC, as has been shown in mammalian cells. Thus, PtdIns(3,4,5)P3 might activate aPKC both by recruitment of PDK1, which directly phosphorylates aPKC, and by recruitment of GEFs, which activate aPKC via Cdc42 and PAR6. Studies on PTEN knockout cells have indeed shown that PTEN inhibits Rac1 and Cdc42. The presence of PTEN in one complex together with aPKC, Cdc42 and PAR-6 should therefore lead to inhibition of both pathways that activate aPKCs, revealing a novel way to control the activity of a key component of the PAR/aPKC complex (von Stein, 2005).

Regulated and polarized PtdIns(3,4,5)P3 accumulation, involving Bazooka and PTEN, is essential for apical membrane morphogenesis in photoreceptor epithelial cells

In a specialized epithelial cell such as the Drosophila photoreceptor, a conserved set of proteins is essential for the establishment of polarity, its maintenance, or both -- in Drosophila, these proteins include the apical factors Bazooka, Atypical protein kinase C, and Par6 together with E-cadherin. However, little is known about the mechanisms by which such apical factors might regulate the differentiation of the apical membrane into functional domains such as an apical-most stack of microvilli or more lateral sub-apical membrane. In photoreceptors Bazooka (D-Par3) recruits the tumor suppressor lipid phosphatase PTEN to developing cell-cell junctions (Zonula Adherens or ZA). ZA-localized PTEN controls the spatially restricted accumulation of optimum levels of the lipid PtdIns(3,4,5)P3 within the apical membrane domain. This in turn finely tunes activation of Akt1, a process essential for proper morphogenesis of the light-gathering organelle, consisting of a stack of F-actin rich microvilli within the apical membrane. Spatially localized PtdIns(3,4,5)P3 has been shown to mediate directional sensing during neutrophil and Dictyostelium chemotaxis. It is concluded that a conserved mechanism also operates during photoreceptor epithelial cell morphogenesis in order to achieve normal differentiation of the apical membrane (Pinal, 2006).

Although localized accumulation of PtdIns(3,4,5)P3 is thought to be an essential player in generating the polarity required for directed cell migration, Par proteins have been shown to play a key role in the establishment of polarity in many other biological contexts. This study has found a direct connection between these two pathways in the retinal epithelium and shows that, in photoreceptors, Baz recruits PTEN to the developing ZA and thus promotes PtdIns(3,4,5)P3 degradation and PtdIns(4,5)P2 biosynthesis in that membrane domain. Importantly, ZA-localized PTEN is required for precise regulation of the accumulation of PtdIns(3,4,5)P3 in the entire apical membrane. Although localized rhabdomeric PtdIns(3,4,5)P3 likely involves localized activation of PI3-Kinase, the juxtaposition of PTEN2 (a splice variant of PTEN that contains a C-terminal PDZ binding domain as does mammalian PTEN) to the source of PtdIns(3,4,5)P3 biosynthesis appears essential for achieving the optimal fine tuning of the PtdIns(3,4,5)P3-dependent Akt1 activation. Activated Akt1 is in turn important for controlling the precise localization of Crumbs (Crb) and D-PATJ within the photoreceptor apical membrane and for achieving proper microvilli morphogenesis. This could be due to a direct regulation of the Crb complex by Akt1 or to a more general role for Akt1 in apical membrane differentiation. Interestingly, Akt1 activation occurs precociously within the photoreceptor apical membrane in the absence of PTEN. This is consistent with a previous report that photoreceptor differentiation is accelerated in the absence of PTEN function, but it may also merely reflect the fact that normal levels of Akt activation at these early stages are too low to be detected in this system. Correlating with the early onset and over-activation of Akt 1 detected in the absence of PTEN function, disruption of the microvilli is readily observed early during photoreceptor differentiation (Pinal, 2006).

Later, during photoreceptor morphogenesis, both Baz and PTEN2 progressively localize to the nascent rhabdomere, at a time when microvilli are extending to reach their mature length. Although the arrangement of microvilli is already defective at this stage, part of the PTEN mutant phenotype (i.e., deformed rhabdomeres with short microvilli) might be due to a role for PTEN during this late phase of morphogenesis. Although PtdIns(4,5)P2 can be detected in the developing ZA before the onset of microvilli induction within the apical membrane, correlating with the location of PTEN2:GFP, this phosphatidylinositol species is also found in the developing rhabdomere later in development. Some PtdIns(4,5)P2 may diffuse into the apical membrane from the ZA, but it is also likely to be produced by PTEN2 in the apical domain itself from 60% pupal development onward, perhaps maintaining a precise balance between PtdIns(3,4,5)P3 and PtdIns(4,5)P2 (Pinal, 2006).

These data indicate that precise regulation of phophoinositide levels in a range of polarized cells is critical for regulating appropriate cytoskeleton-dependent responses such as microvilli morphogenesis or protruding activity in migrating cells. This study has demonstrated an important role for Akt in the process of apical membrane differentiation and, in particular, rhabdomere morphogenesis. It will be interesting to test whether this molecule is also important for the directed migration of neutrophils and macrophages or for defining neuronal polarity, for which localized PtdIns(3,4,5)P3 and Par-3 are also crucial. PTEN and Akt are both major effectors of the insulin signaling pathway regulating cell growth, and it is likely that this pathway is coupled to effectors of the cell cytoskeleton to accommodate modulation of cell size. Interestingly, loss of function of TSC1 or TSC2 (tuberous sclerosis complex), important downstream targets of Akt, leads to rhabdomeric phenotypes that are very similar to that of PTEN and, in particular, to split rhabdomeres interrupted by segments of Crb/D-PATJ stalk-like membrane. This observation raises the possibility that the cytoskeletal or membrane effector(s), or both, of the pathway described in this study lie downstream of the TSC1/2 complex (Pinal, 2006).

PTEN is one of the most frequently mutated genes in human cancer. Based on the results presented in this study, it is proposed that in addition to modulating growth and promoting cell survival, the increase in Akt activity could lead to instabilities within the apical membrane of mutant epithelial cells and that these instabilities might contribute to metastatic and invasive phenotypes by facilitating PtdIns(3,4,5)P3-dependent migratory activity (Pinal, 2006).


Cytoplasmic activated protein kinase Akt regulates lipid-droplet accumulation in Drosophila nurse cells

The insulin/insulin-like growth factor signalling (IIS) cascade performs a broad range of evolutionarily conserved functions, including the regulation of growth, developmental timing and lifespan, and the control of sugar, protein and lipid metabolism. Recently, these functions have been genetically dissected in Drosophila, revealing a crucial role for cell-surface activation of the downstream effector kinase Akt in many of these processes. However, the mechanisms regulating lipid metabolism and the storage of lipid during development are less well characterized. The nutrient-storing nurse cells of the fly ovary were used to study the cellular effects of intracellular IIS components on lipid accumulation. These cells normally store lipid in a perinuclear pool of small neutral triglyceride-containing droplets. Loss of the IIS signalling antagonist PTEN, which stimulates cell growth in most developing tissues, produces a very different phenotype in nurse cells, inducing formation of highly enlarged lipid droplets. Furthermore, the accumulation of activated Akt in the cytoplasm is responsible for this phenotype and leads to a much higher expression of LSD2, the fly homologue of the vertebrate lipid-storage protein perilipin. This work therefore reveals a signalling mechanism by which the effect of insulin on lipid metabolism could be regulated independently of some of its other functions during development and adulthood. It is speculated that this mechanism could be important in explaining the well-established link between obesity and insulin resistance that is observed in Type 2 diabetes (Vereshchagina, 2006).

These data suggest that the effect of cytoplasmic P-Akt on lipid storage may be cell type-specific. Increasing IIS throughout the whole organism in viable Pten mutants surprisingly reduces total lipid content, whereas decreasing IIS elevates lipid levels both in flies and mice. It has not been possible to show biochemically whether triglycerides are increased rather than merely redistributed in Pten mutant ovaries, because only a small minority of the nurse cells is mutant. However, in some late-stage clones, in which cytoplasmic volumes are decreasing, lipid levels often appear elevated in mutant cells. Insulin-stimulated increases in lipid-droplet number and size have been observed in mammals, and, interestingly, when tumours are induced in mouse liver by raising IIS, lipid-storage mechanisms are also activated. It is proposed that these cell type-specific responses are due to selective accumulation of an IIS-modulated, cytoplasmic activated Akt pool that must, to some extent, be controlled independently of cell-surface P-Akt. Indeed, a Drosophila phosphatase has been identified that regulates levels of cytoplasmic P-Akt in nurse cells, and it has been shown that mutations in this gene produce a very similar enlarged lipid-droplet phenotype, confirming this hypothesis (Vereshchagina, 2006).

How could elevated cytoplasmic P-Akt induce such a dramatic lipid-droplet phenotype in nurse cells? In mammals, Akt can promote the transcription of genes involved in lipid biosynthesis and storage pathways. The data indicate LSD2/perilipin is one of these targets. IIS also post-translationally upregulates the activity of mammalian perilipin. Interestingly, ovaries mutant for Lsd2 show altered lipid accumulation, but droplets are still formed. Therefore, LSD2 is almost certainly one, but not the only, target for IIS in the control of lipid-droplet accumulation in nurse cells. In this context, it is interesting to note that, in addition to its proposed role in coating lipid droplets, LSD2 has recently been shown to regulate microtubule-dependent trafficking of these organelles. Since cytoplasmic P-Akt could still be associated with intracellular membranes or the droplet surface, it may be well positioned to modulate this transport process (Vereshchagina, 2006).

Obesity is a well-established predisposing factor in the acquisition of cellular insulin resistance and Type 2 diabetes. Increased levels of circulating free fatty acids (FFAs) associated with obesity appear to be important in this link. However, it is unclear whether other mechanisms are also involved or how reduced insulin sensitivity ultimately impacts on lipid storage. Molecules downstream of Akt are known to regulate cell-surface IIS through at least two negative-feedback loops involving downstream S6 kinase and the transcription factor FOXO. This work therefore raises the possibility that any predisposition towards increased cytoplasmic P-Akt could specifically promote lipid storage and also selectively suppress insulin-dependent events at the cell surface. It will be interesting to investigate further the molecules involved in controlling this P-Akt pool and whether the feedback mechanisms have any role to play in linking obesity and insulin resistance in Type 2 diabetes (Vereshchagina, 2006).

The role of the prothoracic gland in determining critical weight for metamorphosis in Drosophila melanogaster

The timely onset of metamorphosis in holometabolous insects depends on their reaching the appropriate size known as critical weight. Once critical weight is reached, juvenile hormone (JH) titers decline, resulting in the release of prothoracicotropic hormone (PTTH) at the next photoperiod gate and thereby inducing metamorphosis. How individuals determine when they have reached critical weight is unknown. Evidence is presented that in Drosophila, a component of the ring gland, the prothoracic gland (PG), assesses growth to determine when critical weight has been achieved. The GAL4/UAS system was used to suppress or enhance growth by overexpressing PTEN or Dp110 (Pi3K92E), respectively, in various components of the ring gland. Suppression of the growth of the PG and CA, but not of the CA alone, produced larger-than-normal larvae and adults. Suppression of only PG growth resulted in nonviable larvae, but larvae with enlarged PGs produced significantly smaller larvae and adults. Rearing larvae with enlarged PGs under constant light enhanced these effects, suggesting a role for photoperiod-gated PTTH secretion. These larvae are smaller, in part as a result of their repressed growth rates, a phenotype that could be rescued through nutritional supplementation (yeast paste). Most importantly, larvae with enlarged PGs overestimated size so that they initiated metamorphosis before surpassing the minimal viable weight necessary to survive pupation. It is concluded that the PG acts as a size-assessing tissue by using insulin-dependent PG cell growth to determine when critical weight has been reached (Mirth, 2005; full text of article).

These manipulations of insulin-dependent PG growth showed that this growth is inversely related to larval growth. Suppressing the growth of the PG (P0206>PTEN - ectopically driven PTEN) produced larvae that spent more time in each instar and were larger than normal. These effects are presumably due to a combination of reduced ecdysteroid biosynthesis, which is known to delay development, and increased growth rate. Conversely, larvae with enlarged PGs (phm>Dp110; phm is a phantom GAL4 line which was used to drive expression of Dp110) showed accelerated development in the L3. Their growth rate was dependent on nutritional conditions. Whereas phm>Dp110 larvae reared on suboptimal food grew slowly, well-fed phm>Dp110 larvae grew at the same rate as controls. Together, these data indicate that the growth of the PG negatively regulates the growth rate of the whole animal and that this regulation is modulated by nutrition (Mirth, 2005).

In addition, decreasing PG size in P0206>PTEN larvae resulted in premature metamorphosis and the formation of L2 puparia. Similar L2 puparia have been described in larvae with mutations that affect the regulation of ecdysteroid biosynthesis or signaling and in larvae where the Broad isoform Z3 was overexpressed in the ring gland, resulting in its apoptosis. L2 puparia are seen in situations where ecdysone synthesis is compromised because larvae cross the threshold weight for metamorphosis prior to the production of sufficient ecdysone to initiate a larval molt, redirecting their development to the metamorphic pathway (Mirth, 2005).

Reducing PG size resulted in reduced ecdysteroid biosynthesis; P0206>PTEN larvae showed reduced ecdysteroid titers at 44 hr AEL3, and phm>PTEN larvae only molted to L2 when fed 20E. Under conditions of low ecdysteroid synthesis, fast-growing larvae could surpass the threshold for metamorphosis before the ecdysteroid titer was sufficient to induce a molt, resulting in L2 prepupae. Slower-growing larvae would be unable to reach this threshold weight before the rise in ecdysteroid titer induced the molt to L3. Indeed, undernourished, and presumably slow-growing, P0206>PTEN L2 larvae all molted to L3, whereas only 33% of the well-fed P0206>PTEN larvae molted to L3 (Mirth, 2005).

Enlarging the PG of larvae reared under constant light caused larvae to initiate metamorphosis earlier and at smaller sizes. Nevertheless, even though larvae starved early after the L3 molt were able to pupariate, they were unable to survive to pupation unless they had fed for at least 11.5 hr. This suggests that phm>Dp110 larvae starved prior to 11.5 hr AL3E initiated metamorphosis before surpassing the minimal viable weight. Furthermore, although in control larvae, critical weight and minimal viable weight are apparently attained at the same time, they are uncoupled in phm>Dp110 larvae. Therefore, the assessment of critical weight is dependent on PG growth, whereas the minimal viable weight is not (Mirth, 2005).

In Drosophila, the PGs are responsible for a size-assessment event, early in the L3, that induces the onset of metamorphosis once critical weight is surpassed. Enhancing PG growth resulted in an overestimation of body size, thereby causing the larva to initiate metamorphosis early, at a subnormal size. Under LL, the effects of enlarging the PG were enhanced, producing individuals that pupariated even earlier at even smaller sizes, suggesting that when PTTH release was unconstrained by circadian gating, the PTTH delay period was reduced. These data provide the first indication in Drosophila that the post-critical-weight PTTH release may be under photoperiod control, as it is in Manduca (Mirth, 2005).

There has been some discussion in the literature as to whether critical weight as described in Drosophila is the same as critical weight as defined in Manduca. This discussion has arisen because the definition for Manduca states that critical weight is the minimal size at which starvation can no longer delay the onset of metamorphosis, but when Drosophila larvae are starved before critical weight is reached, they die. The current data suggest that this is due to a tight relationship between minimal viable weight and critical weight in Drosophila. Effects more similar to those observed in Manduca can be obtained when pre-critical-weight Drosophila larvae are starved for an interval and then re-fed. Under these conditions, they delay metamorphosis for a period greater than the period of starvation. Much of the confusion surrounding critical weight in Drosophila has arisen because in wild-type larvae, minimal viable weight and critical weight are achieved at the same time (Mirth, 2005).

After critical weight has been surpassed, the metamorphic pathway appears to be partially suppressed by continued feeding in Drosophila. Hence, the nutrition pathway appears to promote growth and suppress metamorphosis, whereas insulin-dependent PG growth suppresses larval growth and promotes differentiation (Mirth, 2005).

The effects of increased growth in the PG are not simply due to increasing cell size, but rather are specific to the nutrition-dependent InR signaling pathway. Studies have indicated that when either dMYC or cyclinD/cdk4 are used to enlarge the PG cells, there is no reduction in overall body size. Overexpression of dMYC, of cyclinD/cdk4, and of Dp110 all enhance cell growth, but they do so in fundamentally different manners by using separate cascades. Whether the size-assessment mechanism operates via increased intracellular PIP3 levels in the PG cells or the accumulation of some other downstream component of the InR cascade in these cells is unknown (Mirth, 2005).

Although no difference in was detected ecdysteroid titers in larvae with enlarged PGs, there is evidence that increased InR signaling in the PG cells can produce mild increases in ecdysteroidogenesis and ecdysone signaling, increases that are below the level of detection of ecdysteroid-titer assays. Larvae with enlarged PGs showed both a mild increase in the transcription of phantom during feeding stages and an increase in the transcription of the early ecdysone response gene E74B. These subtle differences in ecdysteroid titers may be important for determining growth rates and for size assessment. A gradual rise in ecdysteroid titers is coincident with the time that critical weight is reached in Drosophila. Also, subtle shifts in 20E concentrations are important for growth. Basal concentrations of 20E in combination with bombyxin enhance the growth of wing imaginal tissues in vitro; slightly higher concentrations of 20E suppress growth (Mirth, 2005).

Mutations that cause imaginal disc and larval overgrowth often cause delayed pupariation and, in some cases, show low L3 ecdysteroid titers. In the case of the mutant lethal (2) giant larvae, the ring glands are smaller than normal and have the ultrastructural appearance of glands that have low rates of ecdysteroid biosynthesis. Delayed pupariation in these larvae can be rescued by implanting wild-type ring glands. Lastly, hypomorphic mutations in DHR4, a repressor of ecdysone-induced early genes, cause reductions in critical weight and early-pupariation phenotypes similar to those described in this study. Thus, the size-assessment mechanism is likely to involve surpassing a threshold ecdysteroid titer above which the activation of the ecdysone cascade occurs (Mirth, 2005).

These data allow construction of the following model for size assessment in Drosophila. As PG cells grow in response to increased InR signaling, they increase their basal level of ecdysteroid biosynthesis. Critical weight is then reached when systemic ecdysteroid concentrations surpass a threshold that sets into motion the endocrine events that will end the growth phase of larval development and allow the larva to begin metamorphosis (Mirth, 2005).

Studies in the mid-1970s defined a size-assessment event during the final instar of the moth Manduca sexta; termed critical weight, it is the minimal size required for the timely initiation of metamorphosis. How insect larvae determine when they have reached critical weight has long been a mystery. It is hypothesized that a size-assessing tissue determines when critical weight had been reached. Suppressing growth in this size-assessing tissue would cause an underestimation of body size, resulting in metamorphosis at larger than normal sizes, whereas enlarging this tissue would result in subnormal sizes. Studies in Drosophila have shown that manipulation of the growth of the PG via the InR pathway produced these types of effects. Furthermore, larvae with enlarged PGs metamorphosed at even smaller sizes when reared under LL, suggesting a role for PTTH circadian gating in this response. Smaller size arose both as a result of a reduction in growth rate, an effect that could be rescued via nutritional supplementation, and the early onset of metamorphosis. Most importantly, larvae with enlarged PGs had a remarkably reduced critical weight, suggesting that they are severely overestimating their own body size. These results offer a very new perspective on the problem of size control in insects, uniting the recent data exploring the role of nutrition and the insulin-receptor pathway on growth with the classical physiological experiments that defined critical weight (Mirth, 2005).

Effects of Mutation or Overexpression

To gain insight into the functions of Pten during development, an examination was made of clones of cells in the Drosophila eye that lack Pten. Pten mutant ommatidia in the mosaic eyes appear larger in size when compared to their neighboring wild-type ommatidia. Sections of these mosaic eyes reveal that the mutant ommatidia have normal photoreceptor cell composition and orientation. However, the sizes of the individual mutant cells are much larger than their neighboring wild-type cells (the average size of a mutant ommatidium is approximately 2.5 times that in a wild-type cell). Careful examination of the chimeric ommatidia that contain both mutant and wild-type cells reveals that the Pten mutant phenotype is cell autonomous, since every mutant cell has an enlarged cell body while its immediate wild-type neighbors have normal sized cell bodies. Unlike the Drosophila tumor suppressor genes such as lats, no obvious overproliferation defect is seen in Pten mutant clones in the eye. This could be because Pten may not normally function in the eye in regulating cell proliferation, or alternatively, there may be redundancy for Pten-like molecules in the eye (Huang, 1999).

To explore other potential roles for Pten during eye development, the UAS/GAL4 system was used to overexpress both human and fly PTEN in Drosophila. Interestingly, the phenotypes caused by overexpression of human PTEN and Drosophila Pten are indistinguishable, suggesting that the functions of the two homologs are conserved. For simplicity, the name PTEN will be used here. To verify whether the effects that are observed with expression of PTEN are related to its enzymatic activity, a construct, UAS-PTENC124S, was used in which a critical residue in the phosphatase domain has been mutated. This change also leads to elimination of catalytic activity in vitro. While ectopic expression of wild-type PTEN causes specific phenotypes, expression of PTENC124S under the same conditions has no effect. Together with the PTENc494 mutant, these experiments demonstrate that phosphatase activity is necessary for PTEN to exert its functions during development. Since ubiquitous expression of PTEN directed by heat-shock induction of GAL4 causes lethality during embryonic and larval stages, PTEN is expressed specifically in the developing eye using the eyeless GAL4 line (EYE-GAL4). The eyeless enhancer directs gene expression in the young developing eye disc where cells are actively proliferating. Overexpression of PTEN in proliferating cells of the eye disc results in dramatic reduction of eye size in a dosage-dependent manner. In fact, multiple copies of UAS-PTEN can completely eliminate the eye. This small adult eye phenotype could be caused either by the inhibition of cell proliferation by PTEN, which would result in eye discs of smaller than normal size, or by the failure of ommatidium differentiation in eye discs of normal size. The sizes of eye discs dissected from third instar larvae carrying EYE-GAL4/UAS-PTEN are dramatically reduced, suggesting a defect in cell proliferation. Consistent with this idea, sections of these small adult eyes show that all types of photoreceptor cells are present and the pigment lattice is essentially regular, indicating that cell differentiation and pattern formation are not disrupted. Moreover, although these eye discs are much smaller, neural differentiation in the posterior region occurs normally as indicated by neuronal-specific anti-Elav staining (Huang, 1999).

Suppression of cell proliferation in mammalian tissue culture cells by PTEN has been reported to act through blocking cell cycle progression in the G1 phase. To determine whether PTEN blocks G1/S transition in the developing eye, Drosophila eye discs were examined with GMR-GAL4 driven PTEN expression. The GMR promoter directs gene expression in the region posterior to the morphogenetic furrow (MF) in the eye imaginal disc. While most cells in this region are differentiating, a stripe of cells posterior to MF undergoes a synchronized, last round of cell division. GMR-directed expression of p21C1P1/WAF1, a human cyclin-dependent kinase inhibitor, blocks this last round of division by preventing these cells from entering S phase. Unlike the GMR-directed p21 expression, BrdU labeling shows that these PTEN-expressing discs have the characteristic stripe of staining similar to that of the wild-type discs, suggesting that expression of PTEN does not block G1/S transition. In contrast, propidium iodide staining of the EYE-GAL4/UAS-PTEN eye discs reveals that the discs accumulate many cells with brighter nuclear staining consistent with a DNA content of 4C. FACS analysis of dissociated cells from these discs also shows an increased percentage of cells in G2. These results suggest that overexpression of PTEN during eye development causes cell cycle arrest at the G2 or G2/M phase (Huang, 1999).

Overexpression of PTEN under the direction of GMR results in adult eyes that are not only smaller but also rough. Sections of these adult eyes reveal a complex phenotype with varied degrees of severity. While the apical lenses of the ommatidia are smaller, the sizes of photoreceptor cells in the cross sections are larger than normal. Furthermore, these GMR/PTEN adult eyes have a flattened appearance with a narrowed retina, suggesting the mutant phenotype could result from changes in both cell shape and size. In addition, and different from the phenotype caused by inactivation of PTEN, the rhabdomeres of the PTEN-overexpressed photoreceptors are reduced in size. Moreover, these sections show that some ommatidia have missing pigment or photoreceptor cells. Third instar eye discs were therefore stained with the anti-Elav antibody to monitor retinal neuron differentiation. Elav staining reveals a neuronal differentiation pattern comparable to wild type, indicating that photoreceptor differentiation is not affected. To pinpoint the cause of the phenotype, an assessment was made as to whether cell death might have contributed to the GMR-GAL4/UAS-PTEN eye phenotype. These eye discs were stained with acridine orange. Unlike wild-type third instar eye discs, which have few dying cells, the GMR-GAL4/UAS-PTEN eye discs have a substantially increased amount of cell death in the posterior region of the eye disc, where GMR-GAL4 is expressed. To further verify the contribution of cell death to the eye phenotype, GMR-GAL4 was used to coexpress PTEN with the p35 baculovirus gene, which can block apoptotic cell death. The eye phenotype induced by GMR/PTEN is largely rescued by coexpression of p35 (Huang, 1999).

These experiments suggest the possibility that cell death might also contribute to the EYE/PTEN small eye phenotype. However, EYE/PTEN eye discs do not have increased acridine orange staining and the EYE/PTEN small eye phenotype is not suppressed by coexpression of p35. Thus, cell death appears not to be a major factor in PTEN-mediated inhibition of cell proliferation. Instead, these results suggest that cell death is a consequence of cells at different developmental stages in response to the PTEN product. It is concluded that overexpression of PTEN arrests cell-cycle progression in proliferating cells while promoting apoptosis in differentiating cells during eye development. These results suggest that PTEN may suppress tumorigenesis by preventing damaged cells from dividing and/or promoting a response to apoptotic signals in a cell context-dependent manner (Huang, 1999).

Pten, a Drosophila homolog of the mammalian PTEN tumor suppressor gene, plays an essential role in the control of cell size, cell number, and organ size. In mosaic animals, Pten minus cells proliferate faster than their heterozygous siblings, show an autonomous increase in cell size, and form organs of increased size, whereas overexpression of Pten results in opposite phenotypes. The loss-of-function phenotypes of Pten are suppressed by mutations in the PI3K target Dakt1 and the translational initiation factor eif4A, suggesting that Pten acts through the PI3K signaling pathway to regulate translation. Although activation of PI3K and Akt has been reported to increase rates of cellular growth but not proliferation, loss of Pten stimulates both of these processes, suggesting that PTEN regulates overall growth through PI3K/Akt-dependent and -independent pathways. Furthermore, Pten does not play a major role in cell survival during Drosophila development. These results provide a potential explanation for the high frequency of PTEN mutation in human cancer (Gao, 2000).

The observation that clones of Pten mutant cells have a proliferative advantage over their wild-type twin spots indicates that Pten controls progression through the cell division cycle. To determine which phase(s) of the cell cycle Pten regulates, FACS analysis was used to examine the DNA content of dissociated wing imaginal disc cells containing clones of dPTEN mutant cells. Loss of Pten results in a decrease in the percentage of cells in the G1 phase of the cell cycle and a relative increase in the S and G2 population. Clonal overexpression of Pten has a complementary effect, causing a slight decrease in the number of S-phase cells. FACS analysis also reveals complementary changes in cell size in response to Pten levels: loss of Pten causes an increase in average cell size, while Pten overexpression decreases average cell size. Similar effects were observed throughout all phases of the cell cycle and at multiple developmental stages. The cell size and cell cycle FACS profile of Pten mutant cells is remarkably similar to that of cells overexpressing Drosophila PI3K. In both cases, the percentage of cells in G1 is reduced, indicating an acceleration of this phase of the cell cycle. Since overexpression of PI3K does not increase proliferation rates, this shortened G1 appears to be balanced by a commensurate lengthening in the duration of S and/or G2 phases. A similar phenomenon has been described for cells whose G1 phase is accelerated by overexpression of Cyclin E, dMyc, or activated Ras. In contrast, the rapid proliferation rate of Pten mutant cells indicates that S and G2 phases do not lengthen in response to the abbreviated G1 and thus suggest that Pten regulates multiple phases of the cell cycle (Gao, 2000).

Genetic interactions between Pten and PI3K, a component of the insulin signaling pathway in Drosophila, were examined. Overexpression of the PI3K catalytic subunit, Dp110, results in increased wing size, while overexpression of a dominant negative Dp110 construct (PI3KDN) results in the opposite phenotype. These phenotypes are due to changes in cell size. Coexpression of Pten with PI3KDN further reduces wing size. In addition, overexpression of Pten suppresses the increased wing size resulting from PI3K overexpression. To further examine the genetic interaction between Pten and PI3K, Pten mutant cells were examined in a genetic background of overexpressing PI3K or PI3KDN. When PI3K is overexpressed, Pten mutant cells are indistinguishable in cell size from their nonmutant siblings, suggesting that overexpression of PI3K results in increased PIP3 levels and increased signaling that cannot be further activated by removing Pten. Conversely, overexpression of PI3KDN partially suppresses the increased cell size of Pten mutant cells. Thus, PTEN and PI3K antagonize each other in regulating cell size (Gao, 2000).

Certain combinations of loss-of-function alleles of the Drosophila Insulin-like receptor (inr) result in flies with a decreased cell size. This provides an opportunity to examine the genetic epistasis between Pten and inr. The increased cell size of Pten mutant cells can not be reversed in inr mutant animals, and thus loss of Pten function is epistatic to (acts downstream from) mutations in inr (Gao, 2000).

Previous studies have shown that loss of PTEN function promotes cell survival in mammals through activation of Akt. In addition, PTEN acts through Akt in metabolic and longevity control in C. elegans. Dakt1, a Drosophila homolog of Akt, has been suggested to play a role in cell survival in embryogenesis (Staveley, 1998) and cell size control (Verdu, 1999). A hypomorphic allele of Dakt1 has been identified in the large-scale gene disruption project carried out by the Berkeley Drosophila Genome Project. This allele is semilethal, and homozygous survivors show reduced body size and cell size, consistent with a role of Dakt1 in growth control. To examine whether Pten controls cell size through regulating Akt activity, Pten mutant clones were generated in Dakt1 mutant animals. Dakt1 mutation completely suppressed the increase of cell size associated with the Pten mutation. This result provides strong in vivo evidence that Dakt1 functions downstream of (or in parallel to) Pten in the control of cell size. Taken together, these genetic interactions suggest that the role of Pten in opposing signaling through the PI3K/Akt pathway is conserved between flies and vertebrates (Gao, 2000).

eif4A is an ATP-dependent RNA helicase that is an essential component of the eif4F translation initiation complex. Previous studies have identified an allelic series of eif4A mutants that affects larval growth, DNA replication, and cell proliferation (Galloni, 1999). Since the defect in DNA synthesis in eif4A mutants can be bypassed by overexpressing the E2F transcription factor, it has been suggested that eif4A preferentially regulates a specific set of cell-cycle regulatory genes (Galloni, 1999). To examine whether the proliferative advantage of Pten mutant cells requires the same set of cell-cycle regulatory genes as those controlled by eif4A, Pten;eif4A double-mutant clones were examined. A hypomorphic allele and a stronger allele of eif4A were used. The weaker allele confers proliferation disadvantage to the cells, resulting in mutant clones that are smaller than the twin spots without affecting cell size (Galloni, 1999). Hypomorphilc eif4A can partially suppress the overproliferation of Pten mutant cells, but the increased cell size of Pten mutant cells is not suppressed. The stronger eif4A allele completely suppresses the proliferation of Pten mutant cells. Pten;eif4A double-mutant clones are undetectable, as has been observed in the eif4A strong single mutant (Galloni, 1999). It is suggested that modulation of translation initiation is an important aspect of Pten function in regulating cell proliferation (Gao, 2000).

It is proposed that Pten regulates cell proliferation by multiple mechanisms, both PI3K-dependent and -independent. One potential PI3K-independent mechanism is suggested by the domain in Pten related to tensin, an actin filament capping protein that localizes to focal adhesions. Overexpression of tensin can suppress anchorage-independent proliferation of Ras-transformed 3T3 cells, and therefore this domain may provide a growth-regulatory function in mammalian PTEN as well. Moreover, in addition to its role as a lipid phosphatase, PTEN also possesses a dual-specificity protein phosphatase activity. PTEN has been shown to bind and dephosphorylate the focal adhesion kinase FAK and to down-regulate the formation of focal adhesions. Such cell contacts play a critical role in regulating proliferation in Drosophila, and the gene products of several Drosophila tumor suppressors such as expanded, fat, and l(2) discs large all localize to adherens or septate junctions. The results for Pten are thus consistent with a model in which PTEN suppresses cell growth and G1/S progression by down-regulating the PI3K/Akt pathway and inhibiting the G2/M transition through an alternative mechanism, perhaps involving regulation of the cytoarchitecture. The ability to regulate both growth and cell division may explain why PTEN is such a common target in advanced tumors. This model is also consistent with the different mutant phenotypes between a null Pten allele and an allele that carries a point mutation (Huang, 1999). While the point mutation changes an invariant amino acid within the phosphatase active site and is likely to inactivate the lipid phosphatase activity, the other domains of Pten are still intact. Characterization of Pten mutants that are specifically defective in cell growth or proliferation may shed further light on its role in the control of overall growth (Gao, 2000).

Since loss of lilliputian function affects cell size and head size, tests were performed for genetic interactions between lilli and components of the PI3K/PKB pathway. Pten acts as a negative regulator in the PI3K/PKB pathway by dephosphorylating the second messenger phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3; PIP3]. Tissues mutant for Pten show hyperplastic and hypertrophic growth: Pten mutant cells are larger and proliferate at a higher rate than wild-type cells. Removal of Pten function from the eye imaginal disc tissue using the ey-Flp system results in an increase in eye and head size. The increase in eye size is due to an increase in cell number and cell size as indicated by the increase in number and size of ommatidia. To test whether the Pten large-head phenotype is modified by the removal of Lilli function, Pten;lilli double mutant eyes were generated. Indeed, eyes double mutant for Pten and lilli are considerably smaller than Pten mutant eyes due to a reduction in cell number and cell size. Loss of Lilli function, however, does not completely suppress the Pten phenotype. Although these results suggest that Lilli and Pten cooperate in the control of cell and organ growth, the absence of a clear-cut epistasis between the two mutants indicates that Lilli does not act downstream of Pten in a simple linear pathway but it rather acts in a parallel pathway required for growth (Wittwer, 2001).

Retinal cells lacking lilli are significantly smaller than wild-type cells. Despite the small size of individual lilli mutant adult cells, relatively large clones of lilli mutant cells can be generated, suggesting that lilli may affect cell size without changing the overall rate of growth. This was tested by comparing the size of individual lilli mutant clones to their wild-type twinspots in third instar eye and wing imaginal discs. lilli clones induced either at 24-36 hours or 36-48 hours after egg deposition (AED) are indistinguishable in size and number of cells from their wild-type twinspots. Of 75 individual clones examined at 96 hours after induction, the average area of lilli mutant clones was 1440 pixels, compared to 1400 pixels for corresponding wild-type twinspots. Thus, lilli mutant cells grew at 1.03 times the rate of wild-type cells, indicating that lilli is not required for normal rates of cell growth (Tang, 2001).

Interestingly, despite an approximately 50% reduction in the size of lilli photoreceptor cells and wing margin bristles in the adult, other cell types in the adult eye and wing are unaffected. For example, the surface of eyes containing lilli mutant clones appears normal by SEM analysis, suggesting that loss of lilli does not reduce the size of cone cells. The size of lilli cells in developing wing and eye imaginal discs appears normal as well. This was confirmed by FACS analysis of dissociated wing discs, which has revealed no significant difference in size between lilli and wild-type cells. In addition, unlike mutations in components of the PI3K pathway, lilli mutant cells display a normal cell cycle profile. To test whether lilli is required for PI3K-mediated growth, cells doubly mutant for lilli and Pten, an inhibitor of this pathway, were examined. Mutations in Pten increase cell size and advance G1/S progression; these effects are prevented by mutations in downstream components such as torso (tor). In contrast, loss of lilli does not prevent the cell enlargement or cell cycle changes caused by Pten mutation, indicating that lilli is not an essential element of the PI3K pathway. Interestingly, it was noticed that ommatidia containing the enlarged lilli;Pten photoreceptor cells are severely disorganized, and contain malformed rhabdomeres characteristic of cytoskeletal defects. Together, these results indicate that lilli affects the cell size through a growth-independent and PI3K-independent mechanism. It is suggested that mutations in lilli may affect final cell size by disrupting the morphological changes that cells such as rhabdomeres and bristles, which are the specializations of photoreceptor and trochogen cells respectively, undergo during pupal development (Tang, 2001).

Mutations have been characterized in the Drosophila Tsc1 and Tsc2/gigas genes. Inactivating mutations in either gene cause an identical phenotype characterized by enhanced growth and increased cell size with no change in ploidy. Overall, mutant cells spend less time in G1. Coexpression of both Tsc1 and Tsc2 restricts tissue growth and reduces cell size and cell proliferation. This phenotype is modulated by manipulations in cyclin levels. In postmitotic mutant cells, levels of Cyclin E and Cyclin A are elevated. This correlates with a tendency for these cells to reenter the cell cycle inappropriately as is observed in the human lesions (Tapon, 2001).

The enhanced growth observed in the Tsc1 or Tsc2 mutants most resembles the results of inactivating PTEN or increasing Ras1 or dmyc activity. In each of these situations, there is a reduction in the length of the G1 phase. In contrast, increased growth driven by Cyclin D/cdk4 does not alter the distribution of cells in different phases of the cell cycle. The effects of the combined overexpression of Tsc1 and Tsc2 displays genetic interactions with multiple pathways. The phenotype is influenced by alterations in the levels of dS6K, PTEN, Ras1, dmyc, cyclin D, and cdk4. Thus, Tsc1 and Tsc2 may function downstream of the point of convergence of these pathways. Alternatively, Tsc1 and Tsc2 may primarily antagonize one of these pathways, but this effect could be overcome by increasing the activity of one of the others (Tapon, 2001).

A mutation has been isolated in the Drosophila homolog of TSC1 (Tsc1). Cells mutant for Tsc1 are dramatically increased in size yet differentiate normally. Organ size is also increased in tissues that contain a majority of mutant cells. Clones of Tsc1 mutant cells in the imaginal discs undergo additional divisions but retain normal ploidy. Flow cytometry analysis indicates that the increase in cell size is not due to endoreplication. Tsc1 protein is shown to bind to Drosophila Tsc2 in vitro. Overexpression of Tsc1 or Tsc2 alone in the wing and eye has no effect, but co-overexpression leads to a decrease in cell size, cell number, and organ size. Genetic epistasis data are consistent with a model that Tsc1 and Tsc2 function together in the insulin signaling pathway (Potter, 2001).

Recent work has demonstrated that the insulin signaling pathway plays an important role in the regulation of cell size, cell number, and organ size. Mutations of Drosophila PTEN (dPTEN), which functions as a negative regulator of insulin signaling, result in phenotypes that resemble the effects of Tsc1 and Tsc2 mutations. Therefore, genetic epistasis experiments were performed to test whether Tsc1 or Tsc2 might also function to negatively regulate insulin signaling. Overexpression of Drosophila insulin receptor (dinr) using the eyGAL4 driver leads to lethality at 25°C and a dramatic increase in ommatidia number in escapers at room temperature. Co-overexpression of Tsc1 and Tsc2 (but not either Tsc1 or Tsc2 alone) rescues both the lethality and the extra ommatidia phenotype caused by dinr overexpression. Furthermore, overexpression of dinr using the pGMR-GAL4 driver leads to an increase in ommatidium size, which is also suppressed by co-overexpression of Tsc1 and Tsc2. Clones of dinr mutant ommatidia are smaller in size than wild-type. Ommatidia that are mutant for both dinr and Tsc1, however, exhibit the Tsc1 mutant phenotype of increased ommatidium size (Potter, 2001).

Overexpression of dPTEN using the pGMR-GAL4 driver leads to eyes with a decreased ommatidium size. However, overexpression of dPTEN is unable to suppress the clonal Tsc1 mutant phenotype. Similar to dinr, clones of dAkt mutant ommatidia are smaller in size. Ommatidia mutant for both dAkt and Tsc1 display the Tsc1 phenotype. Similarly, ommatidia that contained Tsc2 mutant clones in a dAkt mutant background exhibit the Tsc2 mutant phenotype. These results suggest that in the eye, Tsc1 and Tsc2 function genetically epistatic to (downstream of) dinr, dPTEN, and dAkt (Potter, 2001).

Genetic analyses suggest that the TSC genes act in a parallel pathway that converges on the insulin pathway downstream from Akt. The most convincing evidence for a functional link between the TSC genes and insulin signaling comes from the observation that heterozygosity of TSC1 or TSC2 is sufficient to rescue the lethality of loss-of-function InR mutants. This argues that the TSC genes are intimately linked to insulin signaling, rather than functioning in a totally independent cell-growth pathway. These results suggest that the TSC tumor suppressor genes are novel negative regulators of insulin signaling, and modulating the activities of the TSC genes might provide a potential way to correct insulin signaling defects in certain diseases such as diabetes and obesity (Gao, 2001).

To distinguish between these two possibilities, cells were generated that were doubly mutant for null alleles of PTEN and TSC1. PTEN is a negative regulator of the InR-PI3K-Akt pathway, and loss of PTEN results in increased Akt activity and cellular growth. It was reasoned that if TSC1 acts downstream from Akt within the InR-PI3K-Akt pathway, it might be expected that PTEN;TSC1 double-mutant cells would show a similar cell-size phenotype to either single mutant. However, if TSC1 acts parallel to the InR-PI3K-Akt the pathway, it might be expected that PTEN;TSC1 double-mutant cells would show additive effects on cell size as compared with each single mutant. PTEN;TSC1 double-mutant photoreceptors are 2.9 times the size of wild-type cells, as compared with 1.9 for PTEN - and 1.8 for TSC1 -. This result strongly suggests that the TSC genes function in a parallel pathway that converges on the insulin pathway at a point downstream from Akt (Gao, 2001).

The insulin/insulin-like growth factor-1 signaling pathway promotes growth in invertebrates and vertebrates by increasing the levels of phosphatidylinositol 3,4,5-triphosphate through the activation of p110 phosphatidylinositol 3-kinase. Two key effectors of this pathway are the phosphoinositide-dependent protein kinase 1 (PDK1) and Akt/PKB. Although genetic analysis in C. elegans has implicated Akt as the only relevant PDK1 substrate, cell culture studies have suggested that PDK1 has additional targets. In Drosophila, dPDK1 (FlyBase name: Protein kinase 61C) controls cellular and organism growth by activating Akt1 and S6 kinase, dS6K (FlyBase name: RPS6-p70-protein kinase). Furthermore, dPDK1 genetically interacts with dRSK but not with dPKN (FlyBase name: Protein kinase related to protein kinase N), encoding two substrates of PDK1 in vitro. Thus, the results suggest that dPDK1 is required for dRSK but not dPKN activation and that it regulates insulin-mediated growth through two main effector branches, dAkt and dS6K (Rintelen, 2001).

The pronounced effect of loss of dPDK1 function on head size suggests that it is a dominant constituent in the dInr pathway. To test this possibility, the ability of complete and partial loss-of-function alleles of dPDK1 to reverse phenotypes caused by either overexpression of dInr or by mutations in dPTEN, the 3-phosphatidylinositide phosphatase, was evaluated. Overexpression of a wild-type dInr cDNA under the control of GMR-Gal4 leads to a marked increase in eye size and a slightly rough eye surface, an effect dominantly suppressed by removing one copy of dPDK1. Further reduction of dPDK1 function by the dPDK11/4 heteroallelic combination reduces the eye to almost wild-type size, suggesting that the amount of dPDK1 protein is rate-limiting for the dInr overgrowth phenotype. Null mutations in dPTEN cause lethality, and removal of dPTEN function in clones stimulates cell autonomous growth, suggesting that increased levels of PIP3 promote growth and are the likely cause of lethality. Thus, if dPDK1 is an essential target of PIP3, mutations in dPDK1 may suppress the dPTEN phenotype. Surprisingly, some dPTEN/dPDK1 double mutant flies survive to adulthood, indicating that the presumed PIP3-induced lethality is primarily caused by the hyperactivation of dPDK1 or of one of its targets (Rintelen, 2001).

These results show that dPDK1 is an essential component in the insulin signaling pathway in the control of cell growth and body size through its two substrates, dAkt and dS6K. These results are distinct from the genetic evidence in C. elegans where Akt is the primary target of PDK1 in dauer formation. Because mutations in the insulin signaling pathway do not show an autonomous alteration of cell size in C. elegans, the regulation of the rate of protein synthesis through S6K does not seem to be a primary target of this pathway. However, the fact that dPDK1 may yet have additional substrates is suggested by the genetic interaction with dRSK gain-of-function mutations and because viable dPDK1 males are almost completely sterile. Although mutations in components of the insulin signaling pathway such as dInr, chico, Dp110/PI(3)K, and dAkt cause female sterility, male sterility is not observed. Further genetic dissection of dPDK1 function is required to determine the role of dPDK1 in male fertility. These findings in Drosophila are consistent with the absence of insulin growth factor-1-induced activation of S6K, Akt, and RSK in mammalian PDK1-/- embryonic stem cells, and therefore provide evidence for the functional conservation of branch points in kinase networks during evolution (Rintelen, 2001).

Insulin/IGF signaling during development controls growth and size, possibly by coordinating the activities of the Ras and PI 3-kinase signaling pathways. In vertebrates, the IR and IGFR act through IRS1-IRS4 proteins, which are multifunctional adaptors that link insulin and IGF signaling to the Ras/MAPK and phosphoinositide 3'-kinase (PI 3-kinase) signaling pathways. The pleckstrin homology domain (PH) and phosphotyrosine binding domain (PTB) of the IRS proteins are believed to mediate binding to phosphoinositol phosphates and the juxtamembrane NPXY motif of IR/IGFR, respectively. Grb2 (Drosophila homolog Drk) is an adaptor protein containing SH2 and SH3 domains. It has been suggested that Grb2 may, via its binding to IRS, link insulin/IGF to the Ras/MAPK pathway and thereby control proliferation. The Drosophila homolog of the SH2 domain containing p85 PI 3-kinase adaptor subunit, p60, binds Chico/IRS and thereby recruits the p110 catalytic subunit of PI 3-kinase [which converts phosphoinositol(4,5)P2 (PtdIns(4,5)P2) into phosphoinositol(3,4,5)P3 (PtdIns(3,4,5)P3)] to the plasma membrane. The p110 PI 3-kinase belongs to the class I PI 3-kinases implicated in the metabolic effects of insulin. The classical effectors that mediate the biological outcomes of insulin and IGF downstream of IRS have been divided into two functional branches: the Ras/MAPK proliferation pathway, and the PI 3-kinase metabolic, growth and survival pathway (Oldham, 2002).

To test whether increasing PtdInsP3 levels in an InR or PI 3-kinase p110 mutant background is sufficient to restore growth, the function of a negative regulator of the insulin pathway was eliminated. The 3'-phosphoinositol-specific lipid phosphatase, PTEN acts as a negative regulator of the PI 3-kinase pathway by converting PtdInsP3 generated by PI 3-kinase into PtdInsP2. Used were a null (Pten2L117) and a hypomorphic (Pten2L100) allele of Pten, identified in a screen for genes involved in growth control. As shown by HPLC analysis of the phospholipids in extracts of Pten mutant larvae, the loss of PTEN function results in a 2-fold increase in PtdInsP3 levels. This is consistent with the increase in PtdInsP3 seen in Pten-deleted murine fibroblasts. One prominent biological effect of these increased PtdInsP3 levels in Drosophila is a substantial increase in size in both larvae and pupae. To test whether loss of PTEN function, and consequently increased PtdInsP3 levels, is sufficient to restore growth or viability in InR null mutants, InR and Pten double mutants were generated by creating mosaic animals using the eyeless-Flipase (eyFlp) tissue-specific recombination system. In such animals, the head consists of homozygous mutant tissue, whereas the rest of the body is heterozygous for the same mutation. While loss of PTEN function (Pten2L117) in the head results in a fly with a disproportionately larger head (with more and larger cells), loss of InR function (InR327) results in flies with smaller heads (pinhead) compared to the wild type. Heads doubly mutant for Pten2L117and InR327, however, are almost the size of heads singly mutant for Pten2L117. Also, two different lethal heteroallelic InR combinations (InR304/InR327 or InR304/InR25), which arrest at the second larval instar stage, develop to the pupal stage (15%-17% of 33% expected) and even to pharate adults in the presence of reduced PTEN levels (Pten2L117/Pten2L100). These results demonstrate that complete loss of PTEN function can largely substitute for InR-mediated growth and proliferation in the absence of InR function and that the Ras/MAPK pathway plays little or no role in the InR mediated control of cell growth. This notion is further supported by the observation that complete loss of InR function in the compound eye does not result in a loss of photoreceptors, a hallmark of loss of Ras pathway function (Oldham, 2002).

Since increasing PtdInsP3 levels can rescue loss of InR function, these results suggest that the level of PtdInsP3 may be critical in determining the amount of growth. This possibility was explored by examining genetic interactions between Pten and PI 3-kinase p60 and p110. The lethality associated with the complete loss of PI 3-kinase p110 function, cannot be rescued by Pten2L117/Pten2L100. It is possible that without any PI 3-kinase p110 function, PTEN function becomes obsolete. In order to test this possibility, double mosaic clones were generated with the strong loss of function Pten2L117 allele and a null mutation for PI 3-kinase p110 or its p60 adaptor. Loss of PTEN function (Pten2L117) is unable to rescue the pinhead phenotype caused by loss of PI 3-kinase p110 function. However, clones that are doubly mutant for PI 3-kinase p60 and Pten2L117 are of wild-type size. In the absence of PI 3-kinase p60 function, PI 3-kinase p110 might have residual activity as suggested by the weaker phenotype of the PI 3-kinase p60 null mutant. Indeed, flies doubly mutant for PI 3-kinase p60 and Pten2L117/Pten2L100 flies are viable. These data provide strong genetic support for the close relationship between PTEN and PI 3-kinase and indicate that the intracellular levels of PtdInsP3 define the amount of cellular growth (Oldham, 2002).

Because loss of Chico function results in increased lipid levels as well as a dramatic decrease in body size, attempts were made to determine whether PTEN might also have a function in metabolic and body size control like Chico. Partial loss of PTEN function results in flies that are considerably bigger than controls. They weigh approximately 50 percent more than their heterozygous siblings without showing any apparent effect on cell differentiation. The increase in size is due to an increase in both cell size and number as determined by a morphometric analysis of the wing and eye. When the levels of lipids and glycogen were measured, a decrease per mass in lipid and glycogen was observed compared to Pten mutant flies rescued by a genomic Pten transgene. One biological outcome of this difference is a twofold increase in the rate of mortality under water-only starvation conditions compared to a 2-fold decrease in chico mutant flies. Thus lipid and glycogen levels strongly correlate with the survival time under starvation conditions. Since Chico and PTEN activity regulates growth during development and the accumulation of energy stores in the adult, the effects of InR-mediated growth and lipid/glycogen metabolism must diverge downstream of Pten (Oldham, 2002).

Thus, growth deficency associated with the loss of InR function is fully compensated by loss of PTEN function. This suggests that the levels of PtdInsP3 in the cell control the amount of cellular growth. Also, partial loss of PTEN function increases body size and decreases lipid and glycogen stores in the adult, suggesting that the levels of PtdInsP3 also control metabolism in the adult (Oldham, 2002).

The rescue of lethal, null InR mutant combinations to near viability by reducing PTEN activity strengthens the argument that a PtdInsP3-dependent signaling pathway is the primary effector for InR-derived growth and proliferation. In support of this observation, PI 3-kinase and Akt have been isolated as retroviral oncogenes, suggesting that activation of PI 3-kinase and Akt is sufficient to mediate growth, proliferation, and oncogenesis in vertebrate systems. In Drosophila and mammals, overexpression of PI 3-kinase causes increased growth; but this is not sufficient for proliferation as is the removal of Pten. From this premise, it has been proposed that PI 3-kinase and PTEN regulate similar yet distinct pathways. Alternatively, it is possible that they do function uniquely in the same pathway and that the difference may be due to altered location and function because of overexpression, or to differential feedback of PI 3-kinase versus PTEN. For example, since PI 3-kinase has been shown to act as a serine/threonine protein kinase on IRS, this may have a negative feedback effect on the insulin pathway that might not be evident in Pten loss-of-function mutations. Nevertheless, PI 3-kinase is absolutely critical in controlling size because using an allelic series of PI 3-kinase mutants in combination with the ey-Flp sytem resulted in a range of different head sizes. Furthermore, expressing an activated and dominant-negative form of PI 3-kinase in Drosophila imaginal discs or the heart of the mouse also leads to a corresponding increase or decrease in cell and organ size. Thus, the PI 3-kinase/PTEN cycle can be considered a dedicated growth rheostat, and the InR pathway is an evolutionary conserved module for regulating the range of growth and size (Oldham, 2002).

Loss of PTEN function results in a metabolically similar phenotype as loss of murine PTP1B (Ptpn1), an IR-specific tyrosine phosphatase, in that hyperactivation of the IR pathway causes resistance to high-fat-diet-induced obesity because of increased basal metabolism. These metabolic lipid effects have likely been conserved during evolution because the increased lipid levels in chico mutants are reminiscent of the enhanced lipid content in Irs2 deleted and NIRKO mice (Oldham, 2002).

Collectively, these data firmly establish Drosophila as a valid model organism for the study of metabolic diseases like diabetes and obesity as well as for the study of growth disorders like cancer. Pten mutant flies are larger in size due to increased cell size and number, but have a corresponding decrease in energy stores, a situation completely opposite that of mutations in positive components of the insulin signaling pathway like InR, chico, PI 3-kinase, and dAkt. These large viable Pten mutants show that a reduction of PTEN function is sufficient for increased organism size. This fact suggests that the four-fold size difference between viable InR and Pten mutants can simply be controlled by the amount of PtdInsP3 and this phenomenon may possibly be extended to vertebrate size regulation. Thus, in Drosophila, the InR/PI 3-kinase/PTEN pathway combines both metabolism and growth control into one pathway that later diverged into two separate, yet interacting systems in mammals (Oldham, 2002).

Insulin-IGF receptor (InR) signaling has a conserved role in regulating lifespan, but little is known about the genetic control of declining organ function. This study describes progressive changes of heart function in aging fruit flies: from one to seven weeks of a fly's age, the resting heart rate decreases and the rate of stress-induced heart failure increases. These age-related changes are minimized or absent in long-lived flies when systemic levels of insulin-like peptides are reduced and by mutations of the only receptor, InR, or its substrate, Chico. Moreover, interfering with InR signaling exclusively in the heart, by overexpressing the phosphatase PTEN or the forkhead transcription factor FOXO, prevents the decline in cardiac performance with age. Thus, insulin-IGF signaling influences age-dependent organ physiology and senescence directly and autonomously, in addition to its systemic effect on lifespan. The aging fly heart is a model for studying the genetics of age-sensitive organ-specific pathology (Wessells, 2004).

Pten negatively regulates PKB

Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in one of two tumor suppressor genes, TSC1 and TSC2. Absence of Drosophila Tsc1 and/or Tsc2 (Gigas) leads to constitutive S6k activation and inhibition of PKB, the latter effect being relieved by loss of S6K. In contrast, the Pten tumor suppressor, a negative effector of PI3K, has little effect on S6k, but negatively regulates PKB (Akt1). More importantly, reducing S6k signaling rescues early larval lethality associated with loss of Tsc1/2 function, arguing that the S6k pathway is a promising target for the treatment of TSC (Radimerski, 2002b).

To determine whether loss of Tsc1/2 or Pten directly affected S6k activity, each was depleted in Drosophila Kc167 cells by dsRNAi. Quantitative Real Time PCR showed that such treatment strongly reduced levels of both transcripts. Compared with control cells, depletion of Tsc1 increases S6k activity and T398 phosphorylation, consistent with the reduced electrophoretic mobility of S6k. These results are in agreement with recent findings in TSC1 null mammalian cells (Kwiatkowski, 2002). Insulin treatment of either control cells or Tsc1-depleted cells did not significantly increase these responses beyond that of Tsc1 depletion alone, indicating that loss of Tsc function leads to full S6k activation. RAD001, a rapamycin derivative, blocks S6k activity in both control and Tsc1-depleted cells treated with insulin. However, it was consistently noted that the RAD001 block of insulin-induced S6k activation is not as strong in Tsc1-depleted cells, suggesting that not all the effects of Tsc on S6k are dependent on Tor, the Drosophila target of rapamycin. Similar results to those described here were obtained by Tsc2 depletion. In addition, the effects appear specific, since Tsc1 depletion has no effect on the basal activity of other AGC-kinase family members, such as PKB or Drosophila atypical PKC. However, insulin-induced PKB activation and S505 phosphorylation are repressed in Tsc1-depleted cells as compared with control cells, consistent with S6k acting in a negative feedback loop to dampen PKB signaling. In contrast to loss of Tsc1, depletion of Pten has little effect on S6k activity and T398 phosphorylation, whereas it leads to elevated levels of both basal and insulin-stimulated PKB activity and S505 phosphorylation. Thus, loss of Tsc1/2, but not Pten, leads to constitutive S6k activation (Radimerski, 2002b).

To determine whether the findings above could be corroborated in the animal, S6k activity was measured in extracts of Tsc1, Pten, and S6k null larvae. The results show that S6k activity in extracts derived from Tsc1 null larvae is strongly increased over that of wild-type larvae, whereas it is slightly increased in larvae lacking Pten. The opposite was found for PKB activity, which is strongly repressed in Tsc1 null larvae, and up-regulated in Pten-deficient larvae. Hence, it cannot be excluded that reduced PKB activity contributes to larval lethality of Tsc mutants. Given that loss of Tsc function leads to increased S6k activity, it was reasoned that ectopic expression of Tsc1/2, but not Pten, would inhibit S6k activity. To test this hypothesis, both tumor suppressors were expressed ubiquitously in larvae using the GAL4/UAS system, such that the GAL4 promoter chosen in each case led to developmental arrest at late larval second instar. Extracts from larvae overexpressing Tsc1/2 display strongly reduce S6k activity, whereas those from Pten overexpressing larvae have normal levels of S6k activity. In contrast, PKB activity is strongly suppressed in Pten overexpressing larvae and little affected in extracts from larvae overexpressing Tsc1/2. These data corroborate previous findings that S6k and PKB act in parallel signal transduction pathways (Radimerski, 2002a), and provide compelling evidence that they are negatively controlled by distinct tumor suppressor genes (Radimerski, 2002b).

Despite the fact that S6k and PKB act in parallel signaling pathways, loss of Tsc1/2 function leads to inhibition of PKB activity, suggesting cross-talk between the two pathways. Compatible with such a model, recent studies have shown that rapamycin treatment of adipocytes inhibits a negative feedback loop, which normally functions to dampen insulin-induced PKB activation. Since RAD001 inhibits S6k activity (Radimerski, 2002a) and increases PKB activity (Radimerski, 2002a), it raised the possibility that the effects of Tsc mutants on PKB are mediated through S6k. Consistent with this hypothesis, inhibition of PKB activity due to loss of Tsc function was relieved in the absence of S6k. Similar results were obtained by using dsRNAi in cell culture. Thus, the suppression of PKB by loss of Tsc function requires S6k (Radimerski, 2002b).

To genetically test the specificity of Tsc1/2 and Pten tumor suppressor function, either Tsc1 or Pten were removed in cells giving rise to the adult eye structure, by inducing mitotic recombination with the FLP/FRT system under the control of the eyeless promoter. In a wild-type genetic background, loss of either Tsc1 or Pten within the developing eye causes strong overgrowth of the head. Eye overgrowth by removal of Tsc1 is strongly suppressed in a genetic background null for S6k, as is ommatidia size, in agreement with a previous report analyzing double mutant clones of Tsc2 and S6k in the eye (Potter, 2001). In contrast, removal of Pten in the eyes of S6k null flies still induces overgrowth of clones with enlarged ommatidia. These findings are supported by results showing that eye overgrowth by removal of Tsc1 is still observed in clones devoid of PKB function (Potter, 2001) and overgrowth by removal of Pten is suppressed in a viable PKB mutant genetic background (Stocker, 2002). Thus, Tsc1/2 appears to be specific for the S6k-signaling pathway, whereas Pten antagonizes PI3K signaling to counteract PKB activation by decreasing PIP3 levels (Radimerski, 2002b).

Taken together, these results demonstrate that the tumor suppressor Tsc1/2 is a critical component in controlling S6k activation. Interestingly, this effect may be Tor independent, as insulin-induced S6k activation is more elevated in Tsc1/2-depleted cells pretreated with RAD001 than in control cells, and in preliminary studies, clonal overgrowth in the eye induced by loss of Tsc1 is not suppressed in a semiviable, heterorallelic Tor mutant background. Overexpression of Tsc1/2 selectively suppresses the S6k-signaling pathway, whereas Pten operates on the dPI3K-signaling pathway. Double mutations for Pten and Tsc1 are additive for clonal overgrowth, compatible with S6k and PKB independently mediating growth. Nevertheless, inhibition of PKB by loss of Tsc function shows that there is negative cross-talk between the two signaling pathways. Given this negative cross-talk, the observation that in double mutant clones growth is additive, suggests that in the absence of Pten, inhibition of PKB by loss of Tsc is circumvented. However, despite the observation that double mutations for Pten and Tsc1 are additive for clonal overgrowth, overgrowth induced by absence of Pten is suppressed in clones mutant for Tor. Since S6k does not prevent such overgrowth, it is possible that this suppression actually represents an intermediate phenotype, or that Pten negatively acts on a Tor target distinct from S6k. At this point, it is important to gain a deeper knowledge of the molecular mechanisms by which Tsc1/2 acts to suppress S6k function and how the signaling components of these two pathways cross-talk with one another (Radimerski, 2002b).

Recently, a successful Phase I clinical trial was completed for a rapamycin analog in the treatment of solid tumors. The results of the trial demonstrated that the drug was efficacious at subtoxic doses, and suggested that specific tumor types may be more sensitive to inhibition by rapamycin than others. The question that arose from the trial is, which tumors would be susceptible to rapamycin treatment? Here, it has been demonstrated for the first time in vivo that a mild reduction in S6k signaling, which alone has no blatant phenotype, is sufficient to restore viability of flies devoid of Tsc function. Thus, these findings imply that rapamycin or its derivatives might be very promising pharmaceutical agents in the treatment of tumors arising from TSC (Radimerski, 2002b).

d4E-BP is a direct downstream target of the dInR-dPI3K-dPTEN-dAkt-dTSC-dTOR signaling cascade

The eIF4E-binding proteins (4E-BPs) interact with translation initiation factor 4E to inhibit translation. Their binding to eIF4E is reversed by phosphorylation of several key Ser/Thr residues. In Drosophila, S6 kinase (dS6K) and a single 4E-BP (d4E-BP/Thor) are phosphorylated via the insulin and target of rapamycin (TOR) signaling pathways. Although S6K phosphorylation is independent of phosphoinositide 3-OH kinase (PI3K) and serine/threonine protein kinase Akt, that of 4E-BP is dependent on PI3K and Akt. This difference prompted an examination of the regulation of d4E-BP in greater detail. Analysis of d4E-BP phosphorylation using site-directed mutagenesis and isoelectric focusing-sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the regulatory interplay between Thr37 and Thr46 of d4E-BP is conserved in flies and that phosphorylation of Thr46 is the major phosphorylation event that regulates d4E-BP activity. RNA interference (RNAi) was used to target components of the PI3K, Akt, and TOR pathways. RNAi experiments directed at components of the insulin and TOR signaling cascades show that d4E-BP is phosphorylated in a PI3K- and Akt-dependent manner. Surprisingly, RNAi of dAkt also affects insulin-stimulated phosphorylation of dS6K, indicating that dAkt may also play a role in dS6K phosphorylation (Miron, 2003).

Is d4E-BP regulated by a PI3K/Akt-independent pathway similar to that described for dS6K? Analysis of signaling to d4E-BP using RNAi indicates that it is not. It is more likely that d4E-BP is a direct downstream target of the dInR-dPI3K-dPTEN-dAkt-dTSC-dTOR signaling cascade. Thus, a linear pathway from InR to Akt that is important for 4E-BP regulation is conserved between Drosophila and mammals (Miron, 2003)

dPDK1 is critical for regulating growth by phosphorylating dAkt and dS6K. RNAi of dPDK1 does not significantly affect insulin-induced phosphorylation of d4E-BP. However, consistent with the direct phosphorylation of dS6K by dPDK1, the phosphorylation of dS6K at Thr398 is completely blocked by RNAi of PDK1. Thus, the results favor a model in which d4E-BP regulation is effected through dAkt, even when dPDK1 levels are dramatically reduced, whereas dS6K requires both dAkt and dPDK1. The differential effects of dPDK1 RNAi on d4E-BP and dS6K phosphorylation can be explained as follows: dPDK1 levels may be reduced below a threshold that is required to phosphorylate dS6K but is still adequate to activate dAkt, allowing d4E-BP phosphorylation. Since dS6K requires direct phosphorylation by dPDK1, it may be more susceptible to variations in its levels. In contrast, d4E-BP, which relies on a signal relayed by dAkt, may be less affected by variations in dPDK1. In mammalian PDK1-hypomorphic mutants, a kinase activity that is 10-fold lower than normal still results in normal Akt and S6K1 activation, yet these animals are greatly reduced in size. This observation supports the notion that reduced PDK1 activity may differentially activate downstream targets (Miron, 2003).

In Drosophila, coexpression of dS6K with dPI3K does not cause additive cellular overgrowth, unlike coexpression of dAkt and dPI3K. RNAi of dPTEN in Kc 167 cells and overexpression of dPTEN in Drosophila larvae had little effect on dS6K activity. Moreover, removal of both dS6K and dPTEN in cell clones does not prevent the dPTEN-dependent overgrowth phenotype. Together, these results and the results of dPI3K and dPTEN RNAi experiments would seemingly support the notion that dS6K-dependent cell growth is not influenced by dPI3K and dPTEN. However, a different effect of dPTEN RNAi on dS6K has been reported in another study: increase in dS6K phosphorylation following RNAi of dPTEN. Consistent with this observation RNAi directed against dPI3K and dPTEN has been shown to modulate dS6K phosphorylation. A reasonable explanation for these discrepancies is that the knockdown of dPI3K and dPTEN achieved in the current experiments was not sufficient to completely deplete these proteins and affect dS6K phosphorylation (Miron, 2003 and references therein).

The role of dAkt in regulating dS6K is subject to debate. In Drosophila, Akt plays a predominant role in mediating the effects of increased PIP3 levels, and all Akt-mediated growth signals are thought to be transduced via Tsc1/2. Tsc2 is directly phosphorylated by Akt, implying that S6K is downstream of Akt in the PI3K signaling pathway. The observation that RNAi of dAkt reduces dS6K phosphorylation at Thr398 supports a direct link among dAkt, dTSC, and dS6K but contradicts the finding that TSC modulates dS6K activity in a dAkt-independent manner. Recent data also support the conclusion of a link between dAkt and dS6K. Clones of cells doubly mutant for dPTEN and dTsc1 display an additive overgrowth phenotype, suggesting that the tumor suppressors act on two independent pathways, from dPTEN to dAkt and from dTSC to dS6K. The findings demonstrate clear effects of dPTEN, dAkt, and dTSC on d4E-BP, which does not preclude the possibility that two pathways regulate d4E-BP; however, a simpler interpretation is that a single pathway is important for its regulation. A possibility is that d4E-BP requires higher dAkt activity than dS6K in order to be phosphorylated. In circumstances of low PI3K activation, low levels of PIP3 are produced, resulting in weaker dAkt activity that is sufficient for dS6K activation but not for d4E-BP phosphorylation. A differential threshold of activation could be the source of the discrepancies between the current results and those of others. This model is strongly supported by recent data showing that in cells lacking both Akt1 and Akt2 isoforms, the low level of Akt activity remaining is sufficient for robust S6K1 phosphorylation, but phosphorylation of 4E-BP1 is dramatically reduced (Miron, 2003 and references therein).

Alternatively, the results could also be explained by the existence of a negative feedback loop between dPI3K and dS6K that dampens insulin signaling by suppressing dAkt activity. This negative feedback loop has been described. Similar observations were made in mammals; insulin-induced activation of Akt is inhibited in Tsc2-deficient mouse embryonic fibroblasts. Thus, depletion of dAkt may trigger this negative feedback loop, which diminishes dS6K phosphorylation and activation. Interestingly, engagement of this feedback mechanism can also provide an explanation for the reduction in total d4E-BP levels observed in dPDK1 RNAi-treated cells. Under these conditions, the reduction of dS6K signaling is accompanied by a concomitant reduction in growth signaling on the dPI3K-dAkt branch of the pathway. Thus, a reduced level of d4E-BP is required to accommodate the reduced need for deIF4E inhibition (Miron, 2003).

insulin-PI3K/TOR pathway induces a HIF-dependent transcriptional response in Drosophila by promoting nuclear localization of HIF-alpha/Sima

The hypoxia-inducible factor (HIF) is a heterodimeric transcription factor composed of a constitutively expressed HIF-ß subunit and an oxygen-regulated HIF-alpha subunit. A hypoxia-inducible transcriptional response has been defined in Drosophila that is homologous to the mammalian HIF-dependent response. In Drosophila, the bHLH-PAS proteins Similar (Sima) and Tango (Tgo) are the functional homologues of the mammalian HIF-alpha and HIF-ß subunits, respectively. HIF-alpha/Sima is regulated by oxygen at several different levels that include protein stability and subcellular localization. Insulin can activate HIF-dependent transcription, both in Drosophila S2 cells and in living Drosophila embryos. Using a pharmacological approach as well as RNA interference, it has been determined that the effect of insulin on HIF-dependent transcriptional induction is mediated by PI3K-AKT and TOR pathways. Stimulation of the transcriptional response involves upregulation of Sima protein but not sima mRNA. Finally, the effect of the activation of the PI3K-AKT pathway on the subcellular localization of Sima protein was analyzed in vivo. Overexpression of dAKT and dPDK1 in normoxic embryos provokes a major increase in Sima nuclear localization, mimicking the effect of a hypoxic treatment. A similar increase in Sima nuclear localization was observed in dPTEN homozygous mutant embryos, confirming that activation of the PI3K-AKT pathway promotes nuclear accumulation of Sima protein. It is concluded that regulation of HIF-alpha/Sima by the PI3K-AKT-TOR pathway is a major conserved mode of regulation of the HIF-dependent transcriptional response in Drosophila (Dekanty, 2005).

Insulin stimulation or exposure to hypoxia can induce common target genes; such transcriptional response depends on the Drosophila HIFalpha and HIFß homologues Sima and Tango. Evidence is provided that insulin-stimulated HRE response is transduced by the PI3K-AKT pathway and, furthermore, that the effect depends on TOR and involves an increase in Sima protein levels, whereas mRNA levels are not affected. These results are in good agreement with the reported effect of the PI3K-AKT/TOR pathway on mammalian HIF, because in several cell lines activation of this pathway led to an increase in HIF protein levels or stabilization of the protein (Dekanty, 2005).

In addition, the subcellular localization of Sima depends on oxygen tension in a dose-dependent manner, and activation of the PI3K-AKT pathway also causes a major increase in Sima nuclear localization. This regulatory mechanism might represent another conserved aspect of HIF regulation, because one recent report suggests that HIF-alpha accumulates in the nucleus of retinal epithelial cells upon IGF-1alpha treatment. The molecular bases of HIFalpha nuclear accumulation upon hypoxia or PI3K activation are so far unclear. Nucleo-cytoplasmic localization of many transcription factors results from a steady-state equilibrium between nuclear import and nuclear export, and accumulation in one or the other compartment depends on the relative rate of import versus export. Whether HIF nuclear accumulation upon hypoxia or growth factor stimulation depends on regulated nuclear import or regulated nuclear export remains to be determined (Dekanty, 2005).

All major cellular features of oxygen-dependent regulation of HIF proteins are conserved in Drosophila and, thus, activation of the HRE response in Drosophila by the PI3K-AKT and TOR pathways extends even further the notion of a conserved HIF system in evolution. The functional significance of the regulation exerted by PI3K-AKT and TOR pathways over the HRE response was discussed in the context of cancer biology, because the loss of PTEN or the tuberous sclerosis complex 1 (TSC1) and TSC2 proteins is frequently associated with human tumors. What is the functional meaning of PI3K-AKT pathway regulation of the HRE response in normal cells? The PI3K-AKT and TOR pathways are regulated in part by growth factors and other endocrine signals and thus, endocrine control of the HRE response is conserved among animal species that have diverged 700 million years ago. It seems reasonable to postulate that the main physiological role of HRE induction by the PI3K-AKT pathway is the stimulation of glycolysis, but a function in the regulation of animal body size and growth control is another interesting possibility (Dekanty, 2005).

A cardinal function of the PI3K-AKT and TOR pathways throughout evolution is to regulate growth, and to determine the final size of developing organs and whole organisms. Genetic studies in Drosophila have shown that a reduction of the activity of the PI3K-AKT pathway results in flies with a reduced body size, bearing smaller cells. Likewise, a reduction in TOR signaling provokes growth decrease and, conversely, over-activation of TOR signaling due to loss-of-function of its negative regulators TSC1 and/or TSC2, leads to an increase in cell and body size. The effect of TOR on cell growth was reported to be mediated at least in part by S6K, a kinase that phosphorylates the ribosomal protein S6, leading to translational activation (Dekanty, 2005).

Besides its role in growth control, the insulin-PI3K-AKT pathway has been traditionally implicated in the regulation of circulating glucose levels and anabolic metabolism. It has been demonstrated that the cellular bases of glucose sensing and regulation of serum glucose are conserved between mammals and Drosophila, and it has been proposed that PI3K-AKT signaling in conjunction with the TOR pathway coordinates growth according to environmental conditions and the nutritional status of the organism. The mechanism involved in this coordination is still unclear. Oxygen tension is one environmental factor that has been shown to modulate growth in Drosophila, because hypoxic flies have a reduced body size (Frazier, 2001). A mechanistic explanation to this phenomenon has been provided by showing that overexpression of Sima protein causes a reduction in cell size in an autonomous manner (Centanin, 2005). Consistent with this, it has been shown that hypoxia provokes a reduction in Drosophila TOR pathway activity and that such reduction results from hyperactivation of the TSC1-TSC2 tumor suppressor complex. Similar results have been reported in mammalian cells, implying that TOR is regulated by hypoxia. Furthermore, hypoxia-dependent TSC1-TSC2 stimulation and growth inhibition are mediated by the product of a HIF/Sima-inducible gene called scylla in Drosophila and RTP801/REDD1 in mammals (Dekanty, 2005).

The results establish a direct link between pathways largely implicated in growth regulation (PI3K-AKT and TOR) and the hypoxia-responsive machinery (HIF/Sima). It is suggested that in hypoxia, HIF prolyl hydroxylase (Hph)/Fatiga activity is reduced, resulting in HIF/Sima stabilization and induction of an HRE response. One of the genes induced by hypoxia is scylla/RTP801/REDD1, which in turn activates TSC1-TSC2. Then, stimulation of the TSC complex provokes reduction of TOR activity and decreases S6K phosphorylation, resulting in growth inhibition. According to this model, PI3K-TOR activation of HIF-alpha/Sima might generate a negative feedback loop to limit or downregulate growth; in this scenario, low oxygen levels are expected to enhance Sima-dependent inhibition of growth (Dekanty, 2005).

It has been reported that mitochondrial dysfunction inhibits Hph/Fatiga activity, thereby triggering the transcriptional response to hypoxia, and also that it concomitantly provokes growth defects. It was proposed that Hph/Fatiga operates as an integration node between oxygen levels and growth regulation. The current results have shown that the effect of Hph/Fatiga on growth regulation is conveyed at least in part by Sima. Further studies will reveal whether Hph/Fatiga also plays Sima-independent roles in cell and organ size determination (Dekanty, 2005).

Dual role for Insulin/TOR signaling in the control of hematopoietic progenitor maintenance in Drosophila

The interconnected Insulin/IGF signaling (IIS) and Target of Rapamycin (TOR) signaling pathways constitute the main branches of the nutrient-sensing system that couples growth to nutritional conditions in Drosophila. This study addressed the influence of these pathways and of diet restriction on the balance between the maintenance of multipotent hematopoietic progenitors and their differentiation in the Drosophila lymph gland. In this larval hematopoietic organ, a pool of stem-like progenitor blood cells (prohemocytes) is kept undifferentiated in response to signaling from a specialized group of cells forming the posterior signaling center (PSC), which serves as a stem cell niche. Reminiscent of the situation in human, loss of the negative regulator of IIS Pten results in lymph gland hyperplasia, aberrant blood cell differentiation and hematopoietic progenitor exhaustion. Using site-directed loss- and gain-of-function analysis, it was demonstrated that components of the IIS/TOR pathways control lymph gland homeostasis at two levels. First, they cell-autonomously regulate the size and activity of the hematopoietic niche. Second, they are required within the prohemocytes to control their growth and maintenance. Moreover, it was shown that diet restriction or genetic alteration mimicking amino acid deprivation triggers progenitor cell differentiation. Hence, this study highlights the role of the IIS/TOR pathways in orchestrating hematopoietic progenitor fate and links blood cell fate to nutritional status (Benmimoun, 2012).

To test whether the IIS pathway is cell-autonomously required in the PSC, the col-Gal4 driver, expression of which is strictly confined to the PSC during lymph gland ontogeny, was used as demonstrated by a lineage tracing experiment. In addition, advantage was taken of col-Gal4-driven expression in the wing disc to confirm the specificity of the UAS transgenes used in this study. As observed in Pten larvae, over-activation of IIS in the PSC, induced by expressing either Pten RNAi or an active form of PI3K (PI3Kcaax), led to a strong increase in PSC size. This phenotype correlated with a rise in PSC cell number. Conversely, knocking down InR by RNAi or overexpressing Pten caused a reduction in PSC cell number. As IIS impinges on TOR activity, tests were performed to see whether this pathway also regulates PSC development. PSC cell number diminished when the TOR pathway was inactivated either by overexpressing both TSC1 and TSC2 (gig) or by downregulating raptor by RNAi. Of note, TSC1/TSC2 overexpression seemed to reduce PSC cell size. Conversely, TSC1 RNAi expression, which resulted in a larger PSC, did not significantly affect cell number but increased cell size. This suggests that TOR signaling not only supports PSC cell proliferation but also their growth. Finally a strong drop in PSC cell number was observed when Foxo, which is the main effector of IIS and whose targets are concomitantly regulated by the TOR kinase, was overexpressed. Together, these data indicate that IIS and TOR pathways are required in the PSC to promote niche cell proliferation/maintenance and growth (Benmimoun, 2012).

The results demonstrate that IIS/TOR signaling plays a dual role in the maintenance of the blood cell progenitors by acting both within the hematopoietic niche to control its size and its activity, and within the prohemocytes to control their fate. To gain a comprehensive view of IIS/TOR function in Drosophila hematopoiesis and in light of the recent report showing that differentiated hemocytes can feedback on prohemocyte maintenance, it will be interesting to explore the role of these pathways in the differentiated blood cells. In addition, the data are consistent with a model whereby the IIS/TOR pathways link prohemocyte maintenance to the Drosophila larvae nutritional status. It is speculated that food shortage, by sensitizing blood cell progenitors to differentiation, might affect the cellular immune response. Along this line, the rate of encapsulation of parasitoid wasp eggs, which relies primarily on the differentiation of lamellocytes, has been shown to diminish in larvae that were deprived of yeast before infestation. It is anticipated that future studies will allow further understanding of how developmental and environmental cues are integrated by IIS/TOR signaling to control blood cell homeostasis (Benmimoun, 2012).

Gene regulatory networks controlling hematopoietic progenitor niche cell production and differentiation in the Drosophila lymph gland

Hematopoiesis occurs in two phases in Drosophila, with the first completed during embryogenesis and the second accomplished during larval development. The lymph gland serves as the venue for the final hematopoietic program, with this larval tissue well-studied as to its cellular organization and genetic regulation. While the medullary zone contains stem-like hematopoietic progenitors, the posterior signaling center (PSC) functions as a niche microenvironment essential for controlling the decision between progenitor maintenance versus cellular differentiation. This study used PSC-specific GAL4 driver and UAS-gene RNAi strains, to selectively knockdown individual gene functions in PSC cells. The effect of abrogating the function of 820 genes was assessed as to their requirement for niche cell production and differentiation. 100 genes were shown to be essential for normal niche development, with various loci placed into sub-groups based on the functions of their encoded protein products and known genetic interactions. For members of three of these groups, loss- and gain-of-function phenotypes were characterized. Gene function knockdown of members of the BAP chromatin-remodeling complex resulted in niche cells that do not express the hedgehog (hh) gene and fail to differentiate filopodia believed important for Hh signaling from the niche to progenitors. Abrogating gene function of various members of the insulin-like growth factor and TOR signaling pathways resulted in anomalous PSC cell production, leading to a defective niche organization. Further analysis of the Pten, TSC1, and TSC2 tumor suppressor genes demonstrated their loss-of-function condition resulted in severely altered blood cell homeostasis, including the abundant production of lamellocytes, specialized hemocytes involved in innate immune responses. Together, this cell-specific RNAi knockdown survey and mutant phenotype analyses identified multiple genes and their regulatory networks required for the normal organization and function of the hematopoietic progenitor niche within the lymph gland (Tokusumi, 2012).

The discovery of a stem cell-like hematopoietic progenitor niche in Drosophila represents a significant contribution of this model organism to the study of stem cell biology and blood cell development. Extensive findings support the belief that the PSC functions as the niche within the larval lymph gland, with this cellular domain essential to the control of blood cell homeostasis within this hematopoietic organ. Molecular communication between the PSC and prohemocytes present in the lymph gland medullary zone is crucial for controlling the decision as to maintaining a pluri-potent progenitor state versus initiating a hemocyte differentiation program. This lymph gland cellular organization and the signaling pathways controlling hematopoieis therein have prompted several researchers in the field to point out its functional similarity to the HSC niche present in mammalian (Tokusumi, 2012).

As a means to discover new information on genetic and molecular mechanisms at work within a hematopoietic progenitor niche microenvironment, an RNAi-based loss-of-function analysis was carried out to selectively eliminate individual gene functions in PSC cells. The effect of knocking-down the function of 820 lymph gland-expressed genes was assessed as to their requirement for niche cell production and differentiation, and 100 of these genes were shown to be required for one or more aspects of niche development. The distinguishable phenotypes observed in these analyses included change in number of Hh-expressing cells, change in number of Antp-expressing cells, scattered and disorganized niche cells, rounded cells lacking extended filopodia, and lamellocyte induction in the absence of a normal PSC. The genes were placed into sub-groups based on their coding capacity and known genetic interactions, and the phenotypes associated with the functional knockdown of members of three of these gene regulatory networks were characterized (Tokusumi, 2012).

Previous studies have demonstrated that the PSC-specific ablation of srp function resulted in a lack of expression of the crucial Hh signaling molecule in these cells, the inactivity of the hh-GFP transgene in the niche, failure of niche cells to properly differentiate filopodial extensions, and the loss of hematopoietic progenitor maintenance coupled with the abundant production of differentiated hemocytes. Thus it was intriguing when it was observed that RNAi function knockdown of several members of the BAP chromatin-remodeling complex resulted in the identical phenotypes of lack of hh-GFP transgene expression and absence of filopodia formation in PSC cells. A convincing functional interaction was observed between srp encoding the hematopoietic GATA factor and osa encoding the DNA-binding Trithorax group protein in the inability of niche cells to express hh-GFP in double-heterozygous mutant lymph glands. Thus one working model is that the BAP chromatin-remodeling complex establishes a chromatin environment around and within the hh gene that allows access of the Srp transcriptional activator to the PSC-specific enhancer, facilitating Hh expression in these cells. It will be of interest to determine if there exists a direct physical interaction between Osa and Srp in this positive regulation of hh niche transcription and if so, what are the functional domains of the proteins essential for this critical regulatory event in progenitor cell maintenance. It is also likely that these functional interactions are important for Srp's transcriptional regulation of additional genes needed for the formation of niche cell filapodia (Tokusumi, 2012).

In this study, a total of 33 gain- or loss-of-function genetic conditions were analyzed that enhanced or eliminated the function of various positive or negatively-acting components of the insulin-like growth factor and TOR signaling pathways. A conclusion to be drawn from these analyses is that genetic conditions that have an end effect of enhancing translation activity and protein synthesis result in supernumerary PSC cell numbers in disorganized niche domains, while conditions that promote growth suppression lead to substantially reduced populations of niche cells. The same conclusion was obtained from recent studies performed by Benmimoun (2012). The Wg and Dpp signaling pathways have also been shown to be important for the formation of a PSC niche of normal size and function, and it is possible that the insulin-like growth factor and TOR signaling networks regulate the translation of one or more members of the Wg and/or Dpp pathways. These analyses have also shown that mutation of the Pten, TSC1, and TSC2 tumor suppressor genes results in severely altered blood cell homeostasis in lymph glands and in circulation, including the prolific induction of lamellocytes. A recent report demonstrated that in response to larval wasp infestation, the PSC secretes the Spitz cytokine signal, which triggers an EGFR-mediated signal transduction cascade in the generation of dpERK-positive lamellocytes in circulation. As dpERK activity is known to inhibit TSC2 function, inactivation of the TSC complex may be a downstream regulatory event leading to robust lamellocyte production in larvae in response to wasp immune challenge (Tokusumi, 2012).

To summarize, an RNAi-based loss-of-function analysis has been undertaken to identify new genes and their signaling networks vital for normal PSC niche formation and function. While information has been gained on the requirements of three such networks for PSC development and blood cell homeostasis within the lymph gland, numerous other genes have been discovered that likewise play key roles in these hematopoietic events. Their characterization is warranted as well to further enhance knowledge of genetic and molecular mechanisms at work within an accessible and easily manipulated hematopoietic progenitor niche microenvironment (Tokusumi, 2012).

Insulin signaling mediates sexual attractiveness in Drosophila

Sexually attractive characteristics are often thought to reflect an individual's condition or reproductive potential, but the underlying molecular mechanisms through which they do so are generally unknown. Insulin/insulin-like growth factor signaling (IIS) is known to modulate aging, reproduction, and stress resistance in several species and to contribute to variability of these traits in natural populations. This study shows that IIS determines sexual attractiveness in Drosophila through transcriptional regulation of genes involved in the production of cuticular hydrocarbons (CHC), many of which function as pheromones. Using traditional gas chromatography/mass spectrometry (GC/MS) together with newly introduced laser desorption/ionization orthogonal time-of-flight mass spectrometry (LDI-MS), it was established that CHC profiles are significantly affected by genetic manipulations that target IIS. Manipulations that reduce IIS also reduce attractiveness, while females with increased IIS are significantly more attractive than wild-type animals. IIS effects on attractiveness are mediated by changes in CHC profiles. Insulin signaling influences CHC through pathways that are likely independent of dFOXO and that may involve the nutrient-sensing Target of Rapamycin (TOR) pathway. These results suggest that the activity of conserved molecular regulators of longevity and reproductive output may manifest in different species as external characteristics that are perceived as honest indicators of fitness potential (Kuo, 2012).

Cell mixing induced by myc is required for competitive tissue invasion and destruction

Cell-cell intercalation is used in several developmental processes to shape the normal body plan. There is no clear evidence that intercalation is involved in pathologies. This study used the proto-oncogene myc to study a process analogous to early phase of tumour expansion: myc-induced cell competition. Cell competition is a conserved mechanism driving the elimination of slow-proliferating cells (so-called 'losers') by faster-proliferating neighbours (so-called 'winners') through apoptosis and is important in preventing developmental malformations and maintain tissue fitness. Using long-term live imaging of myc-driven competition in the Drosophila pupal notum and in the wing imaginal disc, this study showed that the probability of elimination of loser cells correlates with the surface of contact shared with winners. As such, modifying loser-winner interface morphology can modulate the strength of competition. Elimination of loser clones requires winner-loser cell mixing through cell-cell intercalation. Cell mixing is driven by differential growth and the high tension at winner-winner interfaces relative to winner-loser and loser-loser interfaces, which leads to a preferential stabilization of winner-loser contacts and reduction of clone compactness over time. Differences in tension are generated by a relative difference in F-actin levels between loser and winner junctions, induced by differential levels of the membrane lipid phosphatidylinositol (3,4,5)-trisphosphate. These results establish the first link between cell-cell intercalation induced by a proto-oncogene and how it promotes invasiveness and destruction of healthy tissues (Levayer, 2015).

To analyse quantitatively loser cell elimination, long-term live imaging was performed of clones showing a relative decrease of the proto-oncogene myc in the Drosophila pupal notum, a condition known to induce cell competition in the wing disc. Every loser cell delamination was counted over 10 h, and the probability of cell elimination was calculated for a given surface of contact shared with winner cells. A significant increase was observed of the proportion of delamination with winner-loser shared contact, whereas this proportion remained constant for control clones. The same correlation was observed in ex vivo culture of larval wing disc. Cell delamination in the notum was apoptosis dependent and expression of flowerlose (fwelose), a competition-specific marker for loser fate, was necessary and sufficient to drive contact-dependent delamination. Moreover it was confirmed that contact-dependent death is based on the computation of relative differences of fwelose between loser cells and their neighbours. Thus, cell delamination in the notum recapitulates features of cell competition (Levayer, 2015).

This suggests that winner-loser interface morphology could modulate the probability of eliminating loser clones. Using the wing imaginal disc, winner-loser contact was reduced by inducing adhesion- or tension-dependent cell sorting and observed a significant reduction of loser clone elimination. This rescue was not driven by a cell-autonomous effect of E-cadherin (E-cad) or active myosin II regulatory light chain (MRLC) on growth, death or cell fitness but rather by a general diminution of winner-loser contact. Competition is ineffective across the antero-posterior compartment boundary, a frontier that prevents cell mixing through high line tension. Accordingly, there was no increase in death at the antero-posterior boundary in wing discs overexpressing fweloseA in the anterior compartment. However, reducing tension by reducing levels of myosin II heavy chains was sufficient to increase the shared surface of contact between cells of the anterior and posterior compartments, and induced fwelose death at the boundary. Altogether, it is concluded that the reduction in surface contact between winners and losers is sufficient to block competition, which explains how compartment boundaries prevent competition (Levayer, 2015).

Loser clones have been reported to fragment more often than controls, whereas winner clones show convoluted morphology, suggesting that winner-y actin levels, and F-actin levels were higher in WT clones than M-/+ cells. This prompted a test of the role of actin organization in winner-loser mixing. Downregulating the formin Diaphanous (Dia, a filamentous actin polymerization factor) by RNA interference (RNAi) or by using a hypomorphic mutant was sufficient to obtain a high proportion of fragmen-cell intercalation independent of death. To assess the contribution of each phenomenon, the proportion of clones fragmented 48 h after clone induction (ACI) was systematically counted. A twofold increase was observed in the frequency of split clones in losers (wild type (WT) in tub-dmyc) versus WT in WT controls. Overexpressing E-cad or active myosin II was sufficient to prevent loser clone splitting, whereas blocking apoptosis or blocking loser fate by silencing fwelose did not reduce splitting. Finally, the proportion of split clones was also increased for winner clones either during myc-driven competition (UAS-myc, UAS-p35) or during Minute-dependent competition (WT clones in M-/+ background). Altogether, this suggested that winner-loser mixing is increased independently of loser cell death or clone size by a factor upstream of fwe, and could be driven by cell-cell intercalation. Accordingly, junction remodelling events leading to disappearance of a loser-loser junction were three times more frequent at loser clone boundaries than control clone boundaries in the pupal notum. The rate of junction remodelling was higher in loser-loser junctions and in winner-winner junctions than in winner-loser junctions. The preferential stabilization of winner-loser interfaces should increase the surface of contact between winner and loser cells over time. Accordingly, loser clone compactness in the notum decreased over time whereas it remains constant on average for WT clones in WT background. Similarly, the compactness of clones in the notum also decreased over time for conditions showing high frequency of clone splitting in the wing disc, whereas clone compactness remained constant for conditions rescuing clone splitting. Altogether, it is concluded that both Minute- and myc-dependent competition increase loser-winner mixing through cell-cell intercalation (Levayer, 2015).

It was then asked what could modulate the rate of junction remodelling during competition. The rate of junction remodelling can be cell-autonomously increased by myc. Interestingly, downregulation of the tumour suppressor PTEN is also sufficient to increase the rate of junction remodelling through the upregulation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3). It was reasoned that differences in PIP3 levels could also modulate junction remodelling during competition. Using a live reporter of PIP3 that could detect modulations of PIP3 in the notum, a significant increase of PIP3 was observed in the apico-lateral membrane of tub-dmyc-tub-dmyc interfaces compared with WT-WT and WT-tub-dmyc interfaces. Moreover, increasing/reducing Myc levels in a full compartment of the wing disc was sufficient to increase/decrease the levels of phospho-Akt (a downstream target of PIP3, whereas fweloseA overexpression had no effect. Similarly, levels of phospho-Akt were relatively higher in WT clones than in the surrounding M-/+ cells. Thus differences in PIP3 levels might be responsible for winner-loser mixing (Levayer, 2015).

Accordingly, reducing PIP3 levels by overexpressing a PI3 kinase dominant negative (PI3K-DN) or increasing PIP3 levels by knocking down PTEN (UAS-pten RNAi) were both sufficient to induce a high proportion of fragmented clones and to reduce clone compactness over time in the notum, whereas increasing PIP3 in loser clones was sufficient to prevent cell mixing. Moreover, abolishing winner-loser PIP3 differences through larval starvation prevented loser clone fragmentation, the reduction of clone compactness over time in the notum and could rescue WT clone elimination in tub-dmyc background. It is therefore concluded that differences in PIP3 levels are necessary and sufficient for loser-winner mixing and required for loser cell elimination (Levayer, 2015).

It was then asked which downstream effectors of PIP3 could affect junction stability. A relative growth decrease can generate mechanical stress that can be released by cell-cell intercalation. Accordingly, growth reduction through Akt downregulation is sufficient to increase clone splitting and could contribute to loser clone splitting. However, Akt is not sufficient to explain winner-loser mixing because, unlike PIP3, increasing Akt had no effect on clone splitting. PIP3 could also modulate junction remodelling through its effect on cytoskeleton and the modulation of intercellular adhesion or tension. No obvious modifications of E-cad, MRLC or Dachs (another regulator of tension) was detected in loser cells. However, a significant reduction of F-actin levels and a reduction of actin turnover/polymerization rate were observed in loser-loser and loser-winner junctions in the notum. Similarly, modifying Myc levels in a full wing disc compartment was sufficient to modify actin levels, and F-actin levels were higher in WT clones than M-/+ cells. This prompted a test of the role of actin organization in winner-loser mixing. Downregulating the formin Diaphanous (Dia, a filamentous actin polymerization factor) by RNA interference (RNAi) or by using a hypomorphic mutant was sufficient to obtain a high proportion of fragmented clones and to reduce clone compactness over time, whereas overexpressing Dia in loser clones prevented clone splitting (UAS-dia::GFP) and compactness reduction. This effect was specific to Dia as modulating Arp2/3 complex (a regulator of dendritic actin network) had no effect on clone splitting. Thus, impaired filamentous actin organization was necessary and sufficient to drive loser-winner mixing. These actin defects were driven by the differences in PIP3 levels between losers and winners. Thus Dia could be an important regulator of competition through its effect on cell mixing. Overexpression of Dia was indeed sufficient to reduce loser clone elimination significantly (Levayer, 2015).

Filamentous actin has been associated with tension regulation. It was therefore asked whether junction tension was modified in winner and loser junctions. The maximum speed of relaxation of junction after laser nanoablation (which is proportional to tension) was significantly reduced in loser-loser and winner-loser junctions compared with winner-winner junctions. This distribution of tension has been proposed to promote cell mixing. Accordingly, decreasing PIP3 in clones reduced tension both in low-PIP3-low-PIP3 and low-PIP3-normal-PIP3 junctions, whereas overexpressing Dia in loser clones or starvation were both sufficient to abolish differences in tension, in agreement with their effect on winner-loser mixing and the distribution of F-actin. Thus the lower tension at winner-loser and loser-loser junctions is responsible for winner-loser mixing. Altogether, it is concluded that the relative PIP3 decrease in losers increases winner-loser mixing through Akt-dependent differential growth and the modulation of tension through F-actin downregulation in winner-loser and loser-loser junctions (Levayer, 2015).

Several modes of tissue invasion by cancer cells have been described, most of them relying on the departure of the tumour cells from the epithelial layer. This study suggests that some oncogenes may also drive tissue destruction and invasion by inducing ectopic cell intercalation between cancerous and healthy cells, and subsequent healthy cell elimination. myc-dependent invasion could be enhanced by other mutations further promoting intercalation (such as PTEN). Stiffness is increased in many tumours, suggesting that healthy cell-cancer cell mixing by intercalation might be a general process (Levayer, 2015).


Adey, N. B., et al. (2000). Threonine phosphorylation of the MMAC1/PTEN PDZ binding domain both inhibits and stimulates PDZ binding. Cancer Res. 60(1): 35-7. PubMed Citation: 10646847

Benmimoun, B., Polesello, C., Waltzer, L. and Haenlin, M. (2012). Dual role for Insulin/TOR signaling in the control of hematopoietic progenitor maintenance in Drosophila. Development 139: 1713-1717. Pubmed: 22510984

Brisbin, S., et al. (2009). A role for C. elegans Eph RTK signaling in PTEN regulation. Dev. Cell 17(4): 459-69. PubMed Citation: 19853560

Bohni, R., et al. (1999). Autonomous control of cell and organ size by CHICO, a Drosophila homolog of vertebrate IRS1-4. Cell 97(7): 865-75. PubMed Citation: 10399915

Byrne, A. B., Walradt, T., Gardner, K. E., Hubbert, A., Reinke, V. and Hammarlund, M. (2014). Insulin/IGF1 sgnaling inhibits age-dependent axon regeneration. Neuron 81: 561-573. PubMed ID: 24440228

Carreno, S., et al. (2008). Moesin and its activating kinase Slik are required for cortical stability and microtubule organization in mitotic cells. J. Cell Biol. 180: 739-746. PubMed Citation: 18283112

Centanin, L., Ratcliffe, P. J. and Wappner, P. (2005). Reversion of lethality and growth defects in Fatiga oxygen-sensor mutant flies by loss of Hypoxia-Inducible Factor-alpha/Sima. EMBO Rep. 6(11): 1070-5. 16179946

Chalhoub, N., Zhu, G., Zhu, X. and Baker, S. J. (2009). Cell type specificity of PI3K signaling in Pdk1- and Pten-deficient brains. Genes Dev. 23(14): 1619-24. PubMed Citation: 19605683

Chen, L., Wang, Z., Ghosh-Roy, A., Hubert, T., Yan, D., O'Rourke, S., Bowerman, B., Wu, Z., Jin, Y. and Chisholm, A. D. (2011). Axon regeneration pathways identified by systematic genetic screening in C. elegans. Neuron 71: 1043-1057. PubMed ID: 21943602

Chen, Z. H., Zhu, M., Yang, J., Liang, H., He, J., He, S., Wang, P., Kang, X., McNutt, M. A., Yin, Y. and Shen, W. H. (2014). PTEN interacts with histone H1 and controls chromatin condensation. Cell Rep 8: 2003-2014. PubMed ID: 25199838

Cheney, I. W., et al., (1998). Suppression of tumorigenicity of glioblastoma cells by adenovirus-mediated MMAC1/PTEN gene transfer. Cancer Res. 58: 2331-2334. PubMed Citation: 9622068

Christensen, R., et al. (2011). A conserved PTEN/FOXO pathway regulates neuronal morphology during C. elegans development. Development 138(23): 5257-67. PubMed Citation: 22069193

Crackower, M. A., et al. (2002). Regulation of myocardial contractility and cell size by distinct PI3K-PTEN signaling pathways. Cell 110: 737-749. 12297047

Dekanty, A., Lavista-Llanos, S., Irisarri, M., Oldham, S. and Wappner, P. (2005). The insulin-PI3K/TOR pathway induces a HIF-dependent transcriptional response in Drosophila by promoting nuclear localization of HIF-alpha/Sima. J. Cell Sci. 118(Pt 23): 5431-41. 16278294

Di Cristofano, A., Pesce, B., Cordon-Cardo, C. and Pandolfi, P. P. (1998). Pten is essential for embryonic development and tumour suppression. Nat. Genet. 19: 348-355. PubMed Citation: 9697695

Di Cristofano, A., et al. (1999). Impaired Fas response and autoimmunity in Pten+/- mice. Science 285(5436): 2122-5. PubMed Citation: 10497129

Feigin, M. E., Akshinthala, S. D., Araki, K., Rosenberg, A. Z., Muthuswamy, L. B., Martin, B., Lehmann, B. D., Berman, H. K., Pietenpol, J. A., Cardiff, R. D. and Muthuswamy, S. K. (2014). Mislocalization of the cell polarity protein Scribble promotes mammary tumorigenesis and is associated with basal breast cancer. Cancer Res [Epub ahead of print]. PubMed ID: 24662921

Frazier, M. R., Woods, H. A. and Harrison, J. F. (2001). Interactive effects of rearing temperature and oxygen on the development of Drosophila melanogaster. Physiol. Biochem. Zool. 74: 641-650. 11517449

Fukuyama, M., Rougvie, A. E. and Rothman, J. H. (2006). C. elegans DAF-18/PTEN mediates nutrient-dependent arrest of cell cycle and growth in the germline. Curr. Biol. 16(8): 773-9. 16631584

Funamoto, S., Meili, R., Lee, S., Parry, L. and Firtel, R. A. (2002). Spatial and temporal regulation of 3-phosphoinositides by PI 3-kinase and PTEN mediates chemotaxis. Cell 109: 611-623. 12062104

Furnari, F. B., et al., (1997). Growth suppression of glioma cells by PTEN requires a functional phosphatase catalytic domain. Proc. Natl. Acad. Sci. 94: 12479-12484. PubMed Citation: 9356475

Furnari, F. B., Huang, H. J. and Cavenee, W. K. (1998). The phosphoinositol phosphatase activity of PTEN mediates a serum- sensitive G1 growth arrest in glioma cells. Cancer Res. 58: 5002-5008. PubMed Citation: 9823298

Galloni, M., and Edgar, B. A. (1999). Cell-autonomous and non-autonomous growth-defective mutants of Drosophila melanogaster. Development 126: 2365-2375. PubMed Citation: 10225996

Gao, X., Neufeld, T. P. Pan, D. (2000). Drosophila PTEN regulates cell growth and proliferation through PI3K-dependent and -independent pathways. Dev. Biol. 221: 404-418. PubMed Citation: 10790335

Gao, X. and Pan, D. (2001). TSC1 and TSC2 tumor suppressors antagonize insulin signaling in cell growth. Genes Dev. 15: 1383-1392. 11390358

Gassama-Diagne, A., et al. (2006). Phosphatidylinositol-3,4,5-trisphosphate regulates the formation of the basolateral plasma membrane in epithelial cells. Nat. Cell Biol. 8(9): 963-70. Medline abstract: 16921364

Ghosh, A. K., et al. (1999). PTEN transcriptionally modulates c-myc gene expression in human breast carcinoma cells and is involved in cell growth regulation. Gene 235(1-2): 85-91. PubMed Citation: 10415336

Gil, E. B., Malone Link, E., Liu, L. X., Johnson, C. D. and Lees, J. A. (1999). Regulation of the insulin-like developmental pathway of Caenorhabditis elegans by a homolog of the PTEN tumor suppressor gene. Proc. Natl. Acad. Sci. 96: 2925-2930. PubMed Citation: 10077613

Goberdhan, D. C., et al. (1999). Drosophila tumor suppressor PTEN controls cell size and number by antagonizing the Chico/PI3-kinase signaling pathway. Genes Dev. 13(24): 3244-58. PubMed Citation: 10617573

Gu, J., Tamura, M. and Yamada, K. M. (1998). Tumor suppressor PTEN inhibits integrin- and growth factor-mediated mitogen-activated protein (MAP) kinase signaling pathways. J. Cell Biol. 143(5): 1375-83. PubMed Citation: 9832564

Gu, J., et al. (1999). Shc and FAK differentially regulate cell motility and directionality modulated by PTEN. J. Cell Biol. 146(2): 389-403. PubMed Citation: 10427092

Guldberg, P., thor Straten, P., Birck, A., Ahrenkiel, V., Kirkin, A. F. and Zeuthen, J. (1997). Disruption of the MMAC1/PTEN gene by deletion or mutation is a frequent event in malignant melanoma. Cancer Res. 57, 3660- 3663. PubMed Citation: 9288767

Guntur, A. R., et al. (2011). Conditional ablation of Pten in osteoprogenitors stimulates FGF signaling. Development 138(7): 1433-44. PubMed Citation: 21385768

Hamada, K., et al. (2005). The PTEN/PI3K pathway governs normal vascular development and tumor angiogenesis. Genes Dev. 19: 2054-2065. 16107612

Higuchi, M., et al. (2001). Akt mediates Rac/Cdc42-regulated cell motility in growth factor-stimulated cells and in invasive PTEN knockout cells. Curr. Biol. 11: 1958-1962. 11747822

Huang, H., et al. (1999). PTEN affects cell size, cell proliferation and apoptosis during Drosophila eye development. Development 126(23): 5365-72. PubMed Citation: 10556061

Iijima, M. and Devreotes, P. (2002). Tumor suppressor PTEN mediates sensing of chemoattractant gradients. Cell 109: 599-610. 12062103

Jiang, B. H., et al. (2001). Phosphatidylinositol 3-kinase signaling controls levels of hypoxia-inducible factor 1. Cell Growth Differ. 12(7): 363-9. 11457733

Jiang, H., et al. (2005). Both the establishment and the maintenance of neuronal polarity require active mechanisms: Critical roles of GSK-3 and its upstream regulators. Cell 120: 123-135. 15652487

Kim, J. W., et al. (2008). Retinal degeneration triggered by inactivation of PTEN in the retinal pigment epithelium. Genes Dev. 22(22): 3147-57. PubMed Citation: 18997061

Kimura, T., et al. (2003). Conditional loss of PTEN leads to testicular teratoma and enhances embryonic germ cell production. Development 130: 1691-1700. 12620992

Kunda, P., Pelling, A. E., Liu, T. and Baum, B. (2008). Moesin controls cortical rigidity, cell rounding, and spindle morphogenesis during mitosis. Curr. Biol. 18: 91-101. PubMed Citation: 18207738

Kuo, T. H., Fedina, T. Y., Hansen, I., Dreisewerd, K., Dierick, H. A., Yew, J. Y. and Pletcher, S. D. (2012). Insulin signaling mediates sexual attractiveness in Drosophila. PLoS Genet 8: e1002684. Pubmed: 22570625

Lee, J. O., et al. (1999). Crystal structure of the PTEN tumor suppressor: implications for its phosphoinositide phosphatase activity and membrane association. Cell 99(3): 323-34. PubMed Citation: 10555148

Leslie, N. R., Yang, X., Downes, C. P. and Weijer, C. J. (2007). PtdIns(3,4,5)P3-dependent and -independent roles for PTEN in the control of cell migration. Curr. Biol. 17(2): 115-25. Medline abstract: 17240336

Mamillapalli, R., et al. (2001). PTEN regulates the ubiquitin-dependent degradation of the CDK inhibitor p27KIP1 through the ubiquitin E3 ligase SCFSKP2. Curr. Bio. 11: 263-267. 11250155

Mirth, C., Truman, J. W., and Riddiford, L. M. (2005). The role of the prothoracic gland in determining critical weight for metamorphosis in Drosophila melanogaster. Curr. Biol. 15: 1796-1807. Medline abstract: 16182527

Levayer, R., Hauert, B. and Moreno, E. (2015). Cell mixing induced by myc is required for competitive tissue invasion and destruction. Nature 524: 476-480. PubMed ID: 26287461

Li, D. M. and Sun, H. (1997). TEP1, encoded by a candidate tumor suppressor locus, is a novel protein tyrosine phosphatase regulated by transforming growth factor beta. Cancer Res. 57: 2124-2129. PubMed Citation: 9187108

Li, D. M. and Sun, H. (1998). PTEN/MMAC1/TEP1 suppresses the tumorigenicity and induces G1 cell cycle arrest in human glioblastoma cells. Proc. Natl. Acad. Sci. 95: 15406-15411. PubMed Citation: 9860981

Li, G., et al. (2002). Conditional loss of PTEN leads to precocious development and neoplasia in the mammary gland. Development 129: 4159-4170. 12163417

Li, J., Yen, C., Liaw, D., Podsypanina, K., Bose, S., Wang, S. I., Puc, J., Miliaresis, C., Rodgers, L., McCombie, R., et al. (1997). PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 275: 1943-1947. PubMed Citation: 9072974

Li, J., Simpson, L., Takahashi, M., Miliaresis, C., Myers, M. P., Tonks, N. and Parsons, R. (1998). The PTEN/MMAC1 tumor suppressor induces cell death that is rescued by the AKT/protein kinase B oncogene. Cancer Res. 58: 5667-5672. PubMed Citation: 9865719

Li, L., Ernsting, B. R., Wishart, M. J., Lohse, D. L. and Dixon, J. E. (1997). A family of putative tumor suppressors is structurally and functionally conserved in humans and yeast. J. Biol. Chem. 272: 29403-29406. PubMed Citation: 9367992

Liaw, D., Marsh, D. J., Li, J., Dahia, P. L., Wang, S. I., Zheng, Z., Bose, S., Call, K. M., Tsou, H. C., Peacocke, M. et al. (1997). Germline mutations of the PTEN gene in Cowden disease, an inherited breast and thyroid cancer syndrome. Nat. Genet. 16: 64-67. PubMed Citation: 9140396

Liliental, J., et al. (2000). Genetic deletion of the Pten tumor suppressor gene promotes cell motility by activation of Rac1 and Cdc42 GTPases. Curr. Biol. 10: 401-404.. PubMed Citation: 10753747

Liu, H., Feng, X., Ennis, K. N., Behrmann, C. A., Sarma, P., Jiang, T. T., Kofuji, S., Niu, L., Stratton, Y., Thomas, H. E., Yoon, S. O., Sasaki, A. T. and Plas, D. R. (2017). Pharmacologic targeting of S6K1 in PTEN-deficient neoplasia. Cell Rep 18(9): 2088-2095. PubMed ID: 28249155

Liu, K., Lu, Y., Lee, J. K., Samara, R., Willenberg, R., Sears-Kraxberger, I., Tedeschi, A., Park, K. K., Jin, D., Cai, B., Xu, B., Connolly, L., Steward, O., Zheng, B. and He, Z. (2010). PTEN deletion enhances the regenerative ability of adult corticospinal neurons. Nat Neurosci 13: 1075-1081. PubMed ID: 20694004

Liu, W., James, C. D., Frederick, L., Alderete, B. E. and Jenkins, R. B. (1997). PTEN/MMAC1 mutations and EGFR amplification in glioblastomas. Cancer Res. 57: 5254-5257. PubMed Citation: 9393744

Ma, L., et al. (2005). Genetic analysis of Pten and Tsc2 functional interactions in the mouse reveals asymmetrical haploinsufficiency in tumor suppression Genes Dev. 19: 1779-1786. 16027168

Maehama, T. and Dixon, J. E. (1998). The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J. Biol. Chem. 273: 13375-13378. PubMed Citation: 9593664

Manning, B. D., et al. (2005). Feedback inhibition of Akt signaling limits the growth of tumors lacking Tsc2. Genes Dev. 19: 1773-1778. 16027169

Mao, J.-H., et al. (2004). Mutually exclusive mutations of the Pten and ras pathways in skin tumor progression. Genes Dev. 18: 1800-1805. 15289454

Mao, J. H., et al. (2008). FBXW7 targets mTOR for degradation and cooperates with PTEN in tumor suppression. Science 321(5895): 1499-502. PubMed Citation: 18787170

Marino, S., et al. (2002). PTEN is essential for cell migration but not for fate determination and tumourigenesis in the cerebellum. Development 129: 3513-3522. 12091320

Marsh, D.J., V. Coulon, K. Lunetta, P. Rocca-Serra, P. Dahia, Z. Zheng, D. Liaw, S. Caron, B. Duboue, A. Lin. (1998). Mutation spectrum agenotype-phenotype analyses in Cowden disease and Bannayan-Zonana Syndrome, two hamartoma syndromes with germline PTEN mutation. Hum. Mol.Genet. 7: 507-515. PubMed Citation: 9467011

Martin-Belmonte, F., et al. (2007). PTEN-mediated apical segregation of phosphoinositides controls epithelial morphogenesis through Cdc42. Cell 128: 383-397. Medline abstract: 17254974

Mihaylova, V. T., Borland, C. Z., Manjarrez, L., Stern, M. J. and Sun, H. (1999). The PTEN tumor suppressor homolog in Caenorhabditis elegans regulates longevity and dauer formation in an insulin receptor-like signaling pathway. Proc. Natl. Acad. Sci. 96: 7427-7432. PubMed Citation: 10377431

Miron, M., Lasko, P. and Sonenberg, N. (2003). Signaling from Akt to FRAP/TOR targets both 4E-BP and S6K in Drosophila melanogaster. Mol. Cell. Biol. 23(24): 9117-26. 14645523

Myers, M. P., Stolarov, J. P., Eng, C., Li, J., Wang, S. I., Wigler, M. H., Parsons, R. and Tonks, N. K. (1997). P-TEN, the tumor suppressor from human chromosome 10q23, is a dual- specificity phosphatase. Proc. Natl. Acad. Sci. 94: 9052-9057. PubMed Citation: 9256433

Myers, M. P., Pass, I., Batty, I. H., Van der Kaay, J., Stolarov, J. P., Hemmings, B. A., Wigler, M. H., Downes, C. P. and Tonks, N. K. (1998). The lipid phosphatase activity of PTEN is critical for its tumor supressor function. Proc. Natl. Acad. Sci. 95: 13513-13518. PubMed Citation: 9811831

Narbonne, P. and Roy, R. (2006). Inhibition of germline proliferation during C. elegans dauer development requires PTEN, LKB1 and AMPK signalling. Development 133(4): 611-9. 16407400

Neshat, M. N., et al. (2001). Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR. Proc. Natl. Acad. Sci. 98(18): 10314-9. 11504908

Ogg, S. and Ruvkun, G. (1998). The C. elegans PTEN homolog, DAF-18, acts in the insulin receptor-like metabolic signaling pathway. Mol. Cell 2: 887-893. PubMed Citation: 9885576

Oinuma, I., Ito, Y., Katoh, H. and Negishi, M. (2010). Semaphorin 4D/Plexin-B1 stimulates PTEN activity through R-Ras GTPase-activating protein activity, inducing growth cone collapse in hippocampal neurons. J. Biol. Chem. 285(36): 28200-9. PubMed Citation: 20610402

Oldham, S., et al. (2002). The Drosophila insulin/IGF receptor controls growth and size by modulating PtdInsP3 levels. Development 129: 4103-4109. 12163412

Paramio, J. M., et al. (1999). PTEN tumour suppressor is linked to the cell cycle control through the retinoblastoma protein. Oncogene 18(52): 7462-8. PubMed Citation: 10602505

Park, K. K., Liu, K., Hu, Y., Smith, P. D., Wang, C., Cai, B., Xu, B., Connolly, L., Kramvis, I., Sahin, M. and He, Z. (2008). Promoting axon regeneration in the adult CNS by modulation of the PTEN/mTOR pathway. Science 322: 963-966. PubMed ID: 18988856

Patel, L., et al. (2001). Tumor suppressor and anti-inflammatory actions of PPARgamma agonists are mediated via upregulation of PTEN. Cur. Biol. 11: 764-768. 11378386

Perry, J. M., et al. (2011). Cooperation between both Wnt/β-catenin and PTEN/PI3K/Akt signaling promotes primitive hematopoietic stem cell self-renewal and expansion. Genes Dev. 25(18): 1928-42. PubMed Citation: 21890648

Persad, S., et al. (2000). Inhibition of integrin-linked kinase (ILK) suppresses activation of protein kinase B/Akt and induces cell cycle arrest and apoptosis in PTEN null prostate cancer cells. Proc. Natl. Acad. Sci. 97: 3207-3212. 10716737

Persad, S., et al. (2001). Tumor suppressor PTEN inhibits nuclear accumulation of ß-Catenin and T cell/Lymphoid enhancer factor 1-mediated transcriptional activation. J. Cell Bio. 153: 1161-1174. 11402061

Pinal, N., Goberdhan, D. C., Collinson, L., Fujita, Y., Cox, I. M., Wilson, C. and Pichaud, F. (2006). Regulated and polarized PtdIns(3,4,5)P3 accumulation is essential for apical membrane morphogenesis in photoreceptor epithelial cells. Curr. Biol. 16(2): 140-9. 16431366

Podsypanina, K., et al. (1999). Mutation of Pten/Mmac1 in mice causes neoplasia in multiple organ systems. Proc. Natl. Acad. Sci. 96(4): 1563-8. PubMed Citation: 9990064

Podsypanina, K., et al. (2001). An inhibitor of mTOR reduces neoplasia and normalizes p70/S6 kinase activity in Pten+/- mice. Proc. Natl. Acad. Sci. 98(18): 10320-5. 11504907

Potter, C. J., Huang, H. and Xu, T. (2001). Drosophila Tsc1 Functions with Tsc2 to antagonize insulin signaling in regulating cell growth, cell proliferation, and organ size. Cell 105: 357-368. 11348592

Radimerski, T., Montagne, J., Rintelen, F., Stocker, H., van Der Kaay, J., Downes, C. P., Hafen, E., and Thomas, G. (2002a). dS6K-regulated cell growth is dPKB/dPI(3)K-independent, but requires dPDK1. Nat. Cell. Biol. 4: 251-255. 11862217

Radimerski, T., et al. (2002b). Lethality of Drosophila lacking TSC tumor suppressor function rescued by reducing dS6K signaling. Genes Dev. 16: 2627-2632. 12381661

Rasheed, B. K., Stenzel, T. T., McLendon, R. E., Parsons, R., Friedman, A. H., Friedman, H. S., Bigner, D. D. and Bigner, S. H. (1997). PTEN gene mutations are seen in high-grade but not in low-grade gliomas. Cancer Res. 57: 4187-4190. PubMed Citation: 9331072

Rintelen, F., Stocker, H., Thomas, G. and Hafen, E. (2001). PDK1 regulates growth through Akt and S6K in Drosophila. Proc. Natl. Acad. Sci. 98: 15020-15025. 11752451

Risinger, J. I., Hayes, A. K., Berchuck, A. and Barrett, J. C. (1997). PTEN/MMAC1 mutations in endometrial cancers. Cancer Res. 57: 4736-4738. PubMed Citation: 9354433

Rouault, J. P., Kuwabara, P. E., Sinilnikova, O. M., Duret, L., Thierry-Mieg,D. and Billaud, M. (1999). Regulation of dauer larva development in Caenorhabditis elegans by daf-18, a homologue of the tumour suppressor PTEN. Curr. Biol. 9: 329-332. PubMed Citation: 10209098

Roubinet, C., et al. (2011). Molecular networks linked by Moesin drive remodeling of the cell cortex during mitosis. J. Cell Biol. 195(1): 99-112. PubMed Citation: 21969469

Saucedo, L. J., et al. (2003). Rheb promotes cell growth as a component of the insulin/TOR signalling network. Nat. Cell Biol. 5(6):566-71. 12766776

Shaw, R. J., et al. (2004). The LKB1 tumor suppressor negatively regulates mTOR signaling. Cancer Cell 6: 91-99. 15261145

Shen, W. H., et al. (2007). Essential role for nuclear PTEN in maintaining chromosomal integrity. Cell 128(1): 157-70. Medline abstract: 17218262

Smith, A., et al. (1999). Alternative splicing of the Drosophila PTEN gene. Biochim. Biophys. Acta. 1447(2-3): 313-7. PubMed Citation: 10542333

Song, Y., Ori-McKenney, K. M., Zheng, Y., Han, C., Jan, L. Y. and Jan, Y. N. (2012). Regeneration of Drosophila sensory neuron axons and dendrites is regulated by the Akt pathway involving Pten and microRNA bantam. Genes Dev 26: 1612-1625. PubMed ID: 22759636

Stambolic, V., Suzuki, A., de la Pompa, J. L., Brothers, G. M., Mirtsos, C.,Sasaki, T., Ruland, J., Penninger, J. M., Siderovski, D. P. and Mak, T. W. (1998). Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell 95: 29-39. PubMed Citation: 9778245

Staveley, B. E., Ruel, L., Jin, J., Stambolic, V., Mastronardi, F. G., Heitzler, P., Woodgett, J. R., and Manoukian, A. S. (1998). Genetic analysis of protein kinase B (AKT) in Drosophila. Curr. Biol. 8: 599-602. PubMed Citation: 9601646

Steck, P. A., Pershouse, M. A., Jasser, S. A., Yung, W. K., Lin, H., Ligon, A. H., Langford, L. A., Baumgard, M. L., Hattier, T., Davis, T., et al. (1997). Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers. Nat. Genet. 15: 356-362. PubMed Citation: 9090379

Stocker, H., Andjelkovic, M., Oldham, S., Laffargue, M., Wymann, M. P., Hemmings, B. A., and Hafen, E. (2002). Living with lethal PIP3 levels: Viability of flies lacking PTEN restored by a PH domain mutation in Akt/PKB. Science 295: 2088-2091. 11872800

Stone, M. C., Nguyen, M. M., Tao, J., Allender, D. L. and Rolls, M. M. (2010). Global up-regulation of microtubule dynamics and polarity reversal during regeneration of an axon from a dendrite. Mol Biol Cell 21: 767-777. PubMed ID: 20053676

Sun, H., Lesche, R., Li, D. M., Liliental, J., Zhang, H., Gao, J., Gavrilova,N., Mueller, B., Liu, X. and Wu, H. (1999). PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3,4,5,-trisphosphate and Akt/protein kinase B signaling pathway. Proc. Natl. Acad. Sci. 96: 6199-6204. PubMed Citation: 10339565

Suzuki, A., de la Pompa, J. L., Stambolic, V., Elia, A. J., Sasaki, T., del Barco Barrantes, I., Ho, A., Wakeham, A., Itie, A., Khoo, W., et al. (1998). High cancer susceptibility and embryonic lethality associated with mutation of the PTEN tumor suppressor gene in mice. Curr. Biol. 8: 1169-1178. PubMed Citation: 9799734

Tamura, M., et al. (1998). Inhibition of cell migration, spreading, and focal adhesions by tumor suppressor PTEN. Science 280(5369): 1614-7. PubMed Citation: 9616126

Tamura, M., et al. (1999a). Tumor suppressor PTEN inhibition of cell invasion, migration, and growth: differential involvement of focal adhesion kinase and p130Cas. Cancer Res. 59(2): 442-9. PubMed Citation: 9927060

Tamura, M., et al. (1999b). PTEN interactions with focal adhesion kinase and suppression of the extracellular matrix-dependent phosphatidylinositol 3-kinase/Akt cell survival pathway. J. Biol. Chem. 274(29): 20693-703. PubMed Citation: 10400703

Tang, A. H., et al. (2001). Transcriptional regulation of cytoskeletal functions and segmentation by a novel maternal pair-rule gene, lilliputian. Development 128(5): 801-813. 11171404

Tapon, N., et al. (2001). The Drosophila Tuberous sclerosis complex gene homologs restrict cell growth and cell proliferation. Cell 105: 345-355. 11348591

Tashiro, H., Blazes, M. S., Wu, R., Cho, K. R., Bose, S., Wang, S. I., Li, J., Parsons, R. and Ellenson, L. H. (1997). Mutations in PTEN are frequent in endometrial carcinoma but rare in other common gynecological malignancies. Cancer Res. 57: 3935-3940. PubMed Citation: 9307275

Tokusumi, Y., Tokusumi, T., Shoue, D. A., Schulz, R. A. (2012). Gene regulatory networks controlling hematopoietic progenitor niche cell production and differentiation in the Drosophila lymph gland. PLoS One 7(7):e41604. PubMed Citation: 22911822

Trimboli, A. J., et al. (2009). Pten in stromal fibroblasts suppresses mammary epithelial tumours. Nature 461(7267): 1084-91. PubMed Citation: 19847259

Trotman, L. C., et al. (2007). Ubiquitination regulates PTEN nuclear import and tumor suppression. Cell 128(1): 141-56. Medline abstract: 17218261

Verdu, J., Buratovich, M. A., Wilder, E. L., and Birnbaum, M. J. (1999). Cell-autonomous regulation of cell and organ growth in Drosophila by Akt/PKB. Nat. Cell Biol. 1: 500-506.. PubMed Citation: 10587646

Vereshchagina, N. and Wilson, C. (2006). Cytoplasmic activated protein kinase Akt regulates lipid-droplet accumulation in Drosophila nurse cells. Development 133(23): 4731-5. Medline abstract: 17079271

von Stein, W., Ramrath, A., Grimm, A., Muller-Borg, M. and Wodarz, A. (2005). Direct association of Bazooka/PAR-3 with the lipid phosphatase PTEN reveals a link between the PAR/aPKC complex and phosphoinositide signaling. Development 132(7): 1675-86. 15743877

Yang, G., et al. (2008). PTEN deficiency causes dyschondroplasia in mice by enhanced hypoxia-inducible factor 1alpha signaling and endoplasmic reticulum stress. Development 135(21): 3587-97. PubMed Citation: 18832389

Wang, S. I., Puc, J., Li, J., Bruce, J. N., Cairns, P., Sidransky, D. and Parsons, R. (1997). Somatic mutations of PTEN in glioblastoma multiforme. Cancer Res. 57: 4183-4186. PubMed Citation: 9331071

Wang, X., et al. (2007). NEDD4-1 is a proto-oncogenic ubiquitin ligase for PTEN. Cell 128(1): 129-39. Medline abstract: 17218260

Weng, L. P., et al. (1999). PTEN suppresses breast cancer cell growth by phosphatase activity-dependent G1 arrest followed by cell death. Cancer Res. 59(22): 5808-14. PubMed Citation: 10582703

Wessells, R. J., Fitzgerald, E., Cypser, J. R., Tatar, M. and Bodmer, R. (2004). Insulin regulation of heart function in aging fruit flies. Nat. Genet. 36(12): 1275-81. 15565107

Wittwer, F., et al. (2001). Lilliputian: an AF4/FMR2-related protein that controls cell identity and cell growth. Development 128(5): 791-800. 11171403

Wu, X., K. Senechal, M.S. Neshat, Y.E. Whang, and C.L. Sawyers. (1998). The PTEN/MMAC1 tumor suppressor phosphatase functions as a negative regulator of the phosphoinositide 3-kinase/Akt pathway. Proc. Natl. Acad. Sci. 95: 15587-15591. PubMed Citation: 9861013

Wu, X., et al. (2000). Evidence for regulation of the PTEN tumor suppressor by a membrane-localized multi-PDZ domain containing scaffold protein MAGI-2. Proc. Natl. Acad. Sci. 97(8): 4233-8.. PubMed Citation: 10760291

Yue, Q., et al. (2005). PTEN deletion in Bergmann glia leads to premature differentiation and affects laminar organization. Development 132: 3281-3291. 15944184

Zhang, C., Comai, L. and Johnson, D. L. (2005). PTEN represses RNA Polymerase I transcription by disrupting the SL1 complex. Mol. Cell. Biol. 25(16): 6899-911. 16055704

Pten: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 30 October 2015

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